Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae), is an important agricultural pest in U.S. cotton and is managed using transgenic hybrids that produce insecticidal proteins from the bacterium, Bacillus thuringiensis (Bt). The reduced efficacy against H. zea caterpillars of Bt plants expressing Cry toxins is increasing in the field. In a first step towards understanding Bt cotton–bollworm–microbiota interactions, we investigated the internal bacterial microbiota of second–third stadium H. zea collected in the field from non-Bt versus Bt (WideStrike) cotton in close proximity . The bacterial populations were analyzed using culture-dependent and -independent molecular approaches. We found that WideStrike samples had a higher bacterial density and diversity per larva than insects collected from non-Bt cotton over two field seasons: 8.42 ± 0.23 and 5.36 ± 0.75 (log10 colony forming units per insect) for WideStrike compared to 6.82 ± 0.20 and 4.30 ± 0.56 for non-Bt cotton for seasons 1 and 2, respectively. Fifteen phyla, 103 families, and 229 genera were identified after performing Illumina sequencing of the 16S rRNA. At the family level, Enterobacteriaceae and Enterococcaceae were the most abundant taxa. The Enterococcaceae family was comprised mostly of Enterococcus species (E. casseliflavus and another Enterococcus sp.). Members of the Enterococcus genus can acidify their environment and can potentially reduce the alkaline activation of some Bt toxins. These findings argue for more research to better understand the role of cotton–bollworm–bacteria interactions and the impact on Bt toxin caterpillar susceptibility.The bollworm,  Gossypium hirsutum L.) is a fiber, feed, and food crop of global significance [Bacillus thuringiensis (Bt) [Pectinophora gossypiella (Saunders) (Lepidoptera: Gelechiidae) and the American cotton bollworm Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae) are important cotton pests in the USA [Cotton  . The toxsis (Bt) . Caterpi the USA .Bt cotton has been in use for over two decades and has provided significant benefits, e.g., reducing the need for chemical insecticides. For example, the number of insecticide applications was reduced by at least 50% compared to non-transgenic cotton in Arizona . Bt cottH. zea to Cry toxins in cotton, was provided recently by Reisig et al. [Pest resistance to the Cry toxins is now well established ,12. The H. zea in particular, Cry resistance has been attributed to several factors. Caccia et al. [H. zea larvae compared to a susceptible strain (LC). They proposed this as the mechanism of resistance to Cry1Ac because of reduced toxin binding and increased degradation by proteolysis. Zhang et al. [H. zea resistant and susceptible strains but instead came to the conclusion that a decrease in Cry1Ac activation by midgut proteases partially contributed to Cry1Ac resistance in their GA (a field-selected population) and GA-R (derived from GA and further selected in the laboratory for increased resistance) strains. Lawrie et al. [H. zea. RNAseq analyses also revealed several additional differences associated with resistance and included the already recognized known mechanisms of Bt resistance in the same bollworm population [In Lepidoptera in general and in a et al.  detectedg et al.  found noe et al.  also sugpulation .Burkholderia confers insecticide resistance in the bean bug, Riptortus pedestris (Fabricius) (Hemiptera: Alydidae), to fenitrothion, a widely used organophosphate. Cheng et al. [Citrobacter sp. (CF-BD), isolated from the oriental fruit fly, Bactrocera dorsalis, conferred resistance to trichlorphon by increased degradation. When it comes to the relationship between Bt and the insect gut microbiota, the majority of studies have focused on the mode of action and whether the septicemia caused by enteric bacteria is mandatory [Another possible contributing factor to tolerance or resistance to Bt could be gut symbionts. In fact, there is evidence bacteria can affect food digestion and host nutrition, provide protection from pathogens and parasitoids, and degrade toxic compounds ,20,21,22g et al.  showed tandatory ,29 or noandatory ,31 for BH. zea in cotton fields. Wang et al. [H. zea larvae but the insects were collected from sweet corn. Additionally, they used culture-dependent techniques and focused only on bacteria found in fresh oral secretions. The aim of the current study was to determine the bacterial community composition of cotton bollworms from insects (2nd to 3rd stadium) collected from non-Bt versus Bt (WideStrike) cotton grown in the same field in North Carolina. Cultivable bacteria were enumerated, and the total H. zea-associated bacterial community structure was analyzed using culture-independent DGGE and next-generation sequencing of 16S rRNA gene amplicons.Despite the growing interest in studying the interactions between gut microbes and insecticides, little is known about the microbial communities associated with g et al.  studied Helicoverpa zea larvae were collected in August 2016 from non-Bt (PHY425RF) and Bt   cotton fields located at the Upper Coastal Plain Research Station, Rocky Mount, NC, USA. The locations of the collections are shown in TM, Becton, Dickson and Company, Sparks, MD, USA). Fourteen larvae (7 per treatment) were separately surface sterilized with 95% ethanol (30 s) followed by 1% bleach (30 s) and finally, washed 5 times with sterile water. The final washes were pooled together per treatment for subsequent verification of sterility through bacterial culture. The larvae were transferred to sterile 2 mL microcentrifuge tubes , each containing 10 sterilized 3 mm solid glass beads  and were homogenized in 300 µL of sterile phosphate-buffered saline   in the FastPrep™ FP120 system  for 45 s. To enumerate total cultivable bacteria, homogenate from each sample was serially diluted up to 10−7 and then drop plated (25 μL/each dilution) on the surface of the medium using the drop plating method [U-test using the R statistical software [The number of cultivable bacteria per caterpillar from non-Bt and Bt cotton was estimated using a Tryptic Soy Agar (TSA) medium  was performed to investigate the bacterial community diversity of the samples collected during season 1. Total DNA was extracted from three samples per treatment using the method previously described by Ponnusamy et al. . BrieflySince differences were found in season 1 both in cultivable bacteria per insect and bacterial diversity between treatments (see Results and Discussion for more detail), this justified repeating the research in season 2 and using a higher resolution method to determine bacteria diversity.H. zea were collected in August 2018 from non-Bt  and Bt  cotton  located at the Upper Coastal Plain Research Station, Rocky Mount, NC, USA from adjacent field plots , commonly used to assess total cultivable heterotrophic bacterial growth.Larvae were separately surface sterilized the same day they were transferred to the laboratory and homogenized in sterile PBS as described in season 1. Homogenates were then used subsequently for quantification of cultivable bacteria and DNA extraction. For cultivable bacteria, homogenates from the different samples were drop plated on the surface of Petri dishes (as described earlier) containing Plate Count Agar  per the manufacturer’s protocol. Two hundred microliters of each larval homogenate were used, and DNA was eluted in 60 µL of elution buffer (AE buffer). The DNA samples were further purified using the Wizard DNA cleanup system . DNA quality and quantity were assessed using a NanoDrop 1000 spectrophotometer . The genomic DNA samples were normalized to 50–100 ng/µL and stored at −40 °C until further use.Bacterial 16 rRNA gene fragments were amplified using universal V3–V4 hypervariable region-specific primers . AmplifiQuality filtering and analyses of the 16S rRNA gene sequence data see  were perU-test using the R statistical software.Statistical analyses for alpha and beta diversities were conducted in QIIME2. The statistical significance of alpha diversity between groups (Bt and non-Bt) was inferred using pairwise Kruskal–Wallis H-tests. To estimate β-diversity between the two groups (Bt and non-Bt), we used the weighted Unifrac distance metric . For bet10 CFUs/insect (see 10 CFUs/insect) for larvae collected in non-Bt cotton . In the second season, the bacterial count in the larvae varied between 2.78 and 8.1 log10 CFUs/insect .In the first season, the estimated total number of cultivable bacteria in the bollworms varied between 6 and 9 logsect see . One nonsect see . Bollwort cotton , with a sect see  with measect see   of the bollworms collected from Bt versus non-Bt cotton in season 1. Each band shown on the gel image  represenAfter quality filtering, low-sequence samples were removed from the data and not processed during the subsequent analyses. These samples were defined as samples with <5000 sequence reads. Thus, a total of 1,667,663 16S rRNA gene sequences were obtained from 18 16S rRNA gene libraries (6 for non-Bt and 12 for Bt) after quality filtering using DADA2 with an average of 92,648 reads. The reads obtained after quality filtering were clustered into 2301 sequence variants and assigned to 15 phyla, 103 families, and 229 genera.H. zea larvae collected from non-Bt cotton harbored more ASVs (OTUs) compared to Bt (WideStrike) samples  (p = 0.05) (p = 0.07).The shape of the rarefaction curves based on the observed OTUs (ASVs) suggests that the sequencing depth of 5000 used in this study allowed us to capture the majority of taxa present in the bollworm samples . Alpha d= 0.004) . However = 0.05) . β-diver = 0.05) . No dist = 0.05) . This waU-test, p = 0.553). In fact, 30% of the reads in the WideStrike (Bt) samples belonged to Enterococcaceae compared to 15% of the reads in non-Bt cotton , Klebsiella (with three species), Enterobacter, and Erwinia (Enterococcus casseliflavus (20% in Bt versus 15% in non-Bt) and a second non-identified Enterococcus species (9.5% in Bt versus 0.58% in non-Bt) were more abundant in WideStrike samples. Klebsiella oxytoca (the most abundant Klebsiella species identified) was also highly present in WideStrike samples (12% of the reads vs. 2% for non-Bt cotton). Enterobacter was the most abundant genus detected in our samples and comprised 19% of the reads in non-Bt cotton and 28% of the reads in WideStrike (Bt)  at the phylum level were Proteobacteria (67.80%) and Firmicutes (27.26%) see . We foun Erwinia . Enterocike (Bt) . Four peike (Bt) .H. zea, as impacted by the cotton variety. Significantly higher numbers of bacteria were cultured from bollworms collected from Bt (WideStrike) compared to those from non-Bt cotton  and were also the most resistant to various insecticides . It is well-known that diet is an important factor in shaping an insect’s microbiome [In this study, we investigated the density and diversity of bacterial communities associated with the internal body of second and third stadium larvae of the American cotton bollworm, t cotton A during t cotton B using acrobiome , and theIn the first year, we used DGGE to quantify the number of DNA bands and band relative intensities to estimate overall OTU diversity. In the second year, we used high-throughput 16S rRNA gene amplicon sequencing which has a much greater resolution in measuring bacteria diversity than DGGE. The sequencing results showed that samples from non-Bt cotton harbored a significantly higher number of OTUs, but the Shannon diversity analysis revealed no significant difference between the two treatments. The principal coordinates analysis (PCoA) plot revealed no distinct clustering of WideStrike and non-Bt cotton collected caterpillars, confirmed by the PERMANOVA test, which was not significant. Even though these observations could, at first, suggest that the bacterial communities present in the two treatments are the same, the presence of several taxa that are selectively more abundant in one treatment over the other is worth noticing and examining. This is discussed in more detail later.Klebsiella oxytoca and Enterobacter spp., highly present in our samples, are diazotrophic bacteria and are known to help insects fix and use nitrogen [Enterobacter spp. have been shown to promote herbivory on chemically defended plants likely because of their ability to detoxify plant xenobiotics [H. zea is yet to be demonstrated.Overall, Proteobacteria and Firmicutes were the predominant phyla in our samples. Proteobacteria accounted for about 68% of the reads. This result is not surprising and is in agreement with findings in other insects, including Lepidoptera ,53,54. Tnitrogen . In addiobiotics . WhetherEnterococci (Enterococcus casseliflavus and another Enterococcus sp.). Enterococci are commonly found in the gut of larval Lepidoptera, and E. casseliflavus has been isolated from other phytophagous lepidopterans such as Manduca sexta [Spodoptera litura [Hyles euphorbiae, and Brithys crini [H. euphorbia and B. crini  were dominated by E. casseliflavus, suggesting that this bacterium is involved in the tolerance developed by these insects to the toxic compounds found in their host plants. It has also been found to be associated with Spodoptera litura feeding on lima beans which are rich in toxic terpenes [Enterococcus sp. were found to enhance resistance in the diamondback moth, Plutella xylostella [E. faecalis) to acidify their local environment [H. zea larval guts might protect the insect against the insecticidal activity of Bt toxins which require an alkaline environment to be processed. The presence of these bacteria might result in Bt toxin tolerance or resistance . Moreover, some studies have previously suggested that gut bacteria have the potential to inhibit Bt growth (not applicable in our studies) and degrade Bt toxins [Enterococci highly present in the WideStrike samples in this study play a role in changing the caterpillar’s susceptibility to Bt. Additional studies are needed to clarify the role of H. zea gut bacteria in Bt susceptibility, especially given the fact that high levels of resistance to Bt have been found in cotton fields in North Carolina where the insects were collected [The most striking observation in our data is the differential abundance of Enterococcaceae between Bt (WideStrike) and non-Bt cotton. Thirty percent (30%) of the reads in WideStrike belonged to this family, essentially composed of two ca sexta , Spodopta litura , Hyles eys crini . The gutterpenes . Enteroclostella , againstironment ,59. The t toxins ,61. At tollected .H. zea, using culture techniques, DGGE and Illumina MiSeq next-generation sequencing (NGS). We found higher levels of internal cultivable bacteria in bollworms from WideStrike in seasons 1 and 2 but the differences were only statistically significant for season 1. The results also showed that a few taxa dominated the microbiota of the caterpillars at each taxonomical classification level. The most intriguing result was the presence, in high abundances, of Enterococcaceae , especially in Bt (WideStrike) samples. Enterococcus spp. have been shown to enhance resistance to conventional insecticides and some members of this genus can acidify their environment, which could increase tolerance towards Bt by decreasing its activation. Therefore, studies with larger sample sizes and with varying collection sites should be conducted to characterize the role that these bacteria may play in H. zea larvae feeding on Bt cotton.In summary, we characterized for the first time the microbiota associated with field-collected (Bt and non-Bt cotton) second and third stadium cotton bollworms,


Colorectal cancer is an increasingly prevalent disease that accounts for substantial mortality and morbidity and is responsible for an impaired quality of life. This scenario highlights the urgent need to better understand the biological mechanisms underlying colorectal cancer onset, progression and spread to improve diagnosis and establish tailored therapeutic strategies. Therefore, understanding tumor microenvironment dynamics could be crucial, since it is where the tumorigenic process begins and evolves under the heavy influence of the complex crosstalk between all elements: the cellular component , the non-cellular component (extracellular matrix) and the interstitial fluids. Bioengineered models that can accurately mimic the tumor microenvironment are the golden key to comprehending disease biology. Therefore, the focus of this review addresses the advanced 3D-based models of the decellularized extracellular matrix as high-throughput strategies in colorectal cancer research that potentially fill some of the gaps between in vitro two-dimensional and in vivo models.More than a physical structure providing support to tissues, the extracellular matrix (ECM) is a complex and dynamic network of macromolecules that modulates the behavior of both cancer cells and associated stromal cells of the tumor microenvironment (TME). Over the last few years, several efforts have been made to develop new models that accurately mimic the interconnections within the TME and specifically the biomechanical and biomolecular complexity of the tumor ECM. Particularly in colorectal cancer, the ECM is highly remodeled and disorganized and constitutes a key component that affects cancer hallmarks, such as cell differentiation, proliferation, angiogenesis, invasion and metastasis. Therefore, several scaffolds produced from natural and/or synthetic polymers and ceramics have been used in 3D biomimetic strategies for colorectal cancer research. Nevertheless, decellularized ECM from colorectal tumors is a unique model that offers the maintenance of native ECM architecture and molecular composition. This review will focus on innovative and advanced 3D-based models of decellularized ECM as high-throughput strategies in colorectal cancer research that potentially fill some of the gaps between in vitro 2D and in vivo models. Our aim is to highlight the need for strategies that accurately mimic the TME for precision medicine and for studying the pathophysiology of the disease. Colorectal cancer (CRC) is an increasingly prevalent disease that accounts for substantial mortality and morbidity and is responsible for an impaired quality of life and high financial resource consumption . DespiteOver the last few years, the ECM has become a hot topic of research since this complex network of macromolecules is much more than a physical and stable structure providing support to tissues. The ECM is an extremely dynamic component of the TME  that modConsidering the relevant role of this cellular–acellular communication, several efforts have been made to develop new CRC models that accurately mimic the interconnections within the TME to understand the disease ,21,22,23Therefore, the focus of this review is to summarize the innovative and advanced 3D-based models of CRC, with a special highlight on the decellularization-based models, which offer the intrinsic native properties of the ECM to accurately resemble and reconstruct the TME to study CRC biology and drug discovery.CRC is the most frequently diagnosed gastrointestinal neoplasia, affecting the colon and rectum . It is rCRC is a highly complex and heterogeneous disease from both histopathologic and molecular standpoints . DiseaseCRC complexity and evolution are not solely dependent on an accumulation of genetic modifications in malignant cells. Currently, it is widely accepted that the microenvironment plays fundamental roles, not only in tumorigenesis, but also in controlling progression and dictating CRC prognosis . In factDue to the high heterogeneity of these types of tumors, a new classification system was recently proposed based on consensus molecular subtypes (CMS) that reflect significant biological differences: CMS1 (MSI Immune), CMS2 , CMS3 (Metabolic) and CMS4  . AcknowlThese features highlight the complexity of the disease and have to be considered in strategies aiming to model and study CRC.Cells composing the TME are within an elaborate and active network of ECM proteins, which provides a scaffold structure in which cells communicate and proliferate . This ECIn general, ECM proteins are common to both normal and malignant tissues. However, while these proteins are homogeneously distributed in normal tissues, they present an extremely irregular and heterogeneous distribution in tumors . Among oType 1 collagen overexpression in tumor tissues has been implicated in the promotion of tumor growth, epithelial to mesenchymal transition (EMT), distant metastasis and increased stemness properties of CRC cells, through integrin α2β1 and the activation of PI3K/AKT/Snail and WNT/PCP signaling pathways ,55,56. FLaminin also displays an altered expression in tumors  and is iHyaluronic acid, a non-sulfated glycosaminoglycan (GAG), is highly represented in tumors and constitutes an important component in promoting tumorigenesis, cell proliferation, migration and blocking apoptosis, namely by binding to CD44 and TLR4 ,63.+/CD44+ colon cancer stem cells (CSCs) require EDA-FN binding to integrin α9β1 for sphere formation, and tumorigenic capacity by triggering the FAK/ERK/β-catenin signaling pathway [Fibronectin is another vital component of the ECM that is upregulated in CRC and promotes cell proliferation through the NF-kB/p53 signaling pathway . The ext pathway .A proteomic analysis of colorectal normal and tumor ECM revealed that collagen IV, V and XIV, fibrilin, emilin, vitronectin, laminin and endomucin have increased expression in tumor ECM, and that periostin, versican, thrombospondin-2 and tenascin were exclusively present in tumor tissue. Interestingly, when compared with available clinical gene expression array data, these signatures correlated with tumor progression and metastasis . For exaThe thrombospondin (THBS) family has been associated with the regulation of angiogenesis and cancer progression by controlling multiple physiological processes . THBS-1 Each malignant tumor exhibits a specific proteoglycan molecular signature, which is closely associated with tumor differentiation and biological behavior . In the Altogether, these studies highlight the influence of ECM composition in cellular modulation and disease progression. However, not only the biochemical composition of the ECM but also its biomechanical properties must be considered in terms of TME dynamics.Besides alterations in composition, the ECM also suffers a structural rearrangement in the TME, with the alignment of fibers that ultimately contribute to an anisotropic configuration ,81. WhilCell mechano-sensing translates biophysical forces into cellular responses, impacting several biological pathways, mechanisms and cell behavior. Solid tumors are constantly affected by mechanical stimuli, such as compression, matrix stiffness and fluid mechanics ,84. TissLysyl oxidase (LOX) plays a vital role in this context by crosslinking collagen fibers, enhancing matrix compressing and stiffening in CRC, which then activates cell migration pathways . SpecifiThe protein cross-linking enzyme transglutaminase-2 (TG2) also has a role in modulating biomechanical properties through the formation of cross-links between glutamine and lysine sidechains of target proteins that are resistant to proteolytic degradation, exhibiting important pathophysiological functions . In cancMechanical strains produced by external forces can initiate the expression of tumor-associated genes in CRC preneoplastic tissues . RemarkaMatrix stiffness can also regulate the metastasis of CRC cells by SFK and MLCK through receptor-type tyrosine-protein phosphatase alpha (RPTPα) that senses mechanical stimulation . HCT-8 cNotably, contrary to normal ECM from the same patient, tumor ECM was able to polarize human macrophages into an M2-like phenotype, anti-inflammatory, and pro-tumor, with the expression of specific markers and the secretion of anti-inflammatory cytokines, such as CCL18 . These mRecently, the influence of matrix stiffness in treatment resistance has also gained attention. During chemotherapy, the dense and heterogenous structure of ECM in solid tumors are critical determinants for blood perfusion and interstitial transportation of the drug . The expMatrix stiffness also plays a role in radiation resistance. Ionizing radiation upregulates β1 integrins activating its downstream signals and increases the adhesion of CRC cells to collagen and fibronectin, contributing to the survival of cancer cells after treatment . These eStiffening of the ECM progressively increases from early to later stages of CRC , emphasiCRC 3D models have been recently reviewed, including hydrogels, patient-derived scaffolds, spheroids, organoids, microfluidic devices, and tumor and organ on-chip devices ,118,119.Biomaterial-based 3D organotypic models can be subdivided into scaffold-free or scaffold-based systems . ScaffolThe most common 3D organotypic models for drug delivery and in situ tissue engineering are based on hydrogel-based scaffolds, frequently using natural polymers, such as collagen and alginate. These platforms are the most well-studied due to their low cost, low immunogenicity, versatility, biocompatibility and similarity to natural ECM . These sThree-dimensional bioprinting technology has gained increased relevance by allowing the standardization of the scaffold model between experiments . This teEven though hydrogel-based scaffolds and 3D bioprinting organotypic models are interesting tools to recreate the dynamics between major key players of CRC TME, they fail to incorporate fluidic dynamics between these components . ScaffolAltogether, the previously mentioned scaffold-based 3D systems represent suitable options for studying CRC TME interactions. Still, the exact native composition and structure of the ECM remains difficult to recreate reproducibly in vitro and, consequently, the recreation of the TME is highly restricted .Decellularized ECM from malignant tissues is gaining attention in the field of organotypic modeling of tumor-stroma interactions by successfully incorporating key biochemical and biophysical characteristics of the native TME ,134,135.®, proprietary knowhow Fraunhofer IGB) [TM. Moreover, HCT-116 cells displayed a higher rate of cell invasion when cultured in mouse colon cancer decellularized matrices than in cells cultured in wild-type mouse colon scaffolds or MatrigelTM [Overall, decellularization protocols aim to eliminate all cell material while maintaining ECM architecture and biochemical components and are mainly a combination of physical, chemical and enzymatic methodologies . The remfer IGB) , is an ifer IGB) . AnothertrigelTM .Three-dimensional ECM-hydrogels from decellularized human normal and tumor colon tissues have already been prepared through lyophilization, powdering and solubilization techniques and were useful for showing that tumor ECM components induced faster growth of HT-29 cells and their shift toward a glycolytic metabolism . In an eDespite the undeniable utility and potential of these works that clearly show the impact of the matrix in CRC progression and metastasis, these approaches lack the native ECM architecture and mechanical properties that exhibit an active role in cell behavior ,19.In human CRC, strategies using decellularized ECM to study TME dynamics have been broadly focused on patient-derived scaffolds. Several protocols have been developed with the aim of efficiently removing cellular components from intestinal tissue while maintaining the architecture and biomechanical/biochemical features . These sIn this field, approaches that consider paired CRC and normal adjacent tissue benefit from the direct comparison of samples from the same individual and allow the consideration of the role of tumor versus normal ECM on various cancer-associated activities and interactions with other TME components. Nevertheless, studies with access to this type of exceptionally valuable sample have been mainly restricted to recellularization with only one type of cell and require a further complex to create a structure that most trustworthily resembles the TME. Beyond proteomic and structural characterization of the decellularized ECM, reports showed that tumor ECM modulates IL-8 expression by HT29 cells  and thatFrom a different perspective, Pinto et al.  implemenDecellularized tissues have also been applied in the study of the CRC metastatic process. D’Angelo and colleagues created a model with decellularized normal and primary tumor CRC tissue, as well as matched CRC liver metastasis with the aim of recapitulating this specific microenvironment in vitro . This syOne of the major drawbacks of CRC patient-derived scaffolds is the limited amount of tumor tissue available from each individual, since it derives from biopsies or surgical resections, from which most tissue is required for further diagnostic molecular and histological characterization. Another question to keep in mind is that normal mucosa adjacent to the tumor, while often considered a healthy control from the same individual, in fact represents an intermediate state between normal and tumor tissues . DespiteThe recellularization of decellularized ECM also presents a few challenges, namely the choice of cell(s), the cells’ distribution within the scaffold and the reproducibility of recellularization efficiencies, even in samples from the same patient . ConcernThese are relevant topics to be considered when establishing an organotypic 3D model for CRC cancer with decellularized tissues, as well as for previously determining if there was previous neoadjuvant therapy. Still, the possibilities of these kinds of systems to incorporate ECM, cancer, stromal and immune cells will allow the study of the dynamic and complex crosstalks between the different components and recapitulate more closely the TME and, eventually, design strategies with potential for predicting clinical outcomes.The future of cancer research relies on the implementation of translational 3D in vitro models that accurately mimic human tissues. Such models will foster an improved knowledge of cancer physiological and pathological processes, as well as facilitate drug discovery and screening. To scrutinize the molecular and cellular mechanisms in CRC, it is imperative that the approach to complex TME is recreated, namely the genetic, cell-to-cell, and cell–ECM cues that instruct cancer development and progression. To move forward on the study of TME interactions, decellularized colorectal matrices are attractive bioactive scaffolds, as they may be repopulated with different cell types and submitted to several soluble factors, pharmacological agents and/or radiation therapy.We believe it is essential to standardize tissue-specific decellularization protocols according to tissue fragment size and to the intended final application. Additionally, a consensus on the methods to assess the decellularization efficiency, as well as the structural characterization of the decellularized ECM is also required. Until now, there is still limited information about effective long-term storage methodologies for these scaffolds, but some reports indicate that slow-freezing could provide an interesting solution ,157. We In conclusion, it is widely accepted that accurate 3D cell culture models that consider interactions between CRC cells–TME–ECM are required for understanding disease biology and developing more advanced therapies regarding precision cancer medicine.


Youth is characterized by testing and crossing natural boundaries, sometimes with the help of performance-enhancing substances. In this context, doping prevention measures play a crucial role to protect individuals both within and outside the context of elite sport. Based on the PRISMA guidelines, a systematic literature search was conducted in the databases ProQuest (ERIC), Scopus, PSYNDEX/PsychInfo, PubMed, and Web of Science Core Collection to provide an overview of the impact of doping prevention measures, with particular attention to the underlying understanding of learning. As a result of the screening process, 30 of the initial 5,591 articles met the previously defined and recorded eligibility criteria. The analysis led to heterogeneous results regarding content, implementation, target group, or outcome variables considered relevant. Two-thirds of the studies related to the competitive sports context. Nevertheless, there has been a growing interest in studying doping prevention and its effects on non-elite athlete target groups in recent years. In terms of effectiveness, many measures did not achieve long-term changes or did not collect any follow-up data. This contrasts with understanding learning as sustained change and reduces the intended long-term protection of prevention measures, especially for adolescent target groups. Even young age groups from 10 years upwards benefited from doping prevention measures, and almost all doping prevention measures enabled their participants to increase their physical and health literacy. No conclusion can be drawn as to whether doping prevention measures based on constructivist ideas are superior to cognitivist approaches or a combination of both. Nevertheless, programs that actively engage their participants appear superior to lecture-based knowledge transfer. Most of the prevention measures offered a benefit-orientation so that participants can achieve added value, besides trying to initiate health-promoting change through rejection. Because of the lack of sustained changes, a further modification in doping prevention seems necessary. The review results support the value of primary prevention. Doping prevention measures should enable tailored learning and development options in the sense of more meaningful differentiation to individual needs. The implementation in a school context or an online setting is promising and sees doping as a problem for society. The review highlights the importance of accompanying evaluation measures to identify efficient prevention components that promote health and protect young people. Doping prevention is a matter for society as a whole and not an exclusive concern of elite sport. This statement is the consequence of considering the desire for performance-enhancement as a societal phenomenon and acknowledging the association of athletic success and appearance with strength, competence, social ability, or beauty use of performance-enhancing substances in the sense of education . Furthermore, anti-doping agencies offer targeted doping prevention measures for schools  offer a favorable opportunity because they appeal to young target groups regardless of their athletic performance. Unlike in elite sport, they do not need to involve personally addressed repressive information components such as punishments and suspensions. Taking Germany as an example, anti-doping education is not a compulsory part of the school's general physical education curriculum. It is only offered to students who wish to gain university entrance and have chosen physical education as an examination subject concerning their high school diploma .In addition to previous recommendations, further practical implications are derived from the results of this review.The systematic review was conducted following the PRISMA recommendations , Scopus, PSYNDEX/PsychInfo, PubMed, and Web of Science Core Collection databases. The search strategy was based on a specification of the thematic content of relevant studies, the population studied, the label of the intervention, including a description of possible outcomes, and references relating to the studies' evaluation. The systematic approach and the search terms used are presented in The definition of the eligibility criteria was based on the PICOS approach to specifying the participants, interventions, comparisons, outcomes, and study designs to be considered in advance . The studies were mainly conducted in western societies  and span a performance spectrum from international elite athletes . One of the notable features here is that only a few studies have previously conducted a power analysis to determine the optimal sample size  or associated variables, such as nutritional supplements  or diet. Less frequently, norms, behavioral control, or values were evaluated. Concerning self-reported doping behavior, it is noticeable that studies that surveyed both behavior and doping intentions and attitudes showed short-term but rarely long-term changes in doping intentions or attitudes . Programs that provided a personal benefit through the topics covered appeared to be more effective. Meanwhile, measures that focused purely on deterrence . Besides, a large part of the prevention measures also offered a benefit character. For example, the ATLAS and ATHENA programs imparted knowledge about healthy eating or efficient training  Doping prevention measures should be scientifically monitored and evaluated in longitudinal or experimental designs. This approach implies the need for long-term research funding opportunities that also include consideration of follow-ups  More international and transdisciplinary collaborative doping prevention networks composed of researchers and individuals from elite sport  should be established  In terms of tailored doping prevention measures, developers of anti-doping interventions should consider a modular system that offers participants opportunities for differentiation within an overarching theme. This differentiation should increase interest and enable more efficient learning.(d)In addition to self-reporting, alternative methods like implicit or indirect procedures should be used to consider the effects of doping prevention measures Online-based prevention interventions offer benefits of increased individualization but should be evaluated in terms of learning success.(f) Prevention measures should be integrated into school curricula at an early stage and with a positive connotation  so that a constructive atmosphere can be created.(G) The perspective on clean athletes and their empowerment should be expanded  can be found in the article/supplementary material.The author confirms being the sole contributor of this work and has approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Emergency Department (ED) attendances with chest pain reduced during the COVID-19 lockdown. We performed a service evaluation project in NHS Lothian to explore how and why the COVID-19 pandemic and public health advice had affected chest pain presentations and help-seeking behaviour at an individual patient level using a qualitative interview approach.We carried out 28 semi-structured telephone interviews with a convenience sample of patients who presented with chest pain during lockdown and in patients with known coronary heart disease under the outpatient care of a cardiologist in April and May 2020. Interviews were audio recorded and voice files listened to while making detailed notes. Salient themes and issues were documented as verbatim extracts. Interviews were analysed thematically.Patient interviews revealed three main themes. 1) pandemic help-seeking behaviour; describing how participants made the decision to seek professional healthcare assessment. 2) COVID-19 exposure concerns; describing how the subthemes of perceived vulnerability, wishing to protect others and adding pressure to the health service shaped their decision making for an episode of acute chest pain. 3) hospital experience; describing the difference between the imagined and actual experience in hospital.Qualitative interviews revealed how the pandemic shaped help-seeking practices, how patients interpreted their personal vulnerability to the virus, and described patient experience of attending hospital for assessment during this time. As patient numbers presenting to hospital appeared to mirror public health messaging, dynamic monitoring of this messaging should evaluate public response to healthcare campaigns to ensure the net impact on health, pandemic and non-pandemic related, is optimised. Symptoms suggestive of acute coronary syndrome are one of the most common reasons for Emergency Department presentation . Reportsrd March 2020 with advice to ‘stay home, protect our NHS, save lives’. Patients admitted to acute medical units in NHS Lothian, Scotland, during the first 31 days after lockdown were of higher medical acuity and had a higher risk of inpatient mortality when compared to patients in the same period in the preceding 5 years [Scotland entered lockdown on 23 5 years . This suPrevious research on decision making in response to chest pain has revealed a complex series of actions. Patients perform a process of symptom interpretation and self-evaluation of coronary candidacy to assess personal risk , they ofInternal audit data from our own centre revealed the average weekly number of Emergency Department attendances with suspected acute coronary syndrome fell from 287/week between January and May 2019 to 233/week in 2020. The lowest number of attendances per week (128) was seen in the last week of March 2020 as lockdown was announced (unpublished data) . Google These data highlighted the need to explore how and why the COVID-19 pandemic and public health advice had affected chest pain presentations and help-seeking behaviour at an individual patient level using a qualitative interview approach.Single semi-structured telephone interviews were conducted with patients attending hospital for the assessment of suspected acute coronary syndrome between the 17 April 2020 and 08 May 2020. Participants were identified using an order request system for cardiac troponin which identifies all patients with suspected acute coronary syndrome in our centre and permits review of the electronic patient record . ConveniThis project was reviewed by NHS Lothian Research and Development Office and the South East Scotland Research Ethics Service. These bodies advised the project was service evaluation therefore research ethics committee approval was not required. It was registered with and approved by the local cardiology Quality Improvement Team according to local practice.This study was guided by the principles of phenomenology  aiming tThe project was conceptualised through discussion with patients admitted to a cardiology ward during the COVID-19 pandemic. Key ideas for interview questions were developed by consultation with the patient group.Interview participants were aged between 39 and 88 years and 54% were female. 14 participants had an admission troponin greater than the diagnostic threshold for myocardial infarction, 7 had an admission troponin less than the diagnostic threshold for myocardial infarction and 7 were under the care of a cardiologist as an outpatient.Telephone conversations revealed three main themes; pandemic shaping of help seeking behaviour, COVID exposure concern, and hospital experience. These are summarised in Table 1.This theme describes how participants made the decision to seek professional healthcare assessment. It is divided into subthemes describing a staged response to help seeking; 1) symptom appraisal, 2) consultation of lay members for advice, and 3) accessing professional healthcare assessment. Quotations illustrating these interpretations are given in Firstly, participants reported performing a symptom appraisal which could lead to symptoms being attributed to other causes, for example indigestion, due to the transient nature of their symptoms or lack of severity. Some participants had experienced myocardial infarction previously and performed a comparison between prior experience and their current symptoms. Based on their self assessment of symptoms, participants then progressed to the next stage.Persistence of symptoms triggered discussion with local neighbourhood networks and family to decide on the next course of action. It was noted that for the participants in this study, these networks often included a healthcare professional who happened to live closeby. The subsequent outcome of this discussion typically involved contacting a GP or the NHS out of hours service (NHS24) for preliminary assessment. No barriers to accessing these services were reported. Very few participants telephoned the Scottish Ambulance Service directly without additional assessment, with only a few self-presenting to a hospital Emergency Department.Some participants reported that gaining access to physical examination by a healthcare professional was limited. Consultations tended to be telephone based which were sometimes viewed as inadequate due to difficulty in describing symptoms over the telephone to a doctor with whom they did not have a relationship. Other participants did have access to face-to-face primary care appointments but similarly these were not viewed positively due to lack of physical examination.One patient  described having three GP phone consultations and an out of hours appointment where she had blood pressure and oxygen saturations recorded but no ECG performed despite describing chest, arm and jaw pain. This patient subsequently self-presented to the Emergency Department with a confirmed acute myocardial infarction.Table 2.Participants were asked whether coronavirus, and the societal-response to it, had affected their decision making to attend hospital. Participants differed in their response with some stating their decision making was completely unaffected. Some participants were very concerned about presentation to hospital due to COVID-19, however, it did not stop them attending. Concerns could largely be attributed to three subthemes; perceived vulnerability to the virus, wishing to protect others, and avoidance of adding pressure to busy health services. These will be considered separately with quotations used to illustrate interpretations given in Participants spoke about how they believed their personal vulnerability to the virus and access to treatment would be influenced by their increased age. Some participants believed attending hospital would increase their exposure to coronavirus and discussed the possible repercussions of this with reduced access to ventilators with increased age. One participant  felt at increased risk of contracting COVID-19 after sharing a hospital room with three elderly patients. He cited media reports that elderly people were more at risk of severe COVID-19 disease and interpreted that to mean a greater risk of contracting COVID-19 by sharing a room with elderly people.Participants also expressed their vulnerability when talking about pre-existing health conditions. One participant  felt vulnerable to COVID-19 due to an impaired immune system and a previous myocardial infarction. A family decision was made to limit her potential exposure to coronavirus from her daughter, a nurse, by leaving the family home to live in temporary accommodation. Whilst this participant did attend hospital for assessment, she stated that she was frightened. Another patient  who attended hospital with myocardial infarction stated that suffering an acute cardiac event made him more vulnerable to coronavirus and he now considered himself to be in a more vulnerable category.A further participant  had two ED chest pain presentations during lockdown. Initially she was assessed and discharged then represented two weeks later with acute myocardial infarction. She knew attending was the most appropriate course of action but described an internal conversation aiming to weigh up the risks of exposing herself to the virus balanced against the risk of not seeking assessment for chest pain.Protecting others was another consideration for participants. One participant  actively wanted to attend hospital to access a test for COVID-19 so she knew she was not putting her carers at risk. Other examples included considering the exposure risk to grandchildren at home, and inadvertently transmitting the virus from the hospital environment to a vulnerable adult in the community by choosing to attend hospital.Some participants explicitly stated they were not concerned about adding pressure to health services by attending the ED. They described feeling so unwell they knew they had to attend hospital. Others had learnt that Emergency Departments were quiet through discussions with their GP or local networks which included health professionals. Participants also stated they knew that hospitals were fully open.For others, media images such as those being reported in Italy were a factor in their reluctance to attend hospital. Daily news reports detailing the number of new cases and deaths, the building of new emergency hospitals with large capacity and images of staff wearing protective suits all contributed to the message of ‘Stay Away’ at the beginning of the pandemic. Participants stated how their perception of this message had changed over time. Publicity about decreased hospital demand was cited as a reason why some participants who would have been reluctant to use services in the beginning were now less concerned about doing so.Table 3.Participants reported a much more positive hospital experience than they had anticipated. They stated the ED assessment areas were quiet and that they were seen quickly. Some were not aware that patients with COVID-19 symptoms entered the hospital and were assessed through a different point of access. Once in hospital they could see they were separated from suspected COVID-19 patients. Some participants were informed by their GP that this would be the case, others said they assumed the NHS would take this action. Quotations used to illustrate these interpretations are given in Many participants reported feeling safe while in hospital due to regular changing of personal protective equipment and hand washing by staff, in addition to highly visible cleaning taking place.One participant commented that nurses in the hospital ward were not social distancing. It was also mentioned that not being able to have anyone accompany you to the ED or visit you in hospital made an already worrying time even more difficult.Fig 1) suggesting that these may have impacted help seeking behaviour for chest pain during the early stages of the COVID-19 pandemic.Key public health strategies were targeted at decreasing community transmission of SARS-CoV-2 included hand washing, social distancing and self-isolation. Mass media campaigns have previously been shown to elicit potentially beneficial behaviour change in response to the SARS and H1N1 epidemics regarding hand washing and social distancing . GovernmThe majority of participants first sought an assessment of chest pain through primary care services. Reluctance to use the emergency services has been seen prior to the COVID-19 pandemic due to concern about appropriate use of the NHS and resources , 11. It As the actual hospital experience was often very different to the imagined experience, and usually positive, it may be useful for future media and government message campaigns to outline clearly a step by step mechanism by which people can access emergency services and to clearly describe safety measures adopted in Emergency Departments to minimise risk for patients that need to attend hospital urgently during future pandemic events. Commercial sectors of society, for example supermarkets, have done this with television campaigns. A ‘Ways we are keeping you safe’ campaign highlighting that hospitals have taken steps to create separate emergency assessment areas into COVID-19-free zones may make patients feel more comfortable attending the hospital.Patient concerns regarding ‘vulnerability to the virus’ has emerged as an important discourse during the pandemic possibly due to a lack of clarity on which categories of patients were and were not included in government-defined vulnerable groups and the coverage of this topic in the media. For example, a government spokesperson gave confusing messages regarding the inclusion of people over 70 years of age in the vulnerable category . ParticiWhile we aimed to capture the experience of patients who chose not to come to hospital with symptoms of chest pain by targeting those with known coronary heart disease from a community setting, none of the participants in the sample had experienced chest pain during the study period for which they would have normally sought hospital assessment. However, this service evaluation project has revealed valuable insights into how the decision to attend hospital was shaped by the pandemic. The first interviews were carried out at or just after the time of the release of a public health messaging campaign to promote attendance to the ED for urgent conditions. While some interviews included participants who had experienced symptoms two weeks earlier, perceived decision making may have changed in response to evolving media and news campaign. We did not explore factors influencing help seeking behaviour and hospital attendance during the early media campaigns advising patients to stay at home and protect the NHS.Future media and public health campaigns associated with subsequent waves of COVID-19 infection should seek to strike a balance between appropriate care-seeking and avoidance behaviour. Such campaigns should be designed to include dynamic monitoring of the public response to healthcare messaging in a way that permits rapid adjustment to ensure that the net impact on health, pandemic and non-pandemic related, is optimised.S1 Appendix(DOCX)Click here for additional data file.


Muscle and bone interactions might be associated with osteoporosis and sarcopenia. Urinary pentosidine and serum 25-hydroxyvitamin D (25(OH)D) might affect muscle and bone interactions. It is unclear whether these biomarkers are affected by age and sex or play a role in muscle and physical functions. We aimed to investigate the association between urinary pentosidine and serum 25(OH)D levels with muscle mass, muscle strength, and physical performance in community-dwelling adults.Two-hundred and fifty-four middle-aged and elderly adults were enrolled. There was no significant difference in age between 97 men (75.0 ± 8.9 years) and 157 women (73.6 ± 8.1 years). The skeletal muscle mass index (SMI), grip strength, and gait speed were assessed. The urinary pentosidine level was measured. We evaluated the association of urinary pentosidine and serum 25(OH)D levels with age and sex (student’s t-test) and correlations between biomarker and each variable (Pearson’s correlation coefficients). Multiple regression analysis was performed with grip strength and gait speed as dependent variables and with age, height, weight, body mass index (BMI), speed of sound (SOS), SMI, glycated hemoglobin (HbA1c), estimated glomerular filtration rate (eGFR), 25(OH)D, and pentosidine as independent variables using the stepwise method.The urinary pentosidine level was negatively correlated with grip strength, gait speed, eGFR, and insulin-like growth factor-1 (IGF-1) in men and with SOS, grip strength, and gait speed in women. The serum 25(OH)D level was positively correlated with IGF-1 in women and grip strength in men. Grip strength was associated with age, height, and pentosidine in men and height and pentosidine in women. Gait speed was associated with age, BMI, and pentosidine in men and age, height, and pentosidine in women.Urinary pentosidine levels are significantly associated with grip strength and gait speed and may serve as a biomarker of muscle and bone interactions. In recent years, there has been a considerable focus on the relationship between osteoporosis and sarcopenia. In previous studies, osteoporosis patients with fragility fractures were found to have a high prevalence of sarcopenia , 2. MuscPentosidine is a representative cross-linked structure of advanced glycation end products (AGEs), which are induced by the oxidation of bone collagen crosslinks . PentosiErgocalciferol and cholecalciferol (D3) are incorporated into the diet; vitamin D3 is also synthesized in the skin and undergoes hydroxylation in the liver to become 25-hydroxyvitamin D (25(OH)D). 25(OH)D is stable in the blood, and its level has recently been reported to be useful for assessing vitamin D sufficiency . 25(OH)DWe found that insulin-like growth factor-1 (IGF-1) is an important biomarker not only of muscle tissues but also of bones in community-dwelling middle-aged and elderly adults . We simiThis cross-sectional study used data of participants enrolled in the Good Aging and Intervention Against Nursing Care and Activity Decline (GAINA) study in the town of Hino, Tottori Prefecture, Japan –18. A toBlood samples were taken before the assessment of body structure and physical function parameters. Serum creatinine and glycated hemoglobin (HbA1c) levels were measured. The estimated glomerular filtration rate (eGFR) was calculated using the following formula:The serum IGF-1 and parathyroid hormone (PTH) levels were measured using a radioimmunoassay and an electrochemiluminescence immunoassay (ECLIA) kit , respectively. Serum 25(OH)D levels were measured using an ECLIA kit . The limit of quantification is the lowest analyte level that can be reproducibly measured with an intermediate precision coefficient of variance of ≤20%.The urinary pentosidine levels were measured using an enzyme-linked immunosorbent assay (ELISA) kit . The ELISA kit consisted of polyclonal anti-pentosidine IgG and a secondary antibody, and the accuracy, precision, and reliability of this kit were evaluated. In brief, the limit of blank and the limit of detection were 4.25 and 6.24 pmol/mL, respectively. The intra-assay and inter-assay coefficients of variation were < 5%. The spiking and dilution recoveries were 101.4 and 100.5%, respectively. An analysis of cross-reactivity against seven compounds representative of AGEs and with structures close to pentosidine revealed no significant cross-reactivity. The comparability between the values obtained from high-performance liquid chromatography (HPLC) and ELISA (in the same urine samples) was r = 0.815 . Serum cp-value of < 0.05 was considered statistically significant.We assessed the association of urinary pentosidine and serum 25(OH)D levels with age and sexes using the student’s t-test and correlations between biomarker and each variable on Pearson’s correlation coefficients . Finally, multiple regression analysis was performed with grip strength and gait speed as dependent variables and with age, height, weight, body mass index (BMI), SOS, SMI, HbA1c, eGFR, 25(OH)D, and pentosidine as independent variables in men and women using the stepwise method. We judged multicollinearity using a variance inflation factor. All statistical analyses were performed by SPSS statistical software . A p = 0.198). BMI was significantly higher in men (23.0 ± 2.5 kg/m2) than in women (22.0 ± 3.1 kg/m2) (p = 0.014). The right calcaneal SOS (1503.6 ± 28.3 vs 1483.4 ± 20.2 m/s), SMI (7.5 ± 0.8 vs 6.1 ± 0.8 kg/m2), and grip strength (34.9 ± 7.4 vs 23.0 ± 4.5 kg) were significantly higher in men than in women (p < 0.001). Gait speed and IGF-1 were not significantly different between sex [There was no significant difference in the mean age between men (75.0 ± 8.9 years) and women (73.6 ± 8.1 years) (p < 0.001), whereas urinary pentosidine levels were significantly higher in women than in men (p = 0.010). Serum 25(OH)D and urinary pentosidine levels are shown in scatter plots by age in Fig. r = − 0.116, p = 0.258) or women . On the other hand, pentosidine was positively correlated with age in both men  and women .The results of the statistical comparison of serum eGFR, HbA1c, PTH, and 25(OH)D and urinary pentosidine levels between men and women are shown in Table Pearson’s correlation coefficients are shown in Table The purpose of this study was to investigate the association between urinary pentosidine and serum 25(OH)D levels with muscle mass, muscle strength, and physical performance in community-dwelling adults. Serum 25(OH)D levels were significantly higher in men than in women; one of the reasons for this sex difference may be general inactivity and lower intake of vitamin D from daily food among Japanese elderly women compared with men .Urinary pentosidine levels are generally measured by HPLC, but this approach cannot be adapted to analyze many clinical samples, and it is also a time-consuming process. Furthermore, the detection of pentosidine using a reported ELISA kit and an HPLC system requires heat pretreatment, which generates artificial pentosidine, leading to overestimation. A novel pentosidine ELISA system that does not require sample pretreatment for analyzing urine samples has been developed . In thisWe found that urinary pentosidine levels were significantly higher in women than in men. There are few studies on the association between urinary pentosidine level and sex in community-dwelling adults; nevertheless, one of the reasons for this difference is that AGEs may be associated with low physical function in elderly women. We found that pentosidine was significantly associated with grip strength and gait speed in men and women. In recent years, the relationship between AGEs and sarcopenia has attracted much attention. However, there has been no report showing the relationship between urinary pentosidine level and specific parameters of physical function.Independent of age and eGFR , the uriOur study is limited by its relatively small sample size, cross-sectional study design, and recruitment of more women than men. The strength of this study is that it is the first to evaluate the association of urinary pentosidine levels with physical function simultaneously. In the future, it will be necessary to conduct longitudinal studies to confirm the findings of this study. These studies may eventually help elucidate the relationship between osteoporosis and sarcopenia.In this study, the urinary pentosidine level was significantly associated with grip strength and gait speed. We consider that urinary pentosidine level may serve as a biomarker affecting muscle and bone interactions in clinical practice.


Late-life depression is a major mental health problem and constitutes a heavy public health burden. Frailty, an aging-related syndrome, is reciprocally related to depressive symptoms. This study investigated the associations of physical frailty and oral frailty with depression in older adults. This large-scale cross-sectional study included 1100 community-dwelling older adults in Taiwan. The participants completed a dental examination and questionnaires answered during personal interviews. The 15-item Geriatric Depression Scale was used to assess depression, and information on physical conditions and oral conditions was collected. Multivariable logistical regression analysis was conducted to examine associations of interest. Significant factors associated with depression were pre-physical frailty (adjusted odds ratio (aOR) = 3.61), physical frailty (aOR = 53.74), sarcopenia (aOR = 4.25), insomnia (aOR = 2.56), pre-oral frailty (aOR = 2.56), oral frailty (aOR = 4.89), dysphagia (aOR = 2.85), and xerostomia (aOR = 1.10). Depression exerted a combined effect on physical frailty and oral frailty (aOR = 36.81). Physical frailty and oral frailty were significantly associated with late-life depression in community-dwelling older adults in a dose–response manner. Developing physical and oral function interventions to prevent depression among older adults is essential. The aging population is growing worldwide. At present, 16% of Taiwanese are older than 65 years. Ten years from now, older adults are projected to constitute one-fourth of Taiwan’s population . As of 2The late-life depression is part of frailty. Frailty is caused by life-course determinants and disease(s), accumulation of physical, psychological and/or social deficits in functioning, increasing the risk of negative health outcomes such as disabilities, admission to health care facilities, and death, is increasingly common in older adults . Jung etDepression has been found to be strongly associated with oral function ,12 and xStudies have demonstrated an association between frailty and depression ,20. AssoThis large-scale cross-sectional study, which was conducted from May 2018 to January 2019, included community-dwelling adults in Taiwan aged ≥65 years. Stratified cluster sampling was performed, with seniors’ recreation centers selected randomly according to their location in urban, rural, and mountainous areas. Individuals were excluded if they had mental disorders or expressive language disorders, as indicated by the possession of integrated circuit card for severe illness; mild cognitive impairment or dementia, as determined using the Short Portable Mental Status Questionnaire (SPMSQ) ; and higData were collected using a structured questionnaire developed by Lu et al. . The queDental examinations were performed by seven dentists based on the World Health Organization criteria . RegardiThe GDS-15 is a self-reported measure of late-life depression in older adults. The 15 items were selected from the Long Form Geriatric Depression Scale because of their high correlation with depressive symptoms. Users respond in a “Yes/No” format. We administered the Chinese version of the GDS-15 . Scores Physical conditions included insomnia, physical frailty, sarcopenia, and comorbidities. All variable data were collected during the study and are listed as follows.InsomniaInsomnia was measured using the Chinese version of the PSQI, a self-rated questionnaire that assesses sleep quality and sleep disturbances. The Chinese version has been validated and determined to have adequate reliability . The 19 Physical frailtyThe SOF criteria are regarded as being as effective as the frailty criteria for predicting adverse health outcomes but are easier to apply . The SOFSarcopeniaSARC-F has high specificity (94–99%) and is a suitable tool for community screening for sarcopenia . The fivComorbiditiesThis variable was identified by self-reports of more than two chronic diseases, such as hypertension, diabetes, heart disease, and chronic obstructive pulmonary disease.Oral condition was examined across six components, namely dysphagia, xerostomia, masticatory performance, the EI , oral diadochokinetic rate, and the Silness–Löe Plaque Index . For each item, a score of 1 was defined as meeting the targeted measures as the presence of dysphagia, xerostomia, poor masticatory performance, EI categories B3–C3, oral diadochokinetic rate (indicated by failure to pronounce the “ta” monosyllable more than six times per second), and a Silness–Löe Plaque Index score >0.95. Total scores of 0, 1–2, and ≥3 points corresponded to non-oral frailty, pre-oral frailty, and oral frailty, respectively. The six components were investigated as follows.DysphagiaDysphagia was assessed through rapid screening by using the Ohkuma questionnaire, which contains 15 items . ExampleXerostomiaA condensed version of the Xerostomia Inventory was administered to identify and classify mouth dryness . The folMasticatory performanceMasticatory performance was evaluated using color-changing chewing gum . This chewing gum contains xylitol, citric acid, and red, yellow, and blue dyes that change color when subjected to masticatory forces from chewing. The red dye is pH sensitive and changes color under neutral or alkaline conditions. Citric acid maintains the internal pH of the initially yellowish-green gum at a low level before chewing commences. As chewing progresses, the yellow and blue dyes seep into the saliva, and the release of citric acid causes the gum to turn red . ParticiOcclusal supportOcclusal support was evaluated using the EI , which iOral diadochokinesis rateOral diadochokinesis was assessed by examining articulatory oral motor skills at sites such as the lips, tip of the tongue, and the dorsum of the tongue . The parOral hygieneMeasurement of the state of oral hygiene by using the Silness–Löe Plaque Index is based on the examination of both soft debris and mineralized deposits on the number 12, 16, 24, 32, and 44 teeth. Each of the four surfaces of the teeth  is given a score from 0 to 3.Data were collected through face-to-face interviews, which were administered by well-trained interviewers in compliance with a standard protocol. The collection process comprised three steps lasting an hour total. First, a dentist performed the dental examination and recorded the dental status. Second, an interviewer administered the structured questionnaire. The entire interview process took approximately 30 to 45 min. Finally, the research personnel collected masticatory performance data, recorded the monosyllable pronunciation data, and physical function as complete five consecutive chair rises, walking 5 m, climbing a flight of 10 stairs, lifting and carrying 4.5 kg.2 test were conducted to assess the relationship between the factors  and outcomes. Variables exhibiting statistically significant associations in univariate analysis were included in the multivariate analysis. The adjusted odds ratio (aOR) and 95% confidence interval (CI) obtained through the exponentiation of the corresponding regression coefficient were employed in evaluating the association between the study variables and depression status after the effects of potential confounders  were adjusted for. Multivariable logistical regression models were used to evaluate the combined effects of physical frailty and oral frailty on depression status.The participants’ depression status was categorized as late-life depression or non-late-life depression. The data are expressed as means and standard deviations or as frequencies and percentages, and the two-sample t test and χp < 0.001). Of the older adults aged ≥75 years, 62.3% had an occlusal condition in the B3–C3 category, and the mean of the Silness–Löe Plaque Index was 1.04. Significantly higher proportions of older adults aged ≥75 years  failed to pronounce the “pa” “ta” and “ka” monosyllables six or more times per second compared with those in the ≤74 years .The characteristics of the participants, categorized according to age, are presented in p for trend <0.001).The results confirm the premise that physical frailty and oral frailty are associated with late-life depression. Notably, physical frailty, sarcopenia, insomnia, oral frailty, dysphagia, and xerostomia exerted significant combined effects in the participants with all these conditions.Older adults with physical frailty, sarcopenia, and insomnia were more likely to have late-life depression than were older adults without all these conditions. The rates of physical frailty and sarcopenia in the participants aged ≥75 years were three and nine times the corresponding rates in the participants aged ≤74 years. Sarcopenia and depressive symptoms are associated with frailty  reportedPre-oral frailty and oral frailty were associated with late-life depression in a dose–response manner. Oral frailty was twice as prevalent among the older adults aged 75 and older compared with among those aged 65 to 74 years, indicating that oral function declines with age. This was also found in previous research. The researchers found a turning point in oral health from the age of 75 . Eating We observed the participants with both dysphagia and xerostomia were more likely to have late-life depression. Polypharmacy is common among older adults. Medication-induced xerostomia was higher with polypharmacy, most notably in those taking antidepressants . In the Examination of combined effects revealed that the participants with both physical and oral conditions such as physical frailty and dysphagia, or physical frailty and xerostomia, are at increased risk of developing late-life depression. Azzolino et al.  identifiThis study has some limitations. First, because the sample comprised community-dwelling older adults from southern Taiwan, the findings may not be generalizable to all older adults. However, the study methodology can be extended to investigations of community-dwelling older adults’ physical and oral function. Second, we did not evaluate lips and tongue muscle strength, which is related to swallowing motion. However, oral diadochokinetic can be applied to evaluate oral motor skills at sites such as the lips and the tip and dorsum of the tongue in community-dwelling older adults. Third, we used subjective Xerostomia Inventory to identify and classify mouth dryness rather than objective instruments to measure xerostomia. However, the scale is a valid measure for discriminative use in clinical and epidemiologic research . Fourth,Physical frailty and oral frailty were significantly associated with late-life depression in community-dwelling older adults in a dose-response manner. Physical frailty, sarcopenia, insomnia, oral frailty, dysphagia, and xerostomia exerted significant combined effects on late-life depression. The findings suggest that developing early intervention strategies is integral to prevent frailty among older adults, which can in turn reduce the likelihood of late-life depression onset among them.