Analysis of Expression of the ANG1, CaSR and FAK Proteins in Uterine Fibroids.

Understanding the molecular factors involved in the development of uterine myomas may result in the use of pharmacological drugs instead of aggressive surgical treatment. ANG1, CaSR, and FAK were examined in myoma and peripheral tissue samples taken from women after myoma surgery and in normal uterine muscle tissue samples taken from the control group. Tests were performed using tissue microarray immunohistochemistry. No statistically significant differences in ANG1 expression between the tissue of the myoma, the periphery, and the normal uterine muscle tissue of the control group were recorded. The CaSR value was reduced in the myoma and peripheral tissue and normal in the group of women without myomas. FAK expression was also lower in the myoma and periphery compared to the healthy uterine myometrium. Calcium supplementation could have an effect on stopping the growth of myomas.

Serum Selenium Concentration as a Potential Diagnostic Marker for Early-Stage Colorectal Cancer: A Comparative Study.

BACKGROUND Selenium deficiency is an established risk factor for colorectal cancer. The aim of the present study was to determine selenium levels in blood samples obtained from colorectal cancer patients compared with the levels of this element in the blood of patients who had undergone hernia repair and cholecystectomy. MATERIAL AND METHODS The study group consisted of 49 patients diagnosed with colorectal cancer at our institution. The comparison group consisted of 29 and 26 patients undergoing hernia repair and cholecystectomy, respectively. The histological staging level was evaluated on a 4-grade scale. Serum selenium concentration was quantified by inductively coupled mass spectrometry using methane to reduce polyatomic interference. RESULTS Colorectal cancer patients had significantly lower serum selenium concentration than the comparison patients (67.24±15.55 µg/L vs 78.81±12.93 µg/L; P<0.001), and selenium concentration was below the reference range in a high percentage of colorectal cancer patients. However, among the colorectal cancer patients, no significant difference in cancer grading was observed according to selenium concentration (P=0.235). Serum selenium concentration in the patients was evaluated on the basis of 5 independent variables (R=0.6250): age (P=0.011), number of leukocytes (P=0.010), family history of cancer (P=0.045), dietary supplements (P=0.023), and exposure to chemical factors (P=0.057). CONCLUSIONS This study supports findings from previous studies that low serum selenium levels are associated with colorectal cancer and that selenium deficiency may be a risk factor for colorectal cancer.

Estrogen Receptor (ER) and Progesterone Receptor (PgR) Expression in Endometrial Cancer-An Immunohistochemical Assessment.

Endometrial cancer (EC) is one of the most common types of cancer in Poland and worldwide. Many risk factors lead to the pathogenesis of this disease, such as lifestyle choices, BMI, the medicines used in breast cancer therapy, and Lynch syndrome. EC cells show the expression of estrogen receptors (ERs) and progesterone receptors (PgR). These receptors occur in multiple isoforms and have a significant influence on the operation of cells. The loss of ER and PgR expression is associated with a poor prognosis. We assessed tissue slides that were obtained from 103 women with EC diagnoses of various grades, stages, and histological types. In this study, we used computer image analyses to increase the objectivity of the assessment. We proved that, in the tissue of patients with high-grade (G3) EC, the expression of PgR is significantly lower than that in the tissues of patients with low-grade EC. We also observed that PgR is significantly expressed in EC with a low FIGO stage and in the endometroid type of EC (which rarely becomes malignant compared to serous type). The expression of ERb1 was lower in patients with EC at the IV FIGO stage than in patients with stage III EC. These findings confirm that the loss of ER and PgR expression is connected with a poor prognosis.

Immunohistochemical Expression of the SERPINA3 Protein in Uterine Fibroids.

BACKGROUND: SERPINA3 (α-1-antichymotrypsin, AACT, ACT) is produced by the liver and released into plasma in an anti-inflammatory response and plays a role as a modulator of extracellular matrix (ECM) by inhibiting serine proteases. Numerous studies proved an increased level of SERPINA3 in many types of cancer, which could be linked to SERPINA3's anti-apoptotic function. AIM: In the context of progressive ECM fibrosis during the development of uterine fibroids, which are one of the most common hypertrophic changes within the uterus, it is interesting to describe the level of SERPINA3 protein in this type of lesion and the surrounding tissues. METHODS: We used immunohistochemical staining of the SERPINA3 protein and compared the intensity of the signal between the myoma tissue and the surrounding normal tissue. RESULTS: We showed a surprising reduction in the amount of the SERPINA3 protein within uterine fibroids compared to surrounding tissues. CONCLUSION: This observation sheds new light on the role of this protein in the formation of proliferative changes and suggests that understanding the mechanism of its action may become the basis for the development of new diagnostic and therapeutic tools.

Prevalence of HPV and Assessing Type-Specific HPV Testing in Cervical High-Grade Squamous Intraepithelial Lesions in Poland.

The prevalence and distribution of oncogenic human papillomavirus (HPV) genotypes in women who underwent screening for cervical cancer in the Wielkopolska region, Poland, were assessed, and the correlation of genotypes with the histological results was evaluated. Cervical samples were collected from 2969 women for cervical cancer screening. Participants were screened by liquid-based cytology and HPV genotyping (n = 1654) and referred to colposcopy and punch biopsy (n = 616) if recommended. HPV genotypes 16, 31, 52, 66, 53, and 51 are the most frequent types in the studied population. Genotypes 16 and 31 account for nearly one-fifth of the infections of diagnosed HPV infections. HPV 16, 31, and 52 are found in nearly 80% of premalignant HSIL lesions (CIN 2 and CIN 3). That leads to the conclusion that vaccination programs should cover as many types of HPV as possible and shows the urgent need to vaccinate the Polish population with a 9-valent vaccine.

SERPINA3: Stimulator or Inhibitor of Pathological Changes.

SERPINA3, also called α-1-antichymotrypsin (AACT, ACT), is one of the inhibitors of serine proteases, one of which is cathepsin G. As an acute-phase protein secreted into the plasma by liver cells, it plays an important role in the anti-inflammatory response and antiviral response. Elevated levels of SERPINA3 have been observed in heart failure and neurological diseases such as Alzheimer's disease or Creutzfeldt-Jakob disease. Many studies have shown increased expression levels of the SERPINA3 gene in various types of cancer, such as glioblastoma, colorectal cancer, endometrial cancer, breast cancer, or melanoma. In this case, the SERPINA3 protein is associated with an antiapoptotic function implemented by adjusting the PI3K/AKT or MAPK/ERK 1/2 signal pathways. However, the functions of the SERPINA3 protein are still only partially understood, mainly in the context of cancerogenesis, so it seems necessary to summarize the available information and describe its mechanism of action. In particular, we sought to amass the existing body of research focusing on the description of the underlying mechanisms of various diseases not related to cancer. Our goal was to present an overview of the correct function of SERPINA3 as part of the defense system, which unfortunately easily becomes the "Fifth Column" and begins to support processes of destruction.

Expression of E4 Protein and HPV Major Capsid Protein (L1) as A Novel Combination in Squamous Intraepithelial Lesions.

We aim to describe the relationship between the immunohistochemical expression patterns of HPV E4 markers and the presence of HPV major capsid protein (L1) in cervical tissues obtained by biopsy of patients with abnormal liquid-based cytology (LBC) results, HR HPV infections, or clinically suspicious cervix. A novel HPV-encoded marker, SILgrade-E4 (XR-E4-1), and an HPV (clone K1H8) antibody were used to demonstrate the expression in terminally differentiated epithelial cells with a productive HPV infection in the material. A semiquantitative analysis was performed based on light microscope images. The level of E4 protein decreased with the disease severity. Patients with LSIL-CIN 1 and HSIL-CIN 2 diagnoses had significantly lower levels of HPV major capsid protein (L1) than those without confirmed cervical lesions. Our analysis confirms a higher incidence of L1 in patients with molecularly diagnosed HPV infections and excluded lesions of LSIL-CIN 1 and HSIL-CIN 2. Further studies on the novel biomarkers might help assess the chances of the remission of lesions such as LSIL-CIN 1 and HSIL-CIN 2. Higher levels of E4 protein and L1 may confirm a greater probability of the remission of lesions and incidental infections. In the cytological verification or HPV-dependent screening model, testing for E4 protein and L1 expression may indicate a group with a lower risk of progression of histopathologically diagnosed lesions.

Evaluation of CD10 expression as a diagnostic marker for colorectal cancer.

Aim: We aimed to determine the potential of CD10 as a marker for the early diagnosis of adenocarcinoma of the colon. Background: Adenocarcinoma is diagnosed in one out of 20 individuals in the USA and western European countries. Its prognosis and treatment depend largely on the severity of the disease at the time of diagnosis. Additional new biological markers are being sought that can help diagnose colon cancer at an early stage. One such marker present in both serum and tumor tissue is CD10. Methods: CD10 concentrations were tested by ELISA and immunohistochemistry in serum and tissue samples, respectively, from 113 patients diagnosed histopathologically and treated for adenocarcinoma of the colon. Additionally, the ROC curve with optimal cut-off point based on Youden's criterion was calculated for CD10. Results: Serum concentrations of CD10 and its tissue expression in patients diagnosed with adenocarcinoma of the colon correlate with cancer staging based on the Astler-Coller-Dukes classification. To ascertain the optimal cut-off point for CD10 as a predictor of belonging to the study group, ROC curve was prepared for CD10. Optimal cut-off point for CD10 was 0.57, with prediction of belonging to the study group for CD10 ≥ 0.57. Conclusion: CD10 can be a useful marker in the early diagnosis of adenocarcinoma of the colon.

Differential physiological and molecular response of barley genotypes to water deficit.

Changes in physiological parameters (relative water content (RWC), biomass, water use efficiency (WUE), net photosynthetic yield (PN) and quantum yield of PSII (Fv/Fm)), in proline and sugar content, and expression profile of genes reported to be associated with the barley response to water deficit, including LEA genes, NHX1, Hsdr4, BLT101 and genes encoding transcription factors (HvDREB1, HvABF1, HvABI5 and HvZIP1), were analyzed in seedlings of nine barley genotypes subjected to a progressive increase in water deficit. Seedlings of all genotypes wilted when the soil water content (SWC) declined from 65% (control conditions) to 10% (severe drought conditions), but recovered turgor within a few hours of re-watering. However, when severe drought conditions were prolonged for a week, large differences in survival characteristics were observed between genotypes after re-watering. Multivariate analysis of the changes in physiological and molecular characteristics allowed several different homogenous groups within the genotypes to be distinguished, depending on stress intensity. Furthermore, integration between the stress-response traits was found and was shown to vary depending on the genotype and the stress level. Based on analysis of physiological traits and survival characteristics, two barley genotypes with high adaptability to the stress conditions (cv. Saida and breeding line Cam/B1), and two with low adaptability (cv. Express and breeding line Harmal), were identified. In addition, only changes in expression of the genes HvZIP1, encoding a b-ZIP-type transcription factor, and Hsdr4, encoding a protein of unknown function, were shown to be linked with adaptability of barley to water deficit. In summary, physiological and molecular data revealed large, stress-level-dependent differences between the barley cultivars and breeding lines tested in their response to water deficit.

Mutant CAG repeats of Huntingtin transcript fold into hairpins, form nuclear foci and are targets for RNA interference.

The CAG repeat expansions that occur in translated regions of specific genes can cause human genetic disorders known as polyglutamine (poly-Q)-triggered diseases. Huntington's disease and spinobulbar muscular atrophy (SBMA) are examples of these diseases in which underlying mutations are localized near other trinucleotide repeats in the huntingtin (HTT) and androgen receptor (AR) genes, respectively. Mutant proteins that contain expanded polyglutamine tracts are well-known triggers of pathogenesis in poly-Q diseases, but a toxic role for mutant transcripts has also been proposed. To gain insight into the structural features of complex triplet repeats of HTT and AR transcripts, we determined their structures in vitro and showed the contribution of neighboring repeats to CAG repeat hairpin formation. We also demonstrated that the expanded transcript is retained in the nucleus of human HD fibroblasts and is colocalized with the MBNL1 protein. This suggests that the CAG repeats in the HTT mRNA adopt ds-like RNA conformations in vivo. The intracellular structure of the CAG repeat region of mutant HTT transcripts was not sufficiently stable to be protected from cleavage by an siRNA targeting the repeats and the silencing efficiency was higher for the mutant transcript than for its normal counterpart.

Trinucleotide repeat system for sequence specificity analysis of RNA structure probing reagents.

Chemical and enzymatic structural probes have been used for decades to obtain rapid and comprehensive information regarding the molecular architecture of various RNAs. Despite their widespread use, the sequence specificity of these RNA structural probing reagents has not yet been thoroughly characterized. In this study, we revisited the properties of commonly used structural probes such as Pb(II) ions, ribonuclease V1, ribonuclease T2, and the S1 and mung bean nucleases by testing them on highly regular triplet repeat sequences representing phosphodiester bonds with every possible combination of 3' and 5' adjacent nucleotides. We show that Pb(II) ions preferentially cleave after pyrimidines and that S1 nuclease possesses a previously overlooked specificity toward phosphodiester bonds following G residues. We also observed that mung bean nuclease shows a preference for cleaving ApN bonds and that RNase V1 mainly recognizes U residues in both single- and double-stranded RNAs. These data are important for accurate interpretation of the results of structure probing experiments and for assignment of the correct structure to individual RNA molecules. The triplet repeat transcript system described here may be considered as a reliable platform for determining the sequence specificity of other reagents used to probe RNA structure.

Trinucleotide repeats in human genome and exome.

Trinucleotide repeats (TNRs) are of interest in genetics because they are used as markers for tracing genotype-phenotype relations and because they are directly involved in numerous human genetic diseases. In this study, we searched the human genome reference sequence and annotated exons (exome) for the presence of uninterrupted triplet repeat tracts composed of six or more repeated units. A list of 32 448 TNRs and 878 TNR-containing genes was generated and is provided herein. We found that some triplet repeats, specifically CNG, are overrepresented, while CTT, ATC, AAC and AAT are underrepresented in exons. This observation suggests that the occurrence of TNRs in exons is not random, but undergoes positive or negative selective pressure. Additionally, TNR types strongly determine their localization in mRNA sections (ORF, UTRs). Most genes containing exon-overrepresented TNRs are associated with gene ontology-defined functions. Surprisingly, many groups of genes that contain TNR types coding for different homo-amino acid tracts associate with the same transcription-related GO categories. We propose that TNRs have potential to be functional genetic elements and that their variation may be involved in the regulation of many common phenotypes; as such, TNR polymorphisms should be considered a priority in association studies.

Structural diversity of triplet repeat RNAs.

Tandem repeats of various trinucleotide motifs are present in the human transcriptome, but the functions of these regular sequences, which likely depend on the structures they form, are still poorly understood. To gain new insight into the structural and functional properties of triplet repeats in RNA, we have performed a biochemical structural analysis of the complete set of triplet repeat transcripts, each composed of a single sequence repeated 17 times. We show that these transcripts fall into four structural classes. The repeated CAA, UUG, AAG, CUU, CCU, CCA, and UAA motifs did not form any higher order structure under any analyzed conditions. The CAU, CUA, UUA, AUG, and UAG repeats are ordered according to their increasing tendency to form semistable hairpins. The repeated CGA, CGU, and all CNG motifs form fairly stable hairpins, whereas AGG and UGG repeats fold into stable G-quadruplexes. The triplet repeats that formed the most stable structures were characterized further by biophysical methods. UV-monitored structure melting revealed that CGG and CCG repeats form, respectively, the most and least stable hairpins of all CNG repeats. Circular dichroism spectra showed that the AGG and UGG repeat quadruplexes are formed by parallel RNA strands. Furthermore, we demonstrated that the different susceptibility of various triplet repeat transcripts to serum nucleases can be explained by the sequence and structural features of the tested RNAs. The results of this study provide a comprehensive structural foundation for the functional analysis of triplet repeats in transcripts.

Ribonuclease dicer cleaves triplet repeat hairpins into shorter repeats that silence specific targets.

Ribonuclease Dicer functions in cells to excise microRNAs from their precursors and process long double-stranded RNAs into short interfering RNAs. We show that transcripts containing long hairpin structures composed of CNG repeats are another class of Dicer targets. The cellular levels of transcripts from mutant genes involved in triplet repeat expansion diseases such as myotonic dystrophy type 1, Huntington's disease, and spinocerebellar ataxia type 1 are under Dicer control. The Dicer-induced downregulation of the mutant transcript in myotonic dystrophy cells is accompanied by the downregulation of transcripts containing long complementary repeats. Short CUG repeats generated from long repeat hairpins act as siRNAs and use the RNA interference pathway to trigger the downstream silencing effects. We demonstrate that synthetic oligonucleotides composed of repeats are highly specific in the silencing of mutant transcripts containing complementary repeats and may be considered as potential therapeutic agents.

Facile FMR1 mRNA structure regulation by interruptions in CGG repeats.

RNA metabolism is a major contributor to the pathogenesis of clinical disorders associated with premutation size alleles of the fragile X mental retardation (FMR1) gene. Herein, we determined the structural properties of numerous FMR1 transcripts harboring different numbers of both CGG repeats and AGG interruptions. The stability of hairpins formed by uninterrupted repeat-containing transcripts increased with the lengthening of the repeat tract. Even a single AGG interruption in the repeated sequence dramatically changed the folding of the 5'UTR fragments, typically resulting in branched hairpin structures. Transcripts containing different lengths of CGG repeats, but sharing a common AGG pattern, adopted similar types of secondary structures. We postulate that interruption-dependent structure variants of the FMR1 mRNA contribute to the phenotype diversity, observed in premutation carriers.

Structures of trinucleotide repeats in human transcripts and their functional implications.

Among the goals of RNA structural and functional genomics is determining structures and establishing the functions of a rich repertoire of simple sequence repeats in transcripts. These repeats are present in transcripts from their 'birth' in the nucleus to their 'death' in cytoplasm and have the potential of being involved in many steps of RNA regulation. The knowledge of their structural features and functional roles will also shed more light on the postulated mechanisms of RNA pathogenesis in a growing list of neurological diseases caused by simple sequence repeat expansions. Here, we discuss several different lines of research to support the hypothesis that the mechanism of RNA pathogenesis may be a more common phenomenon triggered or modulated also by abundant long normal repeats. We propose structures of the repeat regions in transcripts of genes involved in Triplet Repeat Expansion Diseases. We have classified the polymorphic repeat alleles of these genes according to their ability to form hairpin structures in transcripts, and describe the distribution of different structural forms of the repeats in the human population. We have also reported the results of a systematic survey of the human transcriptome to identify mRNAs containing triplet repeats and to classify them according to structural and functional criteria. Based on this knowledge, we discuss the putative wider role of triplet repeat RNA hairpins in human diseases. A hypothetical model is proposed in which long normal RNA hairpins formed by the repeats may also be involved in pathogenesis.

RNA structure of trinucleotide repeats associated with human neurological diseases.

The tandem repeats of trinucleotide sequences are present in many human genes and their expansion in specific genes causes a number of hereditary neurological disorders. The normal function of triplet repeats in transcripts is barely known and the role of expanded RNA repeats in the pathogenesis of Triplet Repeat Expansion Diseases needs to be more fully elucidated. Here we have described the structures formed by transcripts composed of AAG, CAG, CCG, CGG and CUG repeats, which were determined by chemical and enzymatic structure probing. With the exception of the repeated AAG motif, all studied repeats form hairpin structures and these hairpins show several alternative alignments. We have determined the molecular architectures of these co-existing hairpin structures by using transcripts with GC-clamps which imposed single alignments of hairpins. We have provided experimental evidence that CCUG repeats implicated in myotonic dystrophy type 2 also form hairpin structures with properties similar to that composed of the CUG repeats.

[Clinical picture of patients with Huntington's disease in relation to the number of trinucleotide CAG repeats in IT-15 gene]. / Obraz kliniczny pacjentów z choroba Huntingtona w zestawieniu z liczba powtórzen trójnukleotydu CAG w genie IT-15.

Huntington's disease (HD) is a neurological condition of progressive course that results from abnormally increased number of CAG repeats within IT-15 gene, coding for huntington. The main symptoms consist of choraetic movements, dementia, and characteropathy. The aim of the present study was to search for possible correlation between the age of the onset of HD, time from the onset, clinical status of the patients, and CAG repeats number. Ten patients were studied altogether. Modified UHDRS (MUHDRS) was applied for the estimation of patients' clinical status. The number of CAG repeats in examination of the IT-15 gene was determined by polymerase chain reaction (PCR) and separation of radioisotope labelled PCR product against DNA size marker in polyacrylamide gel. A negative significant correlation was found between the CAG repeats number and the disease onset age (r = -0.67; p < 0.05) and MUHDRS score (r = 0.75; p < 0.05), as well. Negative significant correlation between time from the onset and MUHDRS score (r = -0.95; p < 0.05) and negative correlation between summarised: time from the onset and CAG number on the one site and MUHDRS on the other (p = -0.91) were found, as well. Our findings indicate an interdependence between CAG repeats number within the IT-15 gene, the course of the disease and the clinical status of HD patients.