Higher Transcript Expression Research Articles

The central oxytocinergic system of the prairie vole

Oxytocin (OXT) is a peptide hormone and a neuropeptide that regulates various peripheral physiological processes and modulates behavioral responses in the central nervous system. While the humoral release occurs from the axons arriving at the median eminence, the neuropeptide is also released from oxytocinergic cell axons in various brain structures that contain its receptor, and from their dendrites in hypothalamic nuclei and potentially into the cerebrospinal fluid (CSF). Understanding oxytocin’s complex functions requires the knowledge on patterns of oxytocinergic projections in relationship to its receptor (OXTR). This study provides the first comprehensive examination of the oxytocinergic system in the prairie vole (Microtus ochrogaster), an animal exhibiting social behaviors that mirror human social behaviors linked to oxytocinergic functioning. Using light and electron microscopy, we characterized the neuroanatomy of the oxytocinergic system in this species. OXT+ cell bodies were found primarily in the hypothalamus, and axons were densest in subcortical regions. Examination of the OXT+ fibers and their relationship to oxytocin receptor transcripts (Oxtr) revealed that except for some subcortical structures, the presence of axons was not correlated with the amount of Oxtr across the brain. Of particular interest, the cerebral cortex that had high expression of Oxtr transcripts contained little to no fibers. Electron microscopy is used to quantify dense cored vesicles (DCV) in OXT+ axons and to identify potential axonal release sites. The ependymal cells that line the ventricles were frequently permissive of DCV-containing OXT+ dendrites reaching the third ventricle. Our results highlight a mechanism in which oxytocin is released directly into the ventricles and circulates throughout the ventricular system, may serve as the primary source for oxytocin that binds to OXTR in the cerebral cortex.

Glial-Neuronal Crosstalk in the Ageing Murine Nodose Ganglion

Background: Viscerosensory stimuli are transmitted from the peripheral nervous system to the central nervous system for processing, integration, and adequate motor response. Proper signal transmission is guarded by bidirectional interaction between neurons and glia and their dysfunction has been associated with sensory deficits in both aging and visceral disorders. However, compared to the central nervous system, the bidirectional interactions and pathways of communication between glia and neurons remain relatively unknown. The nodose ganglion (NG) houses the cell bodies of visceral sensory nerves, traveling from the periphery, via the vagus nerve, to the central nervous system. Autonomic remodeling in this ganglion is associated with many of the visceral diseases that increase in incidence with age. Given the increasing recognition of the importance of glia in neuronal function and neurotransmission, understanding their inter-cell dynamics are vital to appropriately interpret the neurophysiology of aging. Objectives: This study aimed to better understand subtypes of neurons and glia most involved in neuro-glial communication, the underlying genetic pathways involved in this crosstalk, and how these pathways change with age. Methods: Single-cell RNA sequencing (scRNAseq) was performed on NG of young (12 weeks) and old (16 months) mice (C57BL/6; N = 6 for both groups). Distinct satellite glial cell (SGC) and neuronal populations were clustered based on transcriptomic similarities using dimensionality reduction. Marker gene analyses were used for cell-type identification. Specific pathways responsible for glial-glial, neuron-neuron, and glial-neuronal cross talk, including secreted signaling, extracellular-matrix receptor interactions, and cell-cell contact, were assessed between the different glial and neuronal sub-clusters in young and aged NG. The R-packages Seurat and CellChat were used for cell clustering and cell-cell communication signaling pathway identification, respectively. Results: SGC ( N = 6835 cells total) were identified by high expression of glial-specific transcripts, S100b and Fabp7. Neurons ( N = 2027 neurons total) were identified by expression of Tubb3, Snap25, and Uchl1. Five distinct glial clusters and six distinct neuronal clusters were identified by expression of known functional marker genes. While neuron-to-neuron communication was limited, significant communication within glial subtypes and between glia and sensory neurons was noted, with residential SGCs being most central to this network. Residential SGC interactions were noted most significantly for excitatory, multimodal neuronal subtypes ( Vglut2+, TRPV1/2+, Piezo1/2+). The primary pathways driving this communication were trophic pathways (e.g., vascular endothelial growth factor , Tenascin C, and Thrombospondin-1). Surprisingly, while glia-glia signaling did not change with aging, glial-neuronal communication seemed to increase. Conclusion: These data suggest that, while there is limited communication between neurons in the nodose ganglia, there is extensive glial-glial and glial-neuronal communication. Resident SGCs and excitatory multimodal neurons, which are known to be critical in sensory neurotransmission, are central to this crosstalk. Aging was associated with increased glial-neuronal crosstalk, though the cause and effect of this increased glial-neuronal communication with age requires further investigation. Dr. Vaseghi is supported by NIH R01HL148190 and AHA 970217. VvW is supported by the NWO Rubicon grant. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Coping with salinity extremes: Gill transcriptome profiling in the black-chinned tilapia (Sarotherodon melanotheron)

Steeper and sometimes extreme salinity gradients increasingly affect aquatic organisms because of climate change. Hypersalinity habitats demand powerful physiological adaptive strategies. Few teleost species have the capacity to spend their whole life cycle in salinities way over seawater levels. Focusing on the multifunctional gill, we unraveled the tilapia S. melanotheron key strategies to cope with different environmental conditions, ranging from freshwater up to hypersaline habitats. De novo transcriptome assembly based on RNAseq allowed for the analysis of 40,967 annotated transcripts among samples collected in three wild populations at 0, 40 and 80 ‰. A trend analysis of the expression patterns revealed responses across the salinity gradient with different gene pathways involved. Genes linked to ion transport, pH regulation and cell surface receptor signaling were mainly upregulated in the high salinity habitat. We identified tight junction proteins that were critical in high salinity habitats and that were different from the well-known tightening junctional proteins identified and expressed in fresh water. Expression profiles also suggest a change in the vascular tone that could be linked to an osmorespiratory compromise not only in fresh water, but also in high salinity environments. A striking downregulation of genes linked to the immune system and to the heat shock response was observed suggesting an energetic trade-off between immunity and acclimation/adaptation in the hypersaline habitat. The high expression of transcripts coding for immune and heat shock response in the freshwater habitat suggests the establishment of powerful mechanisms to protect gills from environmental threats and to maintain protein integrity. Non-directional expression trends were also detected with an upregulation of genes only in the hypersaline habitat (80 ‰) or only in the marine habitat (40 ‰). Unravel physiological strategies in S. melanotheron populations will help to better understand the molecular basis of fish euryhalinity in salinity-contrasted environments.

Abstract 2860: High-resolution mapping of oncogenic structural changes in osteosarcoma

Abstract We have leveraged long-read and spatial sequencing modalities to identify cancer-specific oncogenic alterations for treatment of osteosarcomas. Osteosarcoma is archetypal of cancers driven by aneuploidy and structural rearrangement rather than point mutations. The functional consequences of many SVs stem from their effect on multidimensional genome organization. SVs can enable enhancer hijacking, alter boundaries of topologically associated domains (TADs), and move gene loci to different regulatory compartments to affect gene expression. SVs can also lead to the formation of extrachromosomal DNA amplicons (ecDNA), which can be megabases in size and can incorporate both genes and regulatory elements to result in substantial increases in gene transcription and intratumor heterogeneity. Complex SVs may have profound implications for prognosis and treatment in OS and other cancers yet they remain poorly understood. By incorporating long-read optical genome mapping (OGM) and chromatin conformation capture sequencing (HiC), we studied SVs in osteosarcoma with the goal of elucidating their contribution to cancer development. We used a unique panel of 11 OS patient-derived xenograft (PDX) cell lines which have been previously characterized regarding response to targeted therapies as well as genomically via whole genome (WGS), RNA (RNA-seq), and chromatin accessibility (ATAC-seq) sequencing. To understand the complex genomic reorganization that occurs in osteosarcoma, we integrated OGM and WGS data for all 11 cell lines to describe detailed genome-wide SVs including ecDNA formation. PDX lines assayed using OGM exhibited between 106 and 704 translocations each (versus zero detected in germline controls) and we identified dozens of potential ecDNA amplicons, including one amplicon which may explain exceptionally high expression of YAP1 and BIRC3 transcripts. Further, we used HiC to describe the topological environment resulting from these SVs and observed potential enhancer hijacking events affecting dozens of known oncogenes including MYC and CDK4. HiC interaction maps among six cell lines reveal genomic compartments and TAD boundaries in detail, with 4,167 unique TAD boundaries identified among six cell lines using 50kb genomic bins. Despite the observed complexity of these genomes, we note that 71% of TAD boundaries are consistent in at least four of the six HiC assayed cell lines, and 44% are present in all six lines. Changing TAD boundaries are associated with numerous SVs and/or copy number discrepancies, and are associated with genomic regions that contain genes with dysregulated RNA transcript levels. Overall, OGM and HiC modalities add considerably to our ability to detect oncogenic SVs and the genes they affect. Citation Format: Andrew Scott Clugston, Stan Leung, Eunice Fuentes, Leanne Sayles, Megan Ostrowski, Coco Wu, Christina Curtis, Zhicheng Ma, Yanding Zhao, Georgi K. Marinov, Vijay Ramani, Marcus Breese, Alejandro Sweet-Cordero. High-resolution mapping of oncogenic structural changes in osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2860.

Heterologous expression of Arabidopsis laccase2, laccase4 and peroxidase52 driven under developing xylem specific promoter DX15 improves saccharification in populus

BackgroundSecondary cell wall holds considerable potential as it has gained immense momentum to replace the lignocellulosic feedstock into fuels. Lignin one of the components of secondary cell wall tightly holds the polysaccharides thereby enhancing the recalcitrance and complexity in the biomass. Laccases (LAC) and peroxidases (PRX) are the major phenyl-oxidases playing key functions during the polymerization of monolignols into lignin. Yet, the functions of laccase and peroxidases gene families remained largely unknown. Hence, the objective of this conducted study is to understand the role of specific LAC and PRX in Populus wood formation and to further investigate how the altered Lac and Prx expression affects biomass recalcitrance and plant growth. This study of heterologous expression of Arabidopsis Lac and Prx genes was conducted in poplar to avoid any otherwise occurring co-suppression mechanism during the homologous overexpression of highly expressed native genes. In the pursuit of optimizing lignocellulosic biomass for biofuel production, the present study focuses on harnessing the enzymatic potential of Arabidopsis thaliana Laccase2, Laccase4, and Peroxidase52 through heterologous expression.ResultsWe overexpressed selected Arabidopsis laccase2 (AtLac2), laccase4 (AtLac4), and peroxidase52 (AtPrx52) genes, based on their high transcript expression respective to the differentiating xylem tissues in the stem, in hybrid poplar (cv. 717) expressed under the developing xylem tissue-specific promoter, DX15 characterized the transgenic populus for the investigation of growth phenotypes and recalcitrance efficiency. Bioinformatics analyses conducted on AtLac2 and AtLac4 and AtPrx52, revealed the evolutionary relationship between the laccase gene and peroxidase gene homologs, respectively. Transgenic poplar plant lines overexpressing the AtLac2 gene (AtLac2-OE) showed an increase in plant height without a change in biomass yield as compared to the controls; whereas, AtLac4-OE and AtPrx52-OE transgenic lines did not show any such observable growth phenotypes compared to their respective controls. The changes in the levels of lignin content and S/G ratios in the transgenic poplar resulted in a significant increase in the saccharification efficiency as compared to the control plants.ConclusionsOverall, saccharification efficiency was increased by 35–50%, 21–42%, and 8–39% in AtLac2-OE, AtLac4-OE, and AtPrx52-OE transgenic poplar lines, respectively, as compared to their controls. Moreover, the bioengineered plants maintained normal growth and development, underscoring the feasibility of this approach for biomass improvement without compromising overall plant fitness. This study also sheds light on the potential of exploiting regulatory elements of DX15 to drive targeted expression of lignin-modifying enzymes, thereby providing a promising avenue for tailoring biomass for improved biofuel production. These findings contribute to the growing body of knowledge in synthetic biology and plant biotechnology, offering a sustainable solution to address the challenges associated with lignocellulosic biomass recalcitrance.

High CTLA-4 transcriptomic expression correlates with high expression of other checkpoints and with immunotherapy outcome.

CTLA-4 impedes the immune system's antitumor response. There are two Food and Drug Administration-approved anti-CTLA-4 agents - ipilimumab and tremelimumab - both used together with anti-PD-1/PD-L1 agents. To assess the prognostic implications and immunologic correlates of high CTLA-4 in tumors of patients on immunotherapy and those on non-immunotherapy treatments. We evaluated RNA expression levels in a clinical-grade laboratory and clinical correlates of CTLA-4 and other immune checkpoints in 514 tumors, including 489 patients with advanced/metastatic cancers and full outcome annotation. A reference population (735 tumors; 35 histologies) was used to normalize and rank transcript abundance (0-100 percentile) to internal housekeeping gene profiles. The most common tumor types were colorectal (140/514, 27%), pancreatic (55/514, 11%), breast (49/514, 10%), and ovarian cancers (43/514, 8%). Overall, 87 of 514 tumors (16.9%) had high CTLA-4 transcript expression (⩾75th percentile rank). Cancers with the largest proportion of high CTLA-4 transcripts were cervical cancer (80% of patients), small intestine cancer (33.3%), and melanoma (33.3%). High CTLA-4 RNA independently/significantly correlated with high PD-1, PD- L2, and LAG3 RNA levels (and with high PD-L1 in univariate analysis). High CTLA-4 RNA expression was not correlated with survival from the time of metastatic disease [N = 272 patients who never received immune checkpoint inhibitors (ICIs)]. However, in 217 patients treated with ICIs (mostly anti-PD-1/anti-PD- L1), progression-free survival (PFS) and overall survival (OS) were significantly longer among patients with high versus non-high CTLA-4 expression [hazard ratio, 95% confidence interval: 0.6 (0.4-0.9) p = 0.008; and 0.5 (0.3-0.8) p = 0.002, respectively]; results were unchanged when 18 patients who received anti-CTLA-4 were omitted. Patients whose tumors had high CTLA-4 and high PD-L1 did best; those with high PD-L1 but non-high CTLA-4 and/or other expression patterns had poorer outcomes for PFS (p = 0.004) and OS (p = 0.009) after immunotherapy. High CTLA-4, especially when combined with high PD-L1 transcript expression, was a significant positive predictive biomarker for better outcomes (PFS and OS) in patients on immunotherapy.

Ultra-stable mixotrophic denitrification coupled with anammox under organic stress for mainstream municipal wastewater treatment

Sulfur-based autotrophic denitrification (SAD) coupled with anammox is a promising process for autotrophic nitrogen removal in view of the stable nitrite accumulation during SAD. In this study, a mixotrophic nitrogen removal system integrating SAD, anammox and heterotrophic denitrification was established in a single-stage reactor. The long-term nitrogen removal performance was investigated under the intervention of organic carbon sources in real municipal wastewater. With the shortening of hydraulic retention time, the nitrogen removal rate of the mixotrophic system dominated by the autotrophic subsystem reached 0.46 Kg N/m³/d at an organic loading rate of 0.57 Kg COD/m³/d, with COD and total nitrogen removal efficiencies of 82.5 % and 94 %, respectively, realizing an ideal combination of autotrophic and heterotrophic systems. The 15NO3−-N isotope labeling experiments indicated that thiosulfate-driven autotrophic denitrification was the main pathway for nitrite supply accounting for 80.6 %, while anammox exhibited strong competitiveness for nitrite under the dual electron supply of sulfur and organic carbon sources and contributed to 65.1 % of nitrogen removal. Sludge granulation created differential functional distributions in different forms of sludge, with SAD showing faster reaction rate as well as higher nitrite accumulation rate in floc sludge, while anammox was more active in granular sludge. Real-time quantitative PCR, RT-PCR and high-throughput sequencing results revealed a dynamically changing community composition at the gene and transcription levels. The decrease in heterotrophic denitrification bacteria abundance indicated the effectiveness of the operational strategy for introduction of thiosulfate and maintaining the dominance of SAD in denitrification process in suppressing the excessive growth of heterotrophic bacteria in the mixotrophic system. The high transcriptional expression of sulfur-oxidizing bacteria (SOB) (Thiobacillus and Sulfurimonas) and anammox bacteria (Candaditus_Brocadia and Candidatus_Kuenenia) played a crucial role in the stable nitrogen removal.

LAG-3 transcriptomic expression patterns across malignancies: Implications for precision immunotherapeutics.

Lymphocyte activation gene 3 (LAG-3) or CD223 is a transmembrane protein that serves as an immune checkpoint which attenuates T-cell activation. Many clinical trials of LAG-3 inhibitors have had modest effects, but recent data indicate that the LAG-3 antibody relatlimab, together with nivolumab (anti-PD-1), provided greater benefit than nivolumab alone in patients with melanoma. In this study, the RNA expression levels of 397 genes were assessed in 514 diverse cancers at a clinical-grade laboratory (OmniSeq: https://www.omniseq.com/). Transcript abundance was normalized to internal housekeeping gene profiles and ranked (0-100 percentile) using a reference population (735 tumors; 35 histologies). A total of 116 of 514 tumors (22.6%) had high LAG-3 transcript expression (≥75 percentile rank). Cancers with the greatest proportion of high LAG-3 transcripts were neuroendocrine (47% of patients) and uterine (42%); colorectal had among the lowest proportion of high LAG-3 expression (15% of patients) (all p < 0.05 multivariate); 50% of melanomas were high LAG-3 expressors. There was significant independent association between high LAG-3 expression and high expression of other checkpoints, including programmed death-ligand 1 (PD-L1), PD-1, and CTLA-4, as well as high tumor mutational burden (TMB) ≥10 mutations/megabase, a marker for immunotherapy response (all p < 0.05 multivariate). However, within all tumor types, there was inter-patient variability in LAG-3 expression level. Prospective studies are therefore needed to determine if high levels of the LAG-3 checkpoint are responsible for resistance to anti-PD-1/PD-L1 or anti-CTLA-4 antibodies. Furthermore, a precision/personalized immunotherapy approach may require interrogating individual tumor immunograms to match patients to the right combination of immunotherapeutic agents for their malignancy.

Abstract 214: Patient-specific mapping of oncogenic structural changes in pediatric osteosarcoma

Abstract We have leveraged long-read and spatial sequencing modalities to identify patient-specific oncogenic alterations for treatment of pediatric osteosarcomas. Osteosarcoma (OS) is the most common primary bone malignancy in children and young adults, with over 500 cases identified each year in the United States. OS is archetypal of cancers driven by aneuploidy and structural rearrangement rather than point mutations. These structural variants (SVs) can alter gene expression stoichiometrically, but the functional consequences of many SVs stem from their effect on multidimensional genome organization. SVs can enable enhancer hijacking, alter boundaries of topologically associated domains (TADs), and move gene loci to different regulatory compartments affecting transcription. SVs can also form extrachromosomal DNA amplicons (ecDNA), which can be megabases in size and can incorporate both genes and regulatory elements to result in substantial increases in gene transcription and intratumor heterogeneity. Complex SVs may have profound implications for prognosis and treatment in OS and other cancers. By incorporating long-read optical genome mapping (OGM) and chromatin conformation capture sequencing (HiC), our goal is to detect complex tumor-specific SVs that will inform and refine pediatric OS subtypes, revealing new tumor-specific chemotherapy treatment options. We have generated a unique panel of 11 OS patient-derived xenograft (PDX) cell lines which have been previously characterized regarding response to targeted therapies using whole genome (WGS), RNA (RNA-seq), and chromatin accessibility (ATAC-seq) sequencing. To understand the complex genomic reorganization that occurs in OS, we have integrated OGM and WGS data for six cell lines, and OGM, WGS, and HiC data for two. SVs were detected using OGM, HiC, and WGS modalities, and we observed 59-61% concordance between SVs called using OGM and HiC, versus 22-45% when comparing with WGS calls. PDX lines assayed using OGM exhibit between 5,135 and 7,514 SVs each, and translocation numbers ranged from 212 to 1,422. These lines exhibit between dozens of potential ecDNA amplicons, including one amplicon which may explain that line’s specific and exceptionally high expression of YAP1 and BIRC2 transcripts. HiC interaction maps for two PDX lines reveal genomic compartments and TAD boundaries in detail, with 3,713 unique TAD boundaries identified between them using 50kb genomic bins. We observe that 60% of these boundaries are consistent between these lines, and 27% exhibit differences that suggest boundary loss or gain. These changing boundaries are associated with numerous SVs and/or copy number discrepancies, and with genomic regions containing genes with dysregulated RNA transcript levels. Incorporating OGM and HiC modalities adds substantially to our ability to detect oncogenic SVs and the genes they affect. Citation Format: Andrew S. Clugston, Megan Ostrowski, Coco Wu, Leanne Sayles, Stanley G. Leung, Vijay Ramani, E. Alejandro Sweet-Cordero, Marcus R. Breese. Patient-specific mapping of oncogenic structural changes in pediatric osteosarcoma [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 214.

TPL2, a New Prognostic Factor and a Potential Therapeutic Target in ABC-DLBCL

Background: Activated B-cell like diffuse large B-cell lymphoma (ABC-DLBCL) is a subtype of DLBCL, characterized by the constitutive activation of NF-κB and JAK/STAT3 pathways on the background of the chronic active BCR signaling. Being featured by mutations in MYD88 and/or CD79B, it potentially comprises the MCD/C5 subtype in the recent genetics-based categorization. In light of the inferior prognosis of this subtype to germinal center B-cell (GCB) like DLBCL, identification of subtype-specific strategies has been sought. Tumor progression locus 2 (TPL2) is an intracellular serine/threonine kinase that is reported to be activated downstream of TLRs/MYD88 signaling and to play roles in oncogenesis. However, the involvement of TPL2 in B-cell malignancies is less defined despite relatively higher transcript expression than in solid tumors. Results: To address the impact of TPL2 protein levels on DLBCL phenotypes, we performed an IHC analysis on 79 FFPE samples of DLBCL diagnosed and treated at our institution (48 non-GCB and 31 GCB according to Hans algorithm). When divided into two groups, one with strong TPL2 expression (TPL-high) and the other with no/weak expression (TPL2-low), the ratio of TPL2-high did not differ between non-GCB and GCB (67% vs 64%). However, TPL2-high had a significantly higher percentage of positive B symptom (42% vs 11%, p=0.005) and extra-nodal involvement (75% vs 52%, p=0.047) compared to TPL2-low, and these trends were observed more specifically in non-GCB. Moreover, it is noteworthy that TPL2-high showed a significantly lower overall survival in non-GCB (p=0.037), but did not in GCB (p=0.999) (Figure 1). Intriguingly, the analysis combined with pSTAT3 (Y705) staining revealed that TPL2-high accompanied by positive pSTAT3 tended to display particularly poor prognosis in non-GCB. Then, we analyzed the mechanism by which TPL2 is involved in the tumorigenesis of ABC-DLBCL using TMD8, a well-described ABC-DLBCL cell line with MYD88L265P and CD79BY196 mutations. A specific TPL2 inhibitor significantly affected the cell proliferation of TMD8 cells, whereas did not on the cells with GCB phenotype. Of note, TPL2 inhibitor induced G1 cell-cycle arrest via downregulation of CyclinE and CDK2 and upregulation of p27 in western blot analysis, which was confirmed by quantitative PCR. Next, we investigated the downstream signaling of TPL2 in ABC-DLBCL. Western blot analysis revealed that TPL2 inhibitor as well as genetic TPL2 knock down suppressed the activation of NF-κB components and STAT3, and the expression of c-MYC. These results implied the involvement of TPL2 in IL-6/IL-10 autocrine loop and resultant JAK/STAT3 activation. As expected, treatment with TPL2 inhibitor downregulated IL-6/IL-10 mRNA by quantitative PCR and accordingly decreased the levels of IL-6/IL-10 in the cell culture supernatant of treated TMD8 cells. Therefore, we speculated that TPL2 promoted the oncogenic activation of NF-κB and STAT3 pathways via IL-6/IL-10 upregulation in ABC-subtype. Lastly, we evaluated the physiological function of TPL2 by western blot analysis using 293T. Although an overexpression TPL2 alone did not elicit NF-κB and STAT3 activation, when co-expressed with MYD88L265P TPL2 obviously activated these pathways, whereas dominant negative TPL2 failed to demonstrate those effects even with MYD88L265P co-expression. These findings indicated that TPL2 could possibly exert its tumorigenicity in MYD88L265P-dependent manner in ABC-DLBCL. Conclusion: In association with the importance as an unfavorable prognostic factor, our results suggested that TPL2 might mediate the crosstalk between the oncogenic MYD88L265P and the activation of NF-κB and STAT3 pathways, coordinately contributing to the tumorigenesis of ABC-DLBCL. From this standpoint, TPL2 could emerge as a new therapeutic target in ABC-DLBCL. Figure legend Overall survival of non-GCB (A) and GCB (B) DLBCL patients, with regard to the TPL2 expression: TPL2-low (red line) or TPL2-high (black line). Overall survival was determined by the Kaplan-Meier method and compared by using the log-rank test. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The Effect of Different Frequencies of Pulsed Electromagnetic Fields on Cartilage Repair of Adipose Mesenchymal Stem Cell-Derived Exosomes in Osteoarthritis.

The intra-articular injection of mesenchymal stem cell (MSC)-derived exosomes has already been proved to reverse osteoarthritic cartilage degeneration. Pulsed electromagnetic field (PEMF) has been found to regulate the biogenic function of MSCs. However, the effect of PEMF on MSC-derived exosomes has not yet been characterized. The aim of this study was to elucidate the regulatory role of different frequencies of PEMF in promoting the osteoarthritic cartilage regeneration of MSC-derived exosomes. The adipose tissue-derived MSCs (AMSCs) were extracted from the epididymal fat of healthy rats and further exposed to the PEMF at 1 mT amplitude and a frequency of 15, 45, and 75 Hz, respectively, in an incubator. The chondrocytes were treated with interlukin-1β (IL-1β) and the regenerative effect of co-culturing with PEMF-exposed AMSC-derived exosomes was assessed via Western blot, quantitative polymerase chain reaction, and ELISA assays. A rat model of osteoarthritis was established by anterior cruciate ligament transection (ACLT) surgery and received 4 times intra-articular injection of PEMF-exposed AMSC-derived exosomes once a week. After 8 weeks, the knee joint specimens of rats were collected for micro-computed tomography and histologic analyses. PEMF-exposed AMSC-derived exosomes could be endocytosed with IL-1β-induced chondrocytes. Compared with the AMSC-derived exosomes alone, the PEMF-exposed AMSC-derived exosomes substantially suppressed the inflammation and extracellular matrix degeneration of IL-1β-induced chondrocytes as shown by higher expression of transcripts and proteins of COL2A1, SOX9, and ACAN and lower expression of MMP13 and caspase-1. Of these, the 75-Hz PEMF presented a more significant inhibitive effect than the 15-Hz and 45-Hz PEMFs. Furthermore, the intra-articular injection of 75-Hz PEMF-exposed exosomes could obviously increase the number of tibial epiphyseal trabeculae, lead to a remarkable decrease in Osteoarthritis Research Society International score, and upregulate the COL2A1 and ACAN protein level of the degenerated cartilage. The present study demonstrated that PEMF stimulation could effectively promote the regeneration effects of AMSC-derived exosomes on osteoarthritic cartilage. Compared with other frequency parameters, the PEMF at a frequency of 75 Hz showed a superior positive effect on AMSC-derived exosomes in suppressing the IL-1β-induced chondrocyte inflammation and extracellular matrix catabolism, as well as the osteoarthritic cartilage degeneration.

Bioinformatics analyses of rice calmodulin-lysine N-methyltransferases gene (OsCaM KMT) and characterization of its promoter from Oryza sativa L. sub sp. indica through transgenic approach

• Cloning of OsCaM KMT gene and promoter region from “Swarna”, an indica rice variety. • Presence of OsCAM KMT transcript in meristematic zone of rice plants, with higher expression in root tips suggesting they might be involved in auxin responsiveness. • Promoter analysis of OsCaM KMT::GUS construct depicted strong expression of GUS reporter gene in root tip, lateral roots and young leaf tip region. • Sequencing result indicated single nucleotide difference between the OsCAM KMT CDS from indica and japonica rice variety, resulting in silent mutation. Calmodulin-lysine N-methyltransferases (CaM KMT) belong to a family of protein lysine methyltransferases that catalyzes methylation of non-histone proteins. Lysine present at 116 position in the protein sequence of calmodulin is apparently methylated by CaM KMT in most species, however not characterized in rice. In the present study rice calmodulin-lysine N-methyltransferase ( OsCaM KMT ) gene has been isolated from an indica rice variety ‘Swarna’. Derived amino acid sequences of CaM KMT gene from rice and other plants were used in constructing a phylogenetic tree to show the evolutionary relationship among those species. Motif analysis along with comparative protein modeling was done to find out structural relationships among rice, Arabidopsis, and human CaM KMT. Gene expression analysis revealed differential tissue specific expression of OsCaM KMT . The highest transcript expression was revealed in the root tip followed by flower and new leaf, while, the least expression was found in the old leaf. In silico analysis was performed considering the 2 kilobases (kb) upstream promoter region of OsCaM KMT gene to find out various transcription factor binding sites. Analyses of the putative cis-regulatory elements revealed their involvement in tissue specific and stress dependent responses. For further characterization, the promoter region (2 kb) of OsCaM KMT gene was isolated, cloned, and sequenced. Histochemical GUS detection of transgenic rice depicted strong OsCaM KMT promoter expression in midribs, stomata, and tip of leaf tissues, central cylinder as well as tip parts of the primary root and lateral roots, along with significant expression in pollen grains, ovary, and vascular bundles. OsCaM KMT gene expression was corroborated by the histological GUS detection in OsCaM KMT promoter:: GUS fusion construct. Although several putative microRNAs (miRNAs) has been identified targeting OsCaM KMT coding DNA sequence (CDS) and UTR region, further studies are required to elucidate their tissue-specific regulation.

Assessing the Relationship Between Molecular Rejection and Parenchymal Injury in Heart Transplant Biopsies.

The INTERHEART study (ClinicalTrials.gov #NCT02670408) used genome-wide microarrays to detect rejection in endomyocardial biopsies; however, many heart transplants with no rejection have late dysfunction and impaired survival. We used the microarray measurements to develop a molecular classification of parenchymal injury. In 1320 endomyocardial biopsies from 645 patients previously studied for rejection-associated transcripts, we measured the expression of 10 injury-induced transcript sets: 5 induced by recent injury; 2 reflecting macrophage infiltration; 2 normal heart transcript sets; and immunoglobulin transcripts, which correlate with time. We used archetypal clustering to assign injury groups. Injury transcript sets correlated with impaired function. Archetypal clustering based on the expression of injury transcript sets assigned each biopsy to 1 of 5 injury groups: 87 Severe-injury, 221 Late-injury, and 3 with lesser degrees of injury, 376 No-injury, 526 Mild-injury, and 110 Moderate-injury. Severe-injury had extensive loss of normal transcripts (dedifferentiation) and increase in macrophage and injury-induced transcripts. Late-injury was characterized by high immunoglobulin transcript expression. In Severe- and Late-injury, function was depressed, and short-term graft failure was increased, even in hearts with no rejection. T cell-mediated rejection almost always had parenchymal injury, and 85% had Severe- or Late-injury. In contrast, early antibody-mediated rejection (AMR) had little injury, but late AMR often had the Late-injury state. Characterizing heart transplants for their injury state provides new understanding of dysfunction and outcomes and demonstrates the differential impact of T cell-mediated rejection versus AMR on the parenchyma. Slow deterioration from AMR emerges as a major contributor to late dysfunction.

Gene expression of secretory proteins in the nuptial pads of three Lithobates species (Anura: Ranidae)

Abstract Many amphibian species possess male-specific secretory breeding glands. In anurans, such specialized glands are often present as nuptial pads at the first digit of the hand, which are pressed against the female during amplexus. However, the role of nuptial pad secretions remains largely unknown. Here, we investigate the inner morphology as well as the RNA expression patterns of the nuptial pads of several Central American Lithobates species (Ranidae). As shown for the breeding glands of other amphibian species, the Lithobates nuptial pads are composed of large specialized mucus glands, excreting proteinaceous content to the surface. Whole-transcriptome sequencing revealed that for one of the species the most highly expressed transcripts encoding secretory proteins in the nuptial pads are sodefrin precursor-like factor (SPF) proteins, known to have a pheromone function in multiple amphibian species. The other two species, however, lack high expression of SPF transcripts but express other secretory proteins, whose roles in amphibian breeding glands remain unknown. Several of these proteins are highly expressed in all samples, suggesting a direct role during courtship or for the maintenance/development of the glands.

Single Nucleotide Variants in KIF14 Gene May Have Prognostic Value in Breast Cancer.

Human kinesin 14 (KIF14) is one of the 70 prognostic marker genes (so-called Amsterdam profile) previously identified by the microarray of breast carcinomas, and its high transcript expression in tumor specimens indicates a poor prognosis for patients. We performed a pilot study to explore the prognostic and predictive meaning of KIF14 germline genetic variability in breast cancer patients. KIF14 coding sequence, including 5' and 3' untranslated regions and overlaps to introns for identification of splicing sites, was analyzed using next-generation sequencing in the testing set of blood DNA samples from 105 breast cancer patients with clinical follow-up. After rigorous evaluation of major allele frequency, haplotype blocks, in silico predicted functional aspects, expression quantitative trait loci, and clinical associations, eight single nucleotide variants were subsequently validated in the evaluation set of 808 patients. Carriers of minor alleles G (rs17448931) or T (rs3806362) had significantly shorter overall survival than wild type homozygotes (p = 0.010 and p = 0.023, respectively) thus successfully replicating the results of the testing set. Both associations remained significant in the multivariate Cox regression analysis, including molecular subtype and stage as covariates (hazard ratio, HR = 1.7, 95% confidence interval (CI) = 1.1-2.8 for rs17448931 and HR = 1.9, CI 1.2-3.0 for rs3806362). In conclusion, our preliminary data suggest that minor alleles in rs17448931 and rs3806362 of KIF14 represent candidate biomarkers of poor prognosis of breast cancer patients. After pending validation in independent populations and eventual functional characterization, these candidates might become useful biomarkers in the clinics.

Prognostic capacity of the transcriptional expression of lactate dehydrogenase A in patients with head and neck squamous cell carcinoma.

To analyze the relationship between the transcriptional expression of lactate dehydrogenase A (LDHA) and the disease control in patients with a head and squamous cell carcinoma (HNSCC). We determined the transcriptional expression of LDHA in 110 HNSCC patients treated with surgery. Five-year disease-free survival for patients with a high transcriptional expression of LDHA (n= 51) was 39.2% (95% confidence interval [CI]: 25.3%-53.1%), and for patients with a low expression (n= 59), it was 63.6% (95% CI: 51.1%-76.1%) (p= 0.004). According to the results of a multivariate analysis, patients with a high transcriptional expression of LDHA had a 3.4-fold increased risk of tumor recurrence. Patients with a high transcriptional expression of LDHA tended to show a higher intensity of immunohistochemical expression of LDHA at the tumor cells (p= 0.086). In HNSCC patients treated with surgery, a high transcriptional expression of LDHA was associated with a significant decrease in disease-free survival.

Last but not least: BFL-1 as an emerging target for anti-cancer therapies

BFL-1 is an understudied pro-survival BCL-2 protein. The expression of BFL-1 is reported in many cancers, but it is yet to be clarified whether high transcript expression also always correlates with a pro-survival function. However, recent applications of BH3-mimetics for the treatment of blood cancers identified BFL-1 as a potential resistance factor in this type of cancer. Hence, understanding the role of BFL-1 in human cancers and how its up-regulation leads to therapy resistance has become an area of great clinical relevance. In addition, deletion of the murine homologue of BFL-1, called A1, in mice showed only minimal impacts on the well-being of these animals, suggesting drugs targeting BFL-1 would exhibit limited on-target toxicities. BFL-1 therefore represents a good clinical cancer target. Currently, no effective BFL-1 inhibitors exist, which is likely due to the underappreciation of BFL-1 as a potential target in the clinic and lack of understanding of the BFL-1 protein. In this review, the roles of BFL-1 in the development of different types of cancers and drug resistant mechanisms are discussed and some recent advances in the generation of BFL-1 inhibitors highlighted.

Abstract 1991: Characterization of the exome, transcriptome, and immunopeptidome to map alterations in primary and recurrent glioblastoma

Abstract Glioblastoma is known as the most aggressive and most common malignant primary tumor in the central nervous system. Current treatment options comprise maximal surgical resection followed by radiation and/or chemotherapy with temozolomide. However, these therapies are not able to eliminate all tumor cells, which in turn inevitably leads to disease recurrence and an alteration of identified targets in the context of clonal evolution and potential hypermutation. T cell-based immunotherapy holds great promise to target malignant cells with CAR T cell and vaccination strategies, showing first promising results in glioblastoma. These therapies rely on the rejection of cancer cells through recognition of tumor antigens and T cell-mediated cytotoxicity. In previous work, we have characterized such tumor antigens in primary glioblastoma (Neidert et al., Acta Neuropathol, 2018), nonetheless, alterations in relapsed disease have not been addressed thus far. This study investigated the whole exome, transcriptome, and mass-spectrometry-based immunopeptidome of 38 primary and 24 recurrent tumors, including 22 autologous glioblastoma pairs, to determine alterations that occur during glioblastoma progression on multiple comics levels. In concordance with Neftel et al., Cell, 2019, we identified mutations that can be allocated to astrocyte- and mesenchymal-like classified genes. In addition, an increase in the mutation rate in recurrent glioblastoma was observed which is attributed to radiation and chemotherapy pretreatment of tumors. These newly arising tumor-specific mutations give rise to HLA-presented neoepitopes in the primary cohort. Moreover, we identified transcripts that are differentially expressed between the two cohorts, showing a higher expression of transcripts related to immune system responses in the recurrent cohort. Immunopeptidome analysis of the two cohorts revealed high frequent glioblastoma-exclusive HLA class I and class II ligands presented in both the primary and recurrent cohort, serving as universally applicable tumor antigens. Class I and II HLA ligands of each sample were analyzed and revealed 2,146 HLA class I- and 2,753 HLA class II presented antigens that were uniquely identified on primary glioblastoma. A total of 610 and 1,886 source proteins represent recurrence-exclusive antigens presented on HLA class I or II molecules, respectively. Together this work addressed differences in tumor antigen expression and presentation between primary and recurrent glioblastoma using these omics layers to create an overview of the alterations that occur during disease progression. Besides providing a deep insight into the glioblastoma (immuno-)biology during progression, this study yields targets for innovative immunotherapeutic approaches to eliminate residual cells and improve survival in glioblastoma patients. Citation Format: Marissa L. Dubbelaar, Lena K. Freudenmann, Jonas Scheid, Julia Velz, Gioele Medici, Konstantina Kapolou, Malte Mohme, Leon Bichmann, Marie Gauder, Stefan Czemmel, Christopher Mohr, Daniel J. Kowalewski, Manfred Westphal, Katrin Lamszus, Luca Regli, Michael Weller, Hans-Georg Rammensee, Helmut Salih, Marian C. Neidert, Juliane S. Walz. Characterization of the exome, transcriptome, and immunopeptidome to map alterations in primary and recurrent glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1991.

Single-Dose Intrathecal Dorsal Root Ganglia Toxicity of Onasemnogene Abeparvovec in Cynomolgus Monkeys.

Intravenous onasemnogene abeparvovec is approved for the treatment of spinal muscular atrophy in children < 2 years. For later-onset patients, intrathecal onasemnogene abeparvovec may be advantageous over intravenous administration. Recently, microscopic dorsal root ganglion (DRG) changes were observed in nonhuman primates (NHPs) following intrathecal onasemnogene abeparvovec administration. To characterize these DRG findings, two NHP studies evaluating intrathecal onasemnogene abeparvovec administration were conducted: a 12-month study with a 6-week interim cohort and a 13-week study with a 2-week interim cohort. The latter investigated the potential impact of prednisolone or rituximab plus everolimus on DRG toxicity. An additional 6-month, single-dose, intravenous NHP study conducted in parallel evaluated onasemnogene abeparvovec safety (including DRG toxicity) with or without prednisolone coadministration. Intrathecal onasemnogene abeparvovec administration was well tolerated and not associated with clinical observations. Microscopic onasemnogene abeparvovec-related changes were observed in the DRG and trigeminal ganglion (TG) and included mononuclear cell inflammation and/or neuronal degeneration, which was colocalized with high vector transcript expression at 6 weeks postdose. Incidence and severity of DRG changes were generally decreased after 52 weeks compared with 6 weeks postdose. Other onasemnogene abeparvovec-related microscopic findings of axonal degeneration, mononuclear cell infiltrates and/or gliosis in the spinal cord, dorsal spinal nerve root/spinal nerves, and/or peripheral nerves were absent or found at decreased incidences and/or severities after 52 weeks. DRG and/or TG microscopic findings following intravenous onasemnogene abeparvovec dosing included minimal to slight neuronal degeneration and mononuclear cell inflammation at 6 weeks and 6 months postdose. Nervous system microscopic findings following intrathecal onasemnogene abeparvovec (≥1.2 × 1013 vg/animal) trended toward resolution after 52 weeks, supporting nonprogression of changes, including in the DRG. Onasemnogene abeparvovec-related DRG findings were not associated with electrophysiology changes and were not ameliorated by prednisolone or rituximab plus everolimus coadministration. The pathogenesis is possibly a consequence of increased vector genome transduction and/or transgene expression.

Transcriptional expressions of hsa-mir-183 predicted target genes as independent indicators for prognosis in bladder urothelial carcinoma.

Objective: To uncover novel prognostic and therapeutic targets for BLCA, our study is the first to investigate the role of hsa-mir-183 and its up-regulated predicted target genes in bladder urothelial carcinoma.Methods: To address this issue, our study explored the roles of hsa-mir-183 predicted target genes in the prognosis of BLCA via UALCAN, Metascape, Kaplan-Meier plotter, Human Protein Atlas, TIMER2.0, cBioPortal and Genomics of Drug Sensitivity in Cancer databases.Results: High transcriptional expressions of PDCD6, GNG5, PHF6 and MAL2 were markedly relevant to favorable OS in BLCA patients, whereas SLC25A15 and PTDSS1 had opposite expression significance. Additionally, high transcriptional expression of PDCD6, GNG5, PHF6, MAL2, SLC25A15 and PTDSS1 were significantly correlated with BLCA individual cancer stages and molecular subtypes. Furthermore, high mutation rate of PDCD6, MAL2, SLC25A15 and PTDSS1 were observed. Finally, TP53 mutation of PDCD6, GNG5, PHF6, MAL2, SLC25A15 and PTDSS1 has guiding significance for drug selection in BLCA.Conclusions: PDCD6, GNG5, PHF6, MAL2, SLC25A15 and PTDSS1 could be the advanced independent indicators for prognosis of BLCA patients, and TP53-mutation might be a biomarker for drug option in BLCA patients.