cDNA Expression Research Articles

Preferential expression of SCN1A in GABAergic neurons improves survival and epileptic phenotype in a mouse model of Dravet syndrome

The SCN1A gene encodes the alpha subunit of a voltage-gated sodium channel (Nav1.1), which is essential for the function of inhibitory neurons in the brain. Mutations in this gene cause severe encephalopathies such as Dravet syndrome (DS). Upregulation of SCN1A expression by different approaches has demonstrated promising therapeutic effects in preclinical models of DS. Limiting the effect to inhibitory neurons may contribute to the restoration of brain homeostasis, increasing the safety and efficacy of the treatment. In this work, we have evaluated different approaches to obtain preferential expression of the full SCN1A cDNA (6 Kb) in GABAergic neurons, using high-capacity adenoviral vectors (HC-AdV). In order to favour infection of these cells, we considered ErbB4 as a surface target. Incorporation of the EGF-like domain from neuregulin 1 alpha (NRG1α) in the fiber of adenovirus capsid allowed preferential infection in cells lines expressing ErbB4. However, it had no impact on the infectivity of the vector in primary cultures or in vivo. For transcriptional control of transgene expression, we developed a regulatory sequence (DP3V) based on the Distal-less homolog enhancer (Dlx), the vesicular GABA transporter (VGAT) promoter, and a portion of the SCN1A gene. The hybrid DP3V promoter allowed preferential expression of transgenes in GABAergic neurons both in vitro and in vivo. A new HC-AdV expressing SCN1A under the control of this promoter showed improved survival and amelioration of the epileptic phenotype in a DS mouse model. These results increase the repertoire of gene therapy vectors for the treatment of DS and indicate a new avenue for the refinement of gene supplementation in this disease.Key messagesAdenoviral vectors can deliver the SCN1A cDNA and are amenable for targeting.An adenoviral vector displaying an ErbB4 ligand in the capsid does not target GABAergic neurons.A hybrid promoter allows preferential expression of transgenes in GABAergic neurons.Preferential expression of SCN1A in GABAergic cells is therapeutic in a Dravet syndrome model.

Antimicrobial Peptides Originating from Expression Libraries of Aurelia aurita and Mnemiopsis leidyi Prevent Biofilm Formation of Opportunistic Pathogens.

The demand for novel antimicrobial compounds is rapidly growing due to the rising appearance of antibiotic resistance in bacteria; accordingly, alternative approaches are urgently needed. Antimicrobial peptides (AMPs) are promising, since they are a naturally occurring part of the innate immune system and display remarkable broad-spectrum activity and high selectivity against various microbes. Marine invertebrates are a primary resource of natural AMPs. Consequently, cDNA expression (EST) libraries from the Cnidarian moon jellyfish Aurelia aurita and the Ctenophore comb jelly Mnemiopsis leidyi were constructed in Escherichia coli. Cell-free size-fractionated cell extracts (<3 kDa) of the two libraries (each with 29,952 clones) were consecutively screened for peptides preventing the biofilm formation of opportunistic pathogens using the crystal violet assay. The 3 kDa fraction of ten individual clones demonstrated promising biofilm-preventing activities against Klebsiella oxytoca and Staphylococcus epidermidis. Sequencing the respective activity-conferring inserts allowed for the identification of small ORFs encoding peptides (10-22 aa), which were subsequently chemically synthesized to validate their inhibitory potential. Although the peptides are likely artificial products from a random translation of EST inserts, the biofilm-preventing effects against K. oxytoca, Pseudomonas aeruginosa, S. epidermidis, and S. aureus were verified for five synthetic peptides in a concentration-dependent manner, with peptide BiP_Aa_5 showing the strongest effects. The impact of BiP_Aa_2, BiP_Aa_5, and BiP_Aa_6 on the dynamic biofilm formation of K. oxytoca was further validated in microfluidic flow cells, demonstrating a significant reduction in biofilm thickness and volume by BiP_Aa_2 and BiP_Aa_5. Overall, the structural characteristics of the marine invertebrate-derived AMPs, their physicochemical properties, and their promising antibiofilm effects highlight them as attractive candidates for discovering new antimicrobials.

The RNA-binding proteins hnRNP H and F regulate splicing of a MYC-dependent HRAS exon in prostate cancer cells

The MYC proto-oncogene contributes to the pathogenesis of more than half of human cancers. Malignant transformation by MYC transcriptionally up-regulates the core pre-mRNA splicing machinery and causes misregulation of alternative splicing. However, our understanding of how splicing changes are directed by MYC is limited. We performed a signaling pathway-guided splicing analysis to identify MYC-dependent splicing events. These included an HRAS cassette exon repressed by MYC across multiple tumor types. To molecularly dissect the regulation of this HRAS exon, we used antisense oligonucleotide tiling to identify splicing enhancers and silencers in its flanking introns. RNA-binding motif prediction indicated multiple binding sites for hnRNP H and hnRNP F within these cis-regulatory elements. Using siRNA knockdown and cDNA expression, we found that both hnRNP H and F activate the HRAS cassette exon. Mutagenesis and targeted RNA immunoprecipitation implicate two downstream G-rich elements in this splicing activation. Analyses of ENCODE RNA-seq datasets confirmed hnRNP H regulation of HRAS splicing. Analyses of RNA-seq datasets across multiple cancers showed a negative correlation of HNRNPH gene expression with MYC hallmark enrichment, consistent with the effect of hnRNP H on HRAS splicing. Interestingly, HNRNPF expression showed a positive correlation with MYC hallmarks and thus was not consistent with the observed effects of hnRNP F. Loss of hnRNP H/F altered cell cycle progression and induced apoptosis in the PC3 prostate cancer cell line. Collectively, our results reveal mechanisms for MYC-dependent regulation of splicing and point to possible therapeutic targets in prostate cancers.

Englerin A Inhibits T-Type Voltage-Gated Calcium Channels at Low Micromolar Concentrations.

Englerin A (EA) is a potent agonist of tetrameric transient receptor potential canonical (TRPC) ion channels containing TRPC4 and TRPC5 subunits. TRPC proteins form cation channels that are activated by plasma membrane receptors. They convert extracellular signals such as angiotensin II into cellular responses, whereupon Na+ and Ca2+ influx and depolarization of the plasma membrane occur. Via depolarization, voltage-gated Ca2+ (CaV) channels can be activated, further increasing Ca2+ influx. We investigated the extent to which EA also affects the functions of CaV channels using the high-voltage-activated L-type Ca2+ channel CaV1.2 and the low-voltage-activated T-type Ca2+ channels CaV3.1, CaV3.2, and CaV3.3. After expression of cDNAs in human embryonic kidney (HEK293) cells, EA inhibited currents through all T-type channels at half-maximal inhibitory concentrations (IC50) of 7.5 to 10.3 μM. In zona glomerulosa cells of the adrenal gland, angiotensin II-induced elevation of cytoplasmic Ca2+ concentration leads to aldosterone release. We identified transcripts of low- and high-voltage-activated CaV channels and of TRPC1 and TRPC5 in the human adrenocortical (HAC15) zona glomerulosa cell line. Although no EA-induced TRPC activity was measurable, Ca2+ channel blockers distinguished T- and L-type Ca2+ currents. EA blocked 60% of the CaV current in HAC15 cells and T- and L-type channels analyzed at -30 mV and 10 mV were inhibited with IC50 values of 2.3 and 2.6 μM, respectively. Although the T-type blocker Z944 reduced basal and angiotensin II-induced 24-hour aldosterone release, EA was not effective. In summary, we show here that EA blocks CaV1.2 and T-type CaV channels at low-micromolar concentrations. SIGNIFICANCE STATEMENT: In this study we showed that englerin A (EA), a potent agonist of tetrameric transient receptor potential canonical (TRPC)4- or TRPC5-containing channels and currently under investigation to treat certain types of cancer, also inhibits the L-type voltage-gated Ca2+ (CaV) channel CaV1.2 and the T-type CaV channels CaV3.1, CaV3.2, and CaV3.3 channels at low micromolar concentrations.

Atherosclerosis-related biomarker PABPC1 predicts pan-cancer events

BackgroundAtherosclerosis (AS) and tumours are the leading causes of death worldwide and share common risk factors, detection methods and molecular markers. Therefore, searching for serum markers shared by AS and...

An angiotensin converting enzyme homolog is required for volatile pheromone detection, odorant binding protein secretion and normal courtship behavior in Drosophila melanogaster.

In many arthropods, including insects responsible for transmission of human diseases, behaviors that include mating, aggregation, and aggression are triggered by detection of pheromones. Extracellular odorant binding proteins are critical for pheromone detection in many insects and are secreted into the fluid bathing the olfactory neuron dendrites. In Drosophila melanogaster, the odorant binding protein LUSH is essential for normal sensitivity to the volatile sex pheromone, 11-cis vaccenyl acetate (cVA). Using a genetic screen for cVA pheromone insensitivity, we identified ANCE-3, a homolog of human angiotensin converting enzyme that is required for detection of cVA pheromone. The mutants have normal dose-response curves for food odors, although olfactory neuron amplitudes are reduced in all olfactory neurons examined. ance-3 mutants have profound delays in mating, and the courtship defects are primarily but not exclusively due to loss of ance-3 function in males. We demonstrate that ANCE-3 is required in the sensillae support cells for normal reproductive behavior, and that localization of odorant binding proteins to the sensillum lymph is blocked in the mutants. Expression of an ance-3 cDNA in sensillae support cells completely rescues the cVA responses, LUSH localization, and courtship defects. We show the courtship latency defects are not due to effects on olfactory neurons in the antenna nor mediated through ORCO receptors, but instead stem from ANCE-3-dependent effects on chemosensory sensillae in other body parts. These findings reveal an unexpected factor critical for pheromone detection with profound influence on reproductive behaviors.

The impact of circulating tumor cell HOXB13 RNA detection in men with metastatic castrate-resistant prostate cancer (mCRPC) treated with abiraterone or enzalutamide.

5060 Background: It has been shown that HOXB13 is an important androgen receptor (AR) coregulator. The primary objective of this analysis was to correlate HOXB13 expression with clinical outcomes in men with mCRPC. Methods: We conducted a retrospective analysis of the multicenter prospective PROPHECY trial of mCRPC men (n=118) treated abiraterone (abi) or enzalutamide (enza). CTC detection and specific HOXB13 cDNA detection and expression was measured using a modified Adnatest using appropriate controls. CTC HOXB13 status was categorized into 3 groups: undetectable CTCs (CTC=0 based on CTC PSMA &amp; PSA=0); CTC+ but HOXB13 CTC negative (&lt;4 copies) or positive (≥4 copies) where the HOXB13 cutoff point was determined by the maximally selected rank statistic for prognostic associations. The association between HOXB13 and overall survival (OS) and radiographic progression-free survival (rPFS) were explored using the proportional hazards model. We also explored the association between the two endpoints and multigroup of HOXB13 at cutoff 0 and CellSearch CTC at cutoff 1 and 5. Results: 102 men who had sufficient CTC cDNA sample for HOXB13 detection. Of those men with detectable CTCs, 19% (n=19) had no detectable HOXB13, while the majority of men at 67% (n=58) had both CTC and HOXB13 detection. The proportion of patients with CTC=0, CTC+ HOXB13-, and CTC+ HOXB13+ using the optimal HOXB13 threshold were 25%, 31% and 44%, respectively. Men with HOXB13- CTCs were more likely to have low PSA (median 3 vs 79 copies), low CTC PSMA (0 vs 99 copies), low AR-FL (47 vs. 89 copies) and CTC AR-V7 (5.9 vs. 42 copies), and more likely to have liver/lung metastases (41% vs 25%). Median OS were 25.7, 25 and 12.1 months while the median rPFS of the three groups were 9.0, 7.2 and 4.7 months, respectively. Compared to CTC=0, men with HOXB13- CTCs did not have a worse hazard rate for OS (HR: 0.9, 95% CI: 0.4-1.9), while HOXB13+ CTC patients had a worse hazard for OS (HR: 2.0, 95% CI: 1.0-3.9), adjusting for prior abi/enza treatment and Halabi clinical risk score. The positive HOXB13 had also statistically significant increased hazard for progression/death (HR: 2.31, 95% CI: 1.24-4.30). Compared to the group with both HOXB13=0 and CellSearch CTC=0, men with HOXB13&gt;0 and CellSearch CTC≥5 had increased hazard on both OS and rPFS (OS: HR:2.39, 95% CI: 1.06-5.4; rPFS: HR: 2.78, 95% CI: 1.38-5.59). Conclusions: Higher CTC HOXB13 expression is associated with AR dependent biomarkers in CTCs and is adversely prognostic in the context of potent AR inhibition in men with mCRPC, and this assay could be useful to select for patients appropriate for AR or future HOXB13 targeted therapies. Clinical trial information: NCT02269982 .

Pathogenic Role of RAGE in Tau Transmission and Memory Deficits

BackgroundIn tauopathies, brain regions with tau accumulation strongly correlate with clinical symptoms, and spreading of misfolded tau along neural network leads to disease progression. However, the underlying mechanisms by which tau proteins enter neurons during pathological propagation remain unclear. MethodsTo identify membrane receptors responsible for neuronal propagation of tau oligomers, we established a cell-based tau uptake assay and screened complementary DNA expression library. Tau uptake and propagation were analyzed in vitro and in vivo using a microfluidic device and stereotactic injection. The cognitive function of mice was assessed using behavioral tests. ResultsFrom a genome-wide cell-based functional screening, RAGE (receptor for advanced glycation end products) was isolated to stimulate the cellular uptake of tau oligomers. Rage deficiency reduced neuronal uptake of pathological tau prepared from rTg4510 mouse brains or cerebrospinal fluid from patients with Alzheimer’s disease and slowed tau propagation between neurons cultured in a 3-chamber microfluidic device. RAGE levels were increased in the brains of rTg4510 mice and tau oligomer-treated neurons. Rage knockout decreased tau transmission in the brains of nontransgenic mice after injection with Alzheimer’s disease patient-derived tau and ameliorated memory loss after injection with GFP-P301L-tau-AAV. Treatment of RAGE antagonist FPS-ZM1 blocked transsynaptic tau propagation and inflammatory responses and alleviated cognitive impairment in rTg4510 mice. ConclusionsThese results suggest that in neurons and microglia, RAGE binds to pathological tau and facilitates neuronal tau pathology progression and behavioral deficits in tauopathies.

Potential role for a Nematode Pan Allergen (NPA) in Tropical Pulmonary Eosinophilia (TPE)

Abstract Tropical Pulmonary Eosinophilia (TPE) is an asthma-like hyper-responsive syndrome characterized by accumulation of a high number of eosinophils in the lungs leading to severe respiratory morbidity in patients infected with lymphatic filariasis (LF), a helminth parasitic infection that affects 51 million people in 47 different countries worldwide. Subjects showing TPE also have circulating microfilariae (larval stages of LF) and a high titer of parasite-specific IgE in their blood. IgE and eosinophils are thought to play a major role in TPE pathology. However, specific antigens that are released from microfilariae responsible for generating the IgE antibodies in TPE are not known. Therefore, in this study, we screened a phage displayed cDNA expression library of Wuchereria bancrofti microfilaria with human monoclonal IgE antibodies generated from B cells isolated from TPE patients. The iterative screening identified WbNPA (Nematode Pan Allergen) and WbTCTP (Translationally Controlled Tumor Protein) as IgE antibody binding antigens. Subsequent analysis showed that rWbNPA and rWbTCTP can result in the release of histamine from mast cells in an IgE-dependent and independent manner. rWbNPA can induce accumulation of eosinophils in the peritoneal cavity of mice and can trigger the secretion of IL-2 and TNF-α from alveolar macrophages. These results suggest that WbNPA and WbTCTP have a potential role in the pathology of TPE in LF infections.

Abstract 6734: Identification of the novel TENM3-ALK fusion in an AYA case with ALK rearranged neuroblastoma

Abstract Neuroblastoma is the most common extracranial solid tumor that occurs in childhood and the most common cancer in infancy. Fewer than 5% of neuroblastomas occur in adolescents and young adults (AYAs), for whom the disease has an indolent and fatal course. Chen et al. previously reported that missense mutations in the protein- tyrosine kinase domain of anaplastic lymphoma kinase (ALK) occur in approximately 10% of cases of sporadic neuroblastoma, and these missense mutations result in the activation of the ALK protein kinase domain, which plays a key role in the tumorigenic process as seen in ALK fusion proteins. Since the initial discovery of the NPM-ALK fusion protein in human anaplastic lymphoma cell lines, more than 24 different ALK fusion proteins have been discovered in various malignancies. Till date, ALK-containing chimeric proteins have not been reported in neuroblastoma. In this study, a translocation within chromosome 2p and 4q was found to bring about the formation of an in-frame fusion gene that was composed of portions of the teneurin transmembrane protein 3 (TENM3) gene and ALK gene in tumor cells from an AYA patient with neuroblastoma. The patient was 19-year-old female diagnosed with stage 4 neuroblastoma (unfavorable histology, poorly differentiated subtype, MYCN DNA not amplified). Computed tomography revealed a mass in the left adrenal gland with bone, spine, liver, and lung metastasis. ALK rearrangement was confirmed via FISH analysis. Crizotinib resulted in no disease progression for 5 months, except for metastatic spinal disease, but it did not exhibit high efficacy compared with its effects against lymphoma and lung cancer. This 5′ in-frame fusion partner gene in neuroblastoma has not yet been identified, and its role is not well-known in oncogenesis. We generated the cell line with the expression of the full length TENM3-ALK cDNA in NIH-3T3 cells led to the formation of a fusion protein. A cell-based evaluation of this predicted ALK protein variant revealed activation of downstream targets-STAT3, AKT and ERK- and ALK inhibitors, crizotinib or lorlatinib, treatment inhibited the growth of xenograft tumors with stable TENM3-ALK expression. We determined whether TENM3-ALK affects transformation and proliferation using a soft agar colony formation assay. As expected, the cells that expressed an empty vector and wild type ALK line did not produce a considerable number of colonies, whereas TENM3-ALK transfected cells showed anchorage-independent growth, resulting in the production of a higher number of colonies. The evidence for the role of ALK receptor signaling in stimulating neuroblastoma tumor cell growth and the demonstrated in vitro and in vivo efficacy of the ALK selective inhibitors in this study provide biological and clinical justification for the further exploration of this combination and testing in patients with recurrent or refractory neuroblastoma. Citation Format: Mitsuteru Hiwatari, Masafumi Seki, Ryosuke Matsuno, Kenichi Yoshida, Takeshi Nagasawa, Aiko Sato-Otsubo, Shohei Yamamoto, Motohiro Kato, Kentaro Watanabe, Masahiro Sekiguchi, Satoru Miyano, Seishi Ogawa, Junko Takita. Identification of the novel TENM3-ALK fusion in an AYA case with ALK rearranged neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6734.

Transformation and expressional studies of GaZnF gene to improve drought tolerance in Gossypium hirsutum

Drought stress is the major limiting factor in plant growth and production. Cotton is a significant crop as textile fiber and oilseed, but its production is generally affected by drought stress, mainly in dry regions. This study aimed to investigate the expression of Zinc finger transcription factor’s gene (GaZnF) to enhance the drought tolerance in Gossypium hirsutum. Sequence features of the GaZnF protein were recognized through different bioinformatics tools like multiple sequence alignment analysis, phylogenetic tree for evolutionary relationships, Protein motifs, a transmembrane domain, secondary structure and physio-chemical properties indicating that GaZnF is a stable protein. CIM-482, a local Gossypium hirsutum variety was transformed with GaZnF through Agrobacterium-mediated transformation method with 2.57% transformation efficiency. The integration of GaZnF was confirmed through Southern blot showing 531 bp, and Western blot indicated a 95 kDa transgene-GUS fusion band in transgenic plants. The normalized real-time expression analysis revealed the highest relative fold spatial expression of cDNA of GaZnF within leaf tissues at vegetative and flowering stages under drought stress. Morphological, physiological and biochemical parameters of transgenic cotton plants at 05- and 10-day drought stress was higher than those of non-transgenic control plants. The values of fresh and dry biomass, chlorophyll content, photosynthesis, transpiration rate, and stomatal conductance reduced in GaZnF transgenic cotton plants at 05- and 10-day drought stress, but their values were less low in transgenic plants than those of non-transgenic control plants. These findings showed that GaZnF gene expression in transgenic plants could be a valuable source for the development of drought-tolerant homozygous lines through breeding.

Interaction of a Novel Alternatively Spliced Variant of HSD11B1L with Parkin Enhances the Carcinogenesis Potential of Glioblastoma: Peiminine Interferes with This Interaction.

Glioblastoma (GBM) is a primary brain tumor of unknown etiology. It is extremely aggressive, incurable and has a short average survival time for patients. Therefore, understanding the precise molecular mechanisms of this diseases is essential to establish effective treatments. In this study, we cloned and sequenced a splice variant of the hydroxysteroid 11-β dehydrogenase 1 like gene (HSD11B1L) and named it HSD11B1L-181. HSD11 B1L-181 was specifically expressed only in GBM cells. Overexpression of this variant can significantly promote the proliferation, migration and invasion of GBM cells. Knockdown of HSD11B1L-181 expression inhibited the oncogenic potential of GBM cells. Furthermore, we identified the direct interaction of parkin with HSD11B1L-181 by screening the GBM cDNA expression library via yeast two-hybrid. Parkin is an RBR E3 ubiquitin ligase whose mutations are associated with tumorigenesis. Small interfering RNA treatment of parkin enhanced the proliferative, migratory and invasive abilities of GBM. Finally, we found that the alkaloid peiminine from the bulbs of Fritillaria thunbergii Miq blocks the interaction between HSD11B1L-181 and parkin, thereby lessening carcinogenesis of GBM. We further confirmed the potential of peiminine to prevent GBM in cellular, ectopic and orthotopic xenograft mouse models. Taken together, these findings not only provide insight into GBM, but also present an opportunity for future GBM treatment.

Alternative splicing of HDAC7 regulates its interaction with 14-3-3 proteins to alter histone marks and target gene expression

Chromatin regulation and alternative splicing are both critical mechanisms guiding gene expression. Studies have demonstrated that histone modifications can influence alternative splicing decisions, but less is known about how alternative splicing may impact chromatin. Here, we demonstrate that several genes encoding histone-modifying enzymes are alternatively spliced downstream of Tcell signaling pathways, including HDAC7, a gene previously implicated in controlling gene expression and differentiation in Tcells. Using CRISPR-Cas9 gene editing and cDNA expression, we show that differential inclusion of HDAC7 exon 9 controls the interaction of HDAC7 with protein chaperones, resulting in changes to histone modifications and gene expression. Notably, the long isoform, which is induced by the RNA-binding protein CELF2, promotes expression of several critical Tcell surface proteins including CD3, CD28, and CD69. Thus, we demonstrate that alternative splicing of HDAC7 has a global impact on histone modification and gene expression that contributes to Tcell development.

Targeted expression of bgl23-D, a dominant-negative allele of ATCSLD5, affects cytokinesis of guard mother cells and exine formation of pollen in Arabidopsis thaliana.

Targeted expression of bgl23-D, a dominant-negative allele of ATCSLD5, is a useful genetic approach for functional analysis of ATCSLDs in specific cells and tissues in plants. Stomata are key cellular structures for gas and water exchange in plants and their development is influenced by several genes. We found the A. thaliana bagel23-D (bgl23-D) mutant showing abnormal bagel-shaped single guard cells. The bgl23-D was a novel dominant mutation in the A. thaliana cellulose synthase-like D5 (ATCSLD5) gene that was reported to function in the division of guard mother cells. The dominant character of bgl23-D was used to inhibit ATCSLD5 function in specific cells and tissues. Transgenic A. thaliana expressing bgl23-D cDNA with the promoter of stomata lineage genes, SDD1, MUTE, and FAMA, showed bagel-shaped stomata as observed in the bgl23-D mutant. Especially, the FAMA promoter exhibited a higher frequency of bagel-shaped stomata with severe cytokinesis defects. Expression of bgl23-D cDNA in the tapetum with SP11 promoter or in the anther with ATSP146 promoter induced defects in exine pattern and pollen shape, novel phenotypes that were not shown in the bgl23-D mutant. These results indicated that bgl23-D inhibited unknown ATCSLD(s) that exert the function of exine formation in the tapetum. Furthermore, transgenic A. thaliana expressing bgl23-D cDNA with SDD1, MUTE, and FAMA promoters showed enhanced rosette diameter and increased leaf growth. Taken together, these findings suggest that the bgl23-D mutation could be a helpful genetic tool for functional analysis of ATCSLDs and manipulating plant growth.

Dysregulation of miR-577, miR-505-3p, miR-3682-3p, and miR-4661 in Breast Cancer Patients Based on Estrogen Receptor Status

Background: Breast cancer is one of the most common malignancies and the second leading cause of cancer-related death in women. Approximately 75% of all breast cancers are estrogen receptor-positive (ER+ ) and highly responsive to endocrine therapy. MicroRNAs (miRNAs) are short non-coding RNA with a pivotal role in mammal cells by regulating gene expression. Hence, this study aimed to evaluate the miRNAs expression in various breast cancer subtypes. Materials and Methods: In this study, after total RNA extraction and cDNA synthesis, expressions of miR-577, miR-505-3p, miR-3682-3p, and miR-4661-5p were investigated in 36 breast cancer samples of ER+ and ER- types and compared with 18 normal adjacent tissues by real-time polymerase chain reaction. Also, diagnostic values of miRNAs were determined based on receiver operating characteristic (ROC) by calculating the area under the curve (AUC). Results: Downregulation of miR-577 and miR-505-3p were detected in breast cancer samples, significantly in the ER+ subtype compared to ER- subtype (P&lt;0.001). Also, we showed upregulation of miR-3682-3p and miR-4661-5p in breast cancer tissues compared to normal tissues. Compared to the ER+ subtype, the miR-3682-3p expression significantly decreased in the ER- subtype (P&lt;0.001). However, there was no significant difference between ER+ and ER- subtypes in the term of miR-4661-5p (P˃0.05). The ROC analysis demonstrated that miR-577 and miR-505-3p have acceptable diagnostic values, and miR-3682-3p has a relatively proper diagnostic value in diagnosing breast cancer. Conclusion: Our results revealed that miR-577 and miR-505- 3p could be used as biomarkers for the diagnosis of breast cancer, especially in ER+ subtype.

Molecular characterization of Vasa homolog in the pen shell Atrina pectinata: cDNA cloning and expression analysis during gonadal development.

Vasa is an ATP-dependent RNA helicase of the DEAD (Asp-Glu-Ala-Asp) box family and a representative component of the germ plasm. In this study, we cloned the full-length vasa homolog in the bivalve Atrina pectinata (psvasa), and performed phylogenetic analysis, mRNA expression analysis for tissue-specific distributions, and immunostaining analysis to reveal its histological localization. The sequence of psvasa was 3587bp in length and contained a 5' untranslated region of 150bp, an open reading frame of 2214bp, and a 3' untranslated region of 1223bp. The deduced amino acid sequence of psvasa was 737 amino acids long and contained evolutionarily conserved sequences reported in other animals. The mRNA expression analysis showed the highest expression levels in the gonads. Expression was especially high in the ovaries, followed by the testes. The immunostaining analysis showed Vasa-positive cells in the developing gonads, suggesting the presence of putative germ stem cells contributing to the supply of germ cells. Furthermore, characteristic Vasa signals were observed in the basophilic nuclei of the oocytes, suggesting that psvasa plays an important role in the progression of meiosis in oocytes.

Prognostic importance of PD-L1 expression in bladder cancer patients.

PD-L1/PD-1 molecules are known as important mediators in immune-escape mechanisms of tumors. PD-L1 is highly expressed in various malignancies, including bladder cancer. However, the prognostic value of PD-L1 in bladder cancer patients remains controversial. To investigate the prognostic significance of PD-L1 expression in tumor tissues of bladder cancer patients. RNA was isolated from FFPE tumor tissues of 48 bladder cancer patients using the monophasic phenol and guanidine isothiocyanate method. Total RNA was converted to cDNA and gene expression levels were analyzed by qRT-PCR. The differential expression levels of the PD-L1 gene between tumor grade and cancer stage groups were analyzed by independent student's t-test and one-way ANOVA. Statistically significantly increased PD-L1 expression was observed in the high-grade tumor group (p < 0.05). No significant difference in PD-L1 expression was found among pTa, pT1, and pT2 groups. In addition, the difference in overall survival was not significantly different between groups. The results showed that high PD-L1 expression in bladder cancer was associated with tumor aggressiveness and grade. Despite the inability of the qRT-PCR to show the PD-L1 expression at different locations of tumor tissue, evaluation of PD-L1 mRNA expression by qRT-PCR, which is a highly sensitive and specific assay, appears to be a robust approach. Furthermore, these findings may contribute to a rationale for recommending anti-PD-L1 immunotherapy as an alternative to standard therapy for bladder cancer patients who are most likely to benefit from it.

Next-Generation Yeast Two-Hybrid Screening to Discover Protein-Protein Interactions.

Yeast two-hybrid is a powerful approach to discover new protein-protein interactions. Traditional methods involve screening a target protein against a cDNA expression library and assaying individual positive colonies to identify interacting partners. Here we describe a simple approach to perform yeast two-hybrid screens of a cDNA expression library in batch liquid culture. Positive yeast cell populations are enriched under selection and then harvested en masse. Prey cDNAs are amplified and used as input for next-generation sequencing libraries for identification, quantification, and ranking.

Identification of antibody against wingless‑type MMTV integration site family member 7B as a biliary cancer tumor marker.

Biliary cancer has a poor prognosis due to a lack of specific biomarkers and difficulty in diagnosis. The present study aimed to identify serum tumor markers for the diagnosis of biliary cancer via serological identification of antigens by recombinant cDNA expression cloning. Wingless‑type MMTV integration site family, member 7 (WNT7B) was identified as a target antigen, suggesting the presence of serum antibodies against this antigen. Deletion mutants were then prepared to evaluate the response to serum antibodies. When serum antibody levels against WNT7B deletion mutants (WNT7B-92‑2, -92‑260, 2-260 and 184-260) were examined using amplified luminescence proximity homogeneous assay‑linked immunosorbent assay, the levels of the antibody against WNT7B with amino acids 184‑260 were higher in patients with biliary cancer than in healthy donors. Therefore, the region covering residues 184‑260 of WNT7B was decomposed to generate seven peptides, and the levels of antibodies against these peptides were measured. Among them, the levels of antibodies against WNT7B234‑253 and WNT7B244‑260 were higher in patients with biliary cancers than in healthy donors (WNT7B234‑253, P=0.0009; WNT7B244‑260, P=0.0005). The levels of the antibody against the former were specifically high in patients with biliary cancer but not in those with esophageal, gastric, colorectal, pancreatic, or breast cancer. Furthermore, analysis by the cutoff value of WNT7B234‑253 defined by ROC showed a high sensitivity of 70% in patients with biliary cancer. Therefore, the serum levels of the antibody against WNT7B234‑253 may be useful as a marker for biliary cancer diagnosis.

Rare EIF4A2 variants are associated with a neurodevelopmental disorder characterized by intellectual disability, hypotonia, and epilepsy

Eukaryotic initiation factor-4A2 (EIF4A2) is an ATP-dependent RNA helicase and a member of the DEAD-box protein family that recognizes the 5' cap structure of mRNAs, allows mRNA to bind to the ribosome, and plays an important role in microRNA-regulated gene repression. Here, we report on 15 individuals from 14 families presenting with global developmental delay, intellectual disability, hypotonia, epilepsy, and structural brain anomalies, all of whom have extremely rare de novo mono-allelic or inherited bi-allelic variants in EIF4A2. Neurodegeneration was predominantly reported in individuals with bi-allelic variants. Molecular modeling predicts these variants would perturb structural interactions in key protein domains. To determine the pathogenicity of the EIF4A2 variants invivo, we examined the mono-allelic variants in Drosophila melanogaster (fruit fly) and identified variant-specific behavioral and developmental defects. The fruit fly homolog of EIF4A2 is eIF4A, a negative regulator of decapentaplegic (dpp) signaling that regulates embryo patterning, eye and wing morphogenesis, and stem cell identity determination. Our loss-of-function (LOF) rescue assay demonstrated a pupal lethality phenotype induced by loss of eIF4A, which was fully rescued with human EIF4A2 wild-type (WT) cDNA expression. In comparison, the EIF4A2 variant cDNAs failed or incompletely rescued the lethality. Overall, our findings reveal that EIF4A2 variants cause a genetic neurodevelopmental syndrome with both LOF and gain of function as underlying mechanisms.