TLDc Domain-Containing Genes in Autism Spectrum Disorder: New Players in the Oxidative Stress Response.

Oxidative stress (OS) plays a key role in autism spectrum disorder (ASD), a neurodevelopmental disorder characterized by deficits in social communication, restricted interests, and repetitive behaviors. Recent evidence suggests that the TLDc [Tre2/Bub2/Cdc16 (TBC), lysin motif (LysM), domain catalytic] domain is a highly conserved motif present in proteins that are important players in the OS response and in neuroprotection. Human proteins sharing the TLDc domain include OXR1, TLDC1, NCOA7, TBC1D24, and C20ORF118. This study was aimed at understanding whether TLDc domain-containing mRNAs together with specific microRNAs (200b-3p and 32-5p) and long noncoding RNAs (TUG1), known to target TLDc proteins, contributed to regulate the OS response in ASD. Data showed a significant increase in the levels of OXR1 and TLDC1 mRNAs in peripheral blood mononuclear cells (PBMCs) of ASD children compared to their neurotypically developing (NTD) counterparts, along with an increase in TUG1 mRNA expression levels, suggesting its possible role in the regulation of TLDc proteins. A positive correlation between the expression of some TLDc mRNAs and the Childhood Autism Rating Scale (CARS) global score as well as inflammatory gene expression was found. In conclusion, our data suggest a novel biological pathway in the OS response of ASD subjects that deserves further exploration.

Polymorphic variants of IGF2BP3 and SENCR have an impact on predisposition and/or progression of Ewing sarcoma.

Ewing sarcoma (EWS), the second most common malignant bone tumor in children and adolescents, occurs abruptly without clear evidence of tumor history or progression. Previous association studies have identified some inherited variants associated with the risk of developing EWS but a common picture of the germline susceptibility to this tumor remains largely unclear. Here, we examine the association between thirty single nucleotide polymorphisms (SNPs) of the IGF2BP3, a gene that codes for an oncofetal RNA-binding protein demonstrated to be important for EWS patient's risk stratification, and five SNPs of SENCR, a long non-coding RNA shown to regulate IGF2BP3. An association between polymorphisms and EWS susceptibility was observed for three IGF2BP3 SNPs - rs112316332, rs13242065, rs12700421 - and for four SENCR SNPs - rs10893909, rs11221437, rs12420823, rs4526784 -. In addition, IGF2BP3 rs34033684 and SENCR rs10893909 variants increased the risk for female respect to male subgroup when carried together, while IGF2BP3 rs13242065 or rs76983703 variants reduced the probability of a disease later onset (> 14 years). Moreover, the absence of IGF2BP3 rs10488282 variant and the presence of rs199653 or rs35875486 variant were significantly associated with a worse survival in EWS patients with localized disease at diagnosis. Overall, our data provide the first evidence linking genetic variants of IGF2BP3 and its modulator SENCR to the risk of EWS development and to disease progression, thus supporting the concept that heritable factors can influence susceptibility to EWS and may help to predict patient prognosis.

Validation of circular dichroic spectroscopy of synthetic oligonucleotide PS2.M for K + concentration measurements.

The single-stranded synthetic oligonucleotide PS2.M is known to provide a basis for developing sensors since it tends to fold into structures called G-quadruplexes (G4) having characteristic topology and orientation with probabilities that depend on the chemical environment. The presence and concentration of cation species are among the key factors that determine the outcome of such a process. PS2.M and other aptamers have been used in several applications in conjunction with various probes, such as hemin, at the cost of increased technical complexity and applicability limitations. We instead validated the application limits of Circular Dichroic spectroscopy (CD) as only measurement method to assay PS2.M as K + sensor in a variety of solutions having different chemical complexity. The tested solutions range from simple NaCl and KCl solutions to chemically complex solutions like DMEM-Dulbecco's Modified Eagle Medium-which is widely used in a biological laboratory. PS2.M was also evaluated in solutions of KHCO 3 and D-ribose (K:D-rib), an antioxidant potassium compound, to compare its response to the simple KCl solution case. Our findings show that, within specific concentration applicability ranges, CD spectra can estimate the K + concentration in the examined water solutions even at high Na + concentrations with respect to K + and in the presence of antioxidant molecules. Supplementary Information: The online version supplementary material available at 10.1140/epjp/s13360-022-02581-2.

Non-Coding RNAs in the Transcriptional Network That Differentiates Skeletal Muscles of Sedentary from Long-Term Endurance- and Resistance-Trained Elderly.

In a previous study, the whole transcriptome of the vastus lateralis muscle from sedentary elderly and from age-matched athletes with an exceptional record of high-intensity, life-long exercise training was compared-the two groups representing the two extremes on a physical activity scale. Exercise training enabled the skeletal muscle to counteract age-related sarcopenia by inducing a wide range of adaptations, sustained by the expression of protein-coding genes involved in energy handling, proteostasis, cytoskeletal organization, inflammation control, and cellular senescence. Building on the previous study, we examined here the network of non-coding RNAs participating in the orchestration of gene expression and identified differentially expressed micro- and long-non-coding RNAs and some of their possible targets and roles. Unsupervised hierarchical clustering analyses of all non-coding RNAs were able to discriminate between sedentary and trained individuals, regardless of the exercise typology. Validated targets of differentially expressed miRNA were grouped by KEGG analysis, which pointed to functional areas involved in cell cycle, cytoskeletal control, longevity, and many signaling pathways, including AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR), which had been shown to be pivotal in the modulation of the effects of high-intensity, life-long exercise training. The analysis of differentially expressed long-non-coding RNAs identified transcriptional networks, involving lncRNAs, miRNAs and mRNAs, affecting processes in line with the beneficial role of exercise training.

Skeletal Muscle Gene Expression in Long-Term Endurance and Resistance Trained Elderly.

Physical exercise is deemed the most efficient way of counteracting the age-related decline of skeletal muscle. Here we report a transcriptional study by next-generation sequencing of vastus lateralis biopsies from elderly with a life-long high-level training practice (n = 9) and from age-matched sedentary subjects (n = 5). Unsupervised mixture distribution analysis was able to correctly categorize trained and untrained subjects, whereas it failed to discriminate between individuals who underwent a prevalent endurance (n = 5) or a prevalent resistance (n = 4) training, thus showing that the training mode was not relevant for sarcopenia prevention. KEGG analysis of transcripts showed that physical exercise affected a high number of metabolic and signaling pathways, in particular those related to energy handling and mitochondrial biogenesis, where AMPK and AKT-mTOR signaling pathways are both active and balance each other, concurring to the establishment of an insulin-sensitive phenotype and to the maintenance of a functional muscle mass. Other pathways affected by exercise training increased the efficiency of the proteostatic mechanisms, consolidated the cytoskeletal organization, lowered the inflammation level, and contrasted cellular senescence. This study on extraordinary individuals who trained at high level for at least thirty years suggests that aging processes and exercise training travel the same paths in the opposite direction.

Elevated maternal placental protein 13 serum levels at term of pregnancy in postpartum major hemorrhage (>1000 mLs). A prospective cohort study.

PROBLEM: To compare placental protein 13 (PP13) levels in the serum of women with primary postpartum hemorrhage (PPH) with a control population. METHODS: A prospective cohort study was conducted between May 2014 and May 2016 and included 435 pregnant women at term (38 weeks gestation) without any known risk factor and with normal labor. Multiples of median (MoM) were used to evaluate differences of the PP13 values between cases and controls. PP13 concentrations were adjusted for maternal and neonatal weight. Multivariable analysis was used to detect independent contribution of predictors of PPH. RESULTS: Fifteen had a major PPH >1000 mLs and represented the cases of the study. They were matched with 399 controls. Twenty-one patients who had a minor PPH (500-1000 mLs) were excluded. The mean observed rank in the PPH group was higher than that of controls (28.5 vs 13.5, P-value=.01). PP13 MoM values adjusted for maternal weight were higher than expected being 1.44±0.45 in PPH cases and 1.00±0.59 in controls (P-value .008). This difference was still significant even after adjustment for neonatal weight that represented a confounding variable. CONCLUSION: Higher PP13 levels are independently associated with major PPH >1000 mLs.

CD99 polymorphisms significantly influence the probability to develop Ewing sarcoma in earlier age and patient disease progression.

Ewing sarcoma (EWS), the second most common primary bone tumor in pediatric age, is known for its paucity of recurrent somatic abnormalities. Apart from the chimeric oncoprotein that derives from the fusion of EWS and FLI genes, recent genome-wide association studies have identified susceptibility variants near the EGR2 gene that regulate DNA binding of EWS-FLI. However, to induce transformation, EWS-FLI requires the presence of additional molecular events, including the expression of CD99, a cell surface molecule with critical relevance for the pathogenesis of EWS. High expression of CD99 is a common and distinctive feature of EWS cells, and it has largely been used for the differential diagnosis of the disease. The present study first links CD99 germline genetic variants to the susceptibility of EWS development and its progression. In particular, a panel of 25 single nucleotide polymorphisms has been genotyped in a case-control study. The CD99 rs311059 T variant was found to be significantly associated [P value = 0.0029; ORhet = 3.9 (95% CI 1.5-9.8) and ORhom = 5.3 (95% CI 1.2-23.7)] with EWS onset in patients less than 14 years old, while the CD99 rs312257-T was observed to be associated [P value = 0.0265; ORhet = 3.5 (95% CI 1.3-9.9)] with a reduced risk of relapse. Besides confirming the importance of CD99, our findings indicate that polymorphic variations in this gene may affect either development or progression of EWS, leading to further understanding of this cancer and development of better diagnostics/prognostics for children and adolescents with this devastating disease.

Performance of Circulating Placental Growth Factor as A Screening Marker for Diagnosis of Ovarian Endometriosis: A Pilot Study.

BACKGROUND: The aim of this study is to compare the circulating placental growth factor (PlGF) concentration in women with and without endometrioma to verify the performance of this marker to diagnose the disease. MATERIALS AND METHODS: In this case-control study, thirteen women with histological diagnosis of ovarian endometriosis were compared with women without endometriosis disease. PlGF plasma levels of endometriotic patients and controls were investigated using a fluorescence immunoassay technique. RESULTS: PlGF showed a direct correlation with body mass index (BMI) only in the control group (P=0.013). After adjustment for BMI values, PlGF median value in endometriosis group (14.7 pg/mL) resulted higher than in control group (13.8 pg/ mL, P=0.004). CONCLUSION: PlGF is a promising peripheral blood marker that can discriminate between patients with and without ovarian endometriosis.

Possible Gender-Related Modulation by the ROCK1 Gene in Colorectal Cancer Susceptibility.

AIM: In view of accumulating evidence supporting a pivotal role of the Rho/ROCK pathway in cancer, we investigated Rho-kinase polymorphisms as potential susceptibility factors in colorectal cancer (CRC) in a representative sample of the Italian population. METHODS: DNA obtained from the peripheral blood samples of 137 CRC patients and 141 healthy controls was genotyped for four ROCK1 (rs35996865; rs73963110; rs2127958; rs288980) and five ROCK2 (rs12692437; rs7563468; rs35768389; rs17463896; rs16857265) selected single nucleotide polymorphisms. RESULTS: None of the allelic variants of the nine selected markers was associated with the occurrence of CRC or with the development of regional lymph node metastasis. By contrast, the ROCK1 rs35996865 G variant allele was significantly more frequent in male patients (p = 0.028) than in the control group. CONCLUSION: This finding is, at present, the first that points to a possible gender-related modulation by the ROCK1 gene in CRC susceptibility.

Performance of peripheral (serum and molecular) blood markers for diagnosis of endometriosis.

PURPOSE: To quantify the mRNA levels of MMP-3, MMP-9, VEGF and Survivin in peripheral blood and the serum levels of CA-125 and Ca19-9 in women with and without endometriosis and to investigate the performance of these markers to differentiate between deep and ovarian endometriosis. METHODS: A case control study enrolled a series of 60 patients. Twenty controls have been matched with 20 cases of ovarian and 20 cases of deep endometriosis. Univariable and multivariable performance of serum CA125 and CA19-9, mRNA for Survivin, MMP9, MMP3 and VEGF genes have been evaluated by means of ROC curves and logistic regression, respectively. RESULTS: No difference in markers' concentration was detected between ovarian and deep endometriosis. In comparison with controls, serum CA125 and CA19 yielded the better sensitivity followed by mRNA for Survivin gene (81.5, 51.9 and 7.5% at 10% false positive rate, respectively). Multivariable estimated odds of endometriosis yielded a sensitivity of 87% at the same false positive rate. CONCLUSIONS: A combination of serum and molecular markers could allow a better diagnosis of endometriosis.

Gene expression in chorionic villous samples at 11 weeks of gestation in women who develop pre-eclampsia later in pregnancy: implications for screening.

OBJECTIVES: To determine the gene expression profile in chorionic villous samples (CVSs) of women destined to develop pre-eclampsia (PE). METHOD: Case-control study encompassing five women destined to develop PE [cases matched for gestational age with 30 controls]. We quantified mRNA expression on tissue samples from CVS of normal and PE patients. We then assessed mRNA expressions of cathepsin (CTSD), angiopoietin 2 (ANGPT2), interleukin 8, chemokine (C-X-C motif) ligand 10, neurokinin B (NKB), matrix metallopeptidase 9, major histocompatibility complex, class I, C (HLA-C)and human leukocyte antigen-G (HLA-G). Data were analyzed by nonparametric rank analysis. RESULTS: For all the mRNA species considered in this study, except CTSD and ANGPT2, all the mean observed ranks in the PE group were significantly altered compared with the rank expectation among controls. mRNA for NKB and HLA-C were the markers with the highest degree of aberration in PE, compared with those in controls. CONCLUSION: Our study has directly showed that gene expressions relating to trophoblastic cell invasion or utero-placental hemodynamic adaptation are altered in the first trimester trophoblasts that go on to develop PE later. These results posit the use of residual CVS as a possible screening method for PE.

Matrix metalloproteinase-3 mRNA: a promising peripheral blood marker for diagnosis of endometriosis.

BACKGROUND/AIMS: Endometriosis is an invasive disease. Its diagnosis depends on laparoscopy, which is traumatic and associated with potential complications. The aim of this study was to develop a rapid, reliable, and less invasive diagnostic test for endometriosis. We hypothesized that genes related to cell invasion would be transcriptionally upregulated in endometriosis, and tested whether blood levels of their transcripts might be used as biomarkers of endometriosis. METHODS: We used quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) to quantify the mRNA levels of vascular endothelial growth factor A (VEGFA), matrix metalloproteinase-3 (MMP-3), and MMP-9 in peripheral blood from 20 patients with mild/intermediate endometriosis, 20 patients with severe endometriosis and 20 endometriosis-free subjects. RESULTS: Our results indicate that circulating mRNA for MMP-3 is significantly higher in patients with endometriosis than in control patients, regardless of the degree of severity. Conversely, the level of circulating mRNA for VEGFA and MMP-9 did not distinguish patients from controls. CONCLUSION: MMP-3 mRNA is a promising peripheral blood marker that discriminates between patients with endometriosis and healthy subjects. Our results support the possibility of finding genes suitable for diagnostic qRT-PCR for endometriosis in peripheral blood and should be explored further.

Performance of messenger RNAs circulating in maternal blood in the prediction of preeclampsia at 10-14 weeks.

OBJECTIVE: The purpose of this study was to determine whether the combined distribution of a panel of cellular messenger RNA markers can detect preeclampsia long before onset. STUDY DESIGN: We compared blood at 10-14 weeks from 11 women who ultimately experienced preeclampsia with 88 matched control subjects. After multiples of the median conversion of all the markers, logistic regression was used to calculate the risk of the development of preeclampsia. RESULTS: Higher multiples of the median values than expected were found for endoglin, fms-related tyrosine kinase 1, and transforming growth factor-ß1. Lower multiples of the median values were found for placental growth factor and placental protein 13. Endoglin fms-related tyrosine kinase 1 and transforming growth factor-ß1 had the best discriminant power. Messenger RNA species provided independent contributions to the prediction of preeclampsia. In fact, 11 women with preeclampsia scored a median risk of 50% of experiencing preeclampsia. Control subjects scored a median risk of preeclampsia of 0.18%. The detection rate at a 5% false positive rate was 72.3%. CONCLUSION: The messenger RNA dosage in maternal blood would be a useful method for the calculation of the risk of the development of preeclampsia.

Identification of circulating placental mRNA in maternal blood of pregnancies affected with fetal congenital heart diseases at the second trimester of pregnancy: implications for early molecular screening.

OBJECTIVE: To investigate whether a significantly aberrant expression of circulating placental mRNA genes related with cardiogenesis can be detected at the second trimester of pregnancy. METHODS: The study was performed in two stages. First stage (development model group): match of 14 placental tissues at delivery of fetuses with congenital heart disease versus 20 controls. Second stage (validation model group): mRNA amplification of abnormal expressed genes in maternal blood samples from 26 women bearing a fetus with a congenital heart disease matched with 28 controls. RESULTS: We identified four functional categories of genes possibly involved in abnormal heart development: cardiac morphogenesis: tenascin, thioredoxin, salvador homolog 1 protein; extracellular matrix (ECM) and valvular tissue biosynthesis; placental-associated plasma protein, collagen, type I, alpha 2, fibulin-1, heparanase, procollagen-proline, 2-oxoglutarate 4-dioxygenase, alpha polypeptide II, Jumonji, AT rich interactive domain 1B RBP2-like; normal contractile activity: actinin, alpha 4, fascin homolog 1, actin-bundling protein; and congestive heart failure. CONCLUSION: Altered placental genetic expression was found at term delivery in affected fetuses. The aberration was also confirmed in maternal blood at the second trimester of women bearing a fetus with congenital heart disease. Sensitivity for the most aberrant genes ranged between 42% and 95% at a false positive rate (FPR) of 10%.

Gene expression in chorionic villous samples at 11 weeks of gestation in women who develop preeclampsia later in pregnancy: implications for screening.

OBJECTIVES: To determine the gene expression profile in chorionic villous samples (CVS) of women destined to develop preeclampsia. METHOD: cDNA microarray technology was employed. Ten singleton fetuses of women who subsequently developed preeclampsia where compared with a pool of 50 controls. The mRNA expression of some of the genes previously found to be up- or down-regulated were validated by RT-PCR in peripheral blood from 23 pregnant women at term affected with preeclampsia and 23 controls. RESULTS: Altered expression was found among several genes including those involved in invasion of human trophoblasts (Titin), in inflammatory stress (Lactotransferrin), endothelial aberration (Claudin 6), angiogenesis (Vasohibin 1), blood pressure control (Adducin 1). Also the peripheral blood from preeclampsia patients showed significant differences for all the genes studied. CONCLUSION: CVS show an aberrant gene profile prior to preeclampsia onset that may be predictive of the disease.

Gene expression in chorionic villous samples at 11 weeks' gestation from women destined to develop preeclampsia.

OBJECTIVE: To evaluate the direct alterations in mRNA expression among chorionic villous samples from 11 weeks' pregnant women who would develop preeclampsia (PE) later in the pregnancy. METHOD: Case-control study encompassing five women destined to develop PE [cases matched 1:5 for gestational age (GA) with 25 controls]. We quantified mRNA expression on tissue samples from chorionic villous sampling (CVS) of normal and PE patients. We then assessed mRNA expressions of vascular endothelial growth factor (VEGFA), VEGFA receptor 1 (Flt-1), endoglin (Eng), placental growth factor (PlGF), transforming growth factor-beta1 (TGF-beta1), heme oxygenase-1 (HO-1) and superoxide dismutase (SOD). Data were analyzed by nonparametric rank analysis. RESULTS: For all the mRNA species considered in this study, all the mean observed ranks in the PE group were significantly altered compared to the rank expectation among controls. mRNA for Eng and TGF-beta1 were the markers with the highest degree of aberration in PE, in respect to controls. The results are consistent with those already reported for the corresponding circulating proteins. mRNA for HO-1 and SOD were instead associated with the lowest aberration. CONCLUSION: It is assumed that the pathogenesis of PE is associated with pathophysiological alterations to trophoblasts in early gestation. Our study has directly proved that gene expressions relating to angiogenesis or oxidative stress are altered in the first trimester trophoblasts that go on to develop PE later. These results would put the basis for a possible screening method for PE by using residual CVS.

Apoptotic genes as potential markers of metastatic phenotype in human osteosarcoma cell lines.

Metastasis is the most frequent cause of death among patients with osteosarcoma. We have previously demonstrated in independent experiments that the forced expression of L/B/K ALP and CD99 in U-2 OS osteosarcoma cell lines markedly reduces the metastatic ability of these cancer cells. This behavior makes these cell lines a useful model to assess the intersection of multiple and independent gene expression signatures concerning the biological problem of dissemination. With the aim to characterize a common transcriptional profile reflecting the essential features of metastatic behavior, we employed cDNA microarrays to compare the gene expression profiles of L/B/K ALP- and CD99-transfected osteosarcoma clones showing low metastatic ability with those of osteosarcoma cell lines showing contrasting behavior. Changes in gene expression were validated by real-time PCR and immunohistochemistry in independent samples. In our study we identified several differentially expressed genes (GADD45alpha, VCP, DHX9, survivin, alpha-catulin, ARPC1B) related to growth arrest and apoptosis. Most of these genes are functionally related with the nuclear factor (NF)-kappaB cell survival pathway that appeared to be inhibited in the less malignant osteosarcoma cells. Hence, we propose the inhibition of the NF-kappaB pathway as a rational strategy for effective management of human osteosarcoma.

Caveolin-1 reduces osteosarcoma metastases by inhibiting c-Src activity and met signaling.

Caveolin-1 (Cav-1) is highly expressed in normal osteoblasts. This article reports that Cav-1 down-regulation is part of osteoblast transformation and osteosarcoma progression and validates its role as oncosuppressor in human osteosarcoma. A survey of 6-year follow-up indicates a better overall survival for osteosarcoma expressing a level of Cav-1 similar to osteoblasts. However, the majority of primary osteosarcoma shows significantly lower levels of Cav-1 than normal osteoblasts. Accordingly, Met-induced osteoblast transformation is associated with Cav-1 down-regulation. In vitro, osteosarcoma cell lines forced to overexpress Cav-1 show reduced malignancy with inhibited anchorage-independent growth, migration, and invasion. In vivo, Cav-1 overexpression abrogates the metastatic ability of osteosarcoma cells. c-Src and c-Met tyrosine kinases, which are activated in osteosarcoma, colocalize with Cav-1 and are inhibited on Cav-1 overexpression. Thus, Cav-1 behaves as an oncosuppressor in osteosarcoma. Altogether, data suggest that Cav-1 down-modulation might function as a permissive mechanism, which, by unleashing c-Src and Met signaling, enables osteosarcoma cells to invade neighboring tissues. These data strengthen the rationale to target c-Src family kinases and/or Met receptor to improve the extremely poor prognosis of metastatic osteosarcoma.

Comparative study of gene expression by cDNA microarray in human colorectal cancer tissues and normal mucosa.

The causative molecular pathways underlying the pathogenesis of colorectal cancer (CRC) need to be better characterized. The purpose of our study was to better understand the genetic mechanism of oncogenesis for human colorectal cancer and to identify new potential tumor markers of use in clinical practice. We used cDNA microarrays to compare gene expression profiles of colorectal biopsies from 25 CRC patients and 13 normal mucosa from adjacent non-cancerous tissues. Findings were validated by real-time PCR; in addition, western blotting and immunochemistry analysis were carried out as further confirmation of differential expression at a protein level. Comparing cancerous tissues with normal colonic mucosa we identified 584 known genes differentially expressed to a significant degree (p<0.001). Many of the transcripts that were more abundant in tumors than in non-neoplastic tissues appear to reflect important events for colon carcinogenesis. For example, a significant number of these genes serve as apoptotic inhibitors (e.g. BFAR, BIRC1, BIRC6). Furthermore, we observed the simultaneous up-regulation of HLA-E and the down-regulation of beta2-microglobulin; these genes strongly support a potential tumor escape strategy from immune surveillance in colon cancer tissues. Our study provides new gene candidates in the pathogenesis of human CRC disease. From our results we hypothesize that CRC cells escape immune surveillance through a specific gene expression alteration; moreover, over-expression of several survival genes seems to confer a more anti-apoptotic phenotype. These genes are involved in pathways not previously implicated in CRC pathogenesis and they may provide new targets for therapy.