Artesunate treatment caused significant growth inhibition and apoptosis in A549 cells. Mechanistically, artesunate downregulated the activation of AKT/Survivin signaling. In agreement, hyperactivation of AKT signaling restored artesunate-induced growth inhibition in A549 cells. In mouse lung cancer models, artesunate administration significantly reduced the growth of A549 cells and LLC cells in nude mice and immunocompetent mice, respectively. Our findings suggest that artesunate serves as a potential tumor suppressor in lung cancer and hopefully can provide new insight into the development of therapeutic strategies in the clinical lung cancer treatment.NSCLC  is the deadliest cancer in the world. Artesunate is one of the most potent and rapidly acting antimalarial agents. Recently, emerging evidence has suggested the anticancer function of artesunate. In our work, we aimed to investigate the molecular mechanism of artesunate-induced growth inhibition in human lung adenocarcinoma cells and reported that the anticancer effects of artesunate is related to its ability in downregulating AKT/Survivin signaling in A549 cells. The effect of artesunate on the proliferation of A549 cells was determined by CCK-8 assay and colony formation assay; its effect on A549 cell apoptosis was evaluated by lactate dehydrogenase (LDH) release assay. The role of artesunate on the activation of AKT/Survivin signaling was analyzed by western blot and quantitative QPCR. Finally, we used two mouse tumor models to investigate the function of artesunate on the  Artemisinin is a sesquiterpene lactone extracted from Artemisia annua, which is a semisynthetic derivative of artemisinin. Artesunate is a class of antimalarial drug that controls the symptoms of malaria, but its modulatory roles in tumor growth and the underlying molecular mechanisms remain relatively poorly understood. Artemisinin and its derivatives have been included in the anticancer drug screening and anticancer activity research program by the National Cancer Institute (NCI) in the United States due to their proven anticancer effects . Lung caAKT, also known as protein kinase B, is a serine/threonine kinase with a 70% homology between its kinase domain and protein kinases A and C . AKT sigin vitro but also delayed in vivo growth in nude mice and immune-competent mice. The effect of artemisinin was attributed to its ability in downregulating the activation of AKT/Survivin signaling pathway.In this study, we found that artemisinin serves as a tumor suppressor in lung cancer. Artemisinin administration not only inhibited the growth of A549 lung cancer cells β-actin were all obtained from Proteintech.Artesunate and dimethyl sulfoxide (DMSO) were made a purchase from Sigma (USA). Antibodies of p-AKT, AKT, Survivin, c-caspase-3, and 2 atmosphere at 37°C.A549 cells (ATCC® CCL-185) and Lewis lung carcinoma  cells were obtained from American Type Culture Collection (ATCC). Cells were cultured in DMEM and supplemented with 10% FBS, 20 mM HEPES, 100 U/ml penicillin, and 100 mg/ml streptomycin. Cells were maintained in a humidified, 95% COμl of CCK-8 solution at 37°C for another 1 h. Then, the medium was abandoned and DMSO was used to dissolve the formation of formazan crystals. A microplate spectrophotometer is to measure the absorbance. The calculations of cell viability were based on previous study.We used Cell Counting Kit-8  to analyze the cell viability according to the description by Li and Altieri . In brieWithin 24 hours, different concentrations of artesunate treat A549 cells. The cell culture supernatant was collected, and the levels of LDH were evaluated using the LDH Assay Kit  based on the instructions of thr manufacturer. The measurement of OD values was on a Promega™ Microplate Reader .The colony formation assay measured the cytotoxic effects of artesunate. Briefly, A549 cells were seeded onto 6-well plates (2000 cells/well). After incubation overnight, artesunate deal with the cells with different concentrations within 24 hours. After that, cells with complete medium were breezed for another following 10 days.PBS washed the colonies forming on plates, then being fixed in methanol, and lastly being stained with 1% methylrosanilinium chloride. A microscope  was used to count the colonies and to measure the colony's percentage in each treatment group.μM of each primer and EvaGreen Supermix (Bio-Rad).To synthesize first-strand cDNA with a synthesis kit of iScript cDNA  depending on manufacturer's directions, the analyses of real-time qPCR were performed using a QPCR mixture containing 1 A StepOnePlus real-time QPCR system  was used to perform amplifications at 95°C for 15 s and at 60°C for 30 s. Each sample was examined in triplicate, and glyceraldehyde phosphate-3 dehydrogenase mRNA level was regarded as an inner control. Primer sequences are illustrated as follows: GAPDH: forward 5′-GTCTCCTCTGACTTCAACAGCG, reverse 5′-ACCACCCTGTTGCTGTAGCCAA; Bcl2: GAPDH: forward 5′ATCGCCCTGTGGATGACTGAGT, reverse 5′GCCAGGAGAAATCAAACAGA GG; Bax: GAPDH: forward 5′TCAGGATGCGTCCACCAAGAAG, reverse 5′-TGTGTCCACGGCGGCAATCATC; and Survivin: GAPDH: forward 5′CCACTGAGAACGAGCCAGACTT, reverse 5′-GTATTACAGGCGTAAGCCACCG.T and Spearman's rank correlation and one-way ANOVA of Dunnett, we find that there are big and obvious differences among the groups. All statistical analyses were performed with the Statistical Program for Social Sciences (SPSS), version 17.0.The illustration of all results adopted the mean ± SD according to the independent experiments unless without indication. Through the tests of Student 60 mm plates were used to seed A549 cells, and artesunate was used after overnight incubation to treat artesunate. Then, RIPA , containing protease inhibitor cocktail, was used to lyse the cells. The lysate was centrifuged at 15,000 rpm for 15~20 min at 4°C. The BCA protein assay kit  was used to detect the concentration of proteins. The samples of protein were subjected to electrophoresis, and then, they were transferred to the poly-vinylidene difluoride transfer membranes . The blots were blocked with 5% nonfat milk for 1 h at room temperature and incubated with primary antibody at 4°C. Then, the blots were incubated with horseradish peroxidase-conjugated secondary antibodies for 1 hour. An ECL kit was used with the immunoreactive bands. The software of ImageJ was used to analyze the immunoreactive band density.n = 6/group) or C57BL/6 mice (n = 7/group) were purchased from Shanghai Laboratory Animal Center . 8 × 105 A549 or LLC cells were injected subcutaneously into the left flank of mice. One week after tumor inoculation, mice were intratumoral injected with artesunate (80 mg/kg) or the same volume of PBS every 3 days. Tumor volume was monitored with a caliper and then calculated using the formula: V = Length × (Width)2/2. Fifteen to twenty days postinoculation, when tumor volume reached approximately 1500 mm3, mice were used for cervical dislocation in the following experiments.6 ~ 8-week athymic nude mice , 53.5% (50 μg/ml), and 48.8% (100 μg/ml) separately  treatment for another 24 h. As shown in Next, we examined if artesunate could suppress the in vivo growth of lung cancer cells. We further examined the effect of artesunate on tumor growth in immune-competent mice. Lewis lung carcinoma (LLC) cells were subcutaneously inoculated into C57BL/6 mice. Similar to its effect on A549 tumors, artesunate administration significantly inhibited LLC growth in mice Figures . Likely,To this end, A549 cells were inoculated subcutaneously into nude mice, which were then subjected to artesunate gavage. The results presented that artesunate meaningfully inhibited the growth of A549 cells in mice, as evidenced by the reduced tumor volume and tumor weight Figures . FurtherPrevious evidence has implicated that artemisinin and its derivative artesunate can be used as potential anticancer agents , 13. In An earlier study has reported that artesunate induced cell apoptosis by a Bak/AIF-mediated caspase-independent internal signal, in which BH-3/Bim played an active role in activating the Bak pathway . In thisCell proliferation inhibition is a significant toxic effect through which some anticancer drugs decrease the viability of cancer cells. Therefore, it is crucial to examine whether inhibition cell proliferation caused by anticancer drugs is involved in inhibition of antiapoptotic gene expression, such as Survivin, which is associated with malignant cell proliferation, progression, and chemotherapy resistance –17. In oThe AKT signaling pathway is an effective oncogenic regulator, widely involved in tumor cell growth, invasion, migration, and survival, which is frequently low expressed in human tumors such as non-small-cell lung cancer (NSCLC) and head and neck squamous cell carcinoma (HNSCC). The effect of artesunate on phosphorylation of AKT has also been reported in endothelial cells in vitro . There iHowever, the role of AKT pathway in artesunate-induced apoptosis is unclear. In the present study, the results suggest that AKT may be involved in artesunate-induced cell proliferation inhibition. On the other hand, artesunate could significantly decrease the phosphorylation of AKT in a concentration-dependent manner, while the total levels of AKT showed no change. Meanwhile, we enhanced the AKT pathway in artesunate therapy in SC97 cells, a chemical agonist that increases AKT activity, to determine if there is a mechanism of inhibited cell proliferation by artesunate.We found that low nontoxic concentrations of SC97 significantly alleviated the effect of artesunate and rescued the cell proliferation inhibition. Further, pretreatment with SC97 partially inhibited changes in Survivin and the caspase-3 activity. These results clearly indicate the involvement of AKT pathway activation in artesunate-mediated cell proliferation inhibition. In conclusion, artesunate can induce the growth inhibition and apoptosis in lung cancer cells via dampening AKT/Survivin, indicating its potential role in the clinical treatment of lung cancer.


Chestnut seeds are important kinds of edible nuts rich in starch and protein. The characteristics and nutrient contents of chestnut have been found to show obvious metaxenia effects in previous studies. To improve the understanding of the effect of metaxenia on chestnut starch and sucrose metabolism, this study used three varieties of chestnut, ‘Yongfeng 1’, ‘YongRen Zao’ and ‘Yimen 1’, as male parents to pollinate the female parent, ‘Yongfeng 1’, and investigated the mechanisms of starch and sucrose metabolism in three starch accumulation stages (70 (S1), 82 (S2), and 94 (S3) days after pollination, DAP) in chestnut seed kernels.Most carbohydrate metabolism genes were highly expressed in YFF (self-pollinated ‘Yongfeng 1’) in stage S2 and in YFR (‘Yongfeng 1’ × ‘Yongren Zao’) and YFM (‘Yongfeng 1’ × ‘Yimen 1’) in stage S3. In stage S3, hub genes encoding HSF_DNA-binding, ACT, Pkinase, and LIM proteins and four transcription factors were highly expressed, with YFF showing the highest expression, followed by YFR and YFM. In addition, transcriptome analysis of the kernels at 70, 82 and 94 DAP showed that the starch granule-bound starch synthase (EC 2.4.1.242) and ADP-glucose pyrophosphorylase (EC 2.7 .7.27) genes were actively expressed at 94 DAF. Chestnut seeds regulate the accumulation of soluble sugars, reducing sugars and starch by controlling glycosyl transferase and hydrolysis activity during development.These results and resources have important guiding significance for further research on starch and sucrose metabolism and other types of metabolism related to chestnut metaxenia.The online version contains supplementary material available at 10.1186/s12864-021-07923-5. C. mollissima BL.) has been cultivated for its nuts and timber for over 4000 years [http://www.fao.org/). Chinese chestnut is a rich source of vitamin C, protein, and a variety of minerals, with particularly high contents of magnesium, phosphorus, and potassium . However, Chinese chestnut cultivation continues to face serious problems, such as uneven quality, the production of many hollow bracts and low yields [Chinese chestnut  based on changes at the morphological and physiological levels. The seed collection used in this study was obtained from the following different pollination combinations: YFF, YFR and YFM. As shown in Fig. The sugar content, starch content, sucrose synthase (SS) activity and starch synthase (SSS) activity were evaluated in chestnut seeds at different developmental stages Fig. a, b, c. To explore the potential molecular mechanism of starch accumulation, global transcriptome analysis was performed on chestnut seeds at different developmental stages. RNA was isolated from seeds of YFF, YFR and YFM at S1, S2 and S3 , and 27 cDNA libraries  were constructed  of the average FPKM values was performed based on the SCC analysis  | value > 0. The genes that were specifically expressed at each stage of the starch accumulation period in the seeds from these three pollination combinations were identified  between these nine samples were identified using the BH algorithm. The differential expression analysis revealed a large number of candidate DEGs according to the following criteria: DEGs were defined according to the ratio of FPKM expression values, a false discovery rate (FDR) < 0.05 and an absolute |logied Fig. a. A VennThe gene ontology (GO) analysis of DEGs between the seeds of YFF, YFR and YFM at different developmental stages. The results show many GO terms related to seed development and starch accumulation  aggregated into four profiles, including two downregulated modes and two upregulated modes, and the expression patterns of YFF and YFM were consistent. The 6737 DEGs from YFR were also significantly  aggregated into four profiles, including two downregulated modes and two upregulated modes, but the YFR expression pattern was inconsistent with those of YFF and YFM . Specifically, 5892 DEGs from YFF and 3325 DEGs from YFM were significantly . As shown in the tree diagram  was highly expressed during the peak period of starch accumulation (S3). The encoded protein catalyzes the conversion of g-1-p to adp-glucose, which is the direct precursor of starch synthesis. The expression of a gene encoding starch granule-bound starch synthase (EC 2.4.1.242), which is responsible for the synthesis of amylose and amylopectin, was also increased. Moreover, fructose diphosphate aldolase , enolase (EC 4.2.1.11) and pyruvate phosphokinase (EC 2.7.9.1) were significantly regulated by protein phosphorylation. FBA and enolase catalyze reversible reactions in glycolysis. PPDK promotes gluconeogenesis by catalyzing the mutual conversion of pyruvate and PEP. The pyruvate and PEP production steps are initially irreversible in glycolysis. The above results indicated that glycolysis is inhibited while gluconeogenesis is enhanced, resulting in the flux of G-1-P and G-6-P into the starch synthesis pathway in chestnut seeds.To verify the reliability of the RNA sequencing data, qPCR was used to evaluate the expression profiles of five upregulated and two downregulated genes. The expression trends of the seven selected DEGs were consistent with the transcriptome data Fig. , indicatThe chestnut is a member of the Fagaceae family and is an economically important crop species. It has received extensive attention because of its high nutrient content and health-promoting properties . ChestnuWe coupled transcriptomic analysis with qPCR assays for selected genes to understand the global gene expression involved in starch and sucrose metabolism in three different type of Chinese chestnuts . By analyzing the transcriptomes of three chestnut fruits at three different developmental stages, we found that sugar and starch content, SS and SSS activities all showed significant epigenetic effects during the ripening process of chestnut. In the early stage of seed development, energy metabolism, organic acid metabolism and translation processes are activated in large quantities, which promotes the synthesis of ATP, so that the seeds get more energy; and in the middle and late stages, accompanied by the activation of starch and sugar metabolism, promoted the accumulation of carbohydrates in chestnut.Glycosyltransferases (GTFs) are enzymes (EC 2.4) that establish natural glycosidic linkages. They catalyze the transfer of saccharide moieties from an activated nucleotide sugar  to a nucleophilic glycosyl acceptor molecule, the nucleophile of which can be oxygen carbon, nitrogen, or sulfur based , 29. In In addition to the enzymes closely related to sugar metabolism, the results of GO and KEGG enrichment analysis showed specificity in the seeds of different pollination combinations and in different developmental stages. A previous study showed that many metabolic pathways vary significantly during fruit development of the date palm, and carbohydrate metabolism  is particularly prominent during fruit ripening . In stagThe experimental site was located in Weidi Township, Yongren County ), Yunnan Province. ‘Yongfeng 1’  was selected as the female parent. The following pollination combinations were established: ‘Yongfeng 1’ × ‘Yongfeng 1’ (YFF), ‘Yongfeng 1’ × ‘Yongren Zao’ (YFR), and ‘Yongfeng 1’ × ‘Yimen 1’ (YFM). Four sampling directions  were selected for performing the pollination combinations in the above groups. At the early stage of the germination of female flowers (2018/04/29), approximately 80 samples from each pollination combination were bagged. At the flowering stage of male flowers, fresh pollen was collected for direct pollination (2018/05/19) and was removed from the bag prior to thorn ball swelling (2018/05/26).Description of chestnut varieties: ‘Yongfeng 1’ is a real-breeding variety in Yongren County, Yunnan Province, with an average single seed weight of approximately 16 g. ‘Yongren Zao’ is also a real-breeding variety in Yongren County, Yunnan Province, with an average single-grain weight of 12 g, in which the mature period occurs in late July. ‘Yimen 1’ is a well-approved variety in Yunnan Province. Its fruit ripening period occurs in mid-August, and it presents an average single-grain weight of 14.51 g.Starting approximately 20 days before maturation, samples were collected every 11 days, and the last samples obtained consisted of mature fruits with thorn ball dehiscence. Chestnut seeds were collected from 18 pollination trees at three developmental stages  , and an Agilent 2100 system was used to detect RNA. Sequencing libraries were constructed by using the NEBNext® Ultra™ RNA Library Prep Kit for Illumina®  according to the manufacturer’s recommendations. mRNA was enriched with mRNA capture beads and then fragmented. First-strand cDNA was synthesized using random hexamers, followed by synthesis of double-stranded cDNA. After end repair, an A tail and an adapter were added, and cDNA fragments 150–200 bp in length were selected. Sequencing libraries were generated via PCR. Then, we measured the quality of the libraries. The libraries were sequenced using a HiSeq 2000 sequencer , and 150 bp paired-end reads were generated. Adapters and low-quality reads were removed from the raw data to obtain clean data. We also measured the Q20 and Q30 values of the clean data. The high-quality cleaned reads were mapped to the Chinese chestnut reference genome  using HIP-values  ≤ 0.05 and an absolute log2 (fold change) value were identified as DEGs. To observe the gene expression patterns in seeds in different stages, the DEGs were grouped by hierarchical clustering according to their level of expression using the pheatmap R package.PCA was performed to show the correlations between biological replicates using an R package. We performed differential expression analysis based on Cufflinks. Genes with adjusted p value < 0.05 was set as the threshold for significantly enriched GO terms. Similarly, we identified the statistical enrichment of DEGs in KEGG pathways using KOBAS software (v2.0). MapMan (v.3.5.1R2) was used to visualize the expression patterns of DEGs.We performed GO enrichment analysis of the DEGs according to GO:TermFinder . SignifiTo detect patterns of gene connectivity, we analyzed the data via WGCNA with the WGCNA package (v1.51) in R. Genes with a low coefficient of variation of the averaged RPM (CV < 1) among all samples were discarded, and the remaining 12,662 genes were used for the analysis.First, the scale-free gene network condition should be satisfied before conducting WGCNA. Moreover, it is necessary to define the correlation matrix of gene coexpression and adjacency function. A soft threshold (power) of five was chosen in this study. Then, the dissimilarity measurements of different nodes were calculated, and a hierarchical clustering tree was built based on these data. Different dendrogram branches represent various modules. We drew a heatmap via the module eigengene that was used to show the expression pattern in every sample. Therefore, we can study a module that is significantly related to further research via heatmap data. The functional significance of the modules was evaluated through GO and KEGG enrichment analysis according to a previous method. The coexpression networks of selected modules of genes were visualized by Cytoscape (version 3.1.0).The expression patterns of 7 unigenes encoding SSS and SS in developing Chinese chestnut seeds were studied by qRT-PCR. Total RNA was extracted using a Qiagen RNeasy Mini Kit  and was reverse transcribed into cDNA by using random primers. The internal control was 18S rRNA . The 2(−Additional file 1: Figure S1. Pearson correlation between samples. Figure S2. DEGs and GO annotations in different stages of seeds from three pollination combinations. A: DEGs in different stages. B: GO annotations in different stages. Figure S3. WGCNA of all KEGG pathways. A: Hierarchical clustering of unigenes and module identification. B: Relationships between gene modules and sample groups. Table S1. Statistical analysis of transcriptome sequencing in seeds from three pollination combinations. Table S2. The ten most enriched KO terms of each group are listed.


Regardless of APOE4 status, infected subjects presented (i) significantly more microstructural alterations of the parahippocampal cingulum and fornix, (ii) lower hippocampal volumes only when their anti-HSV IgG level was in the highest tercile—reflecting possibly more frequent reactivations of the virus , and (iii) had no increased risk of developing AD. Nevertheless, among APOE4 carriers, infected subjects presented lower hippocampal volumes, although not significant (p = 0.09), and a two or three times higher risk of developing AD (adjusted Hazard ratio (aHR) = 2.72 [1.07–6.91] p = 0.04 for infected subjects and aHR = 3.87 [1.45–10.28] p = 0.007 for infected subjects with an anti-HSV IgG level in the highest tercile) while no association was found among APOE4 noncarriers. Our findings support an association between HSV infection and AD and a potential interaction between HSV status and APOE4. This reinforces the need to further investigate the infectious hypothesis of AD, especially the associated susceptibility factors and the possibility of preventive treatments.While previous studies suggest the implication of herpes simplex virus (HSV) in the onset of Alzheimer’s disease (AD), no study has investigated its association with early neuroimaging markers of AD. In the Three-City and the AMI cohorts, the associations between HSV infection and (i) hippocampal volume ( Alzheimer’s disease (AD) is a neurodegenerative disease characterized by visible changes within the brain several years before the clinical stage of dementia , includiSeveral studies have suggested the implication of infectious agents in AD pathophysiology , with heHowever, results from epidemiological studies are still inconsistent concerning the association between HSV infection and cognitive decline or dementia (reviewed in ) and, ifIn the present study, we aimed to evaluate the association between HSV status and (i) hippocampal volume (HV), (ii) WM alterations in the cingulum and the fornix which are the main afferent and efferent axonal bundles of the hippocampus in two population-based prospective cohorts of older adults. We also complement our previous results  in a larThe study sample comes from two population-based prospective cohorts: the Three-City study (3C) and the Aging Multidisciplinary Investigation study (AMI). A detailed description of both study protocols was previously published , 28. Brin = 1258) and at inclusion for AMI (n = 609)  or if they presented IgG- IgM+ status (n = 5) reflecting either a primary infection (unlikely given the advanced age of the participants) or IgG false negative or IgM false positive  and subjects without any follow-up after the time of serology (n = 171). Analyses were carried out on 1599 participants  was performed using an ACHIEVA 3T scanner  (details in Supplementary Information) on a subsample of 238 participants 6 years after the serology in 3C and 200 participants 4 years after the serology in AMI. Among these subjects, 387 had anti-HSV serologies.3D T1 weighted images were used to segment the hippocampus using FIRST implemented in FMRIB Software Library v5.0 . The qux,y,z), AxD the diffusivity in the main direction and RD the average of the diffusivity in the two remaining directions. High FA and low diffusivity values reflect a high level of structural coherence and undamaged axon fiber architecture.WM integrity was assessed using diffusion tensor imaging (DTI). Of 387 participants initially included in the MRI study, 259 participants had available diffusion-weighted images  was defined as the middle of the time interval between the last visit without dementia and the diagnosis visit. Participants who developed another type of dementia were censored at the age of onset of dementia. Nondemented participants (included the deceased without dementia) were censored at the age of the last visit without dementia. We considered the following confounding factors in addition to age : sex, level of education, marital status, presence of at least one allele of APOE4, hypertension, diabetes, hypercholesterolemia, tobacco use, and cohort. The proportional hazards assumption was assessed with Schoenfeld residuals and by testing interactions with time.For analyses of the incidence of AD, we considered the presence of IgG and IgG levels in terciles. The existence of an interaction with time prevented us from analyzing the IgM status. Cox models, using age as timescale, were performed . The resFor all analyses, we tested the existence of an interaction between APOE4 and HSV status. As we assumed a priori a different impact of HSV infection according to the APOE4 status, analyses were then systematically stratified by APOE4. In the stratified models, due to the small sample sizes, the adjustment variables were restricted to age, sex, and cohort for linear regression models and to age , sex, level of education, and cohort for Cox models. For the same reason, we did not consider the levels of IgG in terciles in the stratified samples.All statistical tests were two-tailed, and the threshold for statistical significance was 5%. Analyses were performed with SAS software (version 9.4 SAS Institute).n = 349), WM integrity (n = 259), and AD incidence (n = 1599). In the latter, participants were 77 (±5) years old, 45% were male and 18% were APOE4 carriers. In this sample 83% (n = 1327) had anti-HSV IgG and these tended to be more often women, widowed, with a lower level of education, more hypertension, and more often non-smoker (Supplementary Table The characteristics of participants are presented in Table ence n = 599. In tn = 281) had anti-HSV IgG. The mean HV expressed as a percentage of the TIV was 0.25 ± 0.03 . Nevertheless, when considering the levels of IgG in terciles, subjects with an IgG level in the highest tercile had a lower HV compared to uninfected subjects  , the association between the presence of IgG and lower HVs was close to significance (β = −0.016 standard deviation = 0.009 p = 0.09). No association was observed among APOE4 noncarriers .The 15 Table . In APOEn = 206) had anti-HSV IgG. FA, MD, AxD, and RD for the parahippocampal cingulum and fornix are presented in Table Among the available sample, 80% n = 06 had anp-values ≥ 0.33). There were associations at least borderline significant (p ranging from 0.006 to 0.09) between anti-HSV IgG and low FA, high MD, and high RD among APOE4 noncarriers for the parahippocampal cingulum and among both ApoE4 carriers and noncarriers for the parahippocampal fornix.There were no significant interaction between the presence of anti-HSV IgG and APOE4 for DTI parameters (Table n = 1327) had anti-HSV IgG. During the follow-up , 293 cases of dementia were diagnosed, including 222 cases of AD, with an incidence rate of 20.6 cases of AD per 1000 person-years. The median time between the serology and diagnosis of AD was 4.3 years .Among the 1599 participants, 83% , the presence of IgG was significantly associated with an increased risk of AD (aHR = 2.72 [1.07–6.91] p = 0.04) and subjects with an IgG level in the highest tercile had an increased risk of AD compared to uninfected subjects (aHR = 3.87 [1.45–10.28] p = 0.007) or compared to infected subjects with an IgG level in the lowest tercile (aHR = 2.30 [1.08–4.91] p = 0.03). In APOE4 noncarriers, no significant association was observed.In non-stratified analyses Table , there wOur findings suggest an increased risk of AD in subjects infected by HSV. Infected subjects with an anti-HSV IgG level in the highest tercile (possibly reflecting more frequent reactivations over time) had lower HV compared to uninfected subjects. Infected subjects presented also more microstructural alterations of the parahippocampal cingulum and fornix regarding FA, MD, and RD. Our results are also in favor of an interaction between being infected with HSV and APOE4 status regarding advanced markers of the disease (HV and then incidence of AD). Among APOE4 carriers, infected subjects presented lower HVs, although not significant, and had a two times higher risk of developing AD and a three times higher risk if their anti-HSV IgG level was in the highest tercile. Among APOE4 noncarriers, no associations were observed between HSV status and HV or incidence of AD.p = 0.06 vs 0.04) and significant among the APOE4 noncarriers for FA (p = 0.048 vs 0.06).Our study has several strengths. The cohorts benefit from a population-based recruitment strategy allowing for the inclusion of participants regardless of their cognitive performance, unlike clinical studies. Nevertheless, some selection bias persists . As expected, the subjects with brain imaging data were younger, more educated, less often widowed, presented less co-morbidities such as hypertension and diabetes compared to the excluded subjects. It should also be noted that the frequency of APOE4 was slightly increased in these groups  presentAlthough white matter alterations can be seen in neurological pathologies other than AD, the fornix and cingulum bundles are connecting tracts of the limbic system that are thought to be altered prematurely in AD processes. Indeed, previous reports have found significant alterations in DTI parameters in the cingulum or fornix among AD patients, patients with mild cognitive impairment (MCI), or cognitively normal adults who developed amnestic MCI, and these alterations were associated with memory decline –5.Moreover, while the interpretation of DTI parameters remains debated , 38, altOur results highlight an association between the presence or the level of anti-HSV IgG and an increased risk of AD among APOE4 carriers while no significant association was found in the whole sample. Previous studies that have not stratified on APOE status report conflicting results regarding the association between the presence of anti-HSV IgG and cognitive decline or incidence of dementia (reported in ) while aTo note, the presence of anti-HSV IgG reflects an infection that occurred decades before serology, excluding a potential reverse causality bias. Regarding anti-HSV IgG levels , the risk of reverse causation also seems low. Nevertheless, for this marker, such bias cannot be fully excluded if early consequences of AD facilitate viral reactivations and consequently increase the anti-HSV IgG level. With this in mind, we performed a sensitivity analysis to assess whether the significant association found between a high anti-HSV IgG level and incidence of AD in APOE4 carriers could be explained by such problems. In the subsample of APOE4 carriers, after excluding subjects who developed an AD dementia within the subsequent 4 years, the associations remain broadly similar  genetic factors as, beyond APOE4, other genetic risk factors of AD are also linked to HSV , 20 or iWhile the underlying mechanism remains uncertain and may involve direct action of the virus and/or the immune response against it, our findings suggest an association between HSV-1 infection and markers of AD pathology. These results reinforce the need to further investigate the infectious hypothesis of AD, especially the associated susceptibility factors such as APOE4 and the possibility of preventive treatments.Supplementary informationSupplementary 1


Evidence is conflicting regarding differential health outcomes in racial and ethnic minorities with cystic fibrosis (CF), a rare genetic disease affecting approximately 28,000 Americans. We performed a cross-sectional analysis of health outcomes in Black/Latinx patients compared with non-Hispanic Caucasian patients cared for in a CF center in New York City. Adult patients enrolled in the CF Foundation Patient Registry at the Columbia University Adult CF Program and seen at least once during 2019 were included. Health metrics were compared between Black/Latinx and non-Hispanic Caucasian patients.p = 0.087). Age of diagnosis did not differ between groups; 9.56 (15.96) years versus 11.59 (15.8) years for non-Hispanic Caucasian versus Black/Latinx respectively (p = 0.464). Pulmonary function, measured as mean forced expiratory volume in one second (FEV1) was 70.6 (22.5) percent predicted in non-Hispanic Caucasian versus 59.50 (27.9) percent predicted in Black/Latinx patients (p = 0.010). Number of visits to the CF clinic were similar between groups. When controlled for age, gender, co-morbidities, median income, and insurance status, there was a continued association between minority status and lower FEV1.262 patients were eligible. 39 patients (15%) identified as Black/Latinx or non-Hispanic Caucasian. Descriptive statistics are reported with mean (standard deviation). Current age was 35.9 (13.3) years for non-Hispanic Caucasian and 32.0 (9.3) years for Black/Latinx patients (Minorities with CF have significantly lower pulmonary function, the major marker of survival, than non-Hispanic Caucasians, even when controlled for a variety of demographic and socioeconomic factors that are known to affect health status in CF. Significant health disparities based on race and ethnicity exist at a single CF center in New York City, despite apparent similarities in access to guideline based care at an accredited CF Center. This data confirms the importance of design of culturally appropriate preventative and management strategies to better understand how to direct interventions to this vulnerable population with a rare disease. Cystic fibrosis is an autosomal recessive disease affecting nearly 28,000 Americans and is the most common lethal genetic disease among white people , 2. The Although CF is predominantly a disease of Caucasians, the proportion of affected patients who are racial and ethnic minorities is increasing, following demographic trends in general in the US , 6. For While poorer health outcomes have been reported in individuals with CF and lower socioeconomic status (SES) , there iAs a CF center in New York City with a relatively large minority population, we sought to determine if racial and ethnic disparities in health outcomes exist and to further understand possible causes for any existing disparities.del mutations; F508del is the most common mutation in CF with approximately 45% of patients homozygous and 45% heterozygous [del is considered a severe mutation in CF and is associated with worse outcomes.The study was approved by the Columbia University Institutional Review Board. This is a retrospective cross-sectional analysis of adult patients enrolled in the Cystic Fibrosis Foundation (CFF) Patient Registry at the Columbia University Adult CF Program and seen at least once during 2019. 96% of patients followed at the Center have provided signed consent for inclusion in the Registry. Patients who underwent lung transplantation at any time prior to or during the year of analysis were excluded. The Registry collects basic demographic, diagnostic, clinical and outcome data for individuals each time they are seen in the outpatient or inpatient setting. Ethnicity is collected by self-report. Genetic mutations are classified by presence of one, two or no F508del mutation, presence of diabetes, presence of Pseudomonas aeruginosa in the lungs and age at diagnosis were compared between BLx and NHC patients. Mean FEV1 and BMI for the year were calculated as the average of the maximal value reported for each quarter. Pseudomonas infection refers to presence during the reported year. Other predictors of outcomes were identified based on social factors most likely to mediate or confound any relationship between race/ethnicity and health outcomes; specifically Medicaid insurance status and median household income estimated by zip code of residence. Markers of medical access such as number of outpatient health related encounters to the CF clinic, hospitalizations, or use of intravenous antibiotics for treatment of CF pulmonary exacerbations over the year were tabulated.The primary predictor of interest was race/ethnicity, categorized as Black/Latinx (BLx) or non-Hispanic Caucasian (NHC). Clinical and biological factors including age, body mass index (BMI), forced expiratory volume in one second (FEV1), F508del mutation). Patients’ gender, age, age at diagnosis, presence of diabetes, median household income based on zip code of residence, and insurance type (private/Medicare vs. public (Medicaid)) were also included in the model to adjust for potential confounding. We report the unadjusted and adjusted regression coefficient of minority status (β) as well as its corresponding 95% confidence interval and p value. We declare the association as statistically significant if p ≤ 0.05. Findings with p > 0.05 but ≤ 0.10 supported a trend toward statistically significant.Descriptive statistics were generated and compared by minority status for samples, characteristics, and outcomes of interest. We reported mean (standard deviation) and frequency (percentage) for continuous and categorical variables respectively. Comparison by minority status was conducted using t-test for continuous variables and Chi-squared test for categorical variables. Linear regression analysis was applied to examine whether minority status was associated with FEV1. The analysis was done on the whole sample and by genotype (presence of F508p = 0.087). Mean age of diagnosis did not differ between groups; 9.56 (15.9) years versus 11.59 (15.8) years, for NHC versus BLx respectively (p = 0.46). One hundred and sixteen (52%) of NHC patients were diagnosed before age 2 years compared with eighteen (46%) of BLx patients. BLx patients had a significantly lower median household income based on zip code of residence  compared with NHC , p < 0.001 (Table 262 eligible patients were enrolled in the Cystic Fibrosis Registry and had at least one visit during 2019. 39 patients (15%) identified as Black (n = 7) or Hispanic/Latinx. Mean current age was 35.9 (13.3) years for NHC and 32.0 (9.3) years for BLx patients with no group difference (p = 0.010). Mean BMI, total number of outpatient clinic encounters, care episodes (treatment with intravenous antibiotics), number of hospitalizations, and number of intravenous antibiotic days did not differ between groups (Table Mean FEV1 percent predicted was 59.50 (27.9) in BLx patients vs. 70.6 (22.5) in NHC, , p = 0.072).When adjusted for gender, age, age at diagnosis, presence of diabetes, median household income based on zip code of residence and insurance type, the association between minority status and lower FEV1 decreased and trended toward statistical significance , del status, with significantly lower FEV1 percent predicted in F508del heterozygous patients in the BLx/NHC group , p = 0.040). When adjusted for the potential confounding variables, the association for patients with 1 copy of F508del still trended toward significant , p = 0.068) but the results remained non-significant for the other two subgroups , p = 0.273; with 2 copies of F508 mutation: β = -0.67, 95% CI = , p = 0.937).Table Pseudomonas infection, two prognostic indicators in CF. Approximately half of patients were diagnosed before age 2 years; most of the reported cohort consists of patients diagnosed after universal implementation of newborn screening for CF. Access to medical care appears to be similar as measured by the number of out-patient visits to the specialized CF clinic, number of hospitalizations, and number of courses of intravenous antibiotics over the year.Disease severity in CF is a result of complex relationships among multiple factors along a patient’s lifespan. Our study finds that minorities with CF have significantly lower FEV1 percent predicted than non-minority patients, even when controlled for factors that are known to affect health outcomes in CF. Others have reported that lung function for Hispanic individuals with CF is lower starting at a very young age, though rate of decline in FEV1 is not different, suggesting that factors early in life may drive this trend . In our del homozygous  in BLx compared with NHC, similar to what has been reported previously [We did find that indicators of low socioeconomic status, including low median income by zip code and state insurance are significantly more prevalent in the BLx population than in the NHC population cared for at our clinic. Low SES is associated with worse disease outcomes for CF , 12. Wheeviously . Our stueviously –12. It aA limitation of our study is that cross sectional analysis does not take into account factors that are present early in life that may impact disease trajectory. We combined Black and Hispanic patients in the category of minority even though these are distinct populations. Also we were not able to analyze adherence to standard CF therapies which could influence lung function. Modulator use was similar between the two groups. Highly effective modulator therapy (HEMT) for the majority of patients with CF was approved in October 2019 and likely does not impact our findings since use did not begin for most patients until very late 2019. Eligibility for recently approved HEMT is similar in both groups in our study.Although there was no difference in number of visits to the CF clinic, there may be contributing individual factors such as adherence to treatment regimens, self-management culture, health literacy and English proficiency that affect outcomes. This datum confirms the importance of design of culturally appropriate preventative and management strategies to better understand how to direct interventions to this vulnerable CF population.


Pregnancy-associated breast cancer (PABC) is a rare disease with increasing incidence. The prognosis, pregnancy outcomes and subsequent ovarian function of PABC patients are attracting attention.Sixty-three PABC patients and 126 age-matched non-PABC patients were obtained in Tongji Hospital from January 2011 to September 2019. The clinical characteristics and ovarian function of PABC patients were compared with those of non-PABC patients. The pregnancy outcomes and neonatal outcomes of patients with breast cancer diagnosed during pregnancy (BCP) were described. Nonparametric tests, the χ2-test Kaplan–Meier, Cox regression and binomial logistic regression were used for analysis.p = 0.003), which caused worse progression-free survival (PFS) (log-rank p = 0.0138) and breast cancer-specific survival (CSS) (log-rank p = 0.0076) than non-PABC patients. Tumour stage (III/IV vs. 0/I/II)  and endocrine therapy  were predictors of PFS. Tumour stage (III/IV vs. 0/I/II) , endocrine therapy  and targeted therapy  were predictors for breast CSS. Among the 15 BCP patients, 11 patients voluntarily continued their pregnancy, and the newborns had no obvious birth defects, either in 5 patients who received chemotherapy or in 6 patients who did not receive chemotherapy during pregnancy. Among the patients who received chemotherapy and did not receive endocrine therapy, 24 PABC patients and 48 non-PABC patients experienced chemotherapy-induced amenorrhea. There was no significant difference in resumption of menstruation between the two groups at 6 months and 12 months after the end of chemotherapy. No potential factors affecting resumption of menstruation were found.PABC patients were diagnosed with a more advanced tumour stage (II: 47.6% vs. 45.2%, III: 33.3% vs. 19.8%, IV 3.2% vs. 0%, Pregnancy at diagnosis or within 1 year after delivery was not a risk factor for a worse prognosis in PABC patients. Compared with non-PABC patients, patients with PABC presented more aggressive tumour characteristics, which could mostly explain the worse prognosis observed in PABC patients. Receiving the appropriate regimen of chemotherapy in the second and third trimesters did not affect the maternal outcomes or neonatal outcomes of BCP patients. The special physiological state during pregnancy and lactation did not interfere with the damage of chemotherapy to ovarian function.The online version contains supplementary material available at 10.1186/s12885-022-09260-6. Pregnancy-associated breast cancer (PABC) is defined as breast cancer that is diagnosed during pregnancy and/or the postpartum period. The duration of the postpartum period, which ranges from 1 to 5 years, is controversial, but the most common is 1 year after delivery . PABC isMany studies have indicated that PABC patients differ from non-PABC patients in clinicopathologic characteristics. For example, patients diagnosed with PABC are younger, suffer a more advanced stage, have larger tumour sizes and have more axillary lymph node metastasis than non-PABC patients , 4. The Considering that more advanced stage, larger tumour sizes, more axillary lymph node metastasis, and TNBC and BRCA1/2 mutations were associated with poor prognosis , 17, it Considering the positive impact of timely antitumor treatments on the prognosis of patients with breast cancer diagnosed during pregnancy (BCP) and the possible negative impact on fetal health, the management of BCP patients faces many challenges to balance those conflicts . Some stIt is widely known that chemotherapy is an important means of breast cancer treatment. A prominent side effect of chemotherapy is damage to ovarian function, leading to premature ovarian failure (POF) . Most stTherefore, the clinical characteristics of Chinese PABC patients and the impact of related treatments on pregnancy outcomes and ovarian function need to be further explored. Here, we report the clinical characteristics of 63 PABC patients and the pregnancy outcomes of 11 BCP patients and compare the resumption of menstruation of 24 PABC and 48 non-PABC patients who had chemotherapy-induced amenorrhea to verify the factors that impact the ovarian function of PABC patients.This retrospective study screened women aged 20 to 45 years old diagnosed with primary breast cancer between January 2011 and September 2019 in Tongji Hospital. In this study, the criteria were as follows: (1) newly treated cases in Tongji Hospital; (2) patients who had undergone surgery or pathological staging; (3) complete medical record information; and (4) follow-up duration after chemotherapy of more than 1 year.Grouping criteria: ‘PABC’ was defined as breast cancer diagnosed during pregnancy or 1 year postpartum. ‘non-PABC’ is defined as breast cancer not diagnosed during that time. The non-PABC patients were matched with PABC patients with similar ages and times (less than 1 year) at diagnosis.Clinical and demographic data were gathered from the patients’ medical records, including patient charts, operative and pathology reports, chemotherapy records, radiotherapy, endocrine therapy records and targeted therapy records, because BCP patients should include caesarean section operation records or delivery records. Data collection included the age at diagnosis, TNM stage, birth weight and Apgar score of newborns, date and type of surgery, radiotherapy, endocrine therapy, targeted therapy, and chemotherapy regimen and cycle numbers. Follow-up data on the date of recurrence and death, date of amenorrhea and resumption of menstruation were completed by telephone interview or multiple letters.Follow-up examinations were performed every 3 months for the first two years, then every 6 months for the following 3 years, and annually thereafter. The follow-up data were documented by the clinical research office. Definitions of disease recurrence included i) local if recurrence was ipsilateral; ii) regional if ipsilateral axillary recurrence; iii) distinct if metastasis to bone, live, brain, long or peritoneum, and for contralateral axillary recurrence. Progression-free survival was defined as the interval from the date of diagnosis to the date of recurrence or the last follow-up. Breast cancer-specific survival was defined as the time from diagnosis to death or the last date of follow-up. All deaths in our included cases were due to breast cancer recurrence or progression. Amenorrhea was defined as regular menstruation stopping more than 2 menstrual periods, which occurred during or after chemotherapy. Menstruation recovery was defined as two consecutive menstrual periods within 21–35 days.P < 0.2 in univariate analysis were included in multivariate analysis. Differences were considered significant at P < 0.05.Statistical Package for the Social Sciences (SPSS) 25.0 statistical software was used for statistical analysis. The distributions of clinicopathologic factors were evaluated using nonparametric tests or the χ2 test as appropriate. Progression-free survival and breast cancer-specific survival were estimated by the Kaplan–Meier model. A Cox proportional hazard regression model and binomial logistic regression model were used for multivariate analysis. Variables with This study was approved by the Ethics or Institutional Review Board of Tongji Hospital, Huazhong University of Science and Technology in Hubei Province (TJ-IRB20210306).In total, 63 women were diagnosed with PABC , and 126 non-PABC patients were included in our research after the age and year of diagnosis were matched with PABC patients at a ratio of 1:2.p = 0.822), the median age at diagnosis , the median age at menarche , the family history of breast cancer or ovarian cancer  or the childbearing history  in PABC patients and non-PABC patients. Patients with PABC were more likely to be diagnosed at an advanced tumor stage  due to a larger tumor size  and more axillary lymph node metastasis  than non-PABC patients. The states of ER/PR/HER-2 of patients with PABC were similar to those of patients with non-PABC, as were the proportions of molecular subtypes.The clinical and demographic data of the 63 PABC patients and 126 non-PABC patients are shown in Table p < 0.001), and fewer PABC patients received endocrine therapy. There was no significant difference in the percentage of patients who received conservative breast surgery , radiotherapy  or targeted therapy  between PABC patients and non-PABC patients.In terms of treatments that patients received, as shown in Table n = 16) and 12.7% (n = 16) in the PABC and non-PABC groups, respectively. The death rates were 20.6% (n = 13) and 7.9% (n = 10) in the PABC and non-PABC groups, respectively. As shown in Fig. p = 0.0138), as did the results of breast cancer-specific survival (log-rank p = 0.0076).The median follow-up was 33 months for PABC patients and 38 months for non-PABC patients. The median breast cancer-free survival was 32 months for the PABC group and 37.5 months for the non-PABC group. The recurrence rates were 25.4% n = 6 and 12.To explore whether pregnancy within 1 year of breast cancer diagnosis is a high-risk factor affecting the prognosis of patients, we performed univariate and multivariate Cox regression to adjust the confounding factors influencing the prognosis of patients with breast cancer Tables  and  4.Tp = 0.017), family history of breast cancer , more advanced TNM stage(III/IV vs. 0/I/II) , positive PR , positive Her-2 , larger Ki-67 , radiotherapy  and endocrine therapy , radiotherapy (HR 5.332.001) were significant factors that influenced progression-free survival, p = 0.001). Then, those factors were included in multivariate Cox analysis, and the results showed that a more advanced TNM stage (III/IV vs. 0/I/II)  was a predictor for poor breast cancer-free survival and patients receiving endocrine therapy  yielded better breast cancer-free survival outcomes , with family history of breast cancer or ovarian cancer , more advanced cancer stage (III/IV vs. 0/I/II) , positive ER , positive PR , positive HR , positive Her-2 , radiotherapy , endocrine therapy  and targeted therapy  were possible risk factors that influencing the outcomes of breast cancer-specific survival, as shown in Table p < 0.001) was a risk factor of breast cancer-specific survival, while endocrine therapy  and targeted therapy  were predictors for better breast cancer-specific survival outcomes  received 2 cycles of AC (DOX + CTX) during pregnancy, 1 (9.1%) received 1 cycle of AC (DOX + CTX) during pregnancy, 1 (9.1%) underwent radical mastectomy in the third trimester, and the remaining 5 (45.5%) patients did not receive any antitumour therapy during pregnancy. Among the 5 BCP patients who received chemotherapy during pregnancy, 2 (20.0%) patients started NACT in the second trimester, and 3 (60.0%) patients started NACT in the third trimester.As shown in Table As a retrospective study, the results of hormones that reflected ovarian function could not be obtained. The resumption of menstruation is a clinical evaluation indicator of recovery of ovulation, and it is also an indirect reflection of the endocrine function of the ovaries after chemotherapy-induced amenorrhea. Therefore, we used the resumption of menstruation to represent the recovery of ovarian function. Considering the impact of endocrine therapy and history of menstrual disorders or surgery on menstruation, we excluded patients who received endocrine therapy, oophorectomy, or had a history of polycystic ovary syndrome. As shown in the supplementary Fig. n = 16) and 75.0% (n = 36), respectively. The PABC patients’ and non-PABC patients’ rates of resumption of menstruation at 12 months after the end of chemotherapy were 95.8% (n = 23) and 83.3% (n = 40), respectively. There was no significant difference in the menstrual recovery rate between the two groups at 6 months and 12 months after ending chemotherapy (p > 0.05).As shown in Fig. To explore the influencing factors of resumption of menstruation, we performed a binomial logistic regression model to adjust the confounding factors influencing menstruation of patients with breast cancer.p > 0.05).As shown in Table Our study reported the clinical characteristics, current treatment statuses, prognostic factors and influence of treatment on pregnancy and ovarian function in Chinese patients with PABC. Compared with non-PABC patients, patients with PABC presented more aggressive tumour characteristics, including more advanced tumour stage and more axillary lymph node metastasis, which could mostly explain the worse prognosis observed in PABC patients. Receiving the appropriate regimen of chemotherapy in the second and third trimesters did not affect the maternal outcomes or neonatal outcomes of BCP patients. The special physiological state during pregnancy and lactation did not reduce the damage of chemotherapy to ovarian function.Several studies reported that compared with non-PABC patients, PABC patients showed lower ER/PR expression and higher Her-2 overexpression , 28. In p = 0.31), with 253 non-PBC patients [Multiple studies have attempted to answer the question of whether pregnancy within 1 year before diagnosis is a negative factor for the prognosis of young patients with breast cancer , 29. DueIn our study, 5 BCP patients underwent NACT in the second and third trimesters, all of which were AC regimens. Only one patient underwent radical mastectomy in the third trimester. No neonates of patients who received NACT and mastectomy were found to have deformities, children below gestational age, or foetal distress. Our results are consistent with published articles. Studies have shown that during early pregnancy, especially during organogenesis, the use of chemotherapy drugs is related to foetal malformations , 32, butp > 0.05) and 95.8% and 83.3% (p > 0.05), respectively. The similar ovarian function of PABC and non-PABC patients after chemotherapy indicated that although PABC patients’ ovaries were in the anovulatory state with no growing follicles during pregnancy or early lactation, the protective effect was limited or useless and was not enough to weaken the ovarian damage caused by chemotherapy. We further analysed the potential factors affecting the resumption of menstruation in patients with breast cancer after chemotherapy. Although previous studies suggested that age, patients’ desire to have children, and utilization of GnRH agonists during chemotherapy could improve ovarian function [To decrease the risk of recurrence, most patients with breast cancer receive adjuvant chemotherapy, which has been reported to result in temporary or permanent damage to ovarian function . Resumptfunction , 41, no There are some limitations to our study. First, this was a small retrospective study in a single centre. Our research provides the tumour characteristics and prognosis of Chinese patients with PABC, which is rarely mentioned in the existing literature. In the future, a large sample of Chinese PABC patients from multiple centres will be needed to verify the conclusions of our study. Second, it is partial to only use menstrual conditions to refer to ovarian function, and it should also include indicators such as AMH, AFC, FSH, E2 and so on. Since this study is a retrospective analysis, this part of the data is difficult to obtain. It was also due to this limitation that this study cannot evaluate the degree of damage or the recovery of ovarian function of PABC patients who had not recovered regular menstruation, and the latter was a more effective indicator for whether pregnancy or lactation could resist chemotherapy-induced ovarian damage. Third, the genotypes of the patients were not included in the survival analysis, mainly because some of the included cases were diagnosed at the time when genetic testing was not popular, thus lacking evidence of genotype. To obtain a credible result, a randomized controlled trial must be carried out in multiple centres.In conclusion, it is not pregnancy but the more aggressive tumour characteristics at diagnosis that cause the poorer prognosis of patients with PABC than that of non-PABC patients. Timely treatment during pregnancy is necessary because appropriate treatment at the right time during pregnancy will not negatively affect the foetus. The special ovulation state during pregnancy or after childbirth cannot effectively reduce the damage of chemotherapy to the ovaries.Additional file 1.


Background: The current paradigm is that fibrosis promotes electrophysiological disorders and drives atrial fibrillation (AF). In this current study, we investigated the relation between the degree of fibrosis in human atrial tissue samples of controls and patients in various stages of AF and the degree of electrophysiological abnormalities. Methods: The degree of fibrosis was measured in the atrial tissue and serum of patients in various stages of AF and the controls. Hereto, picrosirius and H&E staining were performed to quantify degree of total, endo-perimysial fibrosis, and cardiomyocyte diameter. Western blot quantified fibrosis markers: neural cell adhesion molecule, tissue inhibitor of metalloproteinase, lysyl oxidase, and α-smooth muscle actin. In serum, the ratio carboxyl-terminal telopeptide of collagen/matrix-metalloproteinase1 was determined. High-resolution epicardial mapping evaluated low-voltage areas and conduction abnormalities. Results: No significant differences were observed in the degree of fibrosis between the groups. Finally, no significant correlation—absolute nor spatial—was observed between all electrophysiological parameters and histological fibrosis markers. Conclusions: No differences in the degree of fibrosis were observed in patients from various stages of AF compared to the controls. Moreover, electrophysiological abnormalities did not correlate with any of the fibrosis markers. The findings indicate that fibrosis is not the hallmark of structural remodeling in AF. Atrial fibrillation (AF) is the most common progressive tachyarrhythmia in the Western world that is associated with the presence of an extensive arrhythmogenic substrate , which iFibrosis is a common pathological feature described as excessive deposition of ECM proteins (mainly collagen) in the cardiac interstitial space that can be observed during aging of the heart, and almost all forms of heart diseases ,7. DurinImportantly, distribution of interstitial fibrosis seems to influence the cardiomyocyte function differently. Excessive deposition of ECM separating individual cardiomyocytes, defined as endomysial fibrosis, results in altered electrical impulse propagation . AdditioClinical evaluation of fibrosis in AF patients still faces difficulties. Although cardiac magnetic resonance imaging is often used to assess fibrosis non-invasively, there is still a lack of consensus on the utilized protocols . In addiThe aim of this study was to evaluate whether fibrosis represents a key hallmark of electropathology in patients with AF. Hereto, we investigated the association between the degree of fibrosis with (1) AF stage in atrial appendage tissue and serum samples of patients with paroxysmal (ParAF), persistent (PerAF) and long-standing persistent AF (LSPerAF), and (2) the amount of electrical conduction impairment and low-voltage areas. The findings of the current study elucidate that the degree of fibrosis was comparable in the right and left atrial appendages  of patients with ParAF, PerAF, or LSPerAF and controls in sinus rhythm (SR) without history of AF. Furthermore, in our study, the degree of fibrosis did not correlate with the amount of electrical conduction impairment nor with the presence of atrial low-voltage areas. This knowledge indicates that fibrosis is not the unique root cause of electropathology and AF promotion.The study population consisted of a group of 115 patients with or without a history of AF, subdivided in ParAF (N = 21), PerAF (N = 28), LSPerAF (N = 17), and a control group without history of AF (N = 49) undergoing cardiac surgery due to coronary artery disease and/or valvular heart disease or correction of congenital heart defect. All patients were enrolled from the HALT & REVERSE trial MEC-2014-393) [14-393 [2g for 10 min at 4 °C and subsequently frozen in −80 °C until analysis. RAA was obtained from all patients (both with and without AF) from the incision used for extracorporeal circulation cannulation. Furthermore, amputation of the LAA was performed after cardioplegia in a selection of patients. Both RAA and LAA were immediately frozen in liquid nitrogen and stored at −80 °C. Due to limited tissue availability, not all patient samples could be utilized to analyze the various fibrosis endpoints.Before the procedure, blood samples were collected in BD Vacutainer™ SST™ II Advance Tubes , and its serum obtained by centrifugation at 2000× Epicardial high-resolution mapping was performed during open-heart surgery prior to commencement of extracorporeal circulation, as previously described in detail ,22,23,24Part of the frozen RAA was cut into small fragments on dry ice and mixed with ice cold sample buffer . The tissue sample was homogenized using metal beads in the Qiagen Tissuelyser II for 3 min at 30 Hz, left on ice for 30 min for continued cell lysis and homogenized again for 3 min at 30 Hz. The lysates were centrifuged , the supernatant was collected, passed through an insulin syringe, heated for 5 min at 95 °C, and samples were stored at −20 °C until analysis.For western blot analysis, equal amounts (10 µg) of protein were separated on 4–20% Criterion TGX precast gels  and transferred to nitrocellulose membranes (Bio-Rad). Membranes were blocked in 5% skim milk in TBST for 1 h at room temperature. Membranes were incubated overnight at 4 °C with the following primary antibodies in 3% BSA in TBST: anti-LOX , anti-NCAM , anti-TIMP-1 , and anti-αSMA . Subsequently, these were incubated with secondary antibody (in 3% BSA in TBST) for 1 h at room temperature with horseradish peroxidase-conjugated goat-anti-rabbit or goat-anti-mouse antibodies  depending on the species origin of the primary antibody. Signals were detected by the Amersham ECL prime western blotting detection reagent  utilizing the Amersham Imager 600  and quantified by densitometry . Western blot analysis was performed at least in duplicate. Protein amounts were expressed relative to GAPDH for whole protein lysates.For histochemical analysis, cryosections (5 µm) were obtained from frozen LAA and RAA samples, then stained with picrosirius red or hematoxylin and eosin. To determine the amount of fibrosis, cryosections were dried for 30 min, fixed with 4% paraformaldehyde in 0.1 M PBS for 5 min, stained in 0.1% Sirius red for 60 min, differentiated in 0.01 N HCl, immersed in seriate dehydration solutions , then cleared in xylene. For the determination of cardiomyocyte size and endo-perimysial distance, cryosections were dried for 30 min, stained in hematoxylin for 3 min, washed 3× in water, stained in eosin for 3 min, immersed in a series of washes , then quickly immersed in xylene. All cryosections were mounted with non-aqueous DPX medium. Photographs were taken at 10× magnification to recognize the epicardium and endocardium; subsequently, six pictures at magnification 40× were taken from each patient . Cardiomyocyte diameter, endomysial distance—space in between individual cardiomyocytes—and perimysial distance—space in between in between bundle bundles of cardiomyocytes—were analyzed and quantified by histomorphometry Image J 1.48 software .MMP1 and CITP levels in serum samples were determined by ELISA measurements. For both parameters, serum was diluted 6× in 1% BSA in PBS. Levels were measured in triplicate using ELISA kits for MMP1 and CITP  according to the manufacturer’s instructions.Color-coded local activation maps were constructed using dedicated mapping software, AnnotationTool, based on annotations of the steepest negative deflection if the amplitude exceeded the noise level in the channel with a probability of 99.95%, assuming a minimum threshold of 0.05 mV/ms. Atrial extra systolic, aberrant, and ventricular beats were excluded. The RAA mapping site was subdivided in two halves: inter-caval and free wall. The free wall half of the mapping was used to analyze patterns of activation at the epicardial area closest to the tissue sample collection site. The LAA mapping site was uniformized to the distal 16 rows.Consistent with previous mapping studies , differet test, Mann–Whitney test, and Chi-square test; and Yate’s correction was applied for continuous variables. Correlation was performed with the Spearman correlation test. To compare continuous variables with a skewed distribution, the Mann–Whitney U test was applied. Values of p were two-sided and considered significant if <0.05. SPSS version 20 , R Statistical Software  and GraphPad Prism version 8.0  were used for all statistical evaluations.Results are expressed as the mean  ±  standard error of the mean or median (min-max). Biochemical analyses were performed at least in duplicate. All data were tested for Gaussian distribution. Individual group mean differences were evaluated with the two-tailed Student’s The study population consisted of 115 patients  including a control group of 48 (41.7%) patients without AF and a study group of 67 patients with either ParAF , PerAF , and LSPerAF . p < 0.001). From 48 patients without a history of AF, 24 developed post-operative AF. No significant difference in the degree of fibrosis were observed between patients with and without post-operative AF , hypertension, dyslipidemia, diabetes mellitus, thyroid disease, and left ventricular function were similar among the control and AF groups. AF patients more often use statins, digoxin, and anti-arrhythmic drug class III see . The typative AF .p = 0.77), as evident in p = 0.75), as seen in p = 0.12). Furthermore, the level of cardiomyocyte hypertrophy was determined by measuring the diameter of atrial cardiomyocytes. Additionally, for this parameter, the diameter of atrial cardiomyocytes was comparable among ParAF (N = 9), PerAF (N = 12), LSPerAF (N = 8), and the control group (N = 12) , as depicted in Hematoxylin and eosin (H&E) staining was performed in order to assess endomysial and perimysial fibrosis levels and cardiomyocyte size. No significant difference was present regarding endomysial distance in patients with ParAF (N = 8), PerAF (N = 8), LSPerAF (N = 5), and the control group (N = 8) , as shown in p = 0.41), as seen in αSMA is a marker for transdifferentiated cardiac myofibroblasts  and TIMPp = 0.83; and LOX levels in ParAF:0.81 ± 0.36, PerAF: 1.22 ± 0.64, LSPerAF: 1.14 ± 1.04, and SR: 0.81 ± 0.28; p = 0.11), as illustrated in By using western blot analyses, the levels of NCAM, a marker of satellite cells and (hepatic) fibrosis  and lysyp = 0.07), as illustrated in Given that metalloproteinase 1 (MMP1)  degradesGenerally, fibrosis is a culprit factor for impaired cardiac conduction as well as for areas characterized by low potential voltages . Therefop > 0.05) were found between the histological fibrosis markers and any of the electrophysiological parameters measured during SR  in human atrial tissue. By using this innovative approach, we provide final evidence that fibrosis is not underlying electrophysiological impairment in RAA and LAA. Further research is warranted to confirm the absence of a culprit role for fibrosis in electropathology in other atrial locations.To date, areas with low-voltage have been recognized as surrogate markers for fibrosis and indicators of the arrhythmogenic substrate, thereby commonly guiding electrophysiologists on decision making for the identification of suitable target sites for ablation . As the Together, as our findings indicate that fibrosis is not the unique culprit factor driving electropathology and AF, we advocate for exploring new endpoints for structural remodeling.All patients in this study underwent cardiothoracic surgery for underlying heart disease. Together with aging , this fa


Phlebitis, inflammation of tunica intima of venous wall, occurred in 13–56% of hospitalized patients. It is characterized by pain, erythema, swelling, palpable venous cord, and pussy discharge at catheter site. Cannula-related blood stream infection (CRBSI) is recognized complication of phlebitis. Adverse outcomes of phlebitis embrace patient discomfort, longer hospital stay and higher health care cost. This study aimed to determine the incidence and associated factors of peripheral vein phlebitis among hospitalized patients.A hospital-based prospective, observational study was conducted between April 1 and August 31, 2020 at University of Gondar hospital, Northwest Ethiopia. A consecutive sampling method was used to recruit 384 patients. Patients were interviewed to obtain socio-demographic data. Relevant medical history and laboratory parameters were obtained from patients’ records. Presence and severity of phlebitis was identified by Jackson’s Visual Infusion Phlebitis (VIP) Scoring System. The Data were entered into EPI Info version 4.4.1 and transported to SPSS version 20 for analysis. Logistic regression analysis was used to identify associated factors with occurrence of phlebitis. P-value < 0.05 was used to declare significant association.A total of 384 study subjects were included in the study. The mean age of study subjects was 46 years, with a range of 19 to 96 years. The incidence of phlebitis was 70% among study subjects. Mid-stage (grade 3) and advanced-stage (grade 4) phlebitis were noticed in 136/268 (51%) and 89/268 (33%) respectively. Odds of developing phlebitis were twofold higher in patients with catheter-in situ > 96 h  as compared to those with catheter dwell time < 72 h. Female patients were 70%  lower than male patients with risk of developing phlebitis. Patients who use infusates were 53%  less likely to develop phlebitis as compared to those who didn’t use infusates.The cannula must be reviewed on daily basis, and it should be removed if it stayed later than 96 h. Irritant intravenous drugs and hyperosmolar infusate solutions cause vascular endothelial injury, and results in phlebitis. Biological factors embrace bacterial colonization, biofilm formation and infection. Patient-related factors take account of age, gender, nutritional status, immunosuppression and co-existing comorbidities. Those with malnutrition, immunosuppression, co-morbidities, and elderly (age > 65 years) are vulnerable to phlebitis. Implementing aseptic precautions and health professional skill on catheter securement are the frequently implicated health practice-related factors. Poor aseptic technique and improperly securing of cannula are among listed causes of phlebitis.Peripheral intra venous catheter (PIVC) insertion is the most frequently performed procedure in hospital settings. Approximately 33–67% of hospitalized patients require at least one peripheral vein insertion. Peripheral vein catheters are required for administration of intravenous drugs, infusate solutions, blood products and parenteral feeding. It is as well necessary for access to vascular procedures –13. DespThis study was the first of its kind in Ethiopia to determine the incidence and associated factors of peripheral vein phlebitis among adult hospitalized patients.A hospital-based prospective, observational study was conducted between April 1 and August 31, 2020 at emergency unit, medical and surgical wards of University of Gondar hospital. Both wards had a capacity of 92 beds. The hospital is located in Northwest Ethiopia, which is 750 km away from the capital, Addis Ababa. The hospital had a catchment population of 5 million people.All patients admitted to emergency unit, medical and surgical wards during the study period were considered as study population. Patients older than 18 years old, who were admitted to emergency unit, medical and surgical wards and who were on peripheral intravenous catheter, were included in the study. Patients with history of allergy to intravenous cannula, or those unable to give consent were excluded from the study.Dependent variables: Peripheral intravenous catheter (PIVC) phlebitis.Independent variables: (1) Socio-demographic characteristics include age, gender, occupation, marital status, educational level, income level, residence and religion (2) Clinical characteristics include admission diagnosis, duration of hospital stay, cannula insertion site, catheter size (gauge), catheter dwell time, intravenous drugs use, infusates use, blood products use, performance level, and co-existing comorbidities.The sample size was calculated using single population proportion formula with the assumption of 95% confidence level, 5% margin of error, and taking 50% estimated proportion of phlebitis. Consecutive sampling method was used to recruit 384 study subjects.Data were collected through an investigator administered pre-designed questionnaire. Patients were interviewed to obtain socio-demographic data. Relevant medical history and laboratory parameters were obtained from patients’ records. Three investigators  participated in the data collection process and follow-up of patients on indwelling catheter. Insertion of a catheter was performed by clinical nurses. Hygienic hand-rub with 70% alcohol was done after hand washing. Patient’s skin was disinfected using 1% iodine solution and 70% alcohol. Using clean gloves, a catheter was inserted into a peripheral vein of a patient. The catheter was fixed to the patient’s skin with protective dressing and plaster. Patients on indwelling catheter were followed daily by investigators from insertion to developing phlebitis or catheter removal. Those who developed phlebitis were graded based on Jackson’s Visual Infusion Phlebitis (VIP) Scoring System  and wereData were entered into EPI Info version 4.4.1 and transported to SPSS version 20 for analysis.Patient characteristics were reported as counts (percentages) for categorical variables, and mean with standard deviation for continuous variables. Bi-variable and multi-variable logistic regression models were constructed to identify independently associated factors with occurrence of phlebitis. Those variables with a P-value < 0.25 in the bi-variable analysis were exported to multi-variable analysis to control the possible effect of confounders. Crude odds ratio (COR) and adjusted odds ratio (AOR) were reported. P-value < 0.05 was used to declare significant association.The research protocol complied with Declaration of Helsinki, and was approved by local ethics committee . Study subjects were recruited only after informed written consent was obtained. All data obtained were treated confidentially.Occurrence of at least one inflammatory sign or symptom related to the catheter insertion, comprising persistent pain (duration longer than 2 h after end of infusion) and/or erythema and/or swelling (induration) and/or palpable cord and/or pussy discharge at intravenous site and/or pyrexia .Grade 1 phlebitis: one of the following is evident: Pain near IV site or erythema near IV site; Grade 2 phlebitis (early stage of phlebitis): Two of the following are evident: Pain at IV site, erythema or swelling; Grade 3 phlebitis (mid stage of phlebitis): All of the following signs are evident: Pain along path of cannula, erythema and induration; Grade 4 phlebitis (advanced stage of phlebitis or early stage of thrombophlebitis): All of the following signs are evident and extensive: Pain along path of cannula, erythema, induration and palpable venous cord; Grade 5 phlebitis (Advanced stage of thrombophlebitis): All of the following signs are evident and extensive: Pain along path of cannula, erythema, induration, palpable venous cord, and visible pussy discharge at IV site or pyrexia .A total of 384 participants were included in the study. The mean age of study subjects was 46 years, with a range of 19 to 96 years. More than half (52%) of them were males and were rural dwellers (53%). Majority (89%) of respondents were Christian by religion. Fewer than half (43%) of study subjects attended formal education (Table Two-thirds 63%) of the patients were semi-ambulatory. Majority of patients were hospitalized with lung 25%), heart (18%) and CNS (17%) diseases. Two-third (63%) of patients presumed to have bacterial infections. Fewer than 10% of patients each were diagnosed to have diabetes, CKD, HIV/AIDS, and malignancy as co-morbidities. Indications for PIVC insertion were administration of intravenous drugs (73%), infusates (65%), and blood products (14%). Half (53%) of the catheters were inserted in emergency situations. Forearm was used as catheter placement in half (52%) of patients. 20 G sized cannula was used in most (81%) patients. Two-third (66%) of patients had PIVC in-situ for 96 h or less  among study subjects. Among those who developed phlebitis, mid-stage (grade 3) and advanced-stage (grade 4) phlebitis were noticed in 136/268 (51%) and 89/268 (33%) respectively. Advanced stage thrombophlebitis (grade 5) occurred in 4/268 (1.5%) of phlebitis cases  as compared to those with catheter dwell time < 72 h. Female patients were 70%  lower than male patients with risk of developing phlebitis. Patients who used infusates were 53%  less likely to develop phlebitis as compared to those who didn’t use infusates , use of blood products, concomitant bacterial infections, and co-existing comorbidities such as diabetes, CKD and malignancy were not found to be significantly associated with occurrence of phlebitis in bi-variable analysis .The incidence of phlebitis was 70% with use of peripheral intra venous catheters. It was higher than phlebitis rate reported in other studies, which ranged from 13–56% , 16. TheSelection bias couldn’t be avoided as consecutive sampling method was used to recruit study subjects.The incidence of phlebitis was considerably high. High grade phlebitis (grade 3 and 4) occurred in the majority (84%) of patients with phlebitis. Catheter dwell time > 96 h was found to be a risk factor for increased incidence of phlebitis.The cannula must be reviewed on daily basis, and it should be removed if it stayed later than 96 h. Phlebitis protective measures and catheter management strategy should be improved at the study site.