CRC subtypes that can predict the outcome of colorectal cancer (CRC) in patients with immunogenicity seems to be a promising strategy to develop new drugs that target the antitumoral immune response. In particular, the disinhibition of the antitumoral T‐cell response by immune checkpoint blockade has shown remarkable therapeutic promise for patients with mismatch repair (MMR) deficient CRC. In this review, the authors provide the update of the molecular features and immunogenicity of CRC, discuss the role of possible predictive biomarkers, illustrate the modern immunotherapeutic approaches, and introduce the most relevant ongoing preclinical study and clinical trials such as the use of the combination therapy with immunotherapy. Furthermore, this work is further to understand the complex interactions between the immune surveillance and develop resistance in tumor cells. As expected, if the promise of these developments is fulfilled, it could develop the effective therapeutic strategies and novel combinations to overcome immune resistance and enhance effector responses, which guide clinicians toward a more “personalized” treatment for advanced CRC patients.Assessing the  Colorectal cancer (CRC) cells may escape immune surveillance and develop resistance to immunotherapy by acquiring genetic alterations. Consequently, some patients exhibit primary or acquired resistance The previous studies have focused on somatic tumor mutations and tumor immunogenicity, and have known about the expression of gene phenotype that associated regulatory mechanisms on the impact of immune responses in CRC Among these strategies, assessing the CRC subtypes that can predict the outcome of CRC in patients with immunogenicity, seem to be promising to enhance the immune response for antitumor Subtype based on gene expression has been developed as a novel technical method for assessing the stratification of disease Furthermore, it has been proved that the elucidated molecular subtypes can predict therapeutic responses for CRC β) Collectively, CRC is currently classified into four CMS and a fifth unclassified group To improve CRC patient classification and identify putative molecular targets for immunotherapy, large collections of transcriptomic databases from tumor specimens have been generated β signal matrix reconstruction, and upregulation of integrin‐β3 and immune  pathway, upregulation, angiogenesis, TGF‐Despite the immunogenicity of these subtypes, the tumor is known to establish several mechanisms to escape immune surveillance Notably, MSI‐high tumors have a mutational rate 20 times higher than microsatellite stability (MSS) tumors, reflecting the tendency to express a higher load of neoantigens, thus improving the response to immunotherapy γ‐interferon (IFN‐γ)‐dominant immune profile, positively correlated with relapse‐free and overall survival Galon and colleagues first demonstrated the relevance of specific immune signatures in the prognosis of early‐stage CRC A clinical translation of these findings was to establish a scoring system, called “immunoscore,” which based on two distinct types of lymphocyte populations (CD8+ CTLs and CD45RO memory T cells) at the tumor center and at its invasive margin β and CXCL12, and high expression of genes encoding chemokines that attract myeloid cells, including chemokine (C–C motif) ligand 2 (CCL2) and the related cytokines IL‐23 and IL‐17, which are known carcino‐genic drivers in colitis‐associated CRC The biological link between the inflamed immune CRC subtype is characterized by marked upregulation of immunosuppressive factors, such as TGF‐Taken together, these findings suggest that the molecular CRC subtypes might be associated with specific clinical outcomes and the relevance of specific immune signatures in the prognosis of early‐stage CRC, molecular subtype of colorectal cancer may lead to novel approaches and personalized treatments. The biological link between the inflamed immune CRC subtype is characterized by marked upregulation of immunosuppressive factors which may be a promising chemopreventive and/or chemotherapeutic strategy against CRC Fig. . HoweverIn CMS1 subtypes of CRC, there are some studies that showed the reduced expression of the EGFR ligands amphiregulin (AREG) and epiregulin (EREG), and this reduced expression is linked to hypermethylation of the ligands' promoter regions On the contrary, RAS wild‐type tumor with a mesenchymal phenotype seems to be intrinsically resistant to anti‐EGFR agents in preclinical models. In fact, retrospective biomarker analyses of a patient cohort in the chemotherapy‐refractory setting and a randomized clinical trial in the chemonaive setting suggest no benefit of treatment with cetuximab in patients with mesenchymal‐like tumors β signaling inhibitors to block the crosstalk between cancer cells and the microenvironment was shown to halt disease progression of stromal‐enriched poor prognosis CRC tumors β receptor (TGFR) inhibitor has already moved to clinical trials in patients whose tumors test positive for a “TGF‐β activated” signature as part of project in metastatic CRC Notably, the use of TGF‐αv was particularly effective in patients whose tumors displayed high integrin‐αvβ6 expression levels Nevertheless, the effort to discover the potential targets that may increase the efficacy of EGFR mAbs in the RAS wild‐type CMS4 population includes the drivers of EMT and treatment resistance, such as mesenchymal–epithelial transition (MET) and integrins Both at gene and protein level heterogeneously expressed within KRAS‐mutated colorectal tumors, with unique metabolic dependencies in tumors with a CMS3‐dominant phenotype 18F‐FDG accumulation in patients with metastatic CRC Recent studies have shown a strong causal association between KRAS mutations and In the GLUT‐1 mRNA overexpression with KRAS mutation‐positive tumors, the KRAS oncogene is upregulated in the first steps in the pathway of glucose metabolism. Therefore, the global energy metabolism of cancer cells could be controlled by KRAS mutations to promote glucose uptake, while the control toward Warburg effect is critical to connect tumor cells with complex genetic changes, such as PI3K, AKT, Myc, HIF‐1, p53 + conversion. Compared with normal mucosa, it has been found that liver metastatic tissues have the higher levels of LDH‐A expression than in the primary colorectal cancers, with its remarkably upregulated LDH‐A levels Moreover, the increased expression of PKM2 protein is closely related to serum CEA level and TNM stage, and the prognosis of patients with CRC is poor. At the same time, PKM2 protein expression is the independent prognostic factor of the overall survival (OS) of patients with CRC These findings suggested the critical role of LDH‐A in the invasive colorectal cancers to maintain an efficient glycolysis in glycolytic phenotype. The changes in LDH level were correlated with the overall burden of tumor in CRC. At the same time, the level of LDH was observed to be unusually high in the detection of pseudoprogression. Clinical study has reported on the effect of LDH on the pseudo and real progress in immune‐targeting therapy.Remarkably, high‐dose vitamin C has been shown to impair CRC tumor growth in mouse models by causing oxidative stress mediated the increased uptake of the oxidized vitamin C through GLUT‐1 and this inhibited glycolysis at glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) to result in an energy crisis and ultimately cell death Toll‐like receptor (TLR) 3, the family member of the toll‐like receptor family of the host innate immune system, is the pattern recognition theme of dsRNA pathogens There are several TLR agonists that are rarely approved by the FDA as an adjuvant to immune stimulation for patients with cancer The commercial HEK‐BlueTM‐hTLR3 cell is expressed through TLR3, which is the subject of host pattern recognition and is responsible for the detection of the virus. In addition, the investigation also showed that the effective expression of the host TLR3 was established by establishing a robust innate immune response with the KRAS‐mutant HCT116 cell line These findings clearly show that the inhibition of TLR3 expression inhibits the KRAS‐mutant cancer cells into a better therapeutic target for the oncolytic reovirus. This study needs to describe TLR3 at the molecular level in the process of re virus infection and reproduction mechanism, and special emphasis on the condition of KRAS‐mutant CRC, in search of a better method for the treatment of CRC with KRAS mutations.Whether the relevant features of CRC subclassification, stromal and immune cells may still predict the responses of differential drug remains unknown. This may be because the drugs themselves, which can have a promiscuous mechanism of action, may not be tracking and single way descriptors, or we will not be able to correctly define the involved pathways or crosstalk using static omics data These above results laid the foundation for future research, and the study assessed the presumption of tumor‐specific antigen expression of related genes and regards it as the prognosis of metastatic tumor biomarkers, which especially classified as “mesenchymal” or “metabolic” tumors. In addition, studies have shown that personalized immunotherapy can provide treatment opportunities for patients with MSS CRC tumors. With regard to CMS classification in a research setting, the available models need to be optimized for subtype prediction on tissues in which microenvironment content is different from primary colorectal tumors, such as metastatic lesions and PDXs Overall, advances in patient stratification and drug development strategies have to be rapidly translated from the metastatic to the adjuvant setting Immunotherapy has led to clinical benefits in some cancer patients' therapy. However, in cancer treatment, the great challenge is developing disease progression and/or drug‐resistant diseases after treatment. The first kind of resistance is a special form of Darwinian natural selection that comes from before treatment intervention in the tumor mass pre‐existing genetic or epigenetic traits selected γ secretion when induced cancer cells, so the T cell destroys the tumor cells in vivo model An obvious example of this resistance is the PD‐L1 expression in response to the IFN‐The long‐term clinical benefits of the mutation‐derived neoantigens may be predicted by anti‐CTLA4 (ipilimumab) and the anti‐PD‐L1 (pembrolizumab) treatment One of the early examples of such a high degree of microsatellite instability (MSI—high) in CRC is associated with intense T‐cell infiltration, because of the MSI‐high tumor frameshift mutation and truncated protein (neopeptides) caused by mismatch repair defects, the antitumor T‐cell‐mediated adaptive immunity It appears that the tumor cells may escape immune surveillance by acquiring different genetic alterations. Indeed, some patients exhibit an innate resistance to immunotherapy. A higher expression of mesenchymal transition genes , immunosuppressive genes , and chemokines that recruit immunosuppressive cells  may be related to innate anti‐PD‐1 resistance Because anti‐PD‐L1 treatment improved significantly, however, the immune therapy faced many problems Epithelial interstitial transition (EMT) is the basic process of distant metastasis of cancer. During EMT, epithelial cells expressed mesenchymal genes that transformed their phenotypes from the epithelium to mesenchymes, allowing the cells to invade the tumor matrix and blood vessels. Studies have shown that the excessive expression of snail not only accelerated the invasion in cancer cells, but also produced to induce immune regulation of dendritic cells (regDC) induced immunosuppression. The diversity of immunocheckpoint inhibitors for patients with colorectal cancer may be due to heterogeneity and heterogeneity in tumors Immunological scoring, classification of the type, location, and number of tumor infiltrating lymphocytes (TILs), has been revealed as one of the strongest prognostic markers of CRC Interestingly, the number of TILs and MSI status do not fully predict the prognosis of patients with CRC. One set of CRCs is described as having a characteristic of high lymphogenetic expression, and somewhat unexpected and is associated with poor prognosis Studies have identified a unique microenvironment in immunology of CRC hepatic metastasis in the context of increased cytokines and chemokines in macrophages Resistance to chemotherapy and immune‐suppressive milieu around the tumor cells remain major obstacles in effective anticancer treatments β functions but also immune cells are mediated by its effects, such as inhibition of CTLs, TH1 cells, and NK cells, and expansion of Treg cells, B cells, and MDSCs The idea is to overcome multiple mechanisms that mediate immune tolerance to self‐antigens and block the intense immunosuppressive response in the tumor microenvironment γ response β‐mediated mesenchymal tumors include pharmacological elimination of MDSCs or blockade of related immunosuppressive chemokine signaling circuits and pathways, as demonstrated in other malignancies with an immune‐evasive microenvironment CTLs can be found in tumor edge or tumor core For poorly immunogenic or immune‐ignorant CRC tumors, complementary therapeutic approaches to checkpoint inhibitors are also needed More specifically, immunotherapy alone appears to have modest success, likely due to the complexity of the TME. Therefore, recent trials have been evaluating novel combined approaches, such as immune‐chemotherapy or combo immunotherapy, that could be more effective than chemotherapy or immunotherapy alone It has been observed that MEK inhibition induced the accumulation of T cells within the tumor cells and the major histocompatibility complex (MHC) class I upregulation in mouse models, and controlled the synergy and immune checkpoints, promoted sustainable tumor regression It will be interesting to verify whether combining immunotherapy with chemotherapy and/or biological therapies (anti‐EGFR or anti‐VEGF) could produce a synergistic effect in CRC. Obviously, many clinical trials would require to evaluate the efficacy and safety of these novel approaches.It would also be useful to understand how to enhance immunotherapy, increasing the effector response and reducing the inflammatory component. Indeed, tumor cells can exploit inflammation for cancer promotion. COX‐2 deregulation plays a pivotal role in tumor cells. Unlike COX‐1 that is expressed constitutively in most cells, COX‐2 is produced in response to growth factors and cytokines Preclinical studies found that COX inhibition could enhance the efficacy of anti‐PD‐1 blockade Recent studies have shown that photodynamic therapy (PDT) treatment has the ability to activate the tumor‐specific immune responses by producing tumor‐associated antigens from tumor cell residues, which afterward may be processed by APCs such as DCs and then presented to T cells The PDT destruction of primary tumors would generate a pool of tumor‐associated antigens to trigger specific immune responses, which were then amplified by UCNP‐Ce6‐R837‐based PDT as the immune adjuvant, which combined with T‐lymphocyte‐associated protein 4 (CTLA‐4) blockade would effectively induce the generation of TEM‐based immune memory response to prevent tumor relapse, similar to the functions of cancer vaccines. More significantly, PDT with UCNP‐Ce6‐R837 in combination with the CTLA‐4checkpoint blockade not only showed excellent efficacy in eliminating tumors exposed to the NIR laser but also resulted in strong antitumor immunities to inhibit the growth of distant tumors left behind after PDT treatment. Furthermore, such a cancer immunotherapy strategy has a long‐term immune memory function to protect treated mice from tumor cell challenge In summary, this work has demonstrated the great potency of integrating UCNP‐based PDT with cancer immunotherapy to realize a remarkable synergistic therapeutic outcome in eliminating primary tumors, inhibiting distant tumors, and preventing tumor relapse. While immunotherapy has become a highly promising paradigm for cancer treatment in recent years, it has long been recognized that PDT has the ability to trigger antitumor immune responses. However, conventional PDT triggered by visible light has limited penetration depth, and its generated immune responses may not be robust enough to eliminate tumors.Curcuma longa. More and more evidences suggest that curcumin may be an effective chemical reagent, and its targets are the various molecular signaling pathways involved in the carcinogenesis γ expression in CD4+ T cells γ expression in the CD4+ T cells. As Th1 cells are one of the important antitumor effector cells Curcumin is the main active ingredient of the golden spice γ‐producing Th1 cells. Lee et al. have found that such a conversion was mediated by dendritic cell‐derived molecules, which interacted with the Toll‐like receptors on Tregs γ was increased in the cells, and the nuclear translocation of p65 and c‐Rel was markedly decreased, which was critical for Foxp3 and CD25 expression after curcumin stimulation It has been proved that another in‐depth mechanism by which the curcumin therapy suppressed the levels of Foxp3 in Tregs A large number of studies have been conducted on drugs that may block immune factors, such as LAG‐3 or indoleamine 2,3‐dioxygenase (IDO), which were studied in phase I trials, combined with PD‐L1 or PD‐1inhibitors Drugs that act as direct immune stimulators, such as 4‐1BB (CD137) and KIR, have also been studied in a variety of combinations, including PD‐1inhibitor. Anti‐PD1 and anti‐CD137 mAb act on T cells that express these receptors on their plasma membrane presumably as a consequence of an antigencognate activation process There are multiple immunomodulatory compounds and additional checkpoint in the development and phase I survey β and COX‐2 which are both affected on tumor cells and immune cells in CRC via blockade of related immunosuppressive signaling pathways. In addition, the novel combined approaches, such as immune‐chemotherapy or combo immunotherapy, that could be more effective than chemotherapy or immunotherapy alone  coupled with chemotherapeutic melphalan has been shown synergistic therapeutic effects toward different types of tumors The crosstalk between NK and DCs might be a crucial point in the regulation of the whole immune defense against tumors and viruses Taken together, these data indicate that NK/DC crosstalk stimulates DCs to promote Th cell proliferation and maturation, which in turn “license” cytotoxic T cells, which are the final effectors. This NK‐DC‐Th‐Tc mechanism could also be functional when L19mTNF is used in combination with IL‐2 One of the main choice treatments for colorectal cancer is chemotherapy using multiple anticancer drugs, such as irinotecan, oxaliplatin, and 5‐fluorouracil (5FU) It has been observed that both oxaliplatin and 5‐FU are considered to have a good effect Collectively, selective and effective regulating antitumor T‐cell immune‐specific responses for anticancer therapy are essential to inhibit CRC cells, and TNF is used as an immune‐cytokine tumor necrosis factor in combination with chemotherapeutic agents, and could provide clues for sensitizing resistant tumors to immune checkpoint blockade therapy. Moreover, the novel stratergy to combine effects of chemotherapy and anti‐angiogenic agents on immune checkpoint therapy by provoking an adaptive immune response have been listed Table . Future The recently developed immunotherapeutic strategies have yielded remarkable clinical results in many types of tumors including CRC, indicating that indeed a patient's immune system can mount an immune response, which is effective in controlling tumor growth Specifically, DNA MMR and MSI status is now clinically significant to determining whether patients may be eligible for immunotherapy in clinical trials, but the potential predictive factors in MSS patients have been ignored. To date, the predictive role of the differential expression of PD‐1 and PD‐L1 has not been completely clarified, although some evidence suggests that high expression correlates with a better immunotherapy efficacy. As far as we know, only MSI‐H CRC tumors respond to checkpoint inhibition. In this review, the basis for this has been explained and the results are obtained thus far. This apparent limitation could be seen as a point of strength. Although the only available data have come from phase II trials, as phase III trials are currently ongoing, the results achieved so far are exciting Currently, most immunotherapies are still in early‐phase clinical testing for CRC, but their successful use in other types of cancers suggests that they may ultimately prove useful for CRC as well. As the field of immunotherapy continues to evolve, a more comprehensive understanding of drug resistance mechanisms will be mandatory, leading to the development of new strategies to overcome major and acquired resistance to anti‐PD/PDL‐1 antibodies. The inability of the immunotherapeutic strategies used to eradicate cancer‐initiating cells (CICs) that escapeimmune recognition and destruction may give rise to new tumors in the same organ site or through the metastatic colonization in other anatomic sites. Accordingly, identification of novel therapeutic approaches that can eradicate CICs is a major challenge in the CRC therapy area. In consequence, an improved understanding of the interactions of CICs with immune system and with tumor microenvironment may contribute to optimize the available therapies and to design novel combination treatments for CRC therapy.Importantly, rational combinations of targeted therapies will be required to achieve meaningful effects in different subtypes, with overlapping toxic effects that may further complicate the biomarker–drug codevelopment path. In the setting of actionable genomic alterations detected in tumors samples or ctDNA, for example, an additional layer of complexity is the rarity of most events (such as ERBB2 and MEK1 mutations) and the need to adapt therapies accordingly upon progression. To improve clinical benefit further, it is crucial to understand how residual disease is sustained and how it can be therapeutically tackled. The potential impact of immunotherapy on the cells of resistant clones and the lack of target gene modification is being studied extensively. These combinations include small‐molecule, inhibitors of immunosuppression immunomodulators, and T‐cell costimulatory agents, chemokines, vaccination, targeted agents, cytotoxic drugs, and radiation therapy.The regulatory effector T cells have been developed using CRC in treatment and will be more advanced in overcoming immune avoidance mechanisms and survival in the tumor immune‐suppressive environment. In addition to technical challenges, there is also lack of understanding of the specific steps to promote tumor development, tumor malignancy, and its eventual transfer to resistance. There are a number of mechanisms that provide metabolic pathways; however, whether these pathways may be the upstream signal transduction mechanism is not yet understood. Therefore, the future work should not be focused on the casual switch to the T‐cell response, but try to discover the regulatory reaction of the T cells based on the necessary immune mechanisms. This immunotherapeutic strategy when developed could exhibit effective immune response and minimize the undesirable effects. Additionally, future efforts should focus on specific tumor antigen recognition by employing the method of highly personalized, as well as developing efficient lymphodepleting scheme before the T‐cell transfers. This could be effective in combination with other treatments, such as molecular agents against relying on oncogenes of tumor and strong regulators in host immune.The authors report no conflicts of interests in this work.


DNA sequencing data to investigate the interaction patterns between mycoheterotrophic plants and arbuscular mycorrhizal fungi. We find no phylogenetic signal on the number of fungal hosts nor on the fungal hosts shared among mycoheterotrophic plants. However, we observe a potential trend toward increased phylogenetic diversity of fungal hosts among mycoheterotrophic plants with increasing overlap in their fungal hosts. While these patterns remain for groups of plants regardless of location, we do find higher levels of overlap and diversity among plants from the same location. These findings suggest that species coexistence cannot be fully understood without attention to the two sides of ecological interactions.The vast majority of plants obtain an important proportion of vital resources from soil through mycorrhizal fungi. Generally, this happens in exchange of photosynthetically fixed carbon, but occasionally the interaction is mycoheterotrophic, and plants obtain carbon from mycorrhizal fungi. This process results in an antagonistic interaction between mycoheterotrophic plants and their fungal hosts. Importantly, the fungal‐host diversity available for plants is restricted as mycoheterotrophic interactions often involve narrow lineages of fungal hosts. Unfortunately, little is known whether fungal‐host diversity may be additionally modulated by plant–plant interactions through shared hosts. Yet, this may have important implications for plant competition and coexistence. Here, we use  Therefore, this association represents one of the most ancient and abundant mycorrhizal interaction among plants on a global scale . Next‐generation DNA sequencing of each root sample was used to identify the arbuscular mycorrhizal hosts that can be potentially associated with each MH plant species. We sequenced the ITS2 region using the primers fITS7 (5′‐GTGARTCATCGAATCTTTG‐3′) . We combined the resulting trees based on divergence ages taken from Magallón, Gómez‐Acevedo, Sánchez‐Reyes, and Hernández‐Hernández .To generate the host phylogenetic tree, we used an alignment with the 138 Glomeromycota fungal OTUs with MAFFT 7.017 , we computed the Mantel test correlation between the phylogenetic distance matrix between plants and the dissimilarity matrix between the number of fungal hosts per plant. The phylogenetic distances were extracted from the plants phylogenetic tree, and the dissimilarity matrix was calculated by Cij)/(di + dj), where Cij is the number of shared hosts between plant i and j, and di and dj are the number of fungal hosts associated with MH plant i and j, respectively. Note that the Bray–Curtis measure corresponds to the number of shared fungal hosts relative to the total number of fungal hosts. The second measure we used is the overlap measure Cij/min, where the parameters are the same as above and min refers to the smallest of the two values . Separately, phylogenetic signal on the shared fungal hosts was investigated among MH plants species that belong to the same location.To calculate the phylogenetic signal on the shared fungal hosts among MH plants, we computed the Mantel test correlation between the phylogenetic distance matrix between plants and two dissimilarity matrices between the shared hosts. The phylogenetic distance matrix is the same as above, whereas the dissimilarity matrices here were calculated using two different measures. The Bray–Curtis measure 1 − (2PD′=(PD−PDmin)/(PDmax−PDmin), where PDmax and PDmin correspond, respectively, to the maximum and minimum PD values that can be generated from all the possible combinations of fungal hosts. These combinations are generated by creating groups of fungal hosts of the same number as in the observed case, but the identity of the hosts is changed using the pool of the 138 possible fungi. The MH plants from our study were only found to associate with these 138 fungi, which represent a subset of the total fungal diversity available in the soil. Note that this scaling does not assume a particular generative process, rather it compares the observed phylogenetic diversity to all the possible outcomes with the same number of fungal hosts.For each MH plant, the observed fungal‐host diversity was calculated using the phylogenetic diversity (PD) of the observed hosts. Phylogenetic diversity was calculated by summing up the branch lengths in the fungal‐host phylogenetic tree among all the fungal hosts associated with the MH plant or group. Because the number of fungal hosts determines the branch length, we normalized the PD by calculating the scaled PD as 2.3n plant species using the 20 MH collected species. We generated artificial groups with two, three, four, and five MH species (mimicking the size of the observed communities in the field).We investigated the diversity and overlap patterns among observed co‐occurring MH plants in the field, as well as among the artificially generated groups. In particular, we observed six communities of MH plants that were found to be co‐occurring in the field. To maximize the possibility of co‐occurrence and to avoid small‐scale niche segregation of mycorrhizal communities , where Cij represents the number of fungal hosts shared between MH plant i and j that belong to a given community/group, min refers to the smallest of the two values, and the summation is done over all possible pairs of MH plants  of the fungal hosts that can be associated with a given community/group of MH plants. Similarly, to investigate fungal‐host overlap among MH plants, we calculated the overlap of fungal hosts among MH plants in a given community/group. This overlap is again calculated as Finally, to investigate the spatial influence of our sampling in the observed patterns of fungal hosts in MH plants, we compared the scaled PD and scaled overlap between MH plants belonging to the same location and MH plants belonging to different locations. Because in nine of the fifteen localities we visited, we found more than one MH plant species see Fig. , we gene33.1r=−.050, p=.766, df=19) nor on the fungal hosts shared among plants . Looking at the MH plants that belong to the same location  nor on the fungal hosts shared among plants . Overall, these findings reveal an important variability in MH interactions that can be driven by mechanisms other than evolutionary relationships.We found that the number of fungal hosts in each of the 20 MH plant species varies from 2 to 42  lie in the upper half of the range of possible phylogenetic diversity values  and in the artificially generated groups . This positive relationship does not depend on group size .We find that on average the fungal‐host diversity  is proportional to fungal‐host overlap  in groups of MH plants. This pattern was present in both the observed communities in the field Figure a and in The results above are also qualitatively the same if scaled PD and scaled overlap values are replaced by their raw values while controlling for the total number of fungal hosts. Because the number of specimens and the OTU richness per MH species are variable among samples and may influence the results . We have found that individual MH plants have a tendency to exploit more distantly related fungi than expected by chance. This tendency of targeting distantly related fungi has been described in autotrophic plants (Giovannetti, Sbrana, Avio, & Strani, Building on niche theory, our results may reflect a MH plant strategy to increase its fungal‐host diversity or niche width, as species with a wider niche may be more likely to obtain different resources and to establish successfully in new habitats (Levine & HilleRisLambers, We have found that in communities of co‐occurring MH plant species in the field the fungal‐host diversity among MH plants appear to increase proportionally to their fungal‐host overlap. This same tendency was confirmed among the artificially generated groups of MH plants showing that the patterns observed are not an artifact of the reduced number of MH communities observed in the field. Moreover, we have found that both fungal‐host diversity and overlap are significantly higher among plants that belong to the same geographic location, which could provide an explanation for the lack of phylogenetic signal on the fungal hosts among MH plants. These results indicate that fungus‐plant interactions can be better explained by understanding plant–plant interactions generated by sharing resources or fungal hosts. Future studies could explain whether this symmetry between fungal‐host diversity and overlap may respond to an ecological mechanism driven by maximizing co‐occurrence and avoiding competitive exclusion among MH plants.A potential bias in our study is the use of ITS2 sequences and future work should consider expanding these sequences (see The authors declare no competing financial interests.Data are publicly available as Supporting Information.SS designed the overall study. VSFTM designed the sampling and DNA study and provided data. SIFG performed the analysis. SIFG and SS wrote a first draft of the manuscript; all authors contributed to revisions. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file.


ApcMin/+ mouse, an established genetic model for familial adenomatous polyposis. Clinical pathologies related to tumor development as well as intestinal and colon tissue histopathology were studied at ages 8, 12, and 16 weeks of age, which correspond to the approximate ages of development of neoplasia, gut inflammation with polyposis, and cancer progression, respectively, in this animal model. We show that the antibiotics significantly increase the severity of clinical symptoms, including effects on intestinal histology and goblet cell numbers. In addition, they promote small intestinal polyposis. Finally, metagenomic analysis of fecal samples demonstrated that antibiotic exposure is associated with a significant but nonuniform depletion of the animal's natural gut flora. Overall, these findings support the premise that long‐term antibiotic exposure mediates the selected depletion of gut microbial communities and the concomitant thinning of the protective mucus layer, resulting in an increase in tumor development.Recent epidemiological evidence suggests that exposure to antibiotics in early‐to‐middle adulthood is associated with an increased risk of colorectal adenoma. However, mechanistic studies in established preclinical cancer to examine these claims are extremely limited. Therefore, we investigated the effect of long‐term exposure of an antibiotic cocktail composed of Vancomycin, Neomycin, and Streptomycin, on tumor development and progression in the  The gut microbiome is integral to gastrointestinal tract function and is connected to a variety of health issues Colorectal cancer is the second leading cause of cancer‐related death in the United States and ranks fourth in estimated new cases Despite these lines of evidence, there are significant gaps in our understanding of how antibiotics increase the risk of colorectal cancer. Current studies show that tetracycline mediates upregulation of cyclooxygenase‐2 and prostaglandin production ApcMin/+ mouse, an established genetic model for familial adenomatous polyposis In the current study, we address this knowledge gap by examining the effects of long‐term administration of an antibiotic cocktail of Vancomycin, Neomycin, and Streptomycin on gut polyposis in the ApcMin/+ mice were obtained from Jackson Laboratories and bred in‐house at the Animal Resource Facility at the, University of South Carolina. Food (Purina chow) and drinking water were available to the mice ad libidum under a 12:12‐hour light–dark cycle and a low‐stress environment . At 4 weeks of age, littermates were randomly assigned to the following six groups  ApcMin/+ untreated controls sacrificed after 12 weeks, (3) ApcMin/+ untreated controls sacrificed after 16 weeks, (4) ApcMin/+ administered with antibiotics and sacrificed after 8 weeks, (5) ApcMin/+ administered with antibiotics and sacrificed after 12 weeks and (6) ApcMin/+ administered with antibiotics and sacrificed after 16 weeks. All procedures and animal care followed institutional guidelines and were approved by the Institutional Animal Care and Use Committee at the University of South Carolina. Four mice were assigned to each of the six treatment groups, and this sample size was based on statistical power analyses conducted in previous microbiome studies A mixture containing Vancomycin , Neomycin , and Ampicillin  was administered to antibiotic treatment group (+Abx). The antibiotic mixture was added to the drinking water at age 6, 10, and 14 weeks , two‐way repeated‐measure ANOVA, and one‐way ANOVA were used to analyze the data. A Tukey post hoc analysis was used to determine differences in physiological responses upon antibiotic‐administered mice and the controls. All statistical analyses were performed with SigmaStat 3.5 . For fecal bacteriome analyses, Kruskal–Wallis and Mann–Whitney statistical analyses were performed to calculate significance in diversity and relative abundance, respectively. A ApcMin/+ mice were exposed to the antibiotic cocktail beginning at 6, 10, and 14 weeks of age, and polyp numbers and sizes were assessed at 8, 12, and 16 weeks , as there were no significant differences for polyps <1 mm2. Thus, antibiotic exposure promoted development of intestinal polyps.To mimic long‐term antibiotic exposure, As shown in Figure Clinical scores derived from an integral assessment of weight loss, diarrhea, and fecal hemoccult were observed to increase in antibiotic administered as compared to control mice at all three ages Fig. . Crypt‐tAs antibiotics impact the thickness of the intestinal mucus layer ApcMin/+ mice retained a stable microbiome during the time window of 8–16 weeks. However, for all age groups, alpha‐diversity was significantly decreased by antibiotic treatment, as measured by total operational taxonomic units (OTUs), the Chao1 index, or the Shannon index  was measured in response to antibiotic treatment, several probiotic genera  were nearly eliminated. The shifts in bacterial abundance upon antibiotic administration were also reflected by the beta‐diversity patterns .Alterations at the genus level were also assessed Fig. C. While rns Fig. D, in thaBacteroides and several beneficial genera within Firmicutes.Overall, the results demonstrate that administration of antibiotics eliminated the majority of bacterial flora, with the most drastic depletion being observed for the phylum In the current study, we provide direct experimental evidence in support of the notion that long‐term exposure to antibiotics can promote polyp development in the gut of a genetically susceptible mouse strain, as suggested by previous epidemiological reports that linked colorectal carcinogenesis to antibiotic use in humans An intriguing and novel aspect of the current study is the observation that depletion of the natural bacterial flora upon antibiotic exposure correlates with reduction in mucus‐producing goblet cell numbers, along with an increase in both colon histopathological scores and intestinal crypt‐to‐villus ratios. The functional link between antibiotic‐mediated microbial dysbiosis and the inhibitory effects of antibiotics on development of goblet cells is of particular interest, as these cells are integral to protection against inflammation and polyposis. In all, this is consistent with previous findings suggesting that gut microbiota impact the thickness of the mucus layer Our results imply that the intestinal microbiome has a profound impact on the global physical properties of the gut, which in turn determines the severity of inflammation and cancer. This is illustrated by our ‘cyclical microbiome protection model’ depicted in Figure The choice of the antibiotics for this study was based on their known mechanisms of action in bacterial cells and their minimal nontoxic effects on the host. Neomycin is used against both Gram‐positive and Gram‐negative bacteria. To minimize nephrotoxic effects of the antibiotic, it is prescribed as an oral antibiotic. Its inhibits bacterial growth by binding RNA duplexes Bacteroides and some ‘beneficial’ genera such as Lactobacillus and Desulfovibrio. This implicates the possible role of these genera in protection against cancer progression. The increase in larger polyps during cancer progression may be due to selective depletion of these bacterial genera. Alternatively, the dramatic increase in relative abundance of Firmicutes upon antibiotic administration may underlie increased polyposis. Distinguishing between these and other possibilities will require follow‐up studies with gnotobiotic rodent models. Our findings are different from observations described by Hamoya et al. ApcMin/+ mice. It may be that different antibiotic exposures may result in distinct microbial profiles, which in turn, may lead to variation in disease outcomes.Interestingly, the antibiotic cocktail used currently selectively depleted the genus Our follow‐up studies will investigate the effect of these potential ‘therapeutic microbes’ on cancer development and progression. Other aspects to be investigated in our follow‐up studies are use of different antibiotics, and the timing of antibiotic exposures relative to polyp development. Such studies will provide additional mechanistic information on the association of antibiotic exposure, effects on the gut microbiome, and development of colorectal carcinogenesis.The authors declare no conflict of interest.Figure S1. Representative Hemeatoxylin and eosin (H&E) stained colon tissues belonging to control and +Abx mice at different ages.Click here for additional data file.


A new method is developed to directly spinning perfectly uniaxial fibers in an ultrafast manner. Besides, this method can tune the fibers' diameter through adjusting processing parameters such as the feeding rate of precursors. Uniaxial nylon 66 fibers prepared via this method show superior mechanical properties due to the alignment in each level of the structure. In this study, we report draw‐spinning method that can produce aligned polymer ultrathin fibers with diameter down to 200 nm. Apart from creating perfectly aligned fibers, this method can draw fibers at a high speed and can thus reorient molecular chains. Tensile tests show that nylon 66 fiber bundles fabricated by draw‐spinning exhibit >400% elongation at break and ≈250 MPa strength, which are superior to the corresponding values for conventionally fabricated fibers and bulks . The superior performance can be attributed to the relative motions of building blocks in each of the four levels of the structure: the bundle, the fibers within the bundles, microfibrils within the fibers, and aligned molecular chains within the microfibrils. We confirm that each level within the hierarchy is highly aligned and propose a “tetra‐slip” system.Fibers have been existing inside plants and playing structural roles to give mechanical support in harsh living conditions and produced by animals for specific applications.In this regard, fiber diameter and alignment are two of the key factors. However, combining the structural advantages of these factors necessitates the improvement in fiber spinning methods.In this communication, we present the general procedures of draw‐spinning and describe how it controls the diameter of aligned fibers; then, taking this structural advantage, we prepared nylon 66 and polyethylene oxide (PEO) fibers with different diameters for tensile tests; in the Experimental Section, we analyzed the alignment within each level of the tested bundles and proposed a “tetra‐slip” model to explain the mechanical results.Figure–1 for 90 min resulted theoretically in one ≈5 km‐long submicrometer fiber .The basic setup for draw‐spinning is composed of two main parts. One part is a syringe that serves as the reservoir for the precursors and is loaded on a syringe pump, and the other is a collector, which is typically a rotating substrate . As a result, the shape of the liquid pool on the 0.06‐mm nozzle transformed from “jumping sphere” to “constant jet.” When the amount of the precursor is fixed, an increased rotating speed directly leads to an increase in production rate, resulting in thinner fibers, as shown in the scanning electron microscopy (SEM) images in the right‐hand column, and the scatter plot regarding the statistics are depicted in Figure –1.–1, but the lowest spacing is 100 µm.The draw‐spinning process is a balanced result between feeding and consuming rates of the precursors. Under the same feeding rate, the consuming rate can fluctuate within a certain range while maintaining spinability. Deformation of liquid pools and change in fiber diameter can be observed when the feeding and consuming rates are mismatched. Investigations on the diameter's dependence on drawing speed were performed on PEO fibers. The substrate was a silicon wafer attached to a roller with a diameter of ≈3 cm. While maintaining the feeding rate at 0.13 mL hFigureAs mentioned previously, spinability can be maintained even when a mismatch between feeding and consuming is present, although it influences the diameter of the fibers. Thus, we fixed the flux rate while changing the rotating speed to produce a bundle. With the flux rate and fabricating time fixed, we produced two bundles consisting of fibers with different diameters. Specifically, we used a roller to collect nylon 66 fibers (mixed with PEO at the weight ratio of 10:1). The product is a fiber “ring,” as removed from the roller. It was then folded twice and twisted. Draw‐spun nylon 66 fiber bundle can stretch from its original length of 5–25 cm FigureB. The noFigureThe reason for the huge discrepancy between the actual and theoretical mechanical performance is the folding of the polymeric chains. As soon as they are extruded, industrial spun fibers immediately go through a drawing process to increase their strength but at a sacrifice of stretchability. For a better analysis of the mechanical advantage, we revealed the four‐level alignment and propose a “tetra‐slip” system. On macroscopic levels, slipping level‐1 occurs between the four strands, and inside each strand, slipping level‐2 occurs between the draw‐spun fibers Figure4A,B. ATo conclude, draw‐spinning possesses several advantages over current processing methods for micro‐/nanofibers. First, single continuous fibers with limitless length and tunable diameter can be fabricated through this method. Second, the fibers can be arranged into arrays and meshes. Third, this method is adaptable and versatile, and it can be integrated with current processing methods or followed with well‐established post‐treatments seamlessly to create novel structures and expand the draw‐spinable systems. Small fiber diameter and better fiber alignment improve mechanical properties. We conducted tensile tests on draw‐spun nylon 66 fiber bundles to investigate the influence of fiber diameter on mechanics. Because of the structural advantages, our product is tenfold higher than most nylon 66 samples when elongated, demonstrates superior performance in strength. The mechanical improvements can be explained by our tetra‐slip system.Sample Preparation: Polymer solution containing a certain weight ratio (ranging from 4.0 to 0.8 wt%) of PEO  was prepared using acetonitrile  as the solvent. The nylon 66 precursor was prepared using nylon 66 and PEO  as the solutes. Nylon 66 was added into the ≈1 wt% PEO/formic acid  and stirred until dissolution.Draw‐Spinning Procedures: Precursors were loaded into a syringe, which was then mounted onto a syringe pump. A rotating roller was used as the collector of draw‐spun fibers and by manually drawing the liquid cone on the nozzle into a fiber onto the rotating roller, the precursor can be constantly spun into fibers. To construct arrays, the translational motion was added to the rotating roller.Sample Characterization: Morphology and microstructures of fibers were observed using a field‐emission electron scanning microscopy . Energy Dispersive Spectroscopy signals of the fiber arrays were collected using detectors from Oxford Instrument (X‐MaxN Silicon Drift Detector). Optical transmittance was measured using a UV–vis spectroscopy (SHIMADZU UV‐2600). Fourier‐transformed infrared spectrum was obtained using an IR spectrometer from Bruker Corp (VERTEX 70v). Powder samples were mixed with KBr at weight ratio of 1:100; bulk samples were examined with the help of ZnSe as the attenuated total reflectance crystal. Raman intensity was performed using a Raman spectrometer with a 600 gr mm–1 grating and the laser emitting at 532 nm .Tensile Tests: Tensile tests were performed using a Zwick universal testing machine, Zwick Roell Z005, under room temperature and at moisture of ≈30%. The effective area of the samples was calculated via multiplying the average cross sectional area of an individual fiber by the overall amount of fibers. For each sample, more than three measurements were conducted to ensure the repeatability and the plotted curves are representative. The test speeds were 130% strain per minute. Results at other speeds are available in Supporting Information.SupplementaryClick here for additional data file.SupplementaryClick here for additional data file.SupplementaryClick here for additional data file.SupplementaryClick here for additional data file.SupplementaryClick here for additional data file.SupplementaryClick here for additional data file.


To understand the heterogeneity of functional connectivity results reported in the literature, we analyzed the separate effects of grey and white matter damage on functional connectivity and networks in multiple sclerosis. For this, we employed a biophysical thalamo‐cortical model consisting of interconnected cortical and thalamic neuronal populations, informed and amended by empirical diffusion MRI tractography data, to simulate functional data that mimic neurophysiological signals. Grey matter degeneration was simulated by decreasing within population connections and white matter degeneration by lowering between population connections, based on lesion predilection sites in multiple sclerosis. For all simulations, functional connectivity and functional network organization are quantified by phase synchronization and network integration, respectively. Modeling results showed that both cortical and thalamic grey matter damage induced a global increase in functional connectivity, whereas white matter damage induced an initially increased connectivity followed by a global decrease. Both white and especially grey matter damage, however, induced a decrease in network integration. These empirically informed simulations show that specific topology and timing of structural damage are nontrivial aspects in explaining functional abnormalities in MS. Insufficient attention to these aspects likely explains contradictory findings in multiple sclerosis functional imaging studies so far. The mean membrane potential Va,j can be considered as a low‐pass filter. For every group, its dynamics can be described byHere, α and β are constants, which are independent of time and correspond to the synaptic rise and decay rates in s−1; vaa denotes the synaptic densities between the population types and ϵ corresponds to the global structural coupling strength between populations. The last term in Equation ϕnoise,j(t)=σnαβχt, where χt denotes a unit variance Gaussian white‐noise process, and σn is the strength of this process. A number of cortical populations is sampled over the cortex and connected to each other through empirically informed white matter connections  of the simulated data were discarded to exclude any non‐oscillatory data. Only the time series of the 78 excitatory cortical populations ϕe,nt were used as the model output to mimic MEG signals  Mean functional connectivity is defined as phase synchronization measured by the phase locking value (PLV)  Mean functional connectivity and functional network organization is that functional connectivity refers to the strength of connections between brain regions, whereas functional network organization (topology) corresponds to the pattern of connections between regions in the brain. Specifically, we assess network organization by computing two network topology measures based on the minimum spanning tree (MST)  leaf fraction refers to the fraction of regions in the MST with only one connection. Here, diameter captures information about network integration , while leaf fraction captures information about segregation. The rationale to use these MST metrics is that these metrics showed significant associations with cognitive decline and thalamic atrophy in previous empirical network studies in MS . A reduction of 14% was based on the percentage change in structural connectivity strength that can be observed when comparing empirical data of MS patients (n = 39) compared to those of healthy controls (n = 39)    . This inverted U‐curve curve of functional connectivity during the course of increasing white matter damage may explain its inconsistent relationships with clinical outcome measures  and damage between the nodes  on network organization seems to be a generic principle in a diverse range of networks, which is related to the concept of degeneracy  is much smaller , which could explain its sensitivity for network disruption.In contrast to the white matter, damage of the gray matter induced functional network disintegration at an earlier stage, that is, less cortical damage is required to lead to disintegration of functional networks. Apparently, functional network organization is less robust for grey matter damage than for white matter damage. This could explain why cognitive and clinical measures correlate more strongly to grey matter than white matter alterations in MS. The discrepancy between damage Our findings have important implications. First, our results demonstrate that an increase in connectivity and activity can occur as a result of structural damage and thus not solely as a result of a compensatory process. Therefore, caution should be exercised when interpreting increased functional connectivity values in cross‐sectional studies as “compensation” . Second, as demonstrated by the fast transitions in Figures Some methodological aspects of our study warrant discussion. First, our results apply to resting‐state functional networks and not task‐based connectivity findings, which requires a different way of modeling in terms of external inputs that drive the dynamics. Second, all large‐scale computational models lack regional specificity along white matter tracts. Thus we cannot distinguish between lesional damage and diffuse white matter damage along a white matter tract. Finally, we did not investigate the effects of heterogeneity of conduction velocities along white matter tracts to simulate diffuse white matter demyelination, and did not use thalamo‐cortical connectivity as an outcome measure, warranting future studies. Last, the empirical data of the different types of structural damage that has been used to inform the simulations differs in disease duration. This will have influenced the end points (last iteration) in the graphs Figures  and 3, bIn conclusion, we have demonstrated that joint white and grey matter damage can induce concurrent increases and decreases in functional connectivity, warranting caution when interpreting such changes as a compensatory mechanism. In addition, these concurrent increases and decreases in connectivity could also explain the reported heterogeneity of functional connectivity results reported in the literature. Functional network topology measures show a more insightful pattern of change, regardless of the type of damage induced, which enforces their value for future imaging studies in MS.Additional Supporting Information may be found online in the supporting information tab for this article.Supporting InformationClick here for additional data file.


The aim of this study was to describe early breastfeeding practices  according to childbirth location in low‐ and middle‐income countries. Using data from the most recent Demographic and Health Survey (2000–2013) for 57 countries, we extracted information on the most recent birth for women aged 15–49 with a live birth in the preceding 24 months. Childbirth setting was self‐reported by location (home or facility) and subtype . We produced overall world and four region‐level summary statistics by applying national population adjusted survey weights. Overall, 39% of children were breastfed within 1 hr of birth (region range 31–60%), 49% received no prelacteal feeding (41–65%), and 28% benefited from optimal early breastfeeding (21–46%). In South/Southeast Asia and Sub‐Saharan Africa, early breastfeeding outcomes were more favourable for facility births compared to home births; trends were less consistent in Latin America and Middle East/Europe. Among home deliveries, there was a higher prevalence of positive breastfeeding practices for births with a skilled birth attendant across all regions other than Latin America. For facility births, breastfeeding practices were more favourable among those taking place in the public sector. This study is the most comprehensive assessment to date of early breastfeeding practices by childbirth location. Our results suggest that skilled delivery care—particularly care delivered in public sector facilities—appears positively correlated with favourable breastfeeding practices. This study was approved by the Research Ethics Committee of the London School of Hygiene and Tropical Medicine, UK.2.2Breastfeeding indicators and childbirth location for the most recent live birth were examined among women aged 15–49 with a birth in the 24 months before the survey. We restricted the sample to children who survived to at least 1 month in an attempt to exclude infants who were less likely to breastfeed, perhaps because of separation from their mother or inability to breastfeed due to prematurity or poor health condition at birth. Childbirth setting may also differ for these children, for example, mothers who went into labour early may be more likely to seek facility care.2.32.3.1Our outcomes of interest were key infant feeding indicators reflecting breastfeeding practices in early life. We included two WHO‐recommended indicators for assessing infant and young child feeding practices: the proportion of children ever‐breastfed, and the proportion of children who were put to the breast within 1 hr of birth . Where information was missing on breastfeeding indicators, we assumed the infant was not fed optimally .2.5All analyses were conducted using Stata 13. DHS surveys are conducted using a multistage cluster sampling strategy, and women in each DHS survey have an individual sample weight that is used to calculate country‐level representative summary statistics. We used these and further produced region‐level and overall summary statistics by applying weights that accounted for both country‐specific survey design and national population size , to ensure that estimates are representative of the population residing in study countries/regions. To capture the extent of variability across the included countries, we report ranges and medians. Where overall percentages for all regions are reported in the text, these are population‐weighted means of all included countries unless stated otherwise. We present differences in breastfeeding outcomes for home vs. facility‐based deliveries by country, but differences for SBA vs. non‐SBA and public vs. private facilities are aggregated by region only due to small sample sizes in some of the included countries.3The analysis included 194,042 children born in the 24 months preceding the survey. Overall, about half of births took place at home and half at a facility . At the region level, the highest prevalence of both was in Latin America  and the lowest was South/Southeast Asia . Across all regions, 28.4% (weighted mean) of children experienced both early breastfeeding initiation and avoidance of prelacteal feeding—defined as optimal early breastfeeding (country median 39.2%). The proportion of children benefiting from optimal early breastfeeding was highest in Latin America (45.6%) and lowest in South/Southeast Asia (21.1%).3.1In comparing of home and facility childbirth locations, early breastfeeding outcomes were generally more favourable among facility births compared to home births  and at the region level. The prevalence of no prelacteal feeding showed little variation by the presence of a SBA across all regions , but region‐specific comparisons show that no prelacteal feeding was higher among home births attended by a SBA in all regions other than Latin America  were generally more favourable for facility births compared to home births, particularly in Sub‐Saharan Africa and South/Southeast Asia. Within home deliveries, early breastfeeding outcomes were more favourable among deliveries attended by a SBA, with the exception of prelacteal feeding avoidance in Latin America. The prevalence of positive early breastfeeding practices was higher among public sector births compared to private sector births in all four regions.This study is the most comprehensive assessment to date of early breastfeeding practices by childbirth setting. Our analysis was based on a large sample: nearly 200,000 births across 57 LMICs, with data taken from the most recent nationally‐representative surveys (Measure DHS) conducted since 2000.Data on breastfeeding were collected retrospectively. A review of studies assessing the validity and reliability of maternal recall in relation to a range of breastfeeding indicators suggested that in general short‐term recall of breastfeeding is reliable, particularly when the recall period is 3 years or less . A further limitation of our analysis is that when stratifying by setting subtype , small numbers resulted in some extreme, potentially misleading values. For this reason, we avoided presenting country level data for comparisons other than home vs. facility births.We included breastfeeding practices relating to the most recent birth in the 24 month period. This use of the most recent birth would result in the under‐representation of children born to higher‐fertility women, with these women likely to differ from lower‐fertility women in terms of sociodemographic characteristics. However, few women had more than one birth in the preceding 24 months (0.7–6.7% across each of the 57 countries).The classification of childbirth locations into public  and private  facilities was based on DHS response options presented on surveys. Due to the survey limitations, we were unable to further stratify private facilities into for‐profit and not‐for‐profit, or separately consider NGO and faith‐based providers . Specific components of delivery care which are rapidly increasing in many middle‐income countries, such as increased use of analgesia, labour induction and augmentation, and caesarean delivery, may negatively impact on breastfeeding and contribute to a narrowing of the advantage conferred by facility care. An investigation of delivery setting and essential newborn care using data from prospective trials in South Asia reported that the proportion of women experiencing skin‐to‐skin contact with their infant within 30 min of birth was lower in institutional delivery settings compared to home  by country and childbirth location*Click here for additional data file.


HPV) types 16 and 18, which cause approximately 70% of cervical cancers, provides reason for optimism as roll‐out begins with support from Gavi, the Vaccine Alliance. However, for the hundreds of millions of women beyond the target age for HPV vaccination, cervical cancer screening to detect and treat precancerous lesions remains the only form of prevention. Here we describe the challenges that confront screening programs in low‐resource settings, including (1) optimizing screening test effectiveness; (2) achieving high screening coverage of the target population; and (3) managing screen‐positive women. For each of these challenges, we summarize the tradeoffs between resource utilization and programmatic attributes. We then highlight opportunities for efficient and equitable programming, with supporting evidence from recent mathematical modeling analyses informed by data from the PATH demonstration projects in India, Nicaragua, and Uganda.Women in developing countries disproportionately bear the burden of cervical cancer. The availability of prophylactic vaccines against human papillomavirus ( Cervical cancer is eminently preventable. The natural history of disease is well understood — that cervical cancer is caused by persistent infection with oncogenic human papillomavirus (HPV), acquired through sexual activity Yet cervical cancer is a leading cause of cancer mortality in poor women. Due to limited infrastructure and health budgets, the technological advances that can prevent, detect, and treat oncogenic HPV infections are not generally available in low‐ and middle‐income countries (LMICs), where 86% of cervical cancers occur While cytology  testing is the most common cervical cancer screening method worldwide To inform evidence‐based decision making and intensify a global policy focus on cervical cancer screening, we describe the challenges that confront screening programs in low‐resource settings, including (1) optimizing screening test effectiveness; (2) achieving high screening coverage of the target population; and (3) managing screen‐positive women. For each of these challenges, we summarize the tradeoffs between resource utilization and programmatic attributes. We then highlight opportunities for efficient and equitable prevention programming, with supporting evidence from recent mathematical modeling analyses informed by data from the PATH demonstration projects in India, Nicaragua, and Uganda Optimizing screening effectiveness involves selecting the screening test, delivery mechanism, and screening age(s) and interval that are associated with the greatest reductions in cancer burden while taking into account the financial and logistical constraints in a given setting. The World Health Organization (WHO) recommends, where resources are available, a “screen‐and‐treat” strategy with HPV testing and timely cryotherapy for eligible HPV‐positive women aged 30 to 49 years HPV testing has achieved demonstrable reductions in cervical cancer incidence and mortality. A large randomized trial in India found that a single round of HPV testing in women over age 30 years reduced advanced cervical cancer incidence and mortality by 50%, whereas VIA and Pap testing did not yield significant reductions in disease burden careHPV test—a low‐cost HPV test with basic laboratory requirements—in low‐resource settings for both provider‐ and self‐collection The demonstrated health impact of HPV testing is the result of high sensitivity to detect precancer and infections destined to become precancer, which is critical when the number of lifetime screening opportunities is low and the consequences of a false negative are potentially serious. HPV specimens can be collected by a provider  or by the woman herself . Studies of test performance have validated the careHPV) requires at least two visits—first, for administration of the screening test, and second, for delivery of results and treatment of HPV‐positive women. High loss to follow‐up rates will undermine program success if many women do not receive necessary treatment. Realistically, even VIA may require multiple visits, as cryotherapy is unlikely to be available at all primary health facilities. Optimization of screening effectiveness will need to consider these tradeoffs of costs, number of visits, and achievable compliance with recommended follow‐up, as well as test sensitivity and specificity for each screening test or delivery mechanism in a given setting.Despite the high sensitivity and resulting effectiveness of an HPV screen‐and‐treat approach under study conditions, HPV test kits and laboratory processing systems are costly Mathematical simulation models of disease can integrate biologic, epidemiologic, economic, and behavioral data to project the long‐term health and economic impact of interventions Our model results indicate that the most critical ages for screening fall within WHO guidelines across settings with varying burdens of HPV and cervical cancer The WHO recommends that screening begin at 30 years of age, with priority given to maximizing population screening coverage rather than maximizing the number of screening tests in an individual woman's lifetime An analysis of population‐based surveys from 57 countries found that the average cervical cancer screening coverage in developing countries is 19% among women aged 25–64 years, compared to 63% in developed countries A particular advantage of HPV testing is that self‐collection of HPV specimens circumvents the need for a pelvic examination, potentially increasing acceptability and easing the burden on health systems by shifting the task of screening to women and community health workers. In Argentina, when community health workers visited women at home and offered the opportunity to self‐collect an HPV sample, screening uptake was 86%, compared to only 20% among women who were advised to attend a health clinic for screening With respect to screening coverage, well‐intentioned screening guidelines in many countries prescribe screening at routine intervals or a certain number of times per lifetime without acknowledging the reality that few women have access to any screening opportunity. When we used the Uganda model to examine the tradeoff between expanding coverage for a single lifetime screen versus increasing screening frequency to two or three times in a lifetime for a select group of women, we found that—when existing screening coverage was low —expanding access to screening may lead to greater population‐level health gains, greater reduction in health disparities, and could be very cost‐effective, with an incremental cost‐effectiveness ratio below per capita GDP We also used the Uganda model to explore the efficiency and effectiveness of a onetime, community‐based HPV self‐collection campaign under different uptake scenarios. We found that, if most HPV‐positive women can be successfully navigated to treatment, HPV self‐collection is a very cost‐effective alternative to provider‐collection at the clinic, despite slightly reduced test sensitivity The management of screen‐positive women poses challenges for screening programs in LMICs. According to WHO guidelines, treatment with either cryotherapy and/or loop electrosurgical excision procedure (LEEP) are essential components of screen‐and‐treat programs With HPV testing, the high prevalence of oncogenic HPV infections coupled with the relatively low prevalence of precancer leads to a high “false positive rate,” as few women who test positive for HPV will have or go on to develop precancer or invasive cancer in the future. An HPV‐based screening program that sends all HPV‐positive women to cryotherapy may lead to overtreatment. While there are no data to suggest that overtreatment with cryotherapy is harmful to women Linking screen‐positive women to treatment is essential if the health benefits of screening are to be realized. While the primary advantages of VIA are its low cost and logistical advantages that theoretically facilitate a single‐visit approach, the evidence from demonstration projects and pilot studies indicates that its use as a single‐visit strategy may be limited to urban areas and higher level facilities We used the India, Nicaragua, and Uganda models to estimate the comparative‐ and cost‐effectiveness of HPV testing with VIA triage versus HPV testing alone and found that, even under the most optimistic assumptions about VIA performance, HPV testing alone was both more effective and more cost‐effective in Nicaragua and Uganda. In India, HPV testing with VIA triage had the potential to be a cost‐effective alternative to HPV testing alone when we assumed VIA triage test sensitivity was higher than suggested by much of the literature, but yielded lower reductions in cancer risk and was thus a less effective strategy. These findings reinforce the need for more accessible treatment options that can be readily delivered in all primary health facilities or mobile clinics in LMICs, averting the need for triage testing. Alternatively, low‐cost triage strategies with better ability to predict risk of precancer and cancer are needed.Each of the modeling analyses we summarize here is associated with its own limitations (documented elsewhere) In addition to providing estimates of the health and economic impact of screening strategies, we used the models to determine the economic costs that could be additionally incurred for a new point‐of‐care HPV test to be cost‐effective; this is known as the incremental net monetary benefit (INMB). We found that, if screen‐positive women could subsequently be navigated to treatment, the economic value of an HPV test with same‐day results was high, particularly in settings with low rates of compliance to recommended follow‐up. Thus, in addition to providing qualitative insights across countries with diverse epidemiologic profiles and quantitative insights within a particular country, the models can estimate the value of improvements in screening practice Through expanding access to HPV screen‐and‐treat programs that invoke innovative service delivery mechanisms, we have the tools to substantially reduce the global burden of cervical cancer. Lessons from small‐scale demonstration projects should be applied to implementation, with programs continually evaluated and adapted as roll‐out occurs. Modeling analyses can continue to evaluate the cost‐effectiveness of particular strategies, and will be further enriched as data from real‐world implementation become available. Strengthening primary health systems in low‐resource settings is difficult work, but imperative if we are to address health disparities and the growing burden of noncommunicable diseases in LMICs NGC, VT, MM, and JJK declare no conflicts of interest. JJ was the director of the START‐UP demonstration projects and received all tests used in the study as a donation from QIAGEN. JJ was the co‐owner and Deputy Manager of Onco Prev International, a Peruvian company, from 2012 through March 2017. Onco Prev International offers cervical cancer screening services and in 2016 also began positioning for distribution of medical devices including colposcopes and the Liger thermocoagulator. Onco Prev International did not commercialize any medical instrument during the time Dr. Jeronimo was part of the company.


RATE is a brief patient survey focused on shared decision making. This paper aims to (i) provide insight on facilitators and challenges to implementing a real‐time patient survey and (ii) evaluate CollaboRATE scores and response rates across multiple clinical settings with varied patient populations.CollaboRATE post‐visit. To inform key learnings, we aggregated all mentions of unanticipated decisions, problems and administration errors from field notes and email communications. Mixed‐effects logistic regression evaluated the impact of site, clinician, patient age and patient gender on the CollaboRATE score.All adult patients at three United States primary care practices were eligible to complete CollaboRATE score increased only slightly with increasing patient age , female patient gender was associated with significantly higher CollaboRATE scores . Clinician also predicts CollaboRATE score (random effect variance 0.146). Site‐specific factors such as clinical workflow and checkout procedures play a key role in successful in‐clinic implementation and are significantly related to CollaboRATE scores, with Site 3 scoring significantly higher than Site 1  or Site 2 .While CollaboRATE can be used in diverse primary care settings. A clinic's workflow plays a crucial role in implementation. Patient experience measurement risks becoming a burden to both patients and administrators. Episodic use of short measurement tools could reduce this burden.This study demonstrates that Collabo CollaboRATE has previously been validated in a simulation sample2.1.2Field notes regarding implementation challenges and solutions were kept on an ad hoc basis by study staff at all sites throughout the project as an integral part of project management processes. Investigators and research staff from all sites contributed observations through email messages and regular project meetings; meetings were held weekly at Site 1 and quarterly between Sites 1 and 2 and Sites 1 and 3. All contributors were familiar with the study protocol and participated in the design of survey implementation at their respective sites. Field notes were collected, organized and held by the project coordinator based at the Lebanon, NH site. All instances of unanticipated decisions, problems and errors were documented.2.22.2.1Three geographically diverse US primary care practices were included in this study: Lebanon, NH (Site 1); Los Gatos, CA (Site 2); and Chelsea, MA (Site 3). Detailed information on each site is included below.All adult patients ages 18 and older visiting the participating primary care teams were eligible to participate.All sites allowed clinicians to describe the survey to patients, although this behaviour was eventually encouraged as a participation‐enhancing measure only at the Los Gatos site. No clinicians at any site personally delivered the survey to their patients.2.2.2This setting included three primary care teams based in a rural academic medical centre in New Hampshire with approximately 16 000 patients. Clinicians included physicians, physicians’ assistants, nurses, nurse practitioners and pharmacists. The patient population is more than 95% white and non‐Hispanic, and 99% of patients speak English as a primary language.Parents and guardians of patients under 18 were also eligible to complete CollaboRATE on behalf of their children at this site.This site aimed to administer CollaboRATE for 15 consecutive months beginning in April 2014 in order to embed the survey in routine practice. Administrative staff were asked to notify patients at check‐in of the ongoing research study. The study was reviewed by the Dartmouth Committee for the Protection of Human Subjects and written participant consent requirements were waived for all modes of survey administration. Five survey administration modes were implemented consecutively: (i) paper survey in‐clinic from April through July 2014; (ii) patients were alerted to a survey hosted on the online patient portal (MyChart) of the Epic electronic medical record system from July through November 2014; (iii) automated interactive voice response (IVR) telephone calls were made to patients’ cellular telephones from December 2014 through March 2015; (iv) short messaging service (SMS) text messages were sent to patients’ cellular telephones from April through July 2015; (v) a tablet computer survey was presented to patients in‐clinic, with an option for patients to mail back a paper‐based survey if they declined to complete the tablet survey (July through October 2015); see Appendix 2.2.3This primary care clinic in suburban Los Gatos, CA is one of many community‐based clinics of the Palo Alto Medical Foundation, a large, not‐for‐profit health‐care delivery system in the San Francisco Bay Area, and has a patient panel of approximately 13 000. The patient population is predominantly Caucasian (44%) and Asian (34%). Approximately 90% of patients speak English as a primary language, with 46 other language groups also represented.This site aimed to collect 300 completed CollaboRATE surveys during the February 2015‐March 2015 study period. The study was reviewed by the Sutter Health Institutional Review Board and written participant consent requirements were waived. An initial week‐long pilot period involved only the reception staff informing patients about the CollaboRATE survey as they arrived for their visit. After their visit, a receptionist invited all eligible patients to complete a paper survey containing CollaboRATE. The pilot period yielded very low response rates (2%). Recruitment in the subsequent data collection period included asking physicians to encourage participation, additional reminder signage, and medical assistants providing patients with surveys before they left the exam room. A locked collection box was placed at the clinic exit with a sign reminding patients to deposit the completed survey in the box. Thus, the physical clinic layout did not present a barrier to survey completion at this site. The study population at the Los Gatos, CA site included all patients visiting the participating clinics.2.2.4This urban adult medicine practice is based in a Massachusetts General Hospital affiliated community health centre in Chelsea, MA and has a patient panel of approximately 14 000 patients. Fifty percent of patients are Latino, 31% white, 9% black and 4% other. Fifty‐one percent of patients at this site speak English as their primary language, while 38% speak only Spanish and 11% speak one of 14 other languages.This site aimed to administer the CollaboRATE survey for the month of May 2015. The study was reviewed by the Partners Institutional Review Board and written participant consent requirements were waived. Medical assistants (MAs) assigned to meet with patients following their visits delivered the survey to patients. Survey forms were available in both English and Spanish.2.3,.Returned surveys missing one or more CollaboRATE responses were considered incomplete and excluded from analysis. Descriptive statistics compared response rates and CollaboRATE scores across sites. Because patient reported experience measures often show ceiling effects, we decided a priori to conduct a top score analysis which has been shown to enhance variation in scoresAs data were clustered by site and clinician, we used mixed‐effects logistic regression analysis to evaluate the impact of site and clinician on the dichotomous CollaboRATE score outcome variable  while controlling for the survey administration modality and patient‐level demographic characteristics of age and gender. Clinician was included as a random effect within the mixed‐effects regression model (with resulting clinician variance estimate) to account for clustering of responses and to allow our results to be generalizable to the population of all clinicians who would plausibly work at one of the three sites, not just those in our study, as the random effect specification accounts for the possibility that studying a different group of clinicians may have yielded different results. Site was included as a fixed effect because the small number of sites (3) provided insufficient numbers of sites to reliably estimate a site‐level variance component under a random‐effects specification. Thus, we control for the sites in the study rather than generalizing results to a larger population of sites. While the logistic regression model allows us to compare Los Gatos and Chelsea sites to the Lebanon reference group, the logistic regression model does not allow us to test the difference between scores at the Los Gatos and Chelsea sites. Therefore, as a post‐estimation hypothesis test, we calculated a z‐score to compare CollaboRATE scores at the Los Gatos and Chelsea sites. An inverse logit transformation involving the clinician random effect variance parameter allowed the magnitude of the clinician effect to be compared to that of the model's regression parameters on the probability scale.To inform the key learnings and descriptions of site‐level characteristics, we aggregated all mentions of unanticipated decisions, problems, and administration errors from field notes and email communications.33.1Response rates varied across sites, with Site 3  achieving the highest response rate of 73% compared to 25% overall at Site 1  and 30% at Site 2 . Site 1 saw variation in response rates across the various administration modes, described in detail elsewhere.3.2Table P=.007) sites to give all top scores. The clinician random effect variance of 0.146 implies that the distribution of CollaboRATE scores varied substantially between clinicians; for this study the random effect standard deviation of 0.382 translates to a difference of 0.5364 on the probability scale. Thus, a clinician whose scores fall one standard deviation above the mean clinician will have a 53.64% greater probability of obtaining a perfect CollaboRATE score from a randomly selected patient.While Los Gatos patients were similar to Lebanon patients in their propensity to give a top score on all three CollaboRATE items, Chelsea patients were more likely than those at the Lebanon  and Los Gatos  on cellular telephones also revealed logistical challenges due to significant variation in subscriber plans and very limited reception in some rural areas. In the United States, some cellular telephone subscribers pay a fee (<$0.50 USD) for each text message sent or received. Some cellular service providers offer to deliver an organization's outgoing messages to their customers free of charge for an annual $25 000 USD up‐front fee, although that cost was prohibitive in this project. Despite the cost to patients, we used the SMS approach to assess text message patient survey administration in this study.3.3.2At the Los Gatos site, the clinic's physicians and operational leaders strongly supported the project. When the initial attempt to collect responses by asking receptionists to alert patients to the survey led to only 26 surveys completed over a two‐week pilot period, we were able to consult with the two clinic leaders and change the survey administration workflow. The new workflow ensured that both clinicians and reception staff were requesting survey completion, supported by the medical assistants giving surveys to patients as they finished their clinic visits. Using the modified recruitment methods, 323 surveys were completed during a two‐week data collection period.3.3.3We observed significant staff commitment to the data collection process at this site. The clinic workflow and layout facilitated data collection; patients were required to make contact with administrative staff as they left the clinic, which provided an opportunity for staff to confirm receipt of the survey. We also believe that the additional financial incentive to MAs contributed to the 73% response rate achieved in this site.44.1Variation was found across the three sites with regard to both response rates and CollaboRATE scores. Site‐level factors were associated with scores at Site 3 (Chelsea) where response rates were highest, but these factors were not as influential as the clinician seen and the patient's gender in accounting for observed variation in CollaboRATE scores. These generic site‐level and clinician‐level variables would include, as a component, the site or clinician's actual level of performance. The range of scores at the clinician level indicates that the measure discriminates between high and low performing clinicians. Additionally, associations between CollaboRATE scores and patient age and gender, respectively, show that older patients are only slightly more likely to give higher CollaboRATE scores than their younger peers, while women are much more likely than men to do so.Our qualitative assessment found that site‐level factors such as patient flows, physical clinic layout, and staff enthusiasm towards the project led to different response rates. Efforts to collect patient experience data close to clinical encounters need to negotiate two key challenges. First, scores are observed to have a wider range when CollaboRATE is completed outside the clinic environment. This may be due to social desirability bias where patients perceive in‐clinic survey completion to be less private than completion elsewhere. Recency of the visit may also play a role in score differences observed when CollaboRATE is completed inside the clinic immediately after an appointment versus outside the clinic a short time (less than 24 hours) later. Second, efficient interfaces are needed between modern methods of collecting data using online and mobile technologies and the administrative systems of health‐care organizations. Existing systems are not currently designed to allow efficient communication with groups of patients. Adopting methods to assess, analyse and use patient experience data as inputs into quality improvement at the clinician and clinic level will always be difficult unless real‐time survey administration solutions requiring fewer in‐clinic human resources are developed.4.2The participating sites represent a diverse group of rural, urban, and suburban primary care clinics in which CollaboRATE was successfully administered immediately following primary care clinic visits using multiple survey distribution modalities. Few studies have considered the challenges of collecting real‐time patient experience data in such depth. Our data collection processes sought responses from all adult patients in real‐world primary care practice, potentially avoiding selection bias that may result from collecting survey responses only from those patients who formally document written consent to participate in a research study; however, our lack of demographic data on non‐respondents precludes definitive conclusions about selection bias. Additionally, the short length of the survey reduces the time burden placed on patients as a result of routine data collection.Our lack of detailed patient demographic data may inflate estimates of site and clinician impact on CollaboRATE scores.4.3This work contributes to a nascent area of inquiry surrounding real‐time measurement of patient experience, particularly related to shared decision making. Tai‐Seale's4.44.4.1This study demonstrates that a clinic's workflow, especially its patient checkout procedure, plays a key role in successful in‐clinic implementation of a patient experience measure. Ensuring staff commitment to survey administration is a key issue in maximizing response rates to in‐clinic patient surveys. Strong clinical leadership and engagement may play a role in enhancing staff commitment to survey administration, as may financial incentives to staff members required to go above and beyond their usual duties. For technological solutions, efficient and seamless integration with administrative systems is a key requirement. Avoiding the burden of long‐term data collection (as in Site 1) in favour of sampling for shorter time periods (as in Sites 2 and 3) seems to lead to better response rates. Technological survey administration also helps boost response rates.4.4.2There is a significant risk that patient experience measurement could become a burden to both patients and administrators unless efforts are made to make the process efficient, time‐limited, and most of all, relevant. Existing outpatient surveys, such as the Clinician and Group Consumer Assessment of Healthcare Providers & Systems (CG‐CAHPS),We conclude that collecting real‐time data about key aspects of patient experience is not easy. Ensuring that the data are made available for clinicians and management so that it can be used for timely quality improvement is a research frontier yet to be explored.™ patient decision aids collaborative, which produces and publishes patient knowledge tools; he has been a member of teams that have developed measures of shared decision making and care integration.Financial: Glyn Elwyn reports personal fees from Emmi Solutions LLC, National Quality Forum, Washington State Health Department, Oxford University Press and Radcliffe Press. Non‐Financial: Glyn Elwyn initiated the Option Grid