Weak Expression Research Articles

Efficient stereoselective hydroxylation of deoxycholic acid by the robust whole-cell cytochrome P450 CYP107D1 biocatalyst

Deoxycholic acid (DCA) has been authorized by the Federal Drug Agency for cosmetic reduction of redundant submental fat. The hydroxylated product (6β-OH DCA) was developed to improve the solubility and pharmaceutic properties of DCA for further applications. Herein, a combinatorial catalytic strategy was applied to construct a powerful Cytochrome P450 biocatalyst (CYP107D1, OleP) to convert DCA to 6β-OH DCA. Firstly, the weak expression of OleP was significantly improved using pRSFDuet-1 plasmid in the E. coli C41 (DE3) strain. Next, the supply of heme was enhanced by the moderate overexpression of crucial genes in the heme biosynthetic pathway. In addition, a new biosensor was developed to select the appropriate redox partner. Furthermore, a cost-effective whole-cell catalytic system was constructed, resulting in the highest reported conversion rate of 6β-OH DCA (from 4.8% to 99.1%). The combinatorial catalytic strategies applied in this study provide an efficient method to synthesize high-value-added hydroxylated compounds by P450s.

The impact of the association between Val16Ala-SOD2 SNP and SOD2 immunohistochemistry expression in the prognosis of patients with esophageal cancer

IntroductionEsophageal cancer is an extensive public health issue worldwide, warranting the search for biomarkers related to its risk and progression. Previous studies have indicated an association between Val16AlaSOD2 single nucleotide polymorphism in the gene encoding the enzyme superoxide dismutase 2 and esophageal cancer. However, further investigations are needed to clarify its role in disease risk and progression. ObjectiveTo investigate the role of Val16AlaSOD2-SNP in esophageal cancer progression and in the survival of patients MethodsTumor samples were utilized for Val16Ala-SNP genotyping, while SOD2 expression levels in tissue were assessed using immunohistochemistry. A SOD2 Val16Ala-SNP database was used to obtain information on the genotype of healthy individuals. Risk and overall survival analyzes were performed. ResultsThe Val16Ala SNP was associated with an increased risk of esophageal cancer (RR 2.18, 95%CI 1.23–3.86), regardless of age and gender, but did not have a significant effect on patient survival. In contrast, weak SOD2 expression demonstrated a significantly associated with poor overall survival after treatment, independent of other clinicopathological variables (HR, 0.41; 95% CI, 0.22–0.79 P = 0.007). ConclusionsVal16Ala SNP was positively associated with esophageal cancer, and the expression of SOD2 was an independent prognostic marker.

Divergent Lineage Markers in Anaplastic Thyroid Carcinoma.

Anaplastic thyroid carcinoma (ATC) often results from dedifferentiation of differentiated thyroid carcinoma (DTC), and the diagnosis is not difficult, as the tumor is seen to progress from a recognized DTC. However, in some cases, the diagnosis based on biopsy of limited tissue or resection of a completely undifferentiated tumor relies on immunohistochemical biomarkers and is usually a diagnosis of exclusion. To examine the biomarker profile of ATC and to determine whether divergent lineage markers can complicate this process, we examined the expression of a number of biomarkers in a series of ATCs. Cases retrieved from the department laboratory information system were included if there was evidence of an accurate diagnosis based on the presence of a coexisting or antecedent DTC or in cases where the immunoprofile was consistent with thyroid origin in a non-equivocal clinical setting. Questionable cases were excluded. We identified 36 cases for analysis. Tissue sections were stained for PAX8, TTF1, BRAFV600E, NRASQ61R, TRK, and p53, as well as p40, CDX2, SATB2, GATA3, CD117, CD163, SALL4, SMARCA4, PRAME, SOX10, ERG and HEPPAR1. As expected, all 36 ATCs were negative for TTF1 except for one showing focal, weak expression. Thirteen expressed PAX8 with variable intensity. BRAFV600E was positive in 10/34 tumors and equivocal in 3; NRASQ61R was positive in 12, and TRK was positive in 1 case. Staining for p53 was diffusely positive in 14 and completely negative in 19, with only 3 cases showing a wild-type pattern. We found aberrant expression of GATA3 in 11/36 cases, SATB2 in 8/36, CD117 in 2/35, and SALL4 in 1/30. CD163 expression was identified in tumor cells in 10/30 cases with variable intensity; in the other tumors, interpretation was obscured by abundant histiocytes. P40 was positive in 5 cases with squamoid morphology. CDX2 was negative in 35 tested cases. PRAME was identified in 1 of 33 cases. Stains for SOX10, ERG, and HEPPAR1 were negative in 33 cases. Twenty tested cases showed retained SMARCA4 expression. We conclude that ATCs express a number of divergent lineage markers that can cause diagnostic dilemmas, as they are also features of other tumors in the differential diagnosis of high-grade midline neck malignancies.

Th17 cells target the metabolic miR-142-5p-succinate dehydrogenase subunit C/D (SDHC/SDHD) axis, promoting invasiveness and progression of cervical cancers.

During cervical carcinogenesis, T-helper (Th)-17 cells accumulate in the peripheral blood and tumor tissues of cancer patients. We previously demonstrated that Th17 cells are associated with therapy resistance as well as cervical cancer metastases and relapse; however, the underlying Th17-driven mechanisms are not fully understood. Here, using microarrays, we found that Th17 cells induced an epithelial-to-mesenchymal transition (EMT) phenotype of cervical cancer cells and promoted migration and invasion of 2D cultures and 3D spheroids via induction of microRNA miR-142-5p. As the responsible mechanism, we identified the subunits C and D of the succinate dehydrogenase (SDH) complex as new targets of miR-142-5p and provided evidence that Th17-miR-142-5p-dependent reduced expression of SDHC and SDHD mediated enhanced migration and invasion of cancer cells using small interfering RNAs (siRNAs) for SDHC and SDHD, and miR-142-5p inhibitors. Consistently, patients exhibited high levels of succinate in their serum associated with lymph node metastases and diminished expression of SDHD in patient biopsies correlated with increased numbers of Th17 cells. Correspondingly, a combination of weak or negative SDHD expression and a ratio of Th17/CD4+ T cells > 43.90% in situ was associated with reduced recurrence-free survival. In summary, we unraveled a previously unknown molecular mechanism by which Th17 cells promote cervical cancer progression and suggest evaluation of Th17 cells as a potential target for immunotherapy in cervical cancer.

Orbitally-forced meridional shifts of the westerlies modulated the hydroclimate of northeast China during the late Eocene

The mid-latitude westerlies are a fundamental component of the global climate system. There has been extensive research about how meridional shifts of the westerlies influenced the wet-dry paleoclimate regimes of mid-latitude Asia during the Quaternary. However, how the westerlies operated in much older geological times, such as the late Eocene, particularly on an orbital time scale, is not fully understood. Here, we conducted detailed cyclostratigraphic and palynological analyses on a late Eocene (41.2–37.8 Ma) mudstone-shale sedimentary sequence from the Fushun Basin in northeast China, which uniquely records orbitally-forced meridional shifts of the westerlies. Cyclostratigraphic analyses reveal that the mudstone-shale rhythms exhibit a prominent short eccentricity cycle, with a modest expression of obliquity cycles and a weak expression of precession cycles. The palynological results show that the vegetation experienced frequent alternations between semi-desert and swamp forest, and the climate exhibited periodic oscillations between warm-dry and cold-wet climates. We propose that a combination of eccentricity-modulated low-latitude summer insolation and obliquity-forced high-latitude incipient ice sheet and meridional temperature gradient variations drove cyclical vegetation and hydroclimate fluctuations in northeast China during the late Eocene by regulating the intensity and displacement of the westerlies and the subtropical high. Our results imply that continued poleward movement of the westerlies and the subtropical high will lead to increased aridification along the southern margin of the westerlies under a continuous global warming scenario.

Expression analysis suggests that DNMT3L is required for oocyte de novo DNA methylation only in Muridae and Cricetidae rodents

BackgroundDuring early mammalian development, DNA methylation undergoes two waves of reprogramming, enabling transitions between somatic cells, oocyte and embryo. The first wave of de novo DNA methylation establishment occurs in oocytes. Its molecular mechanisms have been studied in mouse, a classical mammalian model. Current model describes DNA methyltransferase 3A (DNMT3A) and its cofactor DNMT3L as two essential factors for oocyte DNA methylation—the ablation of either leads to nearly complete abrogation of DNA methylation. However, DNMT3L is not expressed in human oocytes, suggesting that the mechanism uncovered in mouse is not universal across mammals.ResultsWe analysed available RNA-seq data sets from oocytes of multiple mammals, including our novel data sets of several rodent species, and revealed that Dnmt3l is expressed only in the oocytes of mouse, rat and golden hamster, and at a low level in guinea pigs. We identified a specific promoter sequence recognised by an oocyte transcription factor complex associated with strong Dnmt3l activity and demonstrated that it emerged in the rodent clade Eumuroida, comprising the families Muridae (mice, rats, gerbils) and Cricetidae (hamsters). In addition, an evolutionarily novel promoter emerged in the guinea pig, driving weak Dnmt3l expression, likely without functional relevance. Therefore, Dnmt3l is expressed and consequently plays a role in oocyte de novo DNA methylation only in a small number of rodent species, instead of being an essential pan-mammalian factor. In contrast to somatic cells, where catalytically inactive DNMT3B interacts with DNMT3A, forming a heterotetramer, we did not find evidence for the expression of such inactive Dnmt3b isoforms in the oocytes of the tested species.ConclusionsThe analysis of RNA-seq data and genomic sequences revealed that DNMT3L is likely to play a role in oocytes de novo DNA methylation only in mice, rats, gerbils and hamsters. The mechanism governing de novo DNA methylation in the oocytes of most mammalian species, including humans, occurs through a yet unknown mechanism that differs from the current model discovered in mouse.

Expression of Melan-A in cutaneous granular cell tumours: a diagnostic pitfall

Morphological overlap exists between cutaneous granular cell tumours (GCT) and malignant melanoma, with the melanocyte-specific markers HMB45 and Melan-A commonly used to support the diagnosis of melanoma. We recently encountered several cases of GCT in our practice showing strong expression of Melan-A. The aim of this study was to establish the prevalence of positive immunohistochemical staining for Melan-A and HMB45 in a series of unequivocal GCTs. We also aimed to assess the prevalence of staining for PRAME (PReferentially expressed Antigen in MElanoma), a marker expressed in >80% of primary melanomas as well as many non-melanocytic tumours. A total of 20 cutaneous/subcutaneous GCTs were evaluated using Melan-A, HMB45 and PRAME immunohistochemistry. Staining for Melan-A and HMB45 was scored using a semiquantitative scale from 0 (absent) to 3+ (staining present in >50% of tumour cells). PRAME expression was recorded as either positive (>75% of cell nuclei staining) or negative. Melan-A expression was observed in four GCTs (20%), with strong and diffuse (3+) staining seen in two cases (10%), both from anogenital areas. Weak patchy nuclear PRAME expression was seen in every case, interpreted to be negative. HMB45 was also negative in all cases (100%). Our study demonstrates that Melan-A expression can be strong and diffuse in a subset of otherwise unequivocal cutaneous GCTs, which may cause diagnostic confusion with malignant melanoma. HMB45 and PRAME did not stain any of the GCTs in our series.

The Biological Characteristics of Eutopic and Ectopic Endometrial Progenitor Cells in Endometriosis.

The aim of this study was to identify the biological characteristics and potential roles of endometrial progenitor cells in the pathogenesis of endometriosis. It is generally believed that progenitor cells in human endometrium are responsible for rapid endometrial regeneration. However, the biological characteristics and potential roles of the paired eutopic and ectopic endometrial progenitor cells in endometriosis remain unclear. This study intends to isolate the epithelial progenitor (EP) cells and endometrial mesenchymal stem cells (eMSCs) from the eutopic and ectopic endometria from endometriosis patients, further to reveal their features and functions respectively. The distributions of EP cells and eMSCs and the expression of steroid hormone receptors in the endometrium and endometriotic tissues were assessed by immunohistochemistry. EP cells and eMSCs were sorted from paired eutopic and ectopic endometria with epithelial cell adhesion molecule (EpCAM) magnetic beads. The clonogenicity, cell viability after being treated with estradiol and progesterone, and cell markers expression were evaluated with colony forming on Matrigel, CCK-8 and immunofluorescence staining, respectively. The differentially expressed genes (DEGs) were further identified with RNA sequencing. SSEA-1- and PDGFRβ-positive cells were distributed in the epithelial and stromal layers. The ERβ staining was much more intense in endometriotic tissues, but PR expression was almost absent. The ectopic EP cells exhibit strong clonogenicity and ERβ expression but weak PR expression, leading to progesterone resistance. There are 12604 and 13242 DEGs revealed by RNA sequencing between eutopic and ectopic EP cells or eMSCs. GO and KEGG analyses revealed that the functions and pathways of DEGs enriched in cellular energy metabolism and regulation of the immune response, respectively. Additionally, ERβ targets were mainly enriched in ectopic EP cells. Both EP cells and eMSCs may engage in ectopic lesion formation in endometriosis by modifying the metabolic mode and immune tolerance. These data not only help to understand the molecular mechanism of endometriosis but also could potentially contribute to the discovery of therapeutic targets for endometriosis.

New Insights on the Progesterone (P4) and PGRMC1/NENF Complex Interactions in Colorectal Cancer Progression.

The literature data regarding the risk of colorectal cancer (CRC) in the context of hormone therapy (HT), including both estrogen-progestogen combinations and estrogen alone, are inconclusive. The precise relationship underlying the action of progesterone (P4) and progesterone receptors in CRC has yet to be determined. We characterized the expression profiles of both nuclear and membrane progesterone receptors and their potential cofactors in CRC tissues. Additionally, we analyzed the P4 and NENF treatment effects on the cell proliferation and invasion of DLD-1 and HT-29 colorectal cancer cells. We observed a weak expression of the nuclear P4 receptor (PGR), but an abundant expression of the P4 receptor membrane component 1 (PGRMC1) and neuron-derived neurotrophic factor (NENF) in the CRC tissues. P4 treatment stimulated the proliferation of the DLD-1 and HT-29 CRC cells. The co-treatment of P4 and NENF significantly increased the invasiveness of the DLD-1 and HT-29 cells. A functional analysis revealed that these effects were dependent on PGRMC1. AN immunocytochemical analysis demonstrated a cytoplasmic co-localization of PGRMC1 and NENF in the CRC cells. Moreover, the concentration of serum NENF was significantly higher in CRC patients, and P4 treatment significantly increased the release of NENF in the DLD-1 cells. P4 or NENF treatment also significantly increased the IL-8 release in the DLD-1 cells. Our data may provide novel insights into the action of P4 and PGRMC1/NENF in CRC progression, where NENF may act as a potential PGRMC1 co-activator in non-classical P4 signaling. Furthermore, NENF, as a secreted protein, potentially could serve as a promising circulating biomarker candidate for distinguishing between colorectal cancer patients and healthy individuals, although large-scale extensive studies are needed to establish this.

Control of rice ratooning ability by a nucleoredoxin that inhibits histidine kinase dimerization to attenuate cytokinin signaling in axillary buds

Rice ratooning, the fast outgrowth of dormant buds on stubble, is an important cropping practice in rice production. However, the low ratooning ability (RA) of most rice varieties restricts the application of this cost-efficient system, and the genetic basis of RA remains unknown. In this study, we dissected the genetic architecture of RA by a genome-wide association study in a natural rice population. Rice ratooning ability 3 (RRA3), encoding a hitherto not characterized nucleoredoxin involved in reduction of disulfide bonds, was identified as the causal gene of a major locus controlling RA. Overexpression of RRA3 in rice significantly accelerated leaf senescence and reduced RA, whereas knockout of RRA3 significantly delayed leaf senescence and increased RA and ratoon yield. We demonstrated that RRA3 interacts with Oryza sativa histidine kinase 4 (OHK4), a cytokinin receptor, and inhibits the dimerization of OHK4 through disulfide bond reduction. This inhibition ultimately led to decreased cytokinin signaling and reduced RA. In addition, variations in the RRA3 promoter were identified to be associated with RA. Introgression of a superior haplotype with weak expression of RRA3 into the elite rice variety Guichao 2 significantly increased RA and ratoon yield by 23.8%. Collectively, this study not only uncovers an undocumented regulatory mechanism of cytokinin signaling through de-dimerization of a histidine kinase receptor—but also provides an eximious gene with promising value for ratoon rice breeding.

FUT1 variants responsible for Bombay or para-Bombay phenotypes in a database

Rare individuals with Bombay and para-Bombay phenotypes lack or have weak expression of the ABO(H) antigens on surface of red blood cells due to no or very weak H-type α(1,2)fucosyltransferase activity encoded by FUT1. These phenotypes are clinically important because subjects with these phenotypes can only accept transfusions of autologous blood or blood from subjects with the same phenotypes due to the anti-H antibody. To survey FUT1 alleles involved in Bombay and para-Bombay phenotypes, the effect of 22 uncharacterized nonsynonymous SNPs in the Erythrogene database on the α(1,2)fucosyltransferase activity were examined by transient expression studies and in silico analysis using four different online software tools. Two nonfunctional alleles (FUT1 with c.503C>G and c.749G>C) and one weakly functional allele (with c.799T>C) were identified in transient expression studies, while the software predicted that the proteins encoded by more alleles including these would be impaired. Because both nonfunctional FUT1 alleles appear to link to the nonsecretor alleles, homozygotes of these alleles would be of the Bombay phenotype. The present results suggest that functional assays are useful for characterization of nonsynonymous SNPs of FUT1 when their phenotypes are not available.

ABO and Rh Blood Group Antigens and Natural Anti-A and ANTI-B Antibodies in the Neonates

ABO blood group is determined by the presence or absence of A and B antigens on the surface of RBC and of anti-A and anti-B antibodies in the serum. The relatively weak expression of A and B antigens in newborns due to their developing immune systems poses challenges in accurately detecting naturally occurring IgM antibodies against these antigens. This difficulty in immunoserological methods contributes to the potential for errors in determining the blood groups of newborns. Despite this, the Rh antigen expression in newborns remains comparable to that in adults. Nonetheless, various factors contribute to diverse blood typing results in newborns, including the utilization of alternative testing methods. The complexity of blood typing is magnified when using samples from the umbilical vein. Furthermore, compared to adults, the exploration of ABO antigen expression in newborns is limited, and the identification of specific subgroups such as A1 and A2 is even rarer. This underscores the need for standardized testing procedures and further research to enhance our understanding of antigen expression patterns in newborns. Based on the aforementioned details, the primary objective of our study was to delve into specific aspects related to blood group characterization in newborns. This encompassed exploring the expression of A, B, AB, and D antigens on the surface of red blood cells (RBCs) and detecting anti-A and anti-B antibodies in the plasma of newborns. These analyses were conducted using samples obtained from the heels of 208 newborns and were typed by forward and reverse blood typing methods with monoclonal antibodies and srandart erythrocytes. The distribution of phenotypic groups within the ABO system among the newborns was not uniform. The r allele was identified with the highest frequency in the analyzed samples (0.6), while the prevalence of the p allele significantly lags at 0.3. The q allele has the lowest frequency (0.1). In our study, we propose that for the majority of cases (43.94±3.5%) among the studied newborns, there was an absence of naturally occurring anti-A and anti-B antibodies (n=87). In a specific scenario, within the O(I) blood group nwborns, partial synthesis of these antibodies was detected in 14.14±2.4% (n=28). Meanwhile, 41.92±3.5% of the newborns in our study exhibited natural antibodies similar to those found in adults. We didn’t find any difficulties in typing the Rh blood group antigens in the newborns. In conclusion, our study's findings indicate that newborns, in certain instances, exhibit strongly pronounced natural anti-A and anti-B antibodies within the ABO system. However, in the majority of cases, these antibodies are not evident. Majority of cases erythrocyte A and B antigens were weakly expressed and for detecting these images optic microscopes were used.

Somatostatin Receptor Expression in Lung Neuroendocrine Tumors: An Analysis of DOTATATE PET Scans.

Somatostatin receptor (SSTR) expression in metastatic lung neuroendocrine tumors (NETs) has not been well characterized using PET imaging. Understanding the degree and uniformity of SSTR expression is important to establish the role of SSTR-targeted treatments in lung NETs. Methods: A retrospective institutional review of patients with metastatic lung NETs who underwent DOTATATE PET imaging from March 2017 to February 2023 was performed. Results: In total, 48 patients with metastatic lung NETs who underwent 68Ga- or 64Cu-DOTATATE PET imaging were identified. Four had completely negative SSTR expression, and 10 had very weak expression (less than in a normal liver). Among the remaining 34 patients, 21 had uniformly positive DOTATATE PET scans, and 13 had heterogeneous expression. Only 44% had uniformly positive receptor expression, identifying them as candidates for peptide receptor radionuclide therapy. Conclusion: Most metastatic lung NETs lack uniform SSTR expression and are thus suboptimal candidates for SSTR-targeted therapy. SSTR imaging in lung NETs should be evaluated carefully for uniformity of expression.

Immunohistochemical expression of SOX2 in OKC and ameloblastoma: A comparative study.

Odontogenic, non-inflammatory maxillofacial cysts and tumours vary greatly in their ability to grow and cause local tissue destruction. Despite their common embryologic origin, the biologic mechanisms responsible for this diverse array of clinical behaviour are largely unknown. Unfortunately, even with accurate tissue diagnosis and appropriate surgical management, these tumours have relatively high recurrence rates. While this may be related to surgical technique, it may also be due to intrinsic tumour biology. SOX2 is differentially expressed in odontogenic cysts and tumours, which has an impact over patient prognosis. This could be related to their diverse cells of origin or stages of histogenesis. SOX2 is expressed in OKC and ameloblastoma, and in this study, we look forward to find altered levels and intensity of SOX2 in the above-mentioned lesions. To profile the expression of SOX2 in odontogenic keratocyst (OKC) and ameloblastomaTo compare the intensity of these lesions, analyse their intrinsic feature and predict their recurrence. Histopathologically diagnosed cases of OKC and ameloblastoma will be selected (n = 40). Paraffin-embedded, formalin-fixed sections of these lesions will be stained for SOX2 marker using a standard immunohistochemical technique. Positive control will be taken as oral squamous cell carcinoma and negative control will be taken as normal oral mucosa. A comparison between the stained cell types in odontogenic keratocyst and ameloblastoma revealed statistically significant differences. The immunoreactivity scores of SOX2 were analysed in both groups. The results indicated that 45% of OKC cases exhibited strongly positive reactivity, while 65% of ameloblastoma cases were negative. Statistical analysis demonstrated highly significant differences in the frequency of SOX2 expression between the two groups, with a higher frequency of negative expression in ameloblastoma. Stem cell markers have been observed in these lesions, suggesting the acquisition of stem-like properties by tumour cells, which can affect patient prognosis. Specifically, the marker SOX2 shows differential expression in odontogenic cysts and tumours. High expression of SOX2 in OKC indicates the presence of stem cells with significant self-renewal and proliferative properties, potentially signifying neoplastic behaviour. In contrast, weak or absent expression of SOX2 in ameloblastoma suggests different molecular pathways involved in its neoplastic behaviour.

Genetic Features of Soils on Dense Parent Rocks of the Donetsk Ridge

The present study investigates morphological, physical and chemical properties of Chernozems at dense parent rocks. The research was carried out on the protected areas native soils of the north-west of the Rostov region. This territory is representing eastern slopes of the Donetsk Ridge, which predetermines the dense rocks release to the surface. It has been found that morphological, physical and chemical properties of Chernozems on dense rocks have emphasized their originality and uniqueness, and at the same time clearly have indicate their classification as Chernozem order. In comparison with the zonal soil – Calcic Chernozem – Chernozems on dense rocks are characterized by a reduced thickness of humus-accumulative layers, an increased of humus content, the presence of gravel in profile bottom half, a weak expression of calcareous accumulations, features of the particle-size distribution of soil components, even if it belongs to the same soil texture class. Chernozems on dense carbonate rocks either do not have accumulation or are characterized by a weak manifestation of secondary carbonates (pseudomycelia and nodules). This fact has differed such soil from the same order Chrernozems on fine-grained rocks (Calcic Chernozems) for which it is diagnostic criteria. Therefore, the mention of the suborder of these soils is incorrect in the designation. In the soil covering of the spurs of the Donetsk Ridge, dark humus soils are also occure, previously classified as incomplete developed chernozems, differing from chernozems by the absence of medium part of soil profiles.

Personalized clinical managements through exploring circulating neural cells and electroencephalography.

Since an initial diagnosis of Alzheimer disease (AD) in 1907, early detection, was unavailable through 116 years. Up-regulation of V-Ets erythroblastosis virus E26 oncogene homolog 2 (Ets2) is capable to enhance neuronal susceptibility and degeneration. Protein expression (PE) of Ets2 has functional impact on AD and Down's syndrome, with diverse intensity. PE of Ets2 has an influential pathogenic impact on AD. Clinical aspects of neurological disorders directly interact with psychological maladies. However, deterioration requires an early management including programmed based protection. To include cell biology in neuro-genetics; personalized, prognostics, predictive, preventive, predisposing (5xP) platform, accompanied by stratifying brain channels behavior pre- and post-intervention by light music in the AD-patients. Include exploration of PE assay and electroencephalography of brain channels. The processes are applied according to: (1) Triangle style, by application of cellular network; and (2) PE assay of Ets2 in the peripheral blood of the patients with AD, by Manual single cell based analysis, and Flow-cytometry. (1) Applying the Genetic counselling and pedigree analysis; (2) considering the psychological status of the referral cases; (3) considering the macro-and/or micro-environmental factors; (4) performing the required Genetics' analysis; and (5) applying the required complementary test(s). PE of Ets2 has pathogenic role in AD. PE unmasked the nature of heterogeneity/diversity/course of evolution by exploring Ets2, D1853N polymorphism in Ataxia Telangiectasia mutated gene (ATM), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF) and course of evolution at the single cell level of the brain. Ets2 revealed different cellular behavior in the blood and suggested the strategy as 'Gene Product-Based Therapy' and the personalized managements for the patients. PE reflected weak expression of ATM, mosaic pattern of Ets2; remarkable expression of VEGF and EGF by highlighting an early detective platform, considering circulating neural cells (CNCs) and the required molecular investigation, for the target individual(s) predisposed to AD or other neural disease including brain neoplasia. Brain channels-cooperation with diverse/interactive-ratios lead to strategic balancing for improving the life-quality in AD. We highlighted application of the single CNCs and correlated Ratio based between Brain channels by providing the 5xP personalized clinical management model for an early detection and therapy of the patients with AD and their targeted/predisposed relatives. Novel-evolutionary/hypothetic/heterogenic-results in brain-channels offer personalizd/constructive markers with unlimited cooperation in health and disease.

Role of organic cation transporter 3 (OCT3) in the response of hepatocellular carcinoma to tyrosine kinase inhibitors

Impaired function of organic cation transporter 1 (OCT1) in hepatocellular carcinoma (HCC) has been associated with unsatisfactory response to sorafenib. However, some patients lacking OCT1 at the plasma membrane (PM) of HCC cells still respond to sorafenib, suggesting that another transporter may contribute to take up this drug. The aim of this study was to investigate whether OCT3 could contribute to the uptake of sorafenib and other tyrosine kinase inhibitors (TKIs) and whether OCT3 determination can predict HCC response to sorafenib. Cells overexpressing OCT3 were used to determine the ability of this carrier to transport sorafenib. Immunostaining of OCT3 was performed in HCC samples obtained in the TRANSFER study. Considering the intensity of staining and the number of OCT3-positive cells, tumors were classified as having absent, weak, moderate, or strong OCT3 expression and were also categorized according to the presence or absence of PM staining. Functional in vitro studies revealed that OCT3 is also able to mediate sorafenib uptake. Other TKIs, such as regorafenib, lenvatinib, and cabozantinib can also interact with this transporter. In silico studies suggested that the expression of OCT3 is better preserved in HCC than that of OCT1. In HCC samples, OCT3 was expressed at the PM of cancer cells, and its presence, detected in 26% of tumors, was associated with better outcomes in patients treated with sorafenib. In conclusion, analysis by immunohistochemistry of OCT3 in the PM of tumor cells may help to predict the response of HCC patients to sorafenib and potentially to other TKIs.

Different Expression Pattern of G Protein-Coupled Estrogen Receptor GPER1 in Esophageal Squamous Cell Carcinoma and Adenocarcinoma.

Esophageal carcinoma is a male-dominant malignancy worldwide, and esophageal adenocarcinoma (EAC) shows more significant sex bias than esophageal squamous cell carcinoma (ESCC) in morbidity and mortality. The G protein-coupled estrogen receptor 1 (GPER1) is involved in several sex-related cancers; however, its expression level in esophageal carcinoma has been poorly investigated and its role is not precisely defined, depending on histological types. In the present study, the mRNA levels of GPER1 in esophageal carcinoma were collected from GEPIA and Oncomine databases for meta-analyses. The protein expression levels of GPER1 were detected by immunohistochemistry in the tissue microarray of EAC and ESCC. The GPER1 selective agonist G1, antagonist G15, and siRNA were applied in vitro to investigate their impacts on esophageal cell lines. Analysis of the RNA levels from the databases showed a decreased expression of GPER1 in overall esophageal carcinoma, and low expression levels of GPER1 were found to be associated with low survival of tumor patients. However, in the subgroup of EAC and its precancerous lesion, Barrett's esophagus, overexpression of GPER1 RNA was increased when compared with the normal tissues. The average staining scores of GPER1 protein in the tissue microarray of EAC were significantly higher than normal esophageal samples, and the rate of positive staining increased with the grade of poor tumor differentiation. The scores of GPER1 protein in ESCC tissues were lower than those in the normal tissues. The results from cell line experiments in vitro showed that the GPER1 agonist G1 inhibited proliferation and promoted apoptosis of ESCC cells EC109 with positive expression of GPER1. G1 had no obvious effect on normal esophageal NE2 cells with weak expression of GPER1. In addition, GPER1 RNA knockdown and application of antagonist G15 reversed the effects of G1 on EC109. The results of this study indicate that the expression levels of GPER1 are higher in EAC than in ESCC, which might be correlated with the dimorphic estrogen signaling pathway in different types of esophageal carcinoma.

P12.02.B CHARACTERIZATION AND PROGNOSTIC EVALUATION OF PSTAT3 EXPRESSION IN PERITUMORAL REACTIVE ASTROCYTES OF BRAIN METASTASES FROM RENAL CELL CARCINOMA

Abstract BACKGROUND The signal transducer and activator of transcription 3 (STAT3) shows a key role in regulating the anti-tumor immune response. STAT3 is activated in cancer cells and in the tumor microenvironment thus inhibiting crucial immune regulators expression and promoting immunosuppressive factors. Brain metastatic cells sustain a pro-metastatic signal through the hyperexpression of pSTAT3 in reactive astrocytes (RAs) surrounding metastases and succeed in generating an immunosuppressive signal resulting in a worsened prognosis. Pre-clinical analyses demonstrated that brain metastases (BMs) size might be reduced by blocking STAT3 signal in RAs. pSTAT3 expression in BMs from renal cell carcinoma (RCC) has never been systematically analyzed. Our study aims to investigate pSTAT3 expression in RCC brain metastatic specimens to identify its therapeutic and prognostic role. Materials and METHODS A total of 27 paraffin-embedded human BMs from RCC obtained through surgical resection at the Neurosurgical Units of University of Bari “Aldo Moro” and University of Turin were stained for phosphorylated (Tyr705) STAT3 (pSTAT3) and GFAP, then evaluated and scored by clinical neuropathologists of the University of Turin. Protein expression was semi-quantitatively assessed according to the intensity and the number of positive cells. Overall survival (OS) was intended by the date of BMs onset. A survival curve was generated by grouping patients with high (score 2-3) and low (score 0-1) pSTAT3 in ARs. P values were obtained with the two-sided log-rank test. RESULTS 97% (26 out 27) of BMs were positive for pSTAT3 (pSTAT3+) in RAs, with 41% of them receiving a score above 1. In detail, 1 scored with 3 (moderate and diffuse expression), 10 (38%) with 2 (moderate focal expression or weak diffuse expression) and 15 (56%) with 1 (weak and focal expression) according to the abundance and signal intensity of pSTAT3 in ARs. The median OS from BM onset was 12 months. After grouping patients by the high or low pSTAT3 expression level, no significant survival differences were evidenced (mOS 10 vs 13 months, p= 0.432). CONCLUSIONS Our study represents the first analysis investigating pSTAT3 expression on BMs from RCC. Considering the low number of specimens, which reflects a low rate of surgical resections, our work assumes an exploratory value. RAs surrounding BMs from RCC ubiquitously express pSTAT3, but only 41% of cases with a moderate or diffuse expression seem to be related to a biological pro-metastatic role. Although patients with high pSTAT3 expression showed reduced survival, this result was not statistically significant, possibly due to the limited sample size. If confirmed in larger and prospective cohorts, these data could lay the foundation for new therapeutic strategies involving pSTAT3 signaling inhibition (Silibinin) eventually improving the intracranial response rate.

Development of [225Ac]Ac-DOTA-C595 as radioimmunotherapy of pancreatic cancer: in vitro evaluation, dosimetric assessment and detector calibration

BackgroundPancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy which may benefit from radioimmunotherapy. Previously, [177Lu]Lu-DOTA-C595 has been developed as a beta-emitting radioimmunoconjugate to target cancer-specific mucin 1 epitopes (MUC1-CE) overexpressed on PDAC. However, the therapeutic effect may be enhanced by using an alpha-emitting radionuclide such as Actinium-225 (Ac-225). The short range and high linear energy transfer of alpha particles provides dense cellular damage and can overcome typical barriers related to PDAC treatment such as hypoxia. Despite the added cytotoxicity of alpha-emitters, their clinical implementation can be complicated by their complex decay chains, recoil energy and short-range impeding radiation detection. In this study, we developed and evaluated [225Ac]Ac-DOTA-C595 as an alpha-emitting radioimmunotherapy against PDAC using a series of in vitro experiments and conducted a preliminary dosimetric assessment and cross-calibration of detectors for the clinical implementation of Ac-225.ResultsCell binding and internalisation of [225Ac]Ac-DOTA-C595 was rapid and greatest in cells with strong MUC1-CE expression. Over 99% of PDAC cells had positive yH2AX expression within 1 h of [225Ac]Ac-DOTA-C595 exposure, suggesting a high level of DNA damage. Clonogenic assays further illustrated the cytotoxicity of [225Ac]Ac-DOTA-C595 in a concentration-dependent manner. At low concentrations of [225Ac]Ac-DOTA-C595, cells with strong MUC1-CE expression had lower cell survival than cells with weak MUC1-CE expression, yet survival was similar between cell lines at high concentrations irrespective of MUC1-CE expression. A dosimetric assessment was performed to estimate the dose-rate of 1 kBq of [225Ac]Ac-DOTA-C595 with consideration to alpha particles. Total absorption of 1 kBq of Ac-225 was estimated to provide a dose rate of 17.5 mGy/h, confirmed via both detector measurements and calculations.Conclusion[225Ac]Ac-DOTA-C595 was shown to target and induce a therapeutic effect in MUC1-CE expressing PDAC cells.