SMAD3 (pancreatitis); CD274, SLCO1B1 (hepatitis); PRDM1, CD274 ; PRDM1, CD274, TSHR, FAN1 (myositis). Myositis and encephalitis, both, were associated with alterations of PRDM1 and CD274, which might explain their joined appearance in clinical practice. Our findings can help to assess the risk for the development of IRAE in melanoma patients with ICI.Our aim was to check for possible associations between clinical parameters or NGS-based genetic alterations and the occurrence of immune-related adverse events (IRAE) in melanoma patients with immune checkpoint inhibitors (ICI). We analyzed 95 melanoma patients with ICI and were able to identify several biomarkers associated with the development of IRAE. Female sex was significantly associated with the development of hepatitis, increased total and relative monocytes at ICI initiation were significantly associated with the development of pancreatitis, the same, pre-existing autoimmune diseases. Furthermore, the following genetic alterations were identified being associated with IRAE: AIRE, TERT, SH2B3, LRRK2, IKZF1, SMAD3, JAK2, PRDM1, CTLA4, TSHR, FAN1, SLCO1B1, PDCD1, IL1RN, CD274, UNG. We obtained relevant results: female sex was significantly associated with the development of hepatitis, combined immunotherapy with colitis, increased total and relative monocytes at therapy initiation were significantly associated with the development of pancreatitis, the same, pre-existing autoimmune diseases. Further significant associations were as follows: HLA homozygosity (hepatitis), and VARs on SMAD3 (pancreatitis). Regarding CNVs, significant markers included PRDM1 deletions and IL1RN (IRAE), CD274 duplications and SLCO1B1 (hepatitis), PRDM1 and CD274 , and PRDM1, CD274, TSHR, and FAN1 (myositis). Myositis and encephalitis, both, were associated with alterations of PRDM1 and CD274, which might explain their joined appearance in clinical practice. The association between HLA homozygosity and IRAE was clarified by finding HLA-A homozygosity as determining factor. We identified several genetic alterations hypothesized in the literature to be associated with the development of IRAE and found significant results concerning pre-existing autoimmune diseases and specific blood count parameters. Our findings can help to better understand the development of IRAE in melanoma patients. NGS might be a useful screening tool, however, our findings have yet to be confirmed in larger studies.Immune checkpoint inhibitors (ICI) have revolutionized the therapeutic landscape of metastatic melanoma. However, ICI are often associated with immune-related adverse events (IRAE) such as colitis, hepatitis, pancreatitis, hypophysitis, pneumonitis, thyroiditis, exanthema, nephritis, myositis, encephalitis, or myocarditis. Biomarkers associated with the occurrence of IRAE would be desirable. In the literature, there is only little data available and furthermore mostly speculative, especially in view of genetic alterations. Our major aim was to check for possible associations between NGS-based genetic alterations and IRAE. We therefore analyzed 95 melanoma patients with ICI and evaluated their NGS results. We checked the data in view of potential associations between copy number variations (CNVs), small variations (VARs), human leucocyte antigen (HLA), sex, blood count parameters, pre-existing autoimmune diseases and the occurrence of IRAE. We conducted a literature research on genetic alterations hypothesized to be associated with the occurrence of IRAE. In total, we identified 39 genes that have been discussed as hypothetical biomarkers. We compared the list of these 39 genes with the tumor panel that our patients had received and focused our study on those 16 genes that were also included in the tumor panel used for NGS. Therefore, we focused our analyses on the following genes:  In the past decade, immune checkpoint inhibitors (ICI) have revolutionized the therapeutic landscape of metastatic melanoma and significantly improved prognosis of metastatic melanoma patients . ICI actFurthermore, the knowledge of biomarkers of distinct IRAE might help in the differential diagnosis, for example by distinguishing IRAE from laboratory abnormalities of other cause. Indeed, symptoms of IRAE might be unspecific in some cases, on the other side, early diagnosis is essential and may be life-saving .TSHR, shown to affect the development of central tolerance and associated with autoimmune diseases [PRDM1 found to influence antigen presentation [A few biomarkers have already been identified, such as female sex , and prediseases ,12, or pentation  and assoentation . Some auentation ,16. FinaIn the following study, we sought to determine whether there are biomarkers associated with the occurrence of IRAE under ICI in melanoma patients. For this purpose, we evaluated NGS results and clinical data in view of the occurrence of IRAE in melanoma patients. We searched for the occurrence of IRAE in general as well as for specific IRAE and focused on germline genetic alterations known to play a central role in the regulation of the immune system as we hypothesized their influence also on the occurrence of IRAE.pubmed.ncbi.nlm.nih.gov, search terms “immunogenetics ipilimumab” and “genetics adverse events PD-1” on 27 January 2020), we found 39 genes that have been discussed in the literature. We compared the list of these 39 genes with the two different tumor panels that our patients had received for NGS by CeGat GmbH , IL1RN, CD274 (PD-L1), UNG. Except for AIRE, both panels included all the genes mentioned, and AIRE was removed from the analysis due to lack of data. Therefore, we focused our analyses on these 16 genes. Our objective was also to check for possible associations between patient specific parameters such as sex, blood count, pre-existing autoimmune diseases and the occurrence of IRAE.We analyzed 95 melanoma patients that had been enrolled in a prospective study on the value of liquid biopsy and tumor sequencing between January 2018 and June 2018 who subsequently received immune checkpoint inhibitor (ICI) therapy. Details concerning tumor panel analysis and bioinformatics have already been reported . The aimGmbH see , includiThe patients either received combined immunotherapy or anti-PD-1 monotherapy. If they received both therapies in sequence, for example first anti-PD-1 monotherapy and later combined ICI, we focused on the data of the combined immunotherapy, as IRAE are more likely to occur here. Subsequently, we documented the time point of occurrence of IRAE, as well as the most common affected organs, in particular: colitis, pneumonitis, hepatitis, encephalitis, myocarditis, myositis, pancreatitis, exanthema, hypophysitis, nephritis, and thyroiditis. Regarding IRAE, we documented the date of first occurrence and the highest grade according to the Common Terminology Criteria of Adverse Events (CTCAE) scale from 1 to 5 , date ofn = 95 subjects). Our results support the markers previously suggested in the literature but need to be confirmed and further specified in future more comprehensive studies as correction for multiple testing was not feasible due to lack of power.We then documented potential biomarkers for the occurrence of IRAE such as sex, type of immunotherapy, pre-existing autoimmune diseases, and blood count parameter at start of immunotherapy. Continuous variables, in particular blood count parameter, were divided into categories for better assessability , compared to only 7.4% of male patients, whereas 18.5% of male patients developed pancreatitis, opposed to only 7.3% of female patients.A total of 24.4% of female patients developed hepatitis (p = 0.004) developed colitis. 18.6% of patients with combined therapy had hepatitis, compared to 8.3% of patients with monotherapy. Finally, 16.9% of patients with combined therapy had pancreatitis, compared to 8.3% of patients with monotherapy.Combined immunotherapy led to more IRAE than monotherapy. 67.8% of patients with combined therapy developed IRAE, compared to 52.8% of patients with monotherapy. 30.5% of patients with combined therapy compared to 5.6% with monotherapy (Elevated levels of protein S100 or lactate dehydrogenase (LDH) at therapy initiation were not associated with IRAE.p < 0.0005), whereas increased relative monocytes were only associated with colitis or pancreatitis (p = 0.001). The single patient having had high levels of eosinophils at start of immunotherapy developed colitis, pancreatitis and nephritis during immunotherapy. In the differential blood cell count at therapy initiation, increased leucocytes were associated with colitis, hepatitis or pancreatitis. Increased total neutrophils were associated with colitis or pancreatitis. Decreased relative lymphocytes were associated with pancreatitis. Increased total and relative monocytes were also associated with IRAE. Increased total monocytes were associated with colitis, hepatitis, or pancreatitis .The occurrence of colitis was associated with rheumatoid arthritis (50%), Crohn’s disease (100%), or heparin-induced thrombopenia type II . The development of hepatitis was associated with diabetes mellitus Type 1 (50%), vitiligo (100%), or HIT-II (100%). The development of pancreatitis was associated with rheumatoid arthritis (50%), vitiligo (100%), or HIT-II (100%). Pancreatitis was linked significantly to pre-existing autoimmune diseases . HLA-B homozygosity is seemingly a protective factor in general IRAE, colitis or pancreatitis. A positive correlation between IRAE and homozygosity was found for HLA-A and HLA-C homozygosity for colitis, and HLA-A, HLA-B, and HLA-C homozygosity for hepatitis. HLA homozygosity was significantly linked to the development of hepatitis . This result must be read and interpreted with caution due to the exploratory approach. VARs on IKZF1, FAN1 and IL1RN were linked to IRAE in general, as well as all three main IRAE in particular. Deletions were found to be more frequently associated with IRAE than duplications. CNVs on IL1RN (p = 0.033) and deletions on PRDM1 (p = 0.026) were significantly linked to IRAE. Duplications on CD274 (p = 0.043) and CNVs on SLCO1B1 (p = 0.010) were significantly linked to hepatitis. These results must be interpreted with caution due to the exploratory approach. We distinguished deletions and duplications for each CNV for IRAE in general, as well as colitis, hepatitis and pancreatitis  and myositis (p = 0.014 and p = 0.032). CNVs on TSHR and FAN1 were significantly linked to myositis (p = 0.049 and p = 0.039). These results must be interpreted with caution due to the exploratory approach. Since a distinction between duplications and deletions would have made the sample size too small for statistical statements for less common specific IRAE, this has been omitted here  to be associated with higher survival rates compared to patients homozygous on at least one HLA-I locus . InferioIt seems imperative to differentiate between HLA-A, HLA-B, and HLA-C homozygosity as they seem to have opposite effects on treatment response and IRAE occurrence.SMAD3 were significantly linked to the development of pancreatitis: 33% of patients with SMAD3 variants suffered from pancreatitis while the incidence of pancreatitis in the complete cohort was only 14%. To our best knowledge, SMAD3 has not yet been linked to IRAE. Of note, SMAD3 is known as a key molecule in TGF-β signaling pathway and studies have shown the inhibitory effect of SMAD3 and the TGF-β pathway on natural killer cells in the tumor microenvironment: disruption of SMAD3 in natural killer cells was associated with enhanced activity of natural killer cells and cytokine production [SMAD3 plays an immunosuppressive role, as shown by Wang et al., an affection of SMAD3 by VARs could lead to a dysfunction potentially explaining an IRAE. We observed a correlation between SMAD3 affected by VARs and partial response at first staging, which confirms findings about the association between response and occurrence of IRAE [In our study, we found out that VARs on oduction . If SMAD of IRAE .IL1RN. The same, a higher incidence of toxicity under ICI was found being associated with alterations of CD274.Concerning the CNVs, it is interesting, that higher numbers of CNVs in melanoma tissue have been linked to a higher incidence of metastases, recurrence and death . In the IL1RN were significantly linked to IRAE in general and associated with all three main IRAE in particular, i.e., colitis, hepatitis and pancreatitis. If the role of CNVs on IL1RN concerning the occurrence of IRAE during immunotherapy is not yet fully understood, IL1RN is known for its role in the development of autoimmune diseases. Yoshizaki et al. showed an increased risk for the development of autoimmune aortitis in IL1RN-deficient mice, which they explained by stronger signaling of IL-1 [IL1RN have been found to be associated with the occurrence of Hashimoto’s thyroiditis and to predict severity [CNVs on  of IL-1 . Polymorseverity .CD274 (PD-L1) were a recurrent marker for IRAE, being significantly linked to hepatitis, encephalitis and myositis. Once again, the role of CNVs on CD274 in view of the occurrence of IRAE during immunotherapy has not yet been fully understood. Polymorphisms on CD274 are however known to be associated with multiple autoimmune diseases, such as type 1 diabetes [CNVs on diabetes , ankylosdiabetes , and Gradiabetes .PRDM1 have been associated with systemic lupus erythematodes (SLE) [PRDM1 associated with SLE, decreasing the expression of BLIMP1 (B-lymphocyte-induced maturation proteine-1) in dendritic cells, which is involved in immunological tasks such as antigen presentation [PRDM1 have also been associated with other autoimmune diseases such as rheumatoid arthritis and inflammatory bowel disease [PRDM1 in preventing autoimmunity has been analyzed in mice by Roberts et al. [PRDM1 is expressed by medullary thymic epithelial cells, involved in the deletion of self-reactive T cells and the development of regulatory T cells. Deletion of PRDM1 resulted in autoimmune pathology [PRDM1 resulted in IRAE. Xia et al. revealed that homozygous deletions of PRDM1 correlated with decreased plasma cell differentiation and upregulation of genes involved in tumor cell proliferation in activated B-cell-like diffuse large B-cell lymphoma (ABC-DLBCL) patients [PRDM1 deletions were showing upregulation of transcription factors such as STAT3. STAT3 has been identified as main factor in increased PD-L1 expression on tumor cells [PRDM1 deletions may thus be regarded as factor supporting Anti-PD-1 therapy, and thereby inducing IRAE. Of note, the association of CNVs on PRDM1 to encephalitis and myositis has not been reported in the literature yet. It would certainly be interesting if this could be tested in larger cohorts.Polymorphisms on es (SLE) . More prentation . Polymor disease . The rols et al. . The autathology . Our respatients . Xia et or cells , which iTSHR in enhancer regions, especially rs4411444 and rs4903961, are known to be associated with autoimmune diseases such as Graves’ disease and Hashimoto’s disease [TSHR have been shown to affect the development of central tolerance, thus explaining the occurrence of autoimmune phenomena such as Graves’ disease [Polymorphisms of  disease .SLCO1B1 have not yet been found to be associated with autoimmune diseases or IRAE. SLCO1B1 is a drug transporter and member of the solute carrier family, and polymorphisms of SLCO1B1 have been associated with drug metabolism disorders such as atorvastatin-induced adverse events [SLCO1B1 have also been associated with the occurrence of methimazole-induced liver injury in patients with Grave’s disease [Polymorphisms of e events  or sorafe events . Polymor disease .FAN1 has not yet been associated with autoimmune diseases or IRAE. FAN1 plays an important role in the removal of DNA interstrand crosslinks [osslinks , and hasosslinks .CD274 and PRDM1 in our study, possibly explaining the combined occurrence of these IRAE in clinical practice. Sato et al. also reported the frequent combined occurrence of neurological IRAE such as myasthenia gravis, encephalitis and meningitis with myositis during immunotherapy [The occurrence of myositis as well as encephalitis was associated with CNVs on otherapy . Reynoldotherapy .Although it is currently assumed that interruption or even termination of ICI due to severe side effects has no impact on the prognosis, i.e., the survival of patients , the devOur study can help to estimate the risk of patients to develop IRAE.In summary, to estimate the risk of developing IRAE, on the one hand, we have markers such as gender, blood count parameters, and pre-existing autoimmune diseases, which are easy to obtain; on the other hand, NGS-based results are a more complex and expensive option. However, NGS provides us with additional information and should be considered in risk assessment, especially when multiple therapies are available.Of course there are several limitations in our study. First, results of organ-specific IRAEs and biomarkers with a prevalence outside the interval of 33% to 66% are purely exploratory. This is due to the limited sample size of our work and the number of biomarkers to be detected, especially in the However, there is also great strength in our study. We have carefully evaluated the patient records and can therefore assume with a high degree of confidence that the clinical data are accurate. We used a comprehensive tumor panel, which increases the chance of identifying biomarkers for IRAE. By specifically differentiating distinct IRAE, HLA-I homozygosity, CNVs deletions and duplications, we have been able to obtain important results. Our study can help to define biomarkers associated with the occurrence of IRAE in general and of several specific IRAE. We found a significant association between several genetic markers and the occurrence of IRAE, which were merely hypothesized in the literature. We propose that in future, basic screening for biomarkers associated with the occurrence of IRAE should be carried out before initiation of ICI, in particular in patients for whom a therapeutic alternative, for example with BRAF-/MEK inhibitors is possible, even though ICI is a mainstay of therapy for BRAF-mutated patients as well . Where NAs ICI are used more and more frequently across many different cancer types, further studies on biomarkers associated with the development of IRAE should be performed.


Objective: The 2019 novel coronavirus disease (COVID-19) broke out in Hubei Province and spread rapidly to the whole country, causing huge public health problems. College students are a special group, and there is no survey on insomnia among college students. The purpose of this study was to investigate the incidence and related factors of insomnia in college students during the period of COVID-19.Method: A total of 1,086 college students conducted a cross-sectional study through the questionnaire star platform. The survey time was from February 15 to February 22, 2020. The collected information included demographic informatics and mental health scale, Athens Insomnia Scale (AIS) to assess sleep quality, Self-Reporting Questionnaire-20 (SRQ-20) to assess general psychological symptoms, Chinese perceived stress scale (CPSS) to assess stress. We used logistic regression to analyze the correlation between related factors and insomnia symptoms.Results: The prevalence of insomnia, general psychological symptoms and stress were 16.67, 5.8, and 40.70%, respectively. Multivariate logistic regression analysis showed that gender , general psychological symptoms  and living in an isolation unit  were risk factors for insomnia of college students.Conclusion: Our results show that the insomnia is very common among college students during the outbreak of covid-19, and the related factors include gender, general psychological symptoms and isolation environment. It is necessary to intervene the insomnia of college students and warrants attention for mental well-being of college students. The coronavirus disease 2019 (COVID-19) was broke out began at the end of 2019 and had spread rapidly across the whole country due to its rapid spread, meanwhile, the number of confirmed cases in the world is also increasing, Not only has it caused great public concern and panic . From February 15 to February 22, a total of 1086 questionnaires were collected in this cross-sectional study. The subjects came from college students from multiple provinces of china. In order to ensure repeated filling of the questionnaire, the same IP address can only be submitted once. The demographic includes age, gender, education, occupation, etc. the Athens Insomnia (AIS), Chinese perceived stress scale (CPSS) and Self-Reporting Questionnaire-20 (SRQ-20) were administered to screen insomnia , stress , and the general psychological symptoms , separately. The survey is anonymous and the participants can give up at any time. The information in the questionnaire ensures confidentiality. All subjects provided informed consent to participate in the study.The study was approved by the Clinical Research Ethics Committee of Ningbo Kangning Hospital. The questionnaire was designed by epidemiologists. All the questionnaires were collected online through the questionnaire star platform , an internationally recognized eight-item self-assessment scale used to assess the quality of sleep. Each item of the scale is divided into 0, 1, 2, and 3 grades from nothing to seriousness. An AIS total score ≥6 indicates that insomnia is present, whereas a total score of <6 indicated no sleep disorders .Chinese Perceived Stress Scale(CPSS): Yang Tingzhong revised the Chinese version based on the perceptual pressure scale (PSS) compiled by American scholar Cohen et al.  In 1983.The Self-report Questionnaire (SRQ-20) was used to assess common mental disorders such as anxiety and depression, which consists of 20 “yes/no” questions, including four about physical symptoms and 16 about mental and emotional disorders. Responses were scored using “0” or “1,” where “1” indicates the presence of symptoms in the past month, and “0” indicates the absence of symptoms. The clinical reference index of SRQ-20 is 7, where a score exceeding 7 is considered to reflect emotional pain .The data were analyzed with spss23.0 for Windows, All demographic and clinical data, including insomnia scores, were normally distributed among the investigators, and were expressed by descriptive statistics . Demographic information and clinical variables were tested by chi square test. The incidence of insomnia was described by percentage. The factors related to insomnia were analyzed by binary logistic regression. The correlation between insomnia and demographic and clinical variables was detected by Pearson correlation. Stepwise multiple regression analysis was then used to identify predictors associated with insomnia. All tests were two-tailed with the significance level set at 0.05.A total of 1,086 college students participated in the survey. The prevalence of insomnia, anxiety and depression physical symptoms and stress-related symptoms among students are 16.67% (AIS ≥ 6), 5.80% (SRQ-20 ≥ 8) and 40.70% (CPSS ≥ 29), respectively.x2 = 5.27, P = 0.02) compared with non-insomnia, and they have higher anxiety and depression physical symptoms  and stress symptoms . In addition, whether in an isolation unit  was also a risk factor for insomnia.OR = 1.55, p = 0.044, 95% CI = 1.00–2.41), general psychological symptoms , and whether living in an isolation unit  were closely related to insomnia in students. Pearson correlation analysis showed that Insomnia Scale score was significantly correlated with gender , general psychological symptoms , stress , and isolation . Further linear regression showed that general psychological symptoms , isolation  were still related to Insomnia.Binary logistic regression analysis showed that gender   to underThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The study was approved by the Clinical Research Ethics Committee of Ningbo Kangning Hospital. All subjects provided informed consent to participate in the study.HZ, DZ, and LW designed the study. CY, XL, GQ, WF, and DZ collected the survey data. CY, XL, DZ, LW, and HZ analyzed the results and wrote the manuscript. All authors have read and approved the final version of the manuscript.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.


Cell-free fetal DNA (cffDNA) from maternal plasma has made it possible to develop noninvasive prenatal paternity testing (NIPPT). However, most studies have focused on customized single nucleotide polymorphism (SNP) typing systems and few have used conventional short tandem repeat (STR) markers. Based on massively parallel sequencing (MPS), this study used a widely-accepted forensic multiplex assay system to evaluate the effect of noninvasive prenatal paternity testing with a combination of well-established SNP and STR markers. Using a ForenSeq DNA Signature Prep Kit, NIPPT was performed in 17 real parentage cases with monovular unborn fetuses at 7 to 24 gestational weeks. Different analytical strategies for the identification of paternally inherited allele (PIA) were developed to deal with SNPs and STRs. Combined paternity index (CPI) for 17 real trios as well as 272 unrelated trios was calculated. With the combination of SNPs and A-STRs, 82.35% (14/17), 88.24% (15/17), 94.12% (16/17), and 94.12% (16/17) of real trios could be accurately determined when the likelihood ratio (LR) threshold for paternity inclusion was set to 10,000, 1000, 100, and 10, respectively. This reveals that simultaneous surveys of SNP and STR markers included in the ForenSeq DNA Signature Prep Kit offer a promising method for NIPPT using MPS technology. Prenatal paternity testing is of great importance in certain situations, such as in the investigation of pregnancy due to rape or incest. One possible method is to perform paternity testing through amniocentesis and chorionic villus sampling at the gestational age of week ten and beyond ,2,3. HowFortunately, the discovery of cell-free fetal DNA (cffDNA) in maternal plasma  enables Short tandem repeat (STR) is a conventionally used genetic marker in paternity testing, and capillary electrophoresis (CE)-based STR length polymorphism analysis is the most commonly utilized typing method. Thus, early attempts in NIPPT began with STR; however, investigators were only able to detect few numbers of informative paternally derived alleles from autosomal STRs (A-STRs) ,12. On tIn recent years, many genetic markers, such as single nucleotide polymorphism (SNP) , DNA metSTR markers are the main markers for exclusion-based relationship analysis and many STR DNA databases have been built and are still going on. If the advancement of well-established STR markers could be taken, the application of NIPPT could be broadened. To achieve this goal, both the problem of long amplicon size and high rate of stutters using the conventional CE-based STR typing method should be solved, so that paternally derived alleles could not only be detected but also be distinguished easier from stutters. Fortunately, MPS enables genotyping of combinations of multiple types  and large numbers of markers with shorter amplicon lengths than the CE method. Among MPS panels developed for forensic research, the ForenSeq DNA Signature Prep Kit , which is based on Illumina Miseq FGx platform, was widely used and evaluated . Also, aA total of 17 pregnant women with singleton pregnancies, including eight in their first trimester  and nine in their second trimester , were enlisted at the Sixth Affiliated Hospital of Sun Yat-sen University. Their husbands  were also included in the study. Three milliliters of peripheral blood were collected in EDTA-containing tubes. Amniotic fluid or chorionic villi were collected for medical purpose by professional operations at the hospital. For both the pregnant women and their husbands, blood stain papers were prepared. To obtain the DNA of the child, maternal plasma was isolated from peripheral blood by a two-step centrifugation method ,25. cfDN2) using the ForenSeq DNA Signature Prep Kit  according to the manufacturer’s recommendations. Sequencing was performed on a MiSeq FGx system  using the MiSeq FGx Reagent Kit  following the manufacturer’s instructions.DNA libraries were prepared with 1 ng input DNA or two punched blood stained papers (1.0 mmBoth parents and their fetus (amniotic fluid or chorionic villus) were genotyped using the Goldeneye 25A kit , which included 23 A-STR loci, according to the manufacturer’s instructions. Then, the PCR-products were separated on an AB 3500xL Genetic Analyzer  and analyzed using the GeneMapper ID-X Version 1.5 . Paternity index (PI) values were calculated according to the technical specification for the paternity test (GB/T 37223-2018) implemented in China . PaterniGenotype calling of SNP and STR markers was performed with STRait Razor 3.0  and an iePIA) and observed paternally inherited allele (oPIA). Specifically, when a fetus was identified to had an allele that the mother did not have at a locus, this allele was defined as an ePIA. For example, if the mother showed the homozygous genotype of AA and the fetus showed the heterozygous genotype of AB, allele B was an expected paternally derived allele, or to say ePIA. Then, if the plasma was also identified to have an allele that the mother did not have at that locus, this allele was defined as an oPIA. For the above example, if the number of reads of the minor SNP allele B in corresponding plasma exceeded the noise threshold, allele B was an observed paternally derived allele, or to say, oPIA, and the inferred genotype was supposed as AB.For plasma samples, a minimum depth of 50× was required for SNP allele calling. We set 2% as the threshold to distinguish paternally derived alleles from background noise in cfDNA as studied before . We alsoePIA and oPIA have the same definition as that in the analysis of SNPs, but the process of identifying alleles in plasma was more complicated. A novel strategy, inspired by our previous study about the characteristics of stutter variants in STR genotyping [oPIAs were identified as the combination of the parental alleles and potential alleles minus maternal alleles.For A-STR and X-STR allele calling in plasma samples, a minimum depth of 30× was required, while it was 10× for Y-STR. In STR analysis, notyping , was devnotyping , and founotyping , were elnotyping . AccordioPIAs and ePIAs. The fetal fraction in cfDNA was calculated using the formula:oPIA and maternal allele for one SNP, respectively. The fetal fraction of a plasma was estimated by averaging the fractions of all loci. Detection rate of the ePIA for SNPs was computed as:oPIAs consistent with paternal alleles of the reference fetus while ePIAs.The dropout rate and drop-in rate for STRs and SNPs were obtained from a comparison between http://cran.r-project.org/web/packages/relMix (accessed on 14 December 2020)), in which both mutations and the probabilities of dropout and/or drop-in were considered [To verify discrimination power of the system, the paternity of 16 unrelated subjects  were tested for each case, resulting in a total of 272  negative tests. PI values were calculated using the R package relMix (Available online: nsidered . The CPIThe ForenSeq DNA Signature Prep Kit (DPMA) allows targeted genotyping of 152 forensically relevant genetic markers in a single reaction, including 94 iiSNPs, 27 A-STRs, seven X-STRs, and 24 Y-STRs. As shown in 2 and the average percentage of the clusters passing filter, phasing, and prephasing were 97.95%, 0.17% and 0.11%, respectively. The average number of reads for each sample was 85,899.Prior to library generation, parent–offspring relationships were confirmed by STR genotyping with Goldeneye 25A system for all 17 families. Then, 68 samples from 17 families were sequenced with three sequencing runs. The average cluster density was 563 k/mmePIAs were expected in plasma across 17 families and the average number of ePIAs was 18 (range—10 to 22). The sequencing result of plasma showed that a total of 240 oPIAs were identified across 17 families with and the average number of oPIAs was 14 (range—1 to 21)  observed at rs2076848 in case 9. The overall error rate was 0.4%, similar to the error rate estimated by Christiansen et al. [For SNPs, a total of 313 1 to 21) . That is1 to 21) . Full pa18 range— to 22. TePIAs for A-STRs, X-STRs, and Y-STRs was 17 (range—11 to 21), two (range—0 to 4), and three (range—0 to 24), respectively. The average number of oPIAs of A-STRs, X-STRs, and Y-STRs was eight (range—2 to 17), one (range—0 to 2), and 13 (range—11 to 15), respectively , respectively  when only 94 iiSNPs were applied and was 4.73 (range—−0.64 to 8.86) when only STRs were analyzed  will improve the testing efficiency. For the three cases with paternity that could not be determined with the LR threshold of 10,000, the stage of pregnancy should be the main reason. In fact, they are amongst the earliest gestational weeks within this study. The earlier the gestational age, the higher probability of low fetal fraction in the plasma, which would further determine a lower However, the application of NIPPT should be very cautious. From a technical aspect, either false negative or false positive PIAs might occur, which will further affect the testing result and make it inconclusive. In this condition, additional tests must be done before determining paternity inclusion or exclusion. Furthermore, ethical and social issues should always be considered ,38,39,40


Cells, includes three excellent reviews covering different aspects of the autophagy process, including autophagy in protists, the role of BECLIN1 protein, and the importance of selective autophagy in spermatogenesis and male fertility. These are clear examples of the conservation of the autophagic machinery during evolution, the complexity of its regulation, and the impact of autophagy on human health.Autophagy is a conserved self-degradation process that is activated under a wide variety of stresses and physiological conditions. The hallmark of autophagy is the formation of a double membrane vesicle, called the autophagosome, that envelops cellular material and delivers it to the lysosomes, where degradation takes place, and simple compounds are released into the cytoplasm for recycling or energy production. The Special Issue “Proteins in Autophagic Machinery”, published by S. cerevisiae, whereas in mammalian cells, it is composed of ULK1/2, ATG13, ATG101, and FIP200 [S. cerevisiae and VPS15, VPS34, BECLIN1, NRFB2, and ATG14 in mammalian cells [S. cerevisiae and ATG2A/ATG2B in mammals) are proposed to be responsible for the elongation of the autophagosome membrane originating from the fusion of ATG9-containing vesicles [The degradation of cellular components by autophagy is an essential process for cell homeostasis and survival. Autophagosome biogenesis is regulated by the so-called autophagic machinery, a series of proteins (ATG proteins) organized in complexes that function in a hierarchical manner. The initiation step is regulated by the Atg1/ULK and PI3KC3 kinase complexes. The Atg1/UlK complex consists of Atg1, Atg13, Atg17, Atg29, and Atg31 in the yeast model vesicles ,7. One cvesicles .E. histolytica compared to the non-pathogenic amoeba D. discoideum [Autophagy is a highly conserved process in eukaryotes, including protists, a very large and phylogenetically diverse group of eukaryotic organisms with diverse lifestyles. Among them, parasitic protists are particularly interesting because of their relevance in pathology. They are responsible for human diseases such as leishmaniasis, trypanosomiasis, malaria, toxoplasmosis, etc. In their review, Sukamoto et al. analyze the repertoire of core autophagy proteins in parasitic protists and compare it with that of the opisthokonta group (fungi and Metazoa) . The autscoideum . InteresBECLIN1 is a complex protein involved in a wide variety of functions, including autophagy and other membrane trafficking processes. It is a conserved scaffolding subunit of the PI3KC3 complexes which, together with VPS34 and VPS15, forms the common core of the C1 and C2 complexes. While complex C1 contains ATG14 and is dedicated to autophagy, complex C2 contains UVRAG and regulates other membrane trafficking processes. Tran et al. comprehensively review the different aspects of this complex protein, including structural aspects, interactors, and function . The autSelective autophagy targets specific materials for degradation, such as damaged organelles, misfolded proteins, or pathogens. Recognition of different cargoes requires the function of multiple proteins, called autophagy receptors, that recognize cargoes and regulate the core autophagic machinery. The review by Lv et al. discusses the role of selective autophagy in reproduction, specifically in spermatogenesis and fertilization . The aut


We developed a liquid crystal (LC) aptamer biosensor for the sensitive detection of amoxicillin (AMX). The AMX aptamer was immobilized onto the surface of a glass slide modified with a mixed self-assembled layer of dimethyloctadecyl [3-(trimethoxysilyl) propyl] ammonium chloride (DMOAP) and (3-aminopropyl) triethoxysilane (APTES). The long alkyl chains of DMOAP maintained the LC molecules in a homeotropic orientation and induced a dark optical appearance under a polarized light microscope (POM). In the presence of AMX, the specific binding of the aptamer and AMX molecules induced a conformational change in the aptamers, leading to the disruption of the homeotropic orientation of LCs, resulting in a bright optical appearance. The developed aptasensor showed high specificity and a low detection limit of 3.5 nM. Moreover, the potential application of the developed aptasensor for the detection of AMX in environmental samples was also demonstrated. Therefore, the proposed aptasensor is a promising platform for simple, rapid, and label-free monitoring of AMX in an actual water environment with high selectivity and sensitivity. Amoxicillin (AMX) is a semi-synthetic, penicillin-type antibiotic commonly used for the treatment of infectious diseases, such as otitis media, pneumonia, and urinary tract infection, due to its strong bactericidal capabilities and low cost ,3,4,5,6.Several methods have been presented to detect AMX, such as high-performance liquid chromatography (HPLC) , surfaceLiquid crystals (LCs) are attractive materials that possess various unique properties, such as optical anisotropy, birefringence, orientational behavior, and long-range orientation ,19,20. TIn recent years, aptamers in LC-based biosensors have been rapidly developed because of their excellent ability to recognize and bind to the target molecules, enhancing the selectivity of biosensors ,26,27. AHere, we developed a simple and sensitive LC-based aptasensor for the detection of AMX in aqueous solutions. In the developed biosensor, the surface of the glass substrates was modified with a DMOAP monolayer to align LCs in homeotropic orientation, providing a uniformly dark background. AMX aptamers immobilized on the glass substrates acted as target recognition probes to capture AMX molecules. The specific binding of aptamers and AMX molecules results in conformational changes in aptamers, inducing an orientational change in LCs, and a change in the optical textures of LCs observed under a POM. A low limit of detection (LOD) of 3.5 nM for AMX was obtained using this sensing system. In addition, this biosensor exhibits high specificity for AMX compared to other antibiotics. Most importantly, the developed LC-based aptasensor was used to successfully analyze AMX in potential application environments, including tap water and river water samples. Therefore, this aptasensor is a simple, sensitive, and label-free platform for the real-time monitoring of AMX in water environments.2)-TTA GTT GGG GTT CAG TTG G-3′) was provided by Mbiotech . C2H5OH, H2O2 (30%), CH3OH, and H2SO4 (95%) were provided by Daejung Chemicals & Metals Co., Ltd. . Stock antibiotic solutions were prepared in DI water and protected from light.The glass slides were provided by Matsunami Glass IND., Ltd. . All the chemicals and reagents were used as received, and deionized (DI) water was used in experiments. In addition, 4-cyano-4′-pentylbiphenyl (5CB) was provided from Tokyo Chemical Industrial Co., Ltd. of Japan. Glutaraldehyde ; (3-aminopropyl) triethoxysilane , amoxicillin trihydrate , sulfamethoxazole (≥98%), tetracycline hydrochloride (≥98%), chloramphenicol (≥98%), norfloxacin (≥98%), sulfamethizole (≥99%), ampicillin sodium salt (≥98%), penicillin G sodium salt (≥96%), ethanolamine, dimethyloctadecyl [3-(trimethoxysilyl) propyl] ammonium chloride , and phosphate-buffered saline  were procured from Sigma-Aldrich, St. Louis, MO, USA. Amoxicillin aptamer  in the tapping mode. A digital camera  mounted on the POM  was used to capture the polarized optical images. The images were taken in transmission mode. The gray-scale intensity (GI) of the images was analyzed using MATLAB software.The upper and bottom glass slides were prepared following previous studies ,28. The To prepare for the detection of AMX, 10 μL of AMX aptamer solution at an optimal concentration was dropped onto the GA-modified glass to form a large circular spot, followed by incubating, washing, and drying, as described in the To evaluate the practicability of the proposed biosensor in a real water environment, AMX was determined in tap water and river water samples. Tap water was obtained from our laboratory, while river water was collected from the Tancheon River, Seongnam, Korea. After filtering, the tap and river water samples were spiked with different concentrations of AMX. The spiked samples were then analyzed using the proposed assay.v/v), corresponding to a random orientation of LC molecules . As shown in In addition to the influence of the APTES/DMOAP ratio, the effect of the DMOAP concentration on the upper glass slides on output signals was also investigated. The cleaned glass substrates were coated with various concentrations of DMOAP and used as covering glass slides, while the APTES/DMOAP ratio modified on the bottom glass substrates was fixed at 2:1 (v/v). As shown in v/v). However, completely dark backgrounds were observed when the GA concentration ≤ 0.01% (v/v) (v/v) was used in further experiments.The amount of GA grafted onto the APTES/DMOAP-modified glass substrates may also affect the orientation behavior of LCs, thereby affecting the optical appearance of the LCs ,38. Ther1% (v/v) . TherefoSimilarly, because the surface topography and homeotropic orientation of LCs are also influenced by the concentration of the aptamer immobilized on the glass substrate, the optimal amount of AMX aptamer to be anchored on the GA-modified glass surface was determined. As shown in Next, we examined the feasibility of the aptasensor developed in this study for the detection of AMX under optimal experimental conditions. An LC cell with its bottom glass substrate incubated with 1 µM AMX solution was fabricated and examined. LCs showed a bright circular spot in the region where the AMX solution was dropped, but it remained dark outside the spotted circle a. This iAfter confirming the feasibility of the developed sensing system, the limit of detection of this biosensor for AMX was investigated by decreasing the concentration of AMX. A series of LC cells with their bottom glass substrates incubated with various AMX concentrations ranging from 0 nM to 800 nM was constructed and examined. For AMX concentrations ranging from 50 nM to 800 nM, bright circular spots on the POM images of the LC cells were observed b–g, indi2) of 0.989. The detection limit of AMX was found to be 3.5 nM, which was calculated using the formula 3α/slope [To further investigate the quantitative performance of the LC-based aptasensor, we evaluated the correlation between the AMX concentration and the GI of the POM images calculated using MATLAB software (the MATLAB code is given in the 3α/slope ,40. HoweIn addition to the sensitivity, the specificity of the LC assay was also evaluated. Several commonly used antibiotics, such as SMXZ, TC, CL, NRFX, SMZ, AMP, and PC, that have similar structures with AMX and are difficult to distinguish, were selected as interferences ,9,15. A The applicability of the proposed aptasensor for the determination of AMX in real water samples, including tap water and river water, was tested. The results are shown in In this study, an LC-based biosensing strategy using a specific DNA aptamer as a target recognition probe was developed to detect amoxicillin in aqueous media. The detection principle is based on the specific binding of AMX molecules to the DNA aptamer immobilized on the LC cell, which disrupts the homeotropic alignment of LCs, resulting in a bright optical image observed under a POM. Using this sensing strategy, under optimal conditions, AMX could be quantified in the range of 10 nM to 800 nM with a minimum detection limit of 3.5 nM. In addition, this biosensor exhibited high specificity for AMX compared to other antibiotics. Moreover, the developed biosensor was successfully applied to tap water and river water. This proposed sensing strategy offers a simple, convenient, label-free, and sensitive method for the detection of AMX in aqueous solutions.


Colocasia esculenta (L.) Schott) is a staple food crop in the Asia-Pacific region in areas where rising sea levels are threatening agricultural production. However, little is known about its response to salinity. In this study, we investigated the effects of salinity on the growth, morphology, physiology, and chemical traits of taro to predict the impacts of rising sea levels on taro production and nutritional value in the Pacific. We grew taro (approximately 4 months old) with a range of NaCl treatments (0–200 mM) for 12 weeks. Full nutrient, micronutrient, and secondary metabolite analyses were conducted, including measures of calcium oxalate (CaOx), an irritant that reduces palatability. Significant reductions in growth and biomass were observed at and above 100 mM NaCl. Concentrations of macro- and micronutrients, including sodium, were higher on a per mass basis in corms of plants experiencing salt stress. Foliar sodium concentrations remained stable, indicating that taro may utilize a salt exclusion mechanism. There was a large amount of individual variation in the concentrations of oxalate and phenolics, but overall, the concentrations were similar in the plants grown with different levels of salt. The total contents of CaOx and phenolics decreased in plants experiencing salt stress. Taro’s ability to survive and produce corms when watered with a 200 mM NaCl solution places it among the salt-tolerant non-halophytes. The nutritional quality of the crop is only marginally affected by salt stress. Taro is, therefore, likely to remain a useful staple in the Pacific region in the future.Taro ( Rising sea levels pose a major challenge for the food security and livelihood of communities around the world, particularly in low-lying regions, such as the Pacific ,2. SalinColocasia esculenta (L.) Schott) are a staple food for over 200 million people in the Indo-Pacific region and are the main source of carbohydrates in the Pacific Islands [Root and tuber crops are important staples throughout the world. This is particularly true in the Pacific Islands, where, aside from bananas and breadfruit, the main staples are yams, taro, cassava, and sweet potato . Root an Islands ,9,10,11. Islands . However Islands .+ and Cl− transport to, and accumulation in, above-ground tissues, particularly the leaves. Other plants exhibit tissue tolerance, which prevents cell damage by the compartmentalization of Na+ and Cl− in vacuoles [Plants respond to salinity (NaCl) in multiple ways. Some minimize the deleterious effects through ion exclusion, which prevents Navacuoles ,15,16,17vacuoles ,18. ConvAraceae), a group notable for storing CaOx crystals. CaOx crystals make the leaves and corms highly acrid and can cause irritation and damage to the mouth, throat, and gut unless thoroughly washed and cooked before consumption [Salinity is known to affect plant nutritional quality and palatability by altering the levels of secondary metabolites, protein, and micronutrients . Salt stsumption ,11,21. Ssumption ,23. To esumption ,24.We tested the impact of salinity on the growth and nutritional value of taro plants by watering with five different concentrations of NaCl  for 12 weeks. We found that taro was relatively salt-tolerant in that while high salt concentrations negatively affected growth and plant biomass, plants survived salinities up to 200 mM, with minimal effects on palatability. We conclude that taro is likely to continue to be a suitable food security option in the Pacific region in the future.Salt significantly reduced the growth rate of taro, but plants were not killed . Overallp ≤ 0.001), but no clear effect of salt was shown until week 3  were analysed separately from the main plants to determine whether their formation or biomass was influenced by the salt treatments. The number of suckers per plant, the total sucker biomass, the percentage of the total biomass in suckers, and the ratio of sucker corm mass-to-main corm mass did not differ significantly between the salt treatments .v/Fm) or chlorophyll concentrations were detected between salt treatments either  of the control plants was nearly four-fold greater than that of plants grown with the highest 200 mM NaCl salt treatment a. The sts either , althoug−1 was the median sodium concentration in all the treatments (−1. The findings for sodium were consistent when nutrients were examined as both concentrations (mg g−1) and total contents per plant measures  by the corresponding corm and leaf mass. When the corm nutrients were examined as the total corm nutrients per plant, the eight nutrients that had increased in concentration were no longer significantly different between treatments  between salt treatments , a related edible aroid; plants grew well in locations where salt was ~100 mM NaCl and tolerated levels up to ~200 mM NaCl, but salt levels above 300 mM NaCl could be lethal [Taro exhibited significant reductions in growth and biomass with increasing salinity, although the concentrations of salt at which the effects were manifested differed between the various parameters (between 50 mM and 200 mM). These results are broadly consistent with those from the in vitro studies on taro by Nyman and others who reported significant growth reduction and necrosis in taro cell cultures grown with 175 mM NaCl ,29. ThesThe Fv/Fm decreased with increasing salinity, but the difference was not significant. The net carbon assimilation also decreased significantly with increasing salinity, with a significant decrease in all gas exchange parameters in plants grown with 200 mM NaCl. That the growth was impacted to a greater extent than the net carbon assimilation is not surprising; a small change in photosynthesis over time can result in highly significant reductions in biomass. It is also possible that the plants were expending more energy on other metabolic activities, such as ion exclusion at the level of the leaves , Leaf. RCorm growth was more sensitive to salt than other plant parts, with reductions in biomass of approximately 30% observed in plants grown with the lowest concentration of 50 mM NaCl. This is not surprising, given the reduction in photosynthesis and the availability of carbohydrates for storage . Other p+ and Cl- ions in the soil may compete directly with the uptake of essential nutrients, such as Ca2 +, K+, and NO3− [The concentrations of mineral nutrients recorded in taro corms in this study are broadly comparable to those recorded in previous studies ,36. The and NO3− ,37. This2+, salinity, and reduced CaOx accumulation comes from the study performed by Hunsch et al. [2+ accumulation in the leaves of Grewia tenax  in response to high salinity resulted in the production of fewer CaOx crystals. In the same study, they included another tropical tree that does not accumulate CaOx crystals (Tamarindua indica), but in this case, they did not detect any difference in foliar Ca2+ levels. Thus, decreases in CaOx crystals are likely to be driven by a reduction in oxalate formation.Concentrations of oxalate and calcium were measured as a proxy for calcium oxalate. Foliar oxalate and calcium concentrations decreased both on a per mass and a per organ basis with increasing salinity despite the decrease in plant biomass. The lower concentrations could be due to either a decrease in available Ca due to competition with sodium at the root, reduced transportation of Ca to the leaves as a result of reduced transpiration, or a reduction in oxalate formation. Evidence to support the latter hypothesis regarding Cah et al. , who fouG. tenax) but not in the tree without this capacity supports this hypothesis [Alternatively, it is possible that any crystals in the leaves were remobilized. Remobilization would have the benefit of releasing oxalate back into the primary metabolism . It is ppothesis . If thisThere was a significant reduction in phenolic acids and an increase in flavonoids in the taro leaves in response to increased salinity. This is in direct contrast to previous research on the response of secondary metabolites to stress, which have been observed to increase in concentrations in a wide range of crop plants, including rice, red pepper, and cassava ,19,20,43−1). Our results are broadly consistent with those previously recorded for taro by [−1 [This study confirms that taro is cyanogenic, but the cyanide levels recorded are very low  Schott cv. Samoan Pink), approximately 20 cm in size and sourced from El Arish Tropical Exotics , were grown in 20 L pots containing commercial Debco potting mix and Osmocote® controlled-release fertilizer  under natural light. The plants were watered with a complete nutrient solution  at 0, 30, and 60 days after transplanting (DAT). The temperature in the greenhouse was maintained at 26 and 20 °C during the day and night, respectively, with a range of 22 °C to 30 °C and humidity between 55 and 65%, consistent with the optimal conditions for taro reported in the literature [A glasshouse experiment was conducted at the School of Biological Sciences, Plants Sciences Complex, Monash University, Clayton. Taro plantlets (terature . The plan = 18), such that each treatment group had an equal number of plants from the small, medium, and large groups. Seawater has a concentration of approximately 500 mM NaCl. The salt concentrations were determined based on the studies of Nyman and Arditti [n = 3) were measured halfway through the treatment period (6 weeks) using a portable refractometer to confirm that the allocated salt concentrations were not exceeded (After 18 weeks of growth (12 December 2016 to 20 March 2017), the taro plants were classified into three groups based on height , and 18 plants within each group were randomly allocated to one of 5 salt treatments . The leaves were classified as peeping, rolled, expanded, or senescent following Lu et al. , as modin = 15, equally distributed between height groups). The plants were grown under treatment conditions for 12 weeks before being destructively harvested when they were 7 months old. This time period was chosen because, at this age, plants typically have a well-developed and mature corm, but side shoot development is limited. The plants were separated into the leaves, petioles, roots, and corm. The leaves were further separated into the five classes described above . Measurements on the second fully expanded leaf from each plant (n = 5) were made at 800 μmol quanta m−2 s−1 PAR, 400 ppm CO2, 25 °C, and approximately 50% relative humidity. Dark-adapted chlorophyll fluorescence, Fv/Fm, was measured on the second fully expanded leaf (n = 4) using a Pulse-Amplitude Modulated PAM-210 Chlorophyll Fluorometer . The Fv/Fm ratio is an indicator of the degree of potential photosynthetic ability and nutrient stress, where 0.8–0.9 is indicative of a healthy, unstressed plant [The photosynthetic parameters were measured on four representative plants from each treatment group in the week prior to the final harvest. The photosynthetic rate, transpiration, conductance, and internal COed plant .The chlorophyll concentrations were measured using the method of Gleadow et al. , as modin = 6 per plant part) using inductively coupled plasma mass spectrometry (ICPMS) .Micro- and macronutrient analysis of finely ground freeze-dried tissue (0.2 g) was conducted on a subset of leaf and corm samples (n = 18) using reverse-phase high-performance liquid chromatography (HPLC) following the methods of Uesugi and Kessler [®, North Ryde, New South Wales, Australia) was added to 0.02 g of tissue. The samples were heated at 80 °C for 15 min, centrifuged for 30 min, and filtered through a 0.45 μm size pore membrane. Chromatographic analysis was carried out using an Agilent Technologies HPLC 1260 Infinity  using a reverse-phase Poroshell 120 column . Two solvents were used—0.25% H3PO3 in MQ H20 (A) and acetonitrile (B), starting with 0% B and using a gradient to obtain 12% B at 2 min, 18% B at 3 min, 58% B at 10 min, and 0% B at 20 min. The flow rate was 0.5 mL/min, and the injection volume was 2 μL. Phenolic acids and flavonoids were identified to compound classes using UV spectra, and oxalates were identified based on the retention time and the UV spectra of the standard . Compounds were quantified at 210 nm for oxalates, 320 nm for phenolic acids, and 360 nm for flavonoids. The relative concentration of each compound was expressed as the peak intensity relative to the tissue mass of each sample, and the totals of each compound class were calculated. The concentrations of oxalic acid were quantified using a standard curve of 0.1–0.02 mg ml−1 oxalic acid.Phenolics and oxalates were measured on finely ground freeze-dried corm and leaf tissue for each individual ( Kessler . For extn = 5) were tested for the presence of cyanogenic glycosides using the evolved cyanide and capture method [Prunus amygdalis (L.) Benth. Hook.) and a solution of latex from cassava (Manihot esculenta Cranz), previously shown to be effective against aliphatic amino acids [A subset of plants in the experiment (e method ,57. Trige method , is an ae method . To ensuno acids ,59, wereno acids . HydrogeData analysis was performed using R version 4.1.1 and RStudio version 1.4.1717 . The wee


We identified a significantly higher tumor mutation burden in leiomyosarcomas vs. leiomyomas in terms of somatic single-nucleotide variants , indels (9491 vs. 4098), and copy number variants (8390 vs. 5376). Further, we discovered alterations in specific copy number variant regions that affect the expression of some tumor suppressor genes. A transcriptomic analysis revealed 489 differentially expressed genes between these two conditions, as well as structural rearrangements targeting ATRX and RAD51B. These results allowed us to develop a machine learning approach based on 19 differentially expressed genes that differentiate both tumor types with high sensitivity and specificity. Our findings provide a novel molecular signature for the diagnosis of leiomyoma and leiomyosarcoma, which could be helpful to complement the current morphological and immunohistochemical diagnosis and may lay the foundation for the future evaluation of malignancy risk. The absence of standardized molecular profiling to differentiate uterine leiomyosarcomas versus leiomyomas represents a current diagnostic challenge. In this study, we aimed to search for a differential molecular signature for these myometrial tumors based on artificial intelligence. For this purpose, differential exome and transcriptome-wide research was performed on histologically confirmed leiomyomas ( Uterine leiomyomas (LM) are benign tumors arising in the smooth muscle cells of the uterine wall. They are the most common pelvic tumors in women, with a prevalence of >80% for African American and ~70% for Caucasian women before 50 years of age . AlthougHistological diagnosis is the gold standard option for myometrial tumors ,6. HowevDespite LM and LMS having been previously characterized at a molecular level ,12,13, tIn this study, we identified that LM and LMS have significant mutational heterogeneity and differences in copy number alterations at the DNA level, while a specific transcriptomic profile and multiple structural rearrangements were detected at the RNA level. With these data, an integrated molecular analysis was performed to assess the effect of copy number variants (CNVs) on gene expression. Targeted RNAseq data and artificial intelligence were used to create a predictive model for the comprehensive molecular classification of LMS and LM at the tumor-tissue level.After obtaining informed consent from the eligible patients, tumor samples were collected from women undergoing a hysterectomy or myomectomy as a surgical treatment for a primary myometrial tumor. Following histological diagnoses according to WHO criteria , a totalWe performed whole-exome sequencing in 44 LM and 34 LMS tumors to screen for single nucleotide variants (SNVs) and insertions/deletions (indels). We detected 181,354 small variants in the LMS tumors, of which 171,863 were SNVs and 9491 were small indels. In the LM samples, we detected 85,250 small variants, of which 81,152 were SNVs and 4098 were small indels. Among these variants, 27.63% where shared between LMS and LM, while 34.56% were LMS-exclusive and 37.81% were LM-exclusive (in at least one sample).p < 0.05). Then, we focused on group-exclusive variants present in at least six samples of each group to find exclusive variants involved in the pathogenic process of each group. As a result, we found a total of thirteen variants affecting eight different genes exclusive to LMS  status to predict the outcome in LM and LMS tumors, although no differences were found in the number of alleles or the fragment size .t-test showed a significant difference in the mean values of CNVs per sample between LMS (439.7) and LM (328.8) (p = 5.61 × 10−5). Because some CNVs were present in more than one sample within each group, we filtered the unique CNVs per group, obtaining a total of 8390 CNVs in LMS and 5376 CNVs in LM. In terms of their structural nature, 18.2% of the CNVs in LMS were deletions, while 73.1% were duplications. Specifically, 3.5% were LMS-specific deletions present in more than one sample, and 5.2% were LMS-specific duplications present in more than one sample. In the LM group, 11.7% of the CNVs were deletions and 84.5% were duplications, with only 0.1% tumor-specific deletions and 3.6% tumor-specific duplications (We next compared the somatic copy number variants (CNVs) in LMS and LM. A total of 14,467 CNVs were detected in LM, while 14,950 CNVs were detected in LMS. Despite the similar results, Student’s ications A. While ications B. Kaplan–Meier survival curves were generated to assess the association between LMS-specific CNVs and clinical prognosis based on overall survival. We selected 12 of the most frequent LMS-specific CNVs present in at least 10 out of 34 LMS  and founTRIP13 with a positive correlation between gains and higher expression, while losses were associated with lower expression. However, the expression of other genes increased when there was a loss in the corresponding region (CDKN1C) or decreased when there was a gain , possibly due to different mechanisms of regulating gene expression. The same analysis was performed in the LM samples , while, in the remaining genes, gains in the chromosomal region were associated with lower expression values due to more complex gene regulation.Because CNVs can involve a large region containing multiple genes, we integrated the detected CNV regions with exome-wide gene expression data from RNAseq in LM and LMS tumors . As a re samples E, lower,Further, we identified high-confidence fusion transcripts in 29.5% of the LM cases and 61.8% of LMS arising from chromosomal rearrangements, resembling chromothripsis in some cases, such as LMS25 and LMS26 A. ATRX were identified in LMS, and a known oncogene, RAD51B, was identified in LM. Specifically, ATRX was fused with several gene partners, such as TRAPPC9, RP11-56A10.1, and EZH1, resulting in a non-functional fusion protein lacking the helicase ATP-binding domain and/or the helicase C-terminal domain in the LMS tumors . Some of the most significant upregulated genes in LMS were validated by RT-PCR, confirming significant overexpression and correlation between qPCR and RNAseq . Next, unsupervised hierarchical clustering grouped the LMS samples in a homogeneous cluster of 29 samples, while 30 LM samples were detected in a separate cluster. Of note, another cluster included the remaining LM with some LMS  A. Based To better understand the molecular functions, biological processes, and pathways differentially regulated by the 489 DEGs, we performed Kyoto Encyclopaedia of Genes and Genomes (KEGG), Gene Ontology (GO), and Reactome enrichment analyses. In total, we detected 10 KEGG pathways, 83 GO terms, and 92 Reactome pathways . BrieflyTo classify LM and LMS, we developed a machine learning approach based on the transcriptomic signatures of each group since we found that management, analysis, and biological comprehension were more straightforward when using RNAseq data. After feature pruning, the final model was composed of 19 DEGs and was able to correctly classify all the samples in the validation set.n = 44 and n = 32, respectively, since two LMS samples were filtered due to poor sequencing quality) in addition to new samples (eight LM and ten LMS). Based on this model, we built a targeted sequencing panel using AmpliSeq technology for the 19 selected genes, which was used to re-analyze all the previous LM and LMS tumors . The unsupervised clustering of the samples based on these 19 genes allowed separation through a heatmap of two distinguishable groups of LM and LMS. However, and resembling results from previous RNAseq analysis, two LMS (LMS26 and LMS39) were clustered in a group opposite to those confirmed by pathology . Specifically, the gradient boosting algorithm was used to build a new model, which achieved optimal values of sensitivity and specificity since it was able to correctly classify all the test samples. The feature selection resulted in a final model that consisted only of 19 genes, out of which only three, athology B.Further, the model was used to classify the samples and to calculate class probabilities, allowing a more fine-tuned classification of the samples, where we defined a “warning range” for those tumors where the model was not confident enough, defined as probabilities of <75% for each group C. IntereThe search for molecular criteria to differentiate uterine myometrial tumors represents an important current diagnostic challenge, where molecular profiling could be a powerful complement to current diagnosis based on the clinical presentation, imaging features, and microscopic morphologic characteristics. Since our main aim was to build a preliminary differential diagnostic tool, we have focused on the comparison between the most prevalent benign myometrial tumors, LM, and the most aggressive ones, LMS. While we are aware that excluding other tumor types, such as STUMPs, inflammatory myofibroblastic tumors (IMT), or undifferentiated uterine sarcomas, is a relevant limitation of our study, we decided to discard these samples in order to avoid introducing noise in our molecular analysis, also considering that, given the low incidence of these tumors, the fact that excluding them would not have a significant impact on our preliminary classification tool. Following a similar rationale, we chose to limit our analysis to only conventional LMS and LM given the low prevalence of non-conventional variants and their differences, which may also introduce noise in our analyses. Consequently, in this study, exome- and transcriptome-wide analyses were performed on histologically confirmed conventional LM and LMS to investigate molecular differences between and within these two different entities.REST gene involves the loss of the repressor in LM and promotes the aberrant expression of GPR10 in the PI3K/AKT mTOR pathway [OBSCN gene, which has been proposed as a molecular tool to differentiate LMS from gastrointestinal stromal tumors within a two gene-classifier [CCDC68 involving microtubule organization have been reported in different cancer types [In terms of small variants, we found that only a small percentage of variants (27.63%) were shared by the two groups, while the rest were exclusive of LM or LMS, suggesting independent mechanisms for tumorigenesis. Interestingly, we found the c.2443C > T mutation in the  pathway . Conversassifier . Likewiser types ,18. Moreer types . Additionally, we found differences in the number and type of somatic CNVs, suggesting that they could be used for the differential diagnosis of myometrial tumors. Furthermore, we provided novel insights into the potential usefulness of CNVs as a prognostic biomarker for patients with LMS. However, the low number of samples available for the survival analysis  is a strong limitation, and, thus, the tendency we observed should be carefully interpreted and further explored and validated.CDKN1C, a cyclin/Cdk inhibitor that may act as a tumor suppressor [Because CNVs may cause changes in transcription levels associated with cancer , we perfppressor . ATRX and RAD51B, respectively. The loss of expression of ATRX has been associated to the Alternative Lengthening of Telomeres phenotype, which allows tumor cells to escape programmed cell death. In uterine LMS, this mechanism has been associated with a poor prognosis and overall survival [RAD51B–HMGA2 fusion transcript in LM, which may have a role in tumor progression, as previously described [Further, we screened transcriptomic data to detect fusions in LM and LMS. While no recurrent fusions were detected in LMS or LM, multiple rearrangements targeted survival ,20. Addiescribed .AURKA, SPAG5, NUF2, BUB1B, and KIF14 [CCDC68 gene affected by a deleterious variant mentioned in AURKA, a cell cycle protein that is also overexpressed in cervical [The transcriptomic results revealed 489 differentially expressed in LMS versus LM. Although some of these genes have been reported ,22,23, wnd KIF14 , while tcervical  and ovarcervical  cancers cervical . Further analysis based on hierarchical clustering revealed some LMS samples that unexpectedly clustered with LM samples. Accordingly, after reviewing the clinical features and follow-up, we found that most of these patients exhibit higher overall survivals than reported by previous retrospective studies . These fBRCA2, CHAF1A, E2F7, and EXO1); DNA damage repair (ARHGAP11A and PBK); extracellular matrix formation and interaction , and, lastly, segregation of chromosomes and microtubule–kinetochore–spindle formation , which, as we have mentioned, could be an affected pathway in the tumorigenesis of LMS. As a result, our model could be a representation of the affected pathways that differ between LMS and LM, where the genes associated with alterations in the extracellular matrix suggest the diagnosis of LM , and the genes associated with defective DNA replication or DNA damage repair or segregation of chromosomes suggest the diagnosis of LMS. We finally built a classification model composed of nineteen genes, sixteen overexpressed in LMS and three overexpressed in LM, possibly suggesting that the overexpression of a small subset of genes results in a more disrupted molecular profile that is sufficient to indicate that a tumor is more likely to be a malignant uterine leiomyosarcoma. The genes in this panel are involved in different processes and functions, including DNA replication . It is also of note that, even though the unsupervised hierarchical clustering of global RNAseq showed a mixed cluster of LMS and LM samples, the machine learning algorithm effectively classified these samples, once again demonstrating the potential of the predictive tool. Nevertheless, although promising, the genomic and transcriptomic outcomes are based on the analysis of the entire tumor, which implies a technical limitation due to intratumoral heterogeneity. Additional studies with larger numbers of samples are also needed to validate and clinically use this tumor classification model. In summary, our findings provide a novel molecular profile and candidate gene targets to discriminate LM from LMS at the tumor-tissue level, establishing a potential diagnostic tool that could be helpful to complement morphological and immunohistochemical diagnostic features. Accordingly, this molecular-driven test linked to functional histology might provide a framework for objective and specific diagnosis at the tumor tissue level. The challenge, however, for both the pathologist and the physician, is how best to effectively integrate this morphological and molecular information into a comprehensive diagnosis and treatment plan.Detailed description of the materials and methods used in this study are provided in the Use of human tissue samples was previously approved by the IRB of the hospitals involved: Hospital La Fe, Valencia, Spain (24 July 2019), Hospital Virgen de la Arrixaca Murcia, Spain (17 December 2019), Hospital Santa Lucía Murcia, Spain (28 January 2020), and Fundación Instituto Valenciano de Oncologia (IVO), Valencia, Spain (2 December 2020). All patients signed and provided written informed consent. n = 16). Briefly, formalin-fixed paraffin-embedded (FFPE) tumor samples were collected from 119 women undergoing hysterectomy or myomectomy as surgical treatment for primary myometrial tumors . Before Specifically, Hospital la Fe contributed with 56 LM samples and 13 LMS tumors. Hospital Santa Lucía provided to this study 6 LMS, 12 LMS came through Hospital Virgen de la Arrixaca, while 3 LMS were provided by IVO, and 13 LMS were supplied by Origene Technologies Inc. . Selected LM and LMS samples were then split into two cohorts: the experimental cohort, which was used to study global DNA and RNAseq profiles , and the validation cohort, including additional new samples  to perform targeted sequencing and model validation . EpidemiAfter nucleic acid isolation, DNA libraries were constructed using the KAPA Hyper Prep kit  and enriched using a panel of 571 hematological-associated genes. DNA sequence data were demultiplexed and aligned to the human hg19 genome (CRGCh37) using BWA  and SAMthttps://github.com/freebayes/freebayes, accessed on 31 December 2021) and annotated with SnpEff . Additional assessment of somatic mutational signatures inferred from SNVs was done using MutationalPatterns [https://github.com/etal/cnvkit, accessed on 31 December 2021), for CNV detection. Sequencing data were analyzed for small variants using Freebayes  . RNA libraries from 44 LM and 34 LMS were constructed using Truseq RNA exome  and aligned to the human hg19 genome using STAR (er 2021)  to estimhttps://github.com/suhrig/arriba/, accessed on 31 December 2021) was used for detection of fusions on RNAseq data, which were then validated by immunohistochemistry. Subsequently, differential expression analysis between LMS and LM was performed using edgeR [First, arriba , excluding genes with <20 counts per million (cpm) and CNV regions with <10 samples in a group deviating from 2n, with a 1 Mbp window and p-value < 0.01 . For this purpose, our sample cohort was randomly split, keeping balanced class distributions into a training (75% of samples) and test set (25% of samples). We used the Adaboost algorithm on DEGs cpm to perform a prior selection of predictor genes.Since we found that analysis and clinical interpretation were more straightforward when using RNAseq, we built a classification model with this data using caret package (n = 44) and LMS (n = 34) samples, adding a new set of 8 LM and 10 LMS samples. Briefly, RNA from 96 FFPE tissue sections was used to prepare libraries with a PCR/amplicon-based workflow .Validation of the model was next performed by re-sequencing LM . Two-tailed Student’s t-tests were used to compare quantitative clinical variables in LMS and LM patients and for gene validation using RT-qPCR. All survival analyses were achieved by Cox regression in a multivariate model to account for LMS prognostic factors and compared between groups using the log-rank test. Correlation between RNAseq and qPCR-based logFC was evaluated using Pearson’s correlation test.Statistical analyses were performed using R (In conclusion, this study provides a novel molecular classification for leiomyoma and leiomyosarcoma tumors and may be a helpful tool for current diagnosis, hopefully laying the foundation for the future evaluation of malignancy risk.A patent disclosure has been filed for the study under the inventors A.M., R.A., and C.S. since 11 October 2021.


Methotrexate and leflunomide are classic treatments for rheumatoid arthritis (RA), however, which is the best choice for patients of RA is still an important question clinically, and this meta-analysis is used to systematically evaluate and compare their efficacy and safety.We searched PubMed, Cochrance Library, Embase, SinoMed, China National Knowledge Infrastructure, China Science and Technology Journal Database, WanFang Data databases. The retrieval time was from the establishment to September 7, 2021. Literature screening, data extraction, and quality assessment were performed according to the Cochrane risk of bias tool. Meta-analysis of the included studies was performed using RevMan 5.3 software and Stata 12.0 software.The clinical efficacy and safety of leflunomide and methotrexate are evaluated by American College of Rheumatology (ACR)20/50/70, DAS28, total effective rate, adverse reaction rate, morning stiffness, swollen joint count, tender joint count, erythrocyte sedimentation rate, C-reactive protein, and rheumatoid factor.The results of this meta-analysis will provide reliable evidence clinical efficacy and safety for RA. More high-quality randomized controlled trials are still needed to provide more reliable evidence for the treatment of RA.CRD42021270980 The incidence rate of RA in China is about 0.45% to 1.0%, and the disability rate in 5 years is as high as 43.5%, which could be aggravated with the prolongation of the disease course. Both the European League Against Rheumatism recommendations (updated in 2019) and the 2018 Chinese guidelines for the treatment of RA clearly indicate that traditional synthetic disease-modifying antirheumatic drugs are the cornerstone drugs for the treatment of RA.,5 Methotrexate (MTX) and leflunomide (LEF) are recognized as the first choice disease-modifying antirheumatic drugs for the treatment of RA at home and abroad,,5 with definite clinical efficacy.Rheumatoid arthritis (RA) is an autoimmune disease characterized by progressive synovitis, pannus neoplasia, and many other adverse reactions, such as decreased white blood cells and diarrhea. Therefore, we use meta-analysis to compare the effectiveness and safety of these 2 drugs for a better therapeutic method of RA.However, MTX and LEF have certain hepatotoxicity22.1All analyses were based on previously published studies, this article does not contain any studies with human participants or animals performed by any of the authors, thus ethical approval and patient consent are not applicable.2.2This meta-analysis was conducted according to the guidelines of the Preferred Reporting Items for Systematic Reviews and Meta-Analyses statement. Chinese databases such as China National Knowledge Infrastructure, WanFang, China Science and Technology Journal Database, and English databases such as PubMed, Cochrane Library were searched. The retrieval time was from the establishment to September 7, 2021. The search terms were included: “rheumatoid arthritis” OR “RA” AND “methotrexate” OR “MTX” AND “leflunomide” OR “LEF” AND “clinical trial” OR “randomized controlled trial.”The inclusion criteria were as follows:(1)Participants: RA patients, and the diagnostic criteria referred to the 1987 American College of Rheumatology (ACR) diagnostic criteria for RA;(2)Interventions: The experimental group was treated with MTX;(3)Comparators: the control group was treated with LEF;(4)Outcomes: The primary outcomes were ACR20/50/70, DAS28, total effective rate, and adverse reaction rate; the secondary outcomes were morning stiffness, swollen joint count, tender joint count, erythrocyte sedimentation rate, C-reactive protein, and rheumatoid factor;The exclusion criteria were as follows:(1)Repeated published studies;(2)The data is incomplete, the outcome effect is not clear, and the data cannot be extracted for analysis;(3)Animal experiments, cell experiments and the review literature;(4)Clinical case reports;(5)The control group was combined with other treatments.2.3Two researchers independently performed the data extraction and quality assessment according to the screening criteria, and cross-checked them. If there are conflicts of opinions, resolve them through collective discussion in the research team. In the process of literature screening, the literature with irrelevant titles were excluded, and the abstracts and full texts should be further read to determine the final included literature. Basic information was extracted from the included studies . The primary outcomes were ACR20/50/70, DAS28, total effective rate, and adverse reaction rate; the secondary outcomes were morning stiffness, swollen joint count, tender joint count, erythrocyte sedimentation rate, C-reactive protein, and rheumatoid factor. The quality of the included studies was evaluated according to the Cochrane Risk of Bias tool.2.4I2 and chi-square tests were used to assess the heterogeneity. The results were shown in the forest plot. For all outcomes, Egger and Begg test were used to detect publication bias.RevMan 5.3 software and Stata 12.0 software was used for meta-analysis. Odds ratio was calculated for the dichotomous variable, mean difference was calculated for the continuous variable. All of them were expressed with 95% confidence interval. 33.1The screening process is shown in Figure 3.2Begg and Egger test are used to evaluated the publication bias analysis of this study.3.3The Grading of Recommendation, Assessment, Development, and Evaluation system will be used to appraise the quality of evidence from the studies obtained. The levels of it will be divided into high, moderate, low, very low.4Studies have shown that MTX is an inhibitor of folate reductase, and its mechanisms in the treatment of RA are as follows:(1)It mainly inhibits the reduction of dihydrofolate reductase into tetrahydrofolate, which blocks the transfer of -C group during the biosynthesis of purine nucleotides and pyrimidine nucleotides, inhibits DNA synthesis, and then inhibits the proliferation of synovial cells.(2),12Regulating the balance of Th1/Th2 and Th17/Treg, thus to inhibit chronic synovitis.(3)Regulating adenosine-related pathways and inhibiting synovitis of RA.(4)Inhibiting the expression of MMP-1, MMP-2, and other MMPs, so as to inhibit the destruction of joint bone.(5)MTX can inhibit bone destruction by upregulating serum OPG, decreasing the expression of RANKL, competitively inhibiting the binding of RANK and RANKL, and regulating the RANKL/RANK/OPG pathway.The mechanisms of LEF in the treatment of RA are as follows:(1)The metabolite A771726 inhibits the synthesis of pyrimidines by inhibiting mitochondrial dihydrofolate dehydrogenase, then leading to the proliferation of T cells.(2)A771726 inhibits the infiltration of synovitis by inhibiting p38 MAPK pathway.(3)Inhibiting bone erosion by acting on AHR-CRP signal.(4),19 It can be seen that MTX and LEF work through different targets to improve clinical symptoms.Inhibiting the expression of MMP-1, MMP-9, and other MMPs, thereby inhibiting the destruction of joint bone. Oral administration of LEF also has a fast absorption rate and it changes to A771726 in the liver. Continuous use of LEF may cause elevation of alanine aminotransferase, thus leading to drug-induced liver damage. LEF is cheaper than MTX, so it is considered that LEF is more worthy of being adopted in the clinical treatment of RA.Pharmacokinetic studies have found that after oral administration of MTX, the absorption rate is fast with a half-life of 7 to 10 hours, and a large amount of MTX accumulates in the liver. The continuous use of a small dose may cause the elevation of alanine aminotransferase, which leads to drug-induced liver damage.It is suggested that RCTs should be conducted with the following considerations in the future:(1)Invite experts of RCTs methodology and evidence-based medicine to participate in the design of standardized and rigorous high-quality RCTs research protocols;(2)Register in the Chinese Clinical Trial Registry or ClinicalTrials.gov prior to implementation of the RCTs.Data curation: Weiyu Qi, Yu Xia.Software: Weiyu Qi, Yu Xia.Supervision: Xin Li.Writing – original draft: Weiyu Qi.Writing – review & editing: Jian-Zhong Cao.