Numerous dietary components have been linked to the development of islet autoimmunity (IA) and type 1 diabetes (T1D); however, no associations are firmly established. This systematic review and meta-analysis aimed to synthesize current knowledge on diet and incidence of IA and T1D.Literature search was performed in Medline, Embase, and Cochrane Library, from inception until October 2020. Eligible studies had IA or T1D as outcome; any dietary exposure; case-control, cohort, or randomized controlled trial design; and hazard, risk, or odds ratios as measures of association. Summary relative risks (RR) and 95% confidence intervals (CI) were estimated with random-effects models. Certainty of evidence was assessed with GRADE. PROSPERO registration number: CRD42020212505.2=43%) and exclusive  breastfeeding, later introduction to gluten , cow's milk , and fruit . Higher childhood intake of cow's milk was associated with increased risk of both IA  and T1D . For the remaining dietary factors investigated, there was no association, or the evidence was of low certainty.Among 5935 identified records, 96 were eligible, and pooled estimates could be produced for 26 dietary factors. Evidence with moderate/high certainty indicated lower risk of T1D in relation to longer , Novo Nordisk Foundation, and Swedish Diabetes Foundation. Certainty of evidence was evaluated for each association.Over the past decades, several dietary exposures at different developmental stages have been assessed in relation to type 1 diabetes (T1D), and many of those have been proposed as potential risk factors. Previous meta-analyses assess a limited number of dietary factors and ours is the first attempt to synthesize the totality of evidence regarding the influence of diet on T1D risk. We searched Medline (Ovid), Embase, and Cochrane Library (Wiley) for cohort and case-control studies, as well as randomized controlled trials (RCTs), published until October 15It was possible to synthesize data for 26 dietary exposures in utero, infancy, childhood, and/or adulthood in relation to IA and T1D from 96 observational studies. Evidence rated with moderate or high certainty suggested a reduced risk of T1D in relation to longer breastfeeding and later introduction to gluten, cow's milk, and fruit, and an increased risk of IA conferred by high childhood intake of cow's milk. Associations were also seen between T1D and childhood intakes of carbohydrates, sugar, sugar sweetened beverages, protein, meat, nitrite, vitamins A and C, as well as vitamin D supplementation in infancy. However, the certainty of this evidence was graded as low. The small number of identified RCTs has failed to find beneficial effects of dietary interventions.This study supports that diet may play a role in the development of T1D. However, an important conclusion is that although many dietary factors are associated with T1D, the certainty of this evidence is low. There is a clear need for future high quality observational studies, preferable with objectively assessed nutritional information and detailed adjustments for confounders, to further elucidate the influence of diet in the etiology of T1D. We also need clinical trials to evaluate if diet modification may indeed prevent T1D.Alt-text: Unlabelled box1Type 1 diabetes (T1D) is an autoimmune disease characterized by destruction of the insulin producing beta-cells that results in chronic dependence on exogenous insulin Despite decades of research, the role of environmental factors in the etiology of T1D remains unclear 22.1https://www.crd.york.ac.uk/prospero/display_record.php?ID=CRD42020212505. This study adheres to the PRISMA guidelines. Eligible studies were in English, had IA or T1D as outcome; nutrients, foods, or beverages, or nutritional biomarker levels as exposure; cohort, case-cohort, case-control, nested case-control, or randomized controlled trial (RCT) design; and hazard ratios, risk ratios, or odds ratios with 95% confidence intervals (CI), as measures of association. In the present paper, the term relative risk (RR) was used for all measures of association. Congress papers, editorials, interviews, letters, and animal studies were excluded.A systematic review and meta-analysis was conducted after registration in the International Prospective Register for Systematic Reviews (PROSPERO) th, 2020, by librarians at Karolinska Institutet University Library (for complete search strategy see Supplementary Tables 1-3). Relevant studies were retrieved from the reference lists of eligible articles. Title and abstract of all identified studies were screened and articles that seemed likely to fulfill the eligibility criteria regarding the exposure and outcome definitions, as well as the study design, were fully examined. Study screening, data extraction, and risk of bias and certainty of evidence assessment were performed independently by AML and JEL, and disagreements were resolved by consultation with SC.Medline (Ovid), Embase, and Cochrane Library (Wiley) were searched from inception until October 152.2From eligible articles we extracted name of first author, publication year, country, cohort name (when applicable), study design, sample size, number of cases, sex, age at diagnosis, type and quantity of exposure, reference group, outcome, method of exposure and outcome assessment, presence of IA at baseline, risk genotypes, family history of T1D, follow-up time, RR with 95% CI, and included covariates. When multiple estimates were available, the most adjusted was extracted and when only separate estimates of different population strata  were provided, they were pooled using fixed-effects models before inclusion in meta-analysis. Additionally, when several articles used the same data, only one of these was included (Supplementary Table 4). We contacted the corresponding author when vital information was missing and if no response was obtained, the study was excluded.2.3Risk of bias was assessed with the Risk of Bias in Non-randomized Studies of Interventions (ROBINS-I) 2.4Statistical analyses were performed with Stata Statistical Software Release 16 (StataCorp). Random-effects models were used for estimating summary RR and 95% CI of IA and T1D in relation to the highest versus lowest or continuous exposure to each dietary factor, depending on the availability of estimates. The cut-offs of categorical exposures differed across studies and are therefore presented as ranges in the meta-analyses. Thus, the range represents the cut-offs used in the individual studies, e.g., the cut-off ≥2-3 vs <2-3 months means that some of the included studies used the cut-off of two months, and others used three months. Exposure definitions in individual studies are presented in Supplementary Table 6. If the exposure categorizations were too diverse to pool, we present the RR from the meta-analysis based on the largest number of studies, in 2 statistic . To investigate the source of heterogeneity, subgroup analyses based on study design, risk of bias, and genetic susceptibility, were performed when I2 >50% and at least five studies were available. We also performed post hoc sensitivity meta-analyses restricted to prospective data for the outcome T1D; for the outcome IA all studies had a prospective design. Small study effects were assessed for meta-analyses with at least 10 studies with the Egger's test and contour-enhanced funnel plots with critical regions at 1%, 5% and 10% significance levels were used to discriminate whether small study effects were attributable to publication bias.Heterogeneity across studies was assessed with the Cochran's Q test and the percentage of observed variance due to heterogeneity with the I2.5The certainty of evidence for each meta-analysis was assessed through the Grading of Recommendations, Assessment, Development and Evaluations (GRADE) framework, which classifies the certainty of evidence as ‘very low’, ‘low’, ‘moderate’, and ‘high’ 2.6The funders had no role in study design, data collection, analysis, interpretation, or writing of the report.33.1Out of 5,935 articles initially screened, 96 studies with a cohort (n=42) or case-control (n=54) design could be meta-analyzed . Of thes42=43%). A tendency towards an inverse association was noted for later introduction to infant formula, whereas age at introduction to solid food was unrelated to T1D, although with substantial heterogeneity across studies (Longer durations of any or exclusive breastfeeding were inversely associated with T1D . The lar studies . Neither studies .4.1Later introduction to cow's milk (≥2-3 versus <2-3 months) was inversely associated with T1D , whereas4.2For T1D, positive associations were observed in relation to childhood intake of meat, protein, and nitrite, with no heterogeneity between studies . In cont4.32=98%) and IA  in relation to higher childhood intake of omega-3 fatty acids, but the associations were not significant and there was substantial heterogeneity (2=0%), but no data on a potential association with T1D. There was no indication of a reduced risk of T1D in relation to childhood fish intake.The results were compatible with a reduced risk of T1D , with a similar tendency for age at introduction to vegetables  and childhood intake of fruit juice  . Higher 4.52=0%) with a similar tendency for cereal introduction  and, for gluten introduction, possibly explained by genetic susceptibility . Gluten intake in childhood showed a tendency towards a positive association with both T1D and IA but confidence intervals were wide, and heterogeneity substantial (Later introduction to gluten (3-6 versus <3-5 months) was associated with reduced T1D risk  and without any indication of heterogeneity across studies .Vitamin D supplementation during infancy was inversely related to T1D , but theThe risk of T1D and IA was also investigated in relation to omega-6 fatty acid intake during childhood and maternal coffee intake, but no associations were observed  and 3.4.8The presence of publication bias could be assessed for meta-analyses investigating the risk of T1D in relation to any breastfeeding and cow's milk introduction . No publication bias was observed through visual inspection of the funnel plots, however, the Egger's test indicated presence of small study effects for any breastfeeding (p=0.0059). Meta-analyses restricted to prospective studies of T1D (25 out of 80 studies) could be performed for less than half of the dietary factors, including breastfeeding (any and exclusive); age at infant formula, solid food, meat, fruit, vegetable, cereal, and gluten introduction; gluten and fiber intake during childhood; maternal, infant, childhood, and adult vitamin D levels; and omega-3 and omega-6 intake during infancy or childhood . These analyses revealed significantly reduced risks of T1D in relation to later fruit and gluten introduction and higher 25(OH)D levels during adulthood . Only one prospective study assessed the risk of T1D in relation to longer breastfeeding (≥12 versus <12 months) and showed an inverse association .4.9Associations between T1D and breastfeeding (≥6-12 versus <6-12 months) and age at introduction to gluten, were rated with high certainty, and associations with exclusive breastfeeding, age at introduction to cow's milk, and fruit, with moderate certainty . For IA,4.10Studies that were not meta-analyzed include 20 observational studies and six RCTs that either had overlapping study populations or were evaluating different dietary factors (Supplementary Tables 7-9). The observational studies assessed introduction to probiotics, maternal micronutrient intakes, and nutritional biomarker levels during infancy and childhood. The RCTs assessed the effects of weaning to an extensively hydrolyzed versus cow's milk-based formula, introduction to gluten at 12 months versus six months, and nicotinamide supplementation in childhood. No beneficial effects were seen for these factors (Supplementary Table 7).5This meta-analysis included 26 dietary exposures assessed in 96 studies. The results indicate positive associations between T1D or IA and childhood intake of cow's milk, carbohydrates, sugar, sugar-sweetened beverages, protein, meat, and nitrite. In contrast, inverse associations were observed with breastfeeding, later introduction to cow's milk, fruit, and gluten, vitamin D supplementation in infancy, 25(OH)D levels in adulthood, and childhood intake of vitamin A and C. None of the fetal dietary exposures were related to IA or T1D. The certainty of evidence was high for a reduced risk relating to longer breastfeeding and later introduction to gluten; and moderate for an increased risk associated with higher intake of cow's milk, and a reduced risk with exclusive breastfeeding, and later introduction to cow's milk and fruit. For the remaining dietary factors, certainty was low . This isOur results in favor of beneficial effects of breastfeeding are consistent with previous meta-analyses,,13 and aOur study also indicated adverse effects of cow´s milk both in terms of quantities consumed in childhood and early introduction. It has been speculated that the excess risk of T1D associated with earlier introduction to cow´s milk may reflect lack of breastfeeding Carbohydrates, sugar, and sugar-sweetened beverages were associated with increased risk of T1D, although certainty of this evidence was low primarily owing to serious risk of bias in the individual studies. The underlying mechanism could involve effects on body weight, since adiposity is associated with increased risk of T1D Vitamin D is suggested to play a role in the etiology of T1D, primarily due to its involvement in the regulation of the immune system Intake of antioxidants may hypothetically reduce the risk of T1D by inhibiting oxidative stress. Consistent with this hypothesis, we found a reduced risk of T1D in relation to vitamin A and C exposure during childhood, although the certainty of this evidence was very low.Strengths include a broad and systematic literature search according to an a priori defined protocol, and assessment of different types of bias in the individual studies and of the overall quality of evidence for each dietary factor that was meta-analyzed. By performing study selection, data extraction, and risk of bias assessment in duplicate, we reduced the risk of missing important information and introducing errors. Additionally, we distinguished between exposures in utero, infancy, and childhood. The main limitation is that the number of studies was small for most dietary factors. This reduced power and the possibilities to perform sub-group analyses, assess dose-response relationships, and publication bias. Several of the included studies had serious risk of bias, mainly due to confounding, and potential misclassification of exposure caused by self-reported dietary information. The latter is a particular concern in case-control studies with retrospective information. This resulted in reduced certainty of evidence for most associations. Importantly, biomarker information was used in some studies, which reduces this bias. The GRADE tool does not distinguish between prospective and retrospective evidence. However, we performed sensitivity analyses restricted to prospective studies, which substantially reduced the number of eligible studies but revealed similar associations for the dietary factors that could be analyzed. In parallel, RCTs were few and not possible to synthesize since the interventions differed. A main concern regarding confounding was the lack of adjustment for other dietary factors, considering that no food is consumed in isolation. Associations were generally weaker for IA than for T1D. This could reflect that IA is a less specific outcome, identified by the presence of one or several different autoantibodies with varying positivity cut-offs. The assessment of dietary factors varied within meta-analyses both in terms of definition, timing, and categorization. This implies that some studies could not be included in the meta-analyses, e.g., if they had a unique exposure assessment that did not align with that of other studies. Most importantly, it precludes formulation of specific recommendations, e.g., regarding optimal age at introduction of certain foods, quantities of milk consumption, and duration of breastfeeding. Finally, it should be noted that most studies came from Europe and North America and whether the results apply to other populations with different genetic makeup and dietary habits remains to be explored.Our findings indicate that longer breastfeeding and delayed introduction to gluten, cow's milk, and fruits may prevent T1D. Moreover, we find support for the general recommendation of vitamin D supplementation in early life. The potential harm in terms of T1D risk of high cow's milk consumption in childhood may be considered in risk-benefit analyses underlying dietary guidelines. There were several dietary factors previously linked to T1D for which we did not find associations including iron, fish, and omega-3 fatty acids and as noted above, many of the observed associations were of low certainty. This emphasizes the need for future studies to elucidate the role of diet in the etiology of T1D and these studies should preferably use a prospective design, nutritional biomarkers, and careful adjustment for potential confounders. Moreover, the potential interaction of genetic susceptibility with diet needs to be explored. Few studies investigated dietary factors in relation to progression from IA to T1D. This is an important topic for future studies as it may provide information to be used for prevention. In addition, distinguishing between stage 1 (IA with normoglycemia) and stage 2 (IA with dysglycemia) of presymptomatic T1D, when studying progression, may provide further insights in the etiology of T1D In conclusion, this study supports that infant and childhood diet may influence the risk of T1D. The most convincing evidence was seen for beneficial effects of breastfeeding, later introduction to gluten, cow´s milk, and fruit, and lower consumption of cow´s milk during childhood. Other associations were observed, but the certainty of evidence was low. The results emphasize the need for future, high quality studies on the influence of diet on development of T1D.6The study was conceived by SC together with AML and JEL. AML and JEL independently performed the study selection, data extraction, and risk of bias and certainty of evidence assessment and consulted SC in case of disagreement. AML did the statistical analyses and wrote the first draft with input from JEL and SC, who also edited the manuscript. All authors contributed to the interpretation of the findings, had full access to all the data and final responsibility for the decision to submit for publication.The authors declare no competing interests.


The enzyme L-asparaginase (ASRGL1) catalyzes the hydrolysis of L-asparagine (Asn) to L-aspartic acid (Asp) and ammonia. Numerous studies have shown a strong correlation between ASRGL1 expression and tumorigenesis. However, the expression and biological function of ASRGL1 in hepatocellular carcinoma (HCC) are still unclear.We explored the mRNA expression of ASRGL1 in HCC using the HCCDB, Oncomine, and TIMER 2.0 databases. Western blotting and immunohistochemical analyses were also used to determine the mRNA expression of ASRGL1 in HCC. LinkedOmics was used to analyze the genes co-expressed with ASRGL1 and regulators including kinases, miRNAs, and transcription factors. The Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways of the co-expressed genes were also investigated using LinkedOmics. The correlation between ASRGL1 expression and immune infiltrates was analyzed using the TIMER 2.0 and Gene Expression Profiling Interactive Analysis (GEPIA) databases. The effects of ASRGL1 expression on patient outcome were investigated using the UALCAN and GEPIA databases, and the Kaplan–Meier plotter. c-Bioportal was used to explore the mutations of ASRGL1 in HCC.Compared with the adjacent tissues, ASRGL1 was upregulated in HCC. High ASRGL1 expression in HCC indicated poor relapse-free survival, progression-free survival, disease-specific survival, and overall survival. The expression of ASRGL1 was significantly correlated with infiltrating levels of B cells, CD4+ T cells, macrophages, neutrophils, and dendritic cells in HCC.Our findings suggest that ASRGL1 is overexpressed in HCC and that ASRGL1 expression was significantly correlated with immune infiltration in HCC and prognosis. Therefore, ASRGL1 may serve as a biomarker for the early diagnosis and treatment of HCC. Hepatocellular carcinoma (HCC) is the fourth most common digestive system malignancy and approximately 42,030 new cases of HCC are diagnosed each year . At presMetabolic abnormalities play important roles in the occurrence and progression of tumors . In thisIn this study, by using databases including The Cancer Genome Atlas (TCGA), Oncomine, and TIMER 2.0, we investigated the network of co-expressed genes and potential function of ASRGL1. The results showed that ASRGL1 was overexpressed in HCC and had a negative correlation with patient outcome. Moreover, ASRGL1 may participate in immune infiltration and promote the progression of HCC. Overall, our research revealed a new perspective on the progression of HCC and provided a potential target molecule for the treatment of HCC.The HCCDB database is a public HCC gene expression profiling database that contains 3,917 samples from the Gene Expression Omnibus (GEO) and TCGA database . In thisLiver cancer tissues and the corresponding adjacent tissues were collected from Nanjing Drum Tower Hospital from 2018 to 2020 and approved by the Committee for Ethical Review of Research. Written informed consent regarding tissue collection was obtained from all patients.5 U/L penicillin. The cells were incubated in a 5% CO2-humidified incubator at 37°C. Liver cancer tissues and the corresponding adjacent healthy tissues were collected from patients treated at Nanjing Drum Tower Hospital between 2018 and 2020. The tissues were stored in liquid nitrogen until use. Before sample collection, written informed consent collection was obtained from all patients.The cell lines used in this study, including LO2, SMMC-7721, Hep3B, HepG2, 97H, 97L, HCC-LM3, and Huh7 were obtained from the ATCC. The cells were cultured in DMEM supplemented with 10% FBS, 100 mg/l streptomycin, and 10Proteins were extracted from cells and tissues using a radioimmunoprecipitation assay kit  supplemented with 0.1% protease inhibitors and 0.1% phosphorylase inhibitors. The protein concentration was quantified using the BCA Protein Assay Kit . Subsequently, the protein samples were separated on 10% SDS-PAGE gels, and the separated proteins were transferred to a PVDF membrane (Millipore). The membranes were incubated in 5% milk for 2 h at room temperature to block non-specific binding, and then incubated with appropriate antibodies overnight at 4°C. The following primary antibodies were used: ASRGL1 , GAPDH . The membranes were visualized with an ECL detection system after incubation with secondary antibody at room temperature for 2 h.https://oncomine.org/resource/login.html)  . In thishttp://ualcan.path.uab.edu/), a website based on TCGA, can be used to analyze the relative gene expression in tumor and normal tissues, as well as the relative gene expression based on race, tumor grade, and other clinicopathological features (UALCAN (features . In thishttp://gepia.cancer-pku.cn/) is an interactive online web tool for the analysis of the tissue samples from the TCGA and GTEx projects  provides data analysis of 10,461 samples, including samples of gastric cancer, breast cancer, and HCC  is an open-access online database providing multidimensional analysis of cancer genomics data sets. The multidimensional analysis usually includes mutation, copy number variation (CNV), and gene co-occurrence. In this study, c-BioPortal was used to investigate ASRGL1 mutations and CNV in HCC  from TCGA , progression-free survival (PFS), disease-specific survival (DSS), and overall survival (OS)  of 370 patients with HCC , B cells, tumor-associated macrophages (TAMs), monocytes, neutrophils, macrophages, dendritic cells, and natural killer (NK) cells, was examined in HCC and STAD tissues. The mRNA expression of ASRGL1 was closely related to the infiltration of CD8+ T cells, T cells , B cells, monocyte, TAMs, macrophages, DCs, and Th1 and Treg cells in HCC tissues. In contrast, there was no significant correlation between immune cells and ASRGL1 in STAD tissues Figure 8The correlation between immune cells and ASRGL1 was also analyzed using the GEPIA database. The mRNA expression of ASRGL1 was positively correlated with monocytes, neutrophils, TAM, Th1, Treg, and T cell exhaustion, but not with Th2 cell infiltration, whereas the correlation between immune cells and ASRGL1 was not significant in STAD tissues Table 4.As the progression of HCC is rapid, patients usually have a poor prognosis; the 5-year survival rate is below 10% . TherefoIn this study, through the analysis using the HCCDB and Oncomine databases, we found that ASRGL1 expression was significantly altered in HCC. To verify the above results, we examined the clinical tissue specimens of HCC and found that ASRGL1 expression was significantly higher in tumor tissues than the adjacent tissues. Meanwhile, the investigation of liver cancer cells revealed that ASRGL1 protein expression was significantly higher than that in LO2 healthy liver cells. However, we found that the expression of ASRGL1 in L02 was higher than that in Huh7 cell line. At present, some literature also showed that the expression of some oncogenes, such as DDK1 and KLF8, was increased in normal liver cell line , 26; theThe analysis of the UALCAN database indicated that the expression of ASRGL1 in HCC tissues also varied in different clinical tumor stages, with a significant positive correlation between ASRGL1 expression and the degree of tumor malignancy. The analysis of the KP and GEPIA databases revealed a significant negative correlation between ASRGL1 expression and patient prognosis, which was verified by the GEO dataset. Patients with higher ASRGL1 expression had a poorer prognosis than patients with lower expression. These results suggested that ASRGL1 may participate in the occurrence and progression of the tumor and may be a biomarker for the early diagnosis of HCC.To identify the regulators of ASTGL1 expression, we constructed a co-expression network, which indicated that a network of kinases, including CDK1, PLK1, ATR, PRKCI, and AURKA, may participate in the development of HCC. CDK1 is an oncogene that can promote the development of HCC . A varieFrom the database analysis, we found that ASRGL1 was extensively involved in tumor immune invasion. A close relationship has been demonstrated between tumor occurrence and the uncontrolled immune regulation of tumors . The inaOur study has demonstrated the biological function and expression of ASRGL1 in HCC using bioinformatics analyses, and we verified these results in tissue samples. These results showed that ASRGL1 was overexpressed in HCC and had a significant negative correlation with the outcome of patients with HCC, suggesting that ASRGL1 is a biomarker for HCC. Moreover, our results also indicate the potential role of ASRGL1 in regulating immune cell infiltration. However, these findings need to be verified by large-scale genomics research and molecular mechanism studies for HCC.The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by the Affiliated Changzhou No. 2 People’s Hospital of Nanjing Medical University. The patients/participants provided their written informed consent to participate in this study.XQ and CZ conceived and designed the study. CX and PG collected the human tissues and conducted the experiments. CX, XZ, PG, XC, LJ, and RL performed data mining. CX wrote the manuscript. All authors contributed to the article and approved the submitted version.This study was supported by grants from the National Natural Science Foundation of China (grant no. 81672469), The Social Development Foundation of Science and Technology of Jiangsu (grant no. BE2016658), and the project of Changzhou medical innovation team (CCX201807).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


TRIM59, SEPT3, RAD51AP1, and EXO1 can act as independent protective prognostic factors, but CTSF and ABHD4B can serve as independent bad prognostic factors in patients with basal breast cancer. Remarkably, we establish a robust prognostic model containing the 6 mRNAsi-related genes that can effectively predict the survival rate of patients with the basal breast cancer subtype. Finally, the drug sensitivity analysis reveals that some drug combinations may be effectively against basal breast cancer via targeting the mRNAsi-related genes. Taken together, our study not only identifies novel prognostic biomarkers for basal breast cancers but also provides the drug sensitivity data by establishing an mRNAsi-related prognostic model.Basal breast cancer subtype is the worst prognosis subtypes among all breast cancer subtypes. Recently, a new tumor stemness index-mRNAsi is found to be able to measure the degree of oncogenic differentiation of tissues. The mRNAsi involved in a variety of cancer processes is derived from the innovative application of one-class logistic regression (OCLR) machine learning algorithm to the whole genome expression of various stem cells and tumor cells. However, it is largely unknown about mRNAsi in basal breast cancer. Here, we find that basal breast cancer carries the highest mRNAsi among all four subtypes of breast cancer, especially 385 mRNAsi-related genes are positively related to the high mRNAsi value in basal breast cancer. This high mRNAsi is also closely related to active cell cycle, DNA replication, and metabolic reprogramming in basal breast cancer. Intriguingly, in the 385 genes,  Breast cancer is the most serious primary malignant tumor type in women, and its incidence rate is about 1.3 million, and the death rate is about 0.5 million worldwide . Some ceThe tumor microenvironment is complex and diverse, which is composed of relatively differentiated cancer cells, tumor stem cells, endothelial cells, tumor-related fibroblasts, invasive immune cells, and other cell types or components . NotablyMany studies have revealed that breast cancer stem cells can produce a large number of primary tumors, thereby driving tumor occurrence and metastasis to lead to the poor prognosis for breast cancer patients , 13. EspIn this study, we firstly determined the mRNAsi of each breast cancer subtype and found that the mRNAsi value of the basal subtype was the highest among all subtypes. We thereby determined to investigate the role of mRNAsi in the patients with the basal subtype. We used the WGCNA to screen key genes related to mRNAsi in the patients with the basal subtype. We next explored the potential functions of these mRNAsi-related genes in the basal breast cancer patients. Our results demonstrated that cell cycle might have an important role in the occurrence and development of breast cancer patients of the basal subtype. Especially, we developed a prognostic model based on mRNAsi for breast cancer patients of the basal subtype. The robust prognostic model consists of 6 genes and has a good predictive performance. In conclusion, our work revealed that mRNAsi is highly correlated with the basal subtype; in particular, these mRNAsi-related genes can be hoped to serve as biomarkers for clinical prognosis and therapy of breast cancer patients of the basal subtype.http://xena.ucsc.edu/). The selected samples were adopted by following criteria: (1) clinical data such as overall survival days and survival status are complete; (2) normal samples were selected only for the matching pairs of cancer tissue. After filtering, 185 breast cancer samples and 17 normal tissue samples with both normalized expression fragments per kilobase million (FPKM) and clinical data were retained for next analysis. Differentially expressed genes (DEGs) were screened using the limma R package [http://bioinfo.life.hust.edu.cn/web/GSCALite/). The modified database has integrated the genome-wide drug sensitivity test data of CTRP and GDSC and provides a good resource for exploring the sensitivity of genes to drugs.The mRNAsi of breast cancer based on pluripotent stem cell samples was obtained from the work of Malta et al. . Gene ex package ; the filP value < 0.05 indicates that the correlation is statistically significant.The Weighted Gene Coexpression Network Analysis (WGCNA) is a system biology method of describing gene association expression patterns among different samples and has been widely used to identify highly collaborative gene sets and alternative biomarker genes or therapeutic targets according to the connectivity of gene sets and the association between gene sets and phenotypes . Here, wThe single sample gene set enrichment analysis (ssGSEA) strategy was used to evaluate genome-wide expression and tumor immune cell infiltration distribution based on 782 marker genes of 28 tumor immune infiltrating cells (TIICs) .https://portal.gdc.cancer.gov/), and the R package maftools was used to summarize and analyze the data [The somatic mutation map of the basal breast cancer patients based on the whole exome sequencing platform was downloaded from GDC (the data . Genome P < 0.05 is considered significant unless otherwise noted.Both Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were carried out to reveal these functional roles of genes associated with mRNAsi by clusterProfiler R package , and P <P < 0.05 was selected as the candidate biomarker. Next, the multivariate cox regression was performed on these candidate mRNAs to identify independent prognostic mRNAs, and a risk value was calculated to construct a predictive model for each mRNAs. Based on the above results, the survivalROC R software package was used to plot the receiver operating characteristic (ROC) curve, and the classification model was evaluated according to the area under the curve (AUC). The t-test was used to analyze the relationship between candidate prognosis genes and clinical and biological characteristics.MRNAsi-related genes were used to construct a prognostic model, and the construction method was as follows: the univariate cox regression was used to further evaluate survival-related mRNAs, and only mRNA with https://string-db.org/) and the Cytoscape software were used to construct PPI network. The survival curve was drawn by Kaplan-Meier, and the difference significance was evaluated by log-rank test. P < 0.05 is considered as statistically significant.All data analyses were performed using the R software version 3.5.2. The STRING  (R2 = 0.93), which revealed that the selected β value can establish the gene scale-free network , whereas the highest negative correlation exists between the blue module and mRNAsi   . We thus 0.0001) . We furt 0.0001) . InteresCCNB1, CDC20, CDK1, CDC25C, CHEK2, MYBL2, E2F1, E2F2, and FOXM1  such as chromosome segregation, nuclear division, organelle fission, sister chromatid separation, DNA replication, and cell cycle checkpoint , while Knd FOXM1 , have bend FOXM1  and PPARnd FOXM1 , while tand FASN . This seCTSF and ABHD4B of them are risk factors (HR > 1), whereas the rest 19 genes are all protective factors (HR < 1)  . We next Figures . Our finP < 0.0001)  . In orde 0.0001) , indicat 0.0001) . InteresSEPT3, RAD51AP1, and EXO1 are more highly expressed in older patients than young ones, but TRIM59, CTSF, and ABHD4B have no significant changes between them. The expression of RAD51AP1 is also higher in patients with high T stage than ones with low T stage, but the expression of ABHD14B is lower in patients with high T stage, as well as no significant changes in other genes. Similarly, the expressions of SEPT3, RAD51AP1, and EXO1 are significantly increased in patients with higher AJCC stage and N stage than ones with lower AJCC stage and N stage, and the rest genes are no significant. While the expression levels of SEPT3, RAD51AP1, and EXO1 are higher in tumor-bearing patients, while ABHD14B is lower. These results revealed that the 6 prognostic genes are related to the clinical indicators of the basal patients to varying degrees, implying that the 6 genes might play a crucial role in the development and progression of the basal breast cancer.In order to explore how these 6 genes affect the clinical indicators of the basal patients, we further investigated the relationship within the 6 genes and the patient's age, T stage, American Joint Committee on Cancer (AJCC) stage, lymph nodes present (N stage), and tumor bearing status. As shown in TP53, TTN, MUC16, SPTA1, FAT3, SYNE1, USH2A, and so on in the basal subtype patients, but they have no significant mutation difference between the high-risk and low-risk group  have drug susceptibility data; in particular, both RAD51AP1 and EXO1 present high drug resistance to PD-0325901, RDEA119, trametinib, selumetinib, and so on  , implyinf tumors –52. TakeCCNB1, CDK1, CHEK2, and MYBL2, have been widely reported as oncogenes and used in the clinical diagnosis for breast cancer patients [PPKAR2B, SLC27A1, SCD, FASN, and ACADS. Tumor cells, especially tumor stem cells, need to provide themselves with a large amount of ATP and other energy substances through metabolic reprogramming, which is one of the characteristics of tumor deterioration [Herein, we found that the upregulated mRNAsi-related genes are significantly enriched in the cell cycle, cell division, DNA replication, and p53 signaling pathway . Many stpatients , 38. Esppatients , 37. In ioration , 58. ThiTRIM59, SEPT3, RAD51AP1, and EXO1 can serve as independent protective factors and CTSF and ABHD14B as the risk factors (CTSF gene as a risk factor is resistant to a variety of tested drugs, except for selumetinib, SB590885, PLX4720, and Dabrafenib (CTSF gene may be a potential reason impacting the treatment effect of the basal breast cancer patients, and selumetinib, SB590885, PLX4720, and Dabrafenib can be considered as potential therapy or adjuvant therapy drugs because it has a good effect on the basal breast cancer patients.In this study, we identified 21 potential markers for the survival and prognosis of patients with the basal subtype . Especia factors , and the factors . Surpris factors  and coul factors . More im factors . Remarka factors . These gbrafenib . We therIn summary, our study has revealed that the basal subtype breast cancer has a higher mRNAsi score, which indicates that this subtype can be driven by cancer stem cells. Our results demonstrate mRNAsi-related modular genes mainly involved in cell cycle activation and metabolic reprogramming, which may be internal factors that maintain the survival of the basal breast tumor malignant stem cells. We construct a prediction model containing the 6 mRNAsi-related prognostic genes, which can be used to predict the survival rate of the basal breast cancer patients. We provide the drug sensitivity data of 15 genes related to mRNAsi, which will be helpful for the treatment of the basal breast cancer patients.


Pleuropulmonary blastoma (PPB) is a rare malignant intrathoracic mesenchymal tumour with a variable aggressiveness. It is the most common primary malignancy in the lung during childhood. In this study, we present a case of a 3-year-old male child who complained of persistent dry cough. Radiographs suggested the diagnosis of PPB, which was been confirmed by the histo-pathological examination of a biopsy taken from the tumour under CT guidance. This case was reported to emphasise the importance of radiology, whether diagnotic or interventional, in diagnosing rare cases such as PPB. Despite being rare, they are considered the most common primary cancers in the lung during childhood,2 especially in those who are under 5 years of age.3 The first two years of age remain the highest risk for developing PPB, with about 90% of the cases occuring within this age group.3 PPB is divided into type I, II & III according to histological type of the tumour. Each of the subtypes correlates to the age at diagnosis and the overall prognosis of the patient.4 The main aim of reporting this case is to highlight the importance of both diagnostic and interventional radiology in helping other physicians to reach rare diagnosis correctly.Pleuropulmonary blastoma (PPB) accounts for 0.25 to 0.5% of all primary lung neoplasms.3, an elevated C reactive protein (CRP) level of 108 mg/l, and anaemia with a haemoglobin level of 9.8 g dl−1.A 3-year-old male child presented to our emergency department with a history of persistent non-productive cough of 4 days duration. He had a history of hospitalisation once for acute bronchiolitis at the age of 1 year. His family history was unremarkable. Upon clinical evaluation, he had tachypnea, intercostal retractions, absent air entry on the right lung and abdominal distention. Otherwise, the clinical examination was normal. Laboratory investigations revealed leukocytosis up to 14,900 / mmA chest X-ray  was perf1 The pathogenesis of these tumours remains uncertain, but due to their resemblance to fetal lung tissue, pluripotent pulmonary blastomas have been postulated to be the origin of the disease. They occur mostly in the infants and young children, however, similiar cases had been reported in adults.5–7Lung malignant tumours during childhood are considered a rare entity, representing only 0.5–1% of primary lung cancers. Pulmonary blastomas are uncommon fast-growing lung neoplasms that account for approximately 0.25–0.5% of primary lung malignancies.8 They are also associated with dysplasia syndrome in 33% of the cases.9 The association of DICER1 mutations and PPB is reported in approximately 66% of the cases.2 Therefore, all patients with PPB should be screened for DICER1 mutation. If DICER1 mutation is detected during screening, the possibility for the patient to develop malignancies in other organs must be considered. In 30% of cases, patients with PPB may also present with multilocular cystic nephroma, and, very rarely, Wilms tumor.9 Following the initial description by Barrett and Barnard in 1945 , pulmonary blastomas were classified into three histological subtypes10 : I, II, and III. Type I is a purely cystic lesion (14%), type II tumours have both cystic and solid components (48%) and type III are purely solid (38%). The classification has also a prognostic value, type I being the least malignant and type III the most malignant lesion.Up to date, no definite causative agent has been determined, but some associations have been reported. PPBs are associated with type IV congenital pulmonary airway malformations.10,11 Symptoms, when present, may include fever, dyspnoea, respiratory distress, cough. Rarely, hemoptysis, chest pain, weight loss, anorexia, fatigue, or even neurologic symptoms can occur. Currently, there is no specific serum marker for pulmonary blastomas.10The clinical picture of blastomas is highly variable. There are no specific presenting signs or symptoms of biphasic pulmonary blastomas. Some studies suggest that symptoms present only in 60% of the cases, with the lesions for the rest of the cases found incidentally.12 PPB is often found late on plain film radiographs as a unilateral lung whiteout with a mediastinal shift to the opposite side. Typically, there are no adjacent rib erosions or calcifications. In the majority of cases, the disease is unilateral, but bilateral disease has been reported. In some cases, pleural effusion may be seen.11 Ultrasound is a poor imaging modality for diagnosing PPB as findings are non-specific and may show a large region of consolidation without sonographic air bronchogram. The appearance of PPB on CT scan is variable depending on the histological type of the tumour.8,13,14 Type I lesions manifest as a single cyst or a multicystic lesion (often air-filled). Type II lesions show air- or fluid-filled cavities with possible air-fluid levels along with solid internal nodules. In type III lesions, neoplasms are solid and show low attenuation, with homogeneous or heterogeneous enhancement. On MRI, the tumor manifests as a heterogeneous mass, typically hyperintense on both T1W and T2W sequences.15 A FDG-PET CT scans reveals a hypermetabolic lesion. PET CT is helpful in evaluating metastatic work up. It is also helpful in evaluating tumour vitality and judging the response to neoadjuvant chemotherapy.16Imaging studies have enormous capability on identifying the charactersticis of lung lesions. Usually, PPB appears as a solitary, well-demarcated, peripheral mass that can be large enough to extend beyond 10 cm in size.The differential diagnosis for a chest wall mass identified on imaging of a young child is wide. For type 1 PPB lesions, the differential diagnosis includes other cystic lesions such as bronchogenic cyst, lung cyst, pneumatocele, and pulmonary interstitial emphysema. The differential diagnosis for types II and III PPBs, particularly when they are locally aggressive, includes more common tumours such as neuroblastoma, Ewing’s sarcoma, Askin tumour and rhabdomyosarcoma. However, chest lesions that arise from the chest wall such as Ewing’s sarcoma and Askin tumour usually demonstrate findings of chest wall involvement. Neuroblastoma typically arises in the posterior mediastinum and demonstrates chest wall involvement as rib erosion. Rhabdomyosarcoma can also involve the chest wall.Identifying imaging findings that can make a specific diagnosis more or less likely. Interpetation of imaging exams was important and helped in making the correct diagnosis in this case by identifying the heterogeneousity, low attenuation, the association of pleural effusion and the absence of chest wall invasion. The definitive diagnosis of PPB was made by cytogenic analysis of the tumor cells, which were biopsied under the guidance of CT.Despite being rare, PPB affects mainly young children and can lead to quick clinical detrioration.When a large hemithorax mass is identified in a young child, PPB should be considered in the differential diagnosis.The imaging findings favouring the possibility of PPB on a chest radiograph include large mass lesion with small pleural effusion, contralateral mediastinal shift and lack of chest wall invasion.


In this researchwork, the fabrication of biphasic composite implantshas been investigated. Porous, commercially available pure Ti  has been used as a cortical bone replacement,while different composites based on a polymer blend and bioactive glass (BG) 45S5 have been applied as a soft layer forcartilage tissues. The microstructure, degradation rates, biofunctionality,and wear behavior of the different composites were analyzed to findthe best possible coating. Experiments demonstrated the best micromechanicalbalance for the substrate containing 200–355 μm sizerange distribution. In addition, although the coating prepared fromalginate presented a lower mass loss, the composite containing 50%alginate and 50% gelatin showed a higher elastic recovery, which entailsthat this type of coating could replicate the functions of the softtissue in areas of the joints. Therefore, results revealed that thecombinations of porous commercially pure Ti and composites preparedfrom alginate/gelatin/45S5 BG are candidates for the fabrication ofbiphasic implants not only for the treatment of osteochondral defectsbut also potentially for any other diseases affecting simultaneouslyhard and soft tissues. Among thedisorders that may affect the musculoskeletal system, the most numerousones are those affecting bones and joints, such as osteoporosis, osteochondraldefects, osteoarthritis, or rheumatoid arthritis, obviously in additionto accident-derived injuries. Independently of the disorder origin,the need for implants as bone and/or articular replacements is becominga public health issue nowadays. For example, only in the United States,more than 1 million arthroplasties are performed every year for thetotal replacement of the knee or the hip, and the number of such interventionsis expected to increase to 4 million by 2030.6 Inparticular, diseases affecting both the bone and the cartilage, suchas osteochondral defects, require the development of therapies basedon the replacement of both tissues.10 In fact, different treatments based on autograftor allograft transplantations or bioengineered tissues have been alreadyapplied, but they only address the articular cartilage.11 Therefore, the use of multiphasic implants hasbeen investigated in the past years to substitute the bone and cartilagetissues in one single surgery.13 Such implants are usuallyconstituted of a rigid section to replace the bone and a soft coatingmimicking the cartilage tissue,18 although in some cases, a third layer is added as the interfacebetween the rigid and the soft zones.22According to the WorldHealth Organization (WHO), musculoskeletaldisorders consist of a group of more than 150 conditions affectingthe locomotor system of approximately 1.71 billion people all overthe world.22 gelatin,17 silk fibroin,16 polyglycolide,18 or polycaprolactone23 have been already investigated as bone substitutes. However, justa very reduced number of research works have considered the applicationof metals as the rigid section of the implant,25 considering their suitable mechanical propertiesfor bone tissue replacement. One of the most commonly used metalsin prosthesic applications is Ti, and its corresponding alloys, dueto its exceptional biocompatibility, mechanical characteristics, andexcellent corrosion resistance.28 However, the high Young’smodulus of Ti and Ti alloys is an important limitation, causing astress shielding phenomenon,29 with consequentreduction in the surrounding bone density. To reduce the differencein the Young’s modulus between the implant and the bone andto modulate the substrate stiffness, the introduction of porosityin the construct has been attempted. Although generation of poresin metallic implants has been investigated by means of additive manufacturing,powder metallurgy, or foaming techniques,33 the space holder fabricationprocedure, based on powder metallurgy, has gained high relevance dueto its versatility, reliability, and low cost.38Nowadays, independentlyof their fabrication method, most of thebiphasic implants proposed for the treatment of osteochondral defectsare based on polymeric materials. Thus, polymers such as collagenI,42 in particular, and tissue engineering, in general.45 Polymers and polymeric composites are the most commonly used materialsconsidered for this application48 due to their biocompatibilityand tunable properties,51 which enable development of theirchemical structure or composition according to the final aim. In thissense, among naturally occurring polymers, gelatin and alginate standout for the possibility of tuning their mechanical properties thanksto the degree of cross-linking.53 Their biocompatibilityhave been widely investigated in different in vitro and in vivo experiments for diverse tissues,57 including the bone and the cartilage.62 In addition, they form hydrogels that allow the easypreparation of composites,65 for example, bioactive glasses,66 for the improvement of their osseointegration capacity.68On the other hand, different approaches havebeen tested to developnew biomaterials as cartilage tissue replacement,11 Thus,the main objective of the present research work is the developmentof biphasic implants intended for the treatment of osteochondral defects.The use of porous Ti to substitute the cortical bone tissue is proposedto reduce the stress shielding phenomenon. On the other hand, basedon our previous work,25 a combination ofgelatin and alginate incorporating 45S5 BG is presented as the materiallayer facing the cartilage tissue. Finally, the influence of the substrateporosity and the chemical composition of the polymeric compositeson the tribomechanical and biofunctional behavior of the biphasicpartial implants will be evaluated.To our knowledge, apart from the authors of this manuscript,theonly research work proposing a biphasic implant based on porous Tiwas carried out by Duan et al.2The protocols to obtainthe porous titanium substrates and thebioactive gelatin–alginate–BG composite coatings aresummarized in 2.14HCO3) with different particlesize ranges (Cymit Química S.L.). The mixtures were pressed at 800 MPa,and the spacer was removed using two thermal cycles, both applyinga low vacuum (10–2 mbar): the first one at 60 °Cfor 10 h followed by another treatment at 110 °C for 12 h. Afterward,the porous green samples were sintered in a molybdenum chamber furnace,at 1250 °C for 2 h and high vacuum conditions (∼10–5 mbar). Finally, before depositing the coatings, thesurface of the titanium substrates (discs of 12 mm diameter and 3mm height) was carefully ground and polished with magnesium oxideand hydrogen peroxide, to preserve the porosity fraction, size, andmorphology of the pores inherent to the spacer.Commercially pure (c.p.) titanium powder, gradeIV , was mixed with 50 vol % ammoniumbicarbonate ,after considering the degradability, bioactivity, and processing conditions.After selecting the polymers, a route for the preparation of the solutionsand the cross-linking of the polymers was fixed, as they need to havea longer degradation time. 2·2H2O aqueous solution and freeze-dried again.For the preparation of the gelatinsoft phase, an aqueous solutionof 0.03 g/mL gelatin was assembled by stirring at 60 °C for 1h, until it was completely dissolved. The cross-linking solution consistedof 0.5 vol % (v/v) glutaraldehyde solution, prepared from a concentratedsolution . The cross-linking agent was addedto the gelatin solution in a proportion of 0.05 mL of glutaraldehydesolution per milliliter of gelatin solution. Concerning the alginatephase, an aqueous solution was prepared with 0.03 g/mL sodium alginateunder stirring at room temperature for 1.5 h until full dissolution.The cross-linking process of the alginate was done after freeze-drying;the sample was immersed for 4 h in a 0.5 M CaClThen, differentcompositions of the polymeric soft phase were prepared,always maintaining the concentration of the polymer in the solutionat 0.03 g/mL, to produce biphasic coatings. When preparing the polymerblend solutions, first, the alginate solution was added dropwise ina beaker and next, the uncross-linked gelatin solution. Both solutionswere produced in the proportions shown in 2O, 24.5 wt % CaO, 45 wt % SiO2, and 6 wt% P2O5) was added to the polymer blend. TheBG powder presented a d50 of 4.5 μm,and it was received in an amorphous  form. Severalproportions of the polymer/BG were studied, using only alginate andonly gelatin as polymers. The goal was to reach a uniform distributionwith no sedimentation of BG particles. This was achieved just whenalginate was employed at a concentration of 30 mg/mL and the BG contentwas 5% of the total weight (w/w) (1.58 mg/mL) of the final composite.Two aqueous solutions, one of gelatin and the other of alginate, wereprepared, with a polymer concentration of 30 mg/mL. The gelatin solutionwas stirred at 60 °C for 1 h and the alginate one at room temperaturefor 1.5 h. Then, they were mixed in the proportions shown in 2·2H2O aqueous solution for 4 h and then freeze-dried again.Finally, as the intention was also to increase the bioactivityof the soft phase, bioactive glass  and scanning electron microscopy (SEM) (Jeol JSM 6330Fmicroscope). In particular, SEM images obtained on the cross sectionsof the studied materials allowed us to evaluate the homogeneity ofthe pore distribution, the degree of infiltration, and the adherenceof gelatinous composites to the porous titanium discs.The densityand total and interconnected porosity of the titanium substrates andpolymeric coatings were determined by the Archimedes’ method,carefully cutting small cubes of approximately 27 mmy) anddynamic Young’s modulus (Ed), implementingthe uniaxial compression test according to ASTM E9-09 69 and an ultrasoundtechnique (KRAUTKRAMER USM 35), respectively.On theother hand, the macromechanical behavior of the porous titanium substrateswas evaluated in terms of yield strength (σiW). Samples were also weighted in the dry state (W0). The swelling (Sw) percentage was calculatedaccording to Then, a studyof the swelling, degradation, and bioactivity ofthe biopolymeric composites was carried out. Swelling results wereobtained by introducing the samples to phosphate-buffered solution(PBS) for 15 min, 30 min, 1 h, and 24 h and measuring their weightafter the predetermined time points of immersion  for 3, 7, and 14 days toassess their bioactive behavior. After these periods, the sampleswere removed from the buffer solution, washed in distilled water for1 or 2 min, and freeze-dried. The samples were then characterizedby SEM imaging, energy-dispersive X-ray spectrometry (EDX), and Fouriertransform infrared spectroscopy (FTIR) to look for hydroxyapatiteformation .V isthe volume of the worn material, estimated from the mass loss andthe density of the composites measured via the Archimedes’method, and d is the distance traveled during thetest.Finally, since the polymeric samples were not completelyflat,their surface (top view) was prepared by carefully cutting a thinsheet of the outer layer—removing only the material requiredto obtain a flat and homogeneous area, just to ensure a completelyflat surface—and/or carefully polishing it by hand, to avoiddamage of the surface or interfering in its microstructure and simultaneouslyto obtain a flat area that would allow a correct evaluation of thetribological behavior. In this context, although the wear resistanceresults could be affected by the presence of the substrate dependingon the elastic deformation, the thickness of the composite layer willbe high enough to simulate the cartilage tissue and avoid the substrateeffect. The wear resistance of the coatings was studied by a ball-on-disctest (Microtest MT/30/NI tribometer). The studies were carried outat room temperature, with a radius of 1 mm and with 6 mm diameteralumina balls. A dead load of 1 N and a rotation speed of 300 rpm(31.4 mm/s) were applied, and the distance traveled was between 5and 15 m. Some additional experiments using more critical wear conditions(greater load and distance) were also carried out. These experimentsallowed us to rule out some of the studied coatings (excessive wearwas observed). During the wear tests, the penetration depth was continuouslyrecorded (LVDT vs distance). The results were analyzed in terms ofthe mass loss, wear kinetics (threshold and slope of the curve) ofthe biopolymers, and the sliding contact response that wasgiven in terms of the coefficient of friction (COF). It was computedthrough the normal and tangential force, both measured by a straingauge-type dynamometer. Also, absolute wear rates were calculatedas follows 2:2where 33.171 In general, c.p. Ti substrates were produced following the spaceholder technique as depicted in 3. The totalporosity of all discs ranged from 50 to 51.6%, close to the initial50% space holder volume content. In addition, most of the pores wereinterconnected, showing an interconnected porosity from 45.6 to 50.1%,which is favorable for biopolymer infiltration. The equivalent diameter(Ed) and yield strength (σy)2c. In ge3.225 gelatin was testedas a potential cartilage tissue substitute in a biphasic implant.The main aim of this type of polymeric coating is the substitutionof the cartilage functions in articulate joints. For these applications,the cartilage tissue must possess a good capacity for elastic deformation(pseudo-damping) during tribological solicitations, when damage causedby the applied wear conditions should be minimized. In addition, thecoating porosity would allow its vascularization, while the presenceof a BG would enhance the material’s bioactivity and thereforeosseointegration. In this sense, polymeric blends of porcine gelatinand sodium alginate including BG 45S5 were investigated, using glutaraldehydeand Ca2+ ions as cross-linkers, respectively. Five differentcompositions were proposed, according to 72 In this research, the concentration of glutaraldehyde appliedto the polymeric material was 10–4 mmol per mg ofgelatin. In the case of the material prepared only with gelatin, theglutaraldehyde concentration is much lower than the limit establishedby Lai. In the rest of the polymer blends, the concentration is evenlower. Therefore, the glutaraldehyde concentration used will not producecytotoxicity.As mentioned above, in a previous research workpublished by our group,52 which maintains the structuralintegrity of the polymeric material.On the other hand, the presence of alginate reduced the swelling capacityof the blends, probably owing to the less hydrophilic character comparedto gelatin and the different cross-linking type in each polymer. Thepolymeric chains of alginate remained closer after the ionic cross-linking,which hindered the PBS intake. Knowing these results, it was expectedthat the degradation of polymers with a high content of gelatin wasmore pronounced in PBS, as displayed in 52 under physiological conditions.To find the best candidate for the final aim amongthe five possibilities, a swelling test in PBS was conducted in thepolymeric blends through the measurement of the weight gain in 24h. The swelling behavior in the first 24 h of the different blendsis depicted in In light of these results, first, screening andselection of materialswere conducted. In this sense, blends G and 1A3G, the ones with ahigher content of gelatin, were dismissed for their fast degradation,mainly in the first hours of use. Therefore, the following experimentswere continued with the polymeric materials A, 3A1G, and 1A1G.To increase the bioactivity of the final material, by promotingthe generation of hydroxyapatite, the addition of 45S5 BG was investigated.Thus, new polymer–ceramic composites were attempted by a homogeneousmixture of 95% (w/w) polymer (or polymeric blend) and 5% (w/w) bioactiveglass, which was the maximum concentration of BG that avoided itssedimentation. In Using the microstructural characterizationof the composites, anotherselection was carried out to reduce the number of samples for thefinal characterization. In this case, composites A and 1A1G were selectedsince they possessed a more differentiated pore structure, which couldallow the investigation of the influence of the polymeric compositemicrostructure on its biofunctional and biomechanical behavior.25 On the other hand, SEM pictures of the substrates’cross section exposed the influence of the c.p. Ti substrate on thestructure of both composites, independently of the pore size distribution.A schematic of this effect is displayed in The deposition of the selected composites on top of the differentsubstrates was carried out, as depicted in 3).It should be noted that the density showed by the polymeric blendwas lower than the value presented by the 100% alginate composite,probably due to the cross-linking of the gelatin, which induced aless compact material. This effect was expected as the differencesin the cross-linkers for both polymers, glutaraldehyde for gelatinand calcium cations for alginate, are considerable. Therefore, comparingthe results obtained from the density estimation from the BG surfaces, which reduces the cross-linking degree of thealginate polymer and therefore increases its swelling capacity. Regardingthe degradable behavior, in general terms, the addition of BG 45S5to the polymers led to an enhanced biodegradability in the first hoursof experiments  on wear behavior was first evaluated. The tribological characterizationpresented for the composite from 1A1G + 5% BG with a space holderof 355–500 μm 12 revealOn the other hand, in In the previous figures, 44HCO3 and pore sizesin the range of 250–355 μm are the most recommended interms of guaranteeing a biomechanical balance (Young’s modulusand yield strength) and biofunctionality . In relation to the wear behavior of coatings,a lower mass loss of the 95% alginate + 5% BG coating was confirmed.However, a higher elastic recovery was observed at the end of thewear tests of the compound incorporating gelatin. This last resultis very interesting since this type of coating could replicate thefunctions of the soft tissue in areas of the joints. In this globalscenario, the proposed system (porous substrate coating) has a potentialapplication in the combined replacement/regeneration of hard  and soft (cartilage and skin) tissues (tissue interfaces).Porousc.p. titanium substrates manufactured by the space holdertechnique with 50 vol % NH


Canagliflozin, dapagliflozin, empagliflozin, and ertugliflozin belong to a class of antidiabetic treatments referred to as sodium-glucose cotransporter 2 inhibitors . SGLT2is are currently indicated in North America and in Europe in type 2 diabetes mellitus, especially in patients with cardiovascular (CV) disease, high CV risk, heart failure, or renal disease. In Europe, dapagliflozin is also approved as an adjunct to insulin in patients with type 1 diabetes mellitus. New data provide evidence for benefits in heart failure with reduced ejection fraction and chronic kidney disease, including in patients without diabetes. The use of SGLT2is is expected to increase, suggesting that a growing number of patients will present to the emergency departments with these drugs. Most common adverse events are easily treatable, including mild genitourinary infections and conditions related to volume depletion. However, attention must be paid to some potentially serious adverse events, such as hypoglycemia (when combined with insulin or insulin secretagogues), lower limb ischemia, and diabetic ketoacidosis. We provide an up-to-date practical guide highlighting important elements on the adverse effects of SGLT2is and their handling in some frequently encountered clinical situations such as acute heart failure and decompensated diabetes. Sodium glucose-cotransporter 2 inhibitors (SGLT2is) are a class of oral antihyperglycemic agents that block glucose and sodium reabsorption in the proximal tubule of the kidney, causing glucosuria and osmotic diuresis. They improve glycemic control in patients with type 2 diabetes mellitus (T2DM) and provide cardiovascular (CV) and renal benefits independently of diabetes status ,2,3.Canagliflozin, dapagliflozin, empagliflozin, and ertugliflozin are indicated as first- or second-line treatments in T2DM individuals with CV disease, high CV risk, heart failure, and chronic kidney disease (CKD) both in Europe and North America, provided that in some European countries, their use may be prohibited if kidney clearance is below 45 mL/min ,7. DespiAdverse events are categorized as very frequent (incidence of ≥10%), frequent (≥1% and <10%), uncommon (≥0.1% and <1%), rare (≥0.01% and <0.1%) and very rare (<0.01%), using the Council for Organizations of Medical Sciences working group definitions, and available evidence in literature . An increased risk of genital mycotic infection is associated with SGLT2is, particularly in women (vulvovaginitis) and uncircumcised men . Although it is the most common adverse event, most infections are mild or moderate . In a meConcerning the risk of urinary tract infections (UTIs), data are less consistent. Reports of pyelonephritis and complicated UTIs (urosepsis) have prompted the EMA and FDA to add warnings about an increased risk of UTIs . HoweverSGLT2is inherently present a low risk of hypoglycemia because of their insulin-independent pathway of action . In clinBecause of its effect on osmotic diuresis, SGLT2is may cause symptomatic hypotension or dehydration (incidence of 1.2% to 1.5%), especially in elderly patients or those already taking diuretics . In pati2 is expected when starting a SGLT2i [Regarding the risk of acute kidney injury (AKI), reports have provided conflicting conclusions. In 2016, the FDA issued warnings regarding the use of canagliflozin and dapagliflozin after 101 cases of AKI, some requiring dialysis . Most ocIn patients presenting to the ED with severe AKI, SGLT2i therapy and other potentially nephrotoxic drugs should be withheld, with the correction of any electrolyte imbalance and the initiation of a standard protocol for fluid resuscitation in severe AKI resulting from circulatory hypovolemia and hemodialysis as a last resort, if needed.First reported in 2015 , euglyceIn clinical trials investigating patients with T2DM and those with HFrEF, SGLT2i-associated DKA was rare, with incidence rates ranging from 0.1% (empagliflozin) to 0.6/1000 patient-years (canagliflozin) ,22,23. IEDKA should systematically be considered in patients taking SGLT2is, regardless of blood sugar levels presenting with nausea, vomiting, malaise, or abdominal pain or, in more severe cases, altered consciousness, Kussmaul breathing, or clinical signs of shock . This isIn the ED setting, physicians may be confronted with other forms of acidosis, particularly metformin-associated lactic acidosis (MALA), which should not be confused with eDKA in patients treated by a SGLT2i and metformin at the same time. The difference between eDKA and MALA is the absence of β-hydroxybutyrate elevation in MALA, but the two disorders can coexist in theory, especially in patients with CKD . DiagnosOnce the diagnosis of eDKA is confirmed, SGLT2is should immediately be discontinued, and treatment should be started including fluid resuscitation, insulin with concomitant glucose infusion, careful electrolyte and glycemia monitoring, and treatment of the underlying trigger if feasible . PatientNecrotizing fasciitis of the perineum, also known as Fournier’s gangrene (FG), is a potentially fatal acute necrotic infection of the subcutaneous tissues around the genital or perianal regions. Classic risk factors for FG include hypertension, obesity, tobacco use, immunosuppression, heart failure, and T2DM . Using tFG should be suspected in any patient taking a SGLT2i who presents with fever along with pain, erythema, or swelling of the genital or perianal area. If diagnosed, immediate discontinuation of the SGLT2i, hospitalization, close monitoring, prompt introduction of broad-spectrum anti-biotherapy, and surgical debridement, if necessary, are required .Canagliflozin was the only SGLT2i associated with a slightly higher risk of fractures, most of them nonvertebral, in the CANVAS-Program trial, but this has not been confirmed in subsequent studies . PossiblAn increased risk of lower limb amputations (LLA) with canagliflozin, mostly toe and metatarsal ones, was reported in the CANVAS-Program trial with a HR of 1.97 (95% CI 1.41–2.75) . AlthougSGLT2is should be used with caution in patients with previous amputations, active cutaneous ulcers, or lower extremity artery disease . In the Of note, some data have reported an increased rate of stroke associated with SGLT2is, the mechanism of which is not fully understood . This waIn general, it is advised to discontinue SGLT2is in patients undergoing urgent surgery or hospitalized with any acute serious medical condition  because of the risk of eDKA. In case of scheduled surgery, the FDA recommends discontinuing SGLT2is 72 h prior to intervention (96 h for ertugliflozin) .However, in certain acute clinical situations, discontinuing the SGLT2i may be debatable. In the following section, we discuss the management of patients taking SGLT2is at baseline, presenting to the ED in four different clinical situations.It is estimated that more than 26 million people suffer from heart failure worldwide, with up to 77% presenting at least once to the ED with decompensated acute heart failure (AHF) . As suchLiterature regarding the management of SGLT2is in the setting of patients presenting with atrial fibrillation (AF) with rapid ventricular response is very scarce. However, in authors’ opinion, the decision to discontinue or not the SGLT2i primarily depends on the hemodynamic tolerance of the AF and the volume status. In patients with hemodynamic instability  and in those presenting with hypovolemia based on clinical and biological evaluation, it seems reasonable to withhold the SGLT2i in addition to general AF management .Acute diabetes decompensation is another commonly encountered condition in the ED and includes hyperosmolar hyperglycemic state (HHS), eDKA, and marked hyperglycemia without hyperosmolar or ketosis conditions. Although historically considered as two distinct clinical entities, HHS and eDKA share the same basic pathophysiologic mechanisms: Significant insulin deficiency and increased concentration of counterregulatory hormones such as glucagon, catecholamines, cortisol, and growth hormone. Furthermore, similar to patients with eDKA, those with HHS frequently may present with signs of dehydration, dry mucous membranes and poor skin turgor, or hypotension . AlthougSGLT2is have been shown to consistently reduce uric acid concentrations via increased urinary uric acid excretion by approximately 35 45 μmol/L to 45 μmol/L (0.60–0.75 mg/dL). The lowering of uric acid by SGLT2is may partly explain the beneficial CV and renal effects associated with this class of treatments . When coNo well-controlled studies of SGLT2is have been performed in the context of pregnancy. According to animal studies, these drugs may affect renal development. Therefore, SGLT2is should be used in pregnant women only if the benefits justify the risk to the fetus .SGLT2is are antidiabetic drugs for which indications are currently rapidly expanding, as new data show beneficial effects not only in patients with T2DM, but also in those with HFrEF and CKD, regardless of the diabetes status. Overall, they are well-tolerated treatments, with mild genital mycotic infections and volume depletion being the most common adverse events. However, as the number of patients taking SGLT2is increases, emergency physicians may be faced with rarer adverse events, some of which, if left unrecognized, could be life-threatening. The risk of adverse events may be reduced by careful patient information and education about self-monitoring. Although it is advised to discontinue SGLT2is in patients undergoing urgent surgery or hospitalized with any acute serious medical condition, in certain acute clinical situations, such as acute heart failure, this may be debatable, as SGLT2 inhibition may actually yield beneficial effects.


The primary microvascular outcomes of the present study included chronic kidney disease (CKD) progression, retinopathy, and neuropathy. Cox proportional-hazards models were performed to evaluate the association of FMI with microvascular diseases. A discordant analysis was performed to compare the magnitude of associations of FMI, BMI, and WC with the risk of microvascular diseases. Our study included 10,251 T2DM participants with a median of 5 years  of follow-up. A total of 6,184 participants developed CKD progression, 896 participants had retinopathy, and 3,213 participants developed neuropathy . After the confounding factors were adjusted for, patients in the highest FMI quartile had a higher risk of CKD progression  and neuropathy , except for retinopathy , than those in the lowest quartile. Discordant analyses found that FMI and WC are better in identifying individuals with obesity-related risk of neuropathy, compared with BMI; neither is better in identifying individuals with obesity-related risk of CKD progression and retinopathy. Obesity is associated with CKD progression and neuropathy in T2DM participants. Further randomized trials are needed to test whether obesity control can improve the outcomes of T2DM participants with CKD or neuropathy. FMI and WC are more useful in identifying obesity-related risk of neuropathy compared with BMI in T2DM patients.This study aimed to evaluate the association between obesity, evaluated by fat mass index (FMI) with the risk of microvascular diseases in patients with type 2 diabetes mellitus (T2DM) and compare the magnitude of associations of FMI, body mass index (BMI), and waist circumference (WC) with the risk of microvascular diseases. We performed a http://www.clinicaltrials.gov, NCT00000620. Epidemiological studies show that obesity has become a major problem worldwide and is associated with an increased risk of macrovascular diseases , 2. HoweMost clinical and epidemiological studies mainly use BMI as the preferred measure of obesity or overall adiposity because it is easily measured and highly associated with more objective fat mass (FM) , 10. Recpost-hoc analysis of the Action to Control Cardiovascular Risk in Diabetes (ACCORD) study. Data are available from the Biologic Specimen and Data Repository Information Coordinating Center (BioLINCC). The ACCORD study was a double-parallel treatment trial that included 10,251 T2DM patients from 77 sites in the US and Canada . Intensified blood glucose control did not reduce the incidence of advanced measures of microvascular diseases, such as the initiation of dialysis or end-stage renal disease (ESRD); however, it delayed the onset of early microvascular diseases, such as albuminuria and some measures of retinopathy and neuropathy , and treatment effect was also used as a confounding factor in our post-hoc analysis  CKD progression [defined as the doubling of baseline serum creatinine or greater than 20 ml/min per 1.73 m² decrease in the estimated glomerular filtration rate (GFR)], (2) retinopathy , or (3) neuropathy [a score >2.0 on the Michigan Neuropathy Screening Instrument (MNSI) ]. The MNNo participants were involved in determining the research questions or outcome measures or in designing or implementing the study. No plans exist to involve patients in knowledge dissemination.The relationships between obesity and primary and secondary endpoints were evaluated using the FMI as both a continuous and a categorical variable. Three Cox proportional-hazards regression models were constructed. Model 1 was unadjusted; model 2 was adjusted for age, race, sex, and treatment effect (intensive or standard blood glucose control); and model 3 was adjusted for diabetes duration, proteinuria, current smoking, weekly alcohol consumption, height, and baseline levels of GFR, total cholesterol, low-density lipoprotein cholesterol, high density lipoprotein cholesterol, systolic blood pressure, and hemoglobin A1C, in addition to the four factors adjusted for in model 2. To evaluate FMI as a categorical variable, the patients were divided into sex-specific quartiles of FMI. The first quartile was used as a reference. Tests for linear trends were conducted by treating quartile categories as a continuous variable after assigning the median value for each category . To evalvice versa. Finally, Cox proportional-hazards models adjusted for the factors in model 3 were performed on categories of discordant values of (1) FMI versus WC, (2) FMI versus BMI, and (3) WC versus BMI.We further aimed to use discordance analysis to evaluate and compare the effectiveness of FMI, WC, and BMI to predict the risk of microvascular diseases. First, the correction between FMI and other obesity indicators, such as WC and BMI, was computed using Pearson or Spearman rank correlation. Second, we calculated the sex-specific medians for the FMI, WC, and BMI to classify the included participants into two categories based on less than median value or greater than or equal to median values of each obesity indicator. We categorized the participants into four groups according to having a low or a high value of obesity indicators as follows: low/low, high/low, low/high, and high/high. We define a high FMI with WC or BMI being low as discordance or P-values were two-sided, and values less than 0.05 were considered statistically significant. All statistical analyses were performed using Stata/SE (version 15.1) and R, version 3.4.3 .All The data that support the findings of this study are available from BioLINCC, but restrictions apply to the availability of these data, which were used under license for the current study and therefore are not publicly available.2, 32.7 ± 5.6 kg/m2, 108.0 ± 13.7 cm, and 0.61 ± 0.08, respectively. During the median 5-year  follow-up period, 6,184 participants developed CKD progression , 896 participants had retinopathy requiring retinal photocoagulation or vitrectomy , and 3,213 participants developed neuropathy (MNSI >2.0) . The detailed characteristics of the participants are presented in The included participants had a mean age of 62 years and a T2DM duration of median 10 years. A median of six  measurements of the obesity indicators had been performed. The mean  time between baseline FMI and the last measurement was 4.15 ± 1.47 years. The mean (SD) FMI, BMI, WC, and W-t-H values were 12.4 ± 4.1 kg/mThe risk of CKD progression and neuropathy (MNSI >2.0) increased with the increased FMI quartiles in the fully adjusted model (model 3). Compared to patients in the lowest FMI quartile, those in the highest quartile were 93 and 26% more likely to develop neuropathy and CKD progression, respectively Table 3.2) increased the risk of CKD progression  and neuropathy (MNSI >2.0) , with a numerical increase in retinopathy  without statistical significance (When FMI was used as a continuous covariate in the fully adjusted model (model 3), each one SD increase in FMI . However, both the lower and higher ranges of FMI showed a higher risk of CKD progression. Regarding the inverted U-shaped relationship between FMI and retinopathy, the plot showed a substantial increase in the risk of retinopathy within the lower range of FMI, reaching the highest risk at around 16.4 kg/m2 and decreasing thereafter . The risk of neuropathy increased sharply to approximately 18.5 kg/m2 and then leveled off . A “plateau phenomenon” was found in the relationship between FMI and the risk of CKD progression or neuropathy (MNSI >2.0).In 2, or those with proteinuria  compared with concordant FMI and BMI below the medians; similar results were found for discordant FMI above the median and WC below  and discordant WC above the median with BMI below  was found in intensive glucose control, female, or white race subgroup. Fourth, discordance analysis demonstrated that FMI or WC was a more accurate indicator of obesity-related risk of neuropathy compared with BMI.This study is a Consistent with the current study, previous studies have pointed out an increased incidence of microalbuminuria, macroalbuminuria, deterioration of renal function, and neuropathy in T2DM patients who are obese , 21. TheWe did not find an exact relationship between obesity and the risk of retinopathy. A previous study has found that weight control may be less useful in T2DM patients with retinopathy. Although bariatric surgery reduces the risk of nephropathy and neuropathy significantly in T2DM patients, it did not reduce the risk of retinopathy, which was consistent with our present study –25. AnotConsistent with previous studies, obesity was associated with an increased risk of neuropathy. However, our present study extends the knowledge on the relationship between obesity and risk of neuropathy. FMI and WC are a more accurate indicators of obesity-related risk of neuropathy compared with BMI. However, no studies have performed discordance analyses on T2DM patients as done in our analyses. In cohorts with T2DM, discordance analyses also favor FMI and WC in identifying individuals with obesity-related risk of neuropathy. FMI and WC reflect different aspects of obesity with added effect on each other. Therefore, when we evaluate the risk of neuropathy, FMI or WC may be more appropriate than BMI. A subgroup among obese patients with lower FM/WC, called metabolically healthy obesity, has recently been recognized, which does not express metabolic disorders and cardiovascular risk factors. A subgroup of normal-weight patients with higher FM/WC has metabolic disorders and cardiovascular risk factors. Importantly, abdominal obesity is a modifiable risk factor, and thus the question arises whether weight reduction in individuals who are overweight or with obesity by interventions, such as lifestyle modification or bariatric surgery, have an influence on the prevention of neuropathy.This study has several limitations. First, the FM that we used was calculated with equations rather than the true FM, which is measured by dual-energy radiographic examination. However, the prediction equations, including factors such as age, sex, height, weight, and WC, showed a high predictive ability for FM and have been widely used in epidemiological studies. Furthermore, we obtained similar results using different anthropometric indicators suggesting a robust association between obesity and microvascular diseases. Second, the short follow-up duration might explain why we could not find out whether obese patients with T2DM had a high risk of renal failure. Finally, this study only included patients from the United States and Canada; hence, these results may not apply to other populations with different body habitus.Obesity is associated with microalbuminuria, macroalbuminuria, neuropathy, and deterioration of renal function in participants with T2DM. Further randomized trials are needed to test whether obesity control can improve the outcomes of T2DM participants with CKD or neuropathy. The FMI and WC are more useful in identifying individuals with obesity-related risk of neuropathy compared with BMI in T2DM patients.Data are available from the Biologic Specimen and Data Repository Information Coordinating Center (BioLINCC).post-hoc analysis of ACCORD study. No participants were involved in setting the research questions or outcome measures or in the design and implementation of the study. No plans exist to involve patients in dissemination.This is a ZX designed the study and provided methodological expertise. ZX and SG drafted the manuscript. HZ and CL revised the manuscript. ZX is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. All authors contributed to the article and approved the submitted version.This work was supported in part by the National Science Foundation of China project 82000298, the Natural Science Foundation of Hunan Province 2021JJ40883 to ZX, the Natural Science Foundation of Hunan Province 2019JJ40451 to HZ, and the Natural Science Foundation of Hunan Province 2021JJ40850 to CL.The authors declare 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.