Expression Patterns Research Articles

Proteomic profiling of extracellular vesicles derived from human serum for the discovery of biomarkers in Avascular necrosis

BackgroundAvascular necrosis (AVN) is a medical condition characterized by the destruction of bone tissue due to a diminished blood supply. When the rate of tissue destruction surpasses the rate of regeneration, effective treatment becomes challenging, leading to escalating pain, arthritis, and bone fragility as the disease advances. A timely diagnosis is imperative to prevent and initiate proactive treatment for osteonecrosis. We explored the potential of differentially expressed proteins in serum-derived extracellular vesicles (EVs) as biomarkers for AVN of the femoral head in humans. We analyzed the genetic material contained in serum-derived exosomes from patients for early diagnosis, treatment, and prognosis of avascular necrosis.MethodsEVs were isolated from the serum of both patients with AVN and a control group of healthy individuals. Proteomic analyses were conducted to compare the expression patterns of these proteins by proteomic analysis using LC-MS/MS.ResultsOur results show that the levels of IGHV3-23, FN1, VWF, FGB, PRG4, FCGBP, and ZSWIM9 were upregulated in the EVs of patients with AVN compared with those of healthy controls. ELISA results showed that VWF and PRG4 were significantly upregulated in the patients with AVN.ConclusionsThese findings suggest that these EV proteins could serve as promising biomarkers for the early detection and diagnosis of AVN. Early diagnosis is paramount for effective treatment, and the identification of new osteonecrosis biomarkers is essential to facilitate swift diagnosis and proactive intervention. Our study provides novel insights into the identification of AVN-related biomarkers that can enhance clinical management and treatment outcomes.

Lead Decreases Bone Morphogenetic Protein-7 (BMP-7) Expression and Increases Renal Cell Carcinoma Growth in a Sex-Divergent Manner.

Both tissue and blood lead levels are elevated in renal cell carcinoma (RCC) patients. These studies assessed the impact of the subchronic lead challenge on the progression of RCC in vitro and in vivo. Lead challenge of Renca cells with 0.5 μM lead acetate for 10 consecutive passages decreased E-cadherin expression and cell aggregation. Proliferation, colony formation, and wound healing were increased. When lead-challenged cells were injected into mice, tumor size at day 21 was increased; interestingly, this increase was seen in male but not female mice. When mice were challenged with 32 ppm lead in drinking water for 20 weeks prior to tumor cell injection, there was an increase in tumor size in male, but not female, mice at day 21. To investigate the mechanism underlying the sex differences, the expression of sex hormone receptors in Renca cells was examined. Control Renca cells expressed estrogen receptor (ER) alpha but not ER beta or androgen receptor (AR), as assessed by qPCR, and the expression of ERα was increased in tumors in both sexes. In tumor samples harvested from lead-challenged cells, both ERα and AR were detected by qPCR, yet there was a significant decrease in AR seen in lead-challenged tumor cells from male mice only. This was paralleled by a plate-based array demonstrating the same sex difference in BMP-7 gene expression, which was also significantly decreased in tumors harvested from male but not female mice; this finding was validated by immunohistochemistry. A similar expression pattern was seen in tumors harvested from the mice challenged with lead in the drinking water. These data suggest that lead promotes RCC progression in a sex-dependent via a mechanism that may involve sex-divergent changes in BMP-7 expression.

Transcriptional Comparison Reveals Differential Resistance Mechanisms between CMV-Resistant PBC688 and CMV-Susceptible G29.

The Cucumber mosaic virus (CMV) presents a significant threat to pepper cultivation worldwide, leading to substantial yield losses. We conducted a transcriptional comparative study between CMV-resistant (PBC688) and -susceptible (G29) pepper accessions to understand the mechanisms of CMV resistance. PBC688 effectively suppressed CMV proliferation and spread, while G29 exhibited higher viral accumulation. A transcriptome analysis revealed substantial differences in gene expressions between the two genotypes, particularly in pathways related to plant-pathogen interactions, MAP kinase, ribosomes, and photosynthesis. In G29, the resistance to CMV involved key genes associated with calcium-binding proteins, pathogenesis-related proteins, and disease resistance. However, in PBC688, the crucial genes contributing to CMV resistance were ribosomal and chlorophyll a-b binding proteins. Hormone signal transduction pathways, such as ethylene (ET) and abscisic acid (ABA), displayed distinct expression patterns, suggesting that CMV resistance in peppers is associated with ET and ABA. These findings deepen our understanding of CMV resistance in peppers, facilitating future research and variety improvement.

Identification and expression profiling of microRNAs in leaf tissues of Foeniculum vulgare Mill. under salinity stress

ABSTRACT Foeniculum vulgare Mill. commonly known as fennel, is a globally recognized aromatic medicinal plant and culinary herb with widespread popularity due to its antimicrobial, antioxidant, carminative, and diuretic properties, among others. Although the phenotypic effects of salinity stress have been previously explored in fennel, the molecular mechanisms underlying responses to elevated salinity in this plant remain elusive. MicroRNAs (miRNAs) are tiny, endogenous, and extensively conserved non-coding RNAs (ncRNAs) typically ranging from 20 to 24 nucleotides (nt) in length that play a major role in a myriad of biological functions. In fact, a number of miRNAs have been extensively associated with responses to abiotic stress in plants. Consequently, employing computational methodologies and rigorous filtering criteria, 40 putative miRNAs belonging to 25 different families were characterized from fennel in this study. Subsequently, employing the psRNATarget tool, a total of 67 different candidate target transcripts for the characterized fennel miRNAs were predicted. Additionally, the expression patterns of six selected fennel miRNAs (i.e. fvu-miR156a, fvu-miR162a-3p, fvu-miR166a-3p, fvu-miR167a-5p, fvu-miR171a-3p, and fvu-miR408-3p) were analyzed under salinity stress conditions via qPCR. This article holds notable significance as it identifies not only 40 putative miRNAs in fennel, a non-model plant, but also pioneers the analysis of their expression under salinity stress conditions.

HER2-neu Expression and Survival in Colorectal Cancer in the South of Egypt; Immunohistochemistry and Genetic Study.

Colorectal cancer (CRC) is a major public health problem and one of leading cancer related death all over the world. One of the prognostic parameters that play a role in different types of cancer is HER2. However, the role of HER2 in CRC and its relation with clinicopathological features and survival is conflicting. We hypothesize that HER2 has different patterns of expression in CRC which may affect the prognosis of patients. We studied sixty specimens of colorectal carcinoma for HER2 immunohistochemistry and gene amplification and correlate it with clinicopathological features and patients` survival. Our data showed that negative HER2 expression was statistically associated with female gender (P = 0.010) and low & intermediate tumor budding (P = 0.030). There was a statistically significant relation between HER2 IHC and HER2 FISH amplification (P=0.000). Although neither HER2 immunoexpression and FISH amplification showed significant relation with overall survival nor disease free survival, HER2 amplified CRCs tended to have a worse survival compared with negative CRCs (40 months versus 50 months). The presence of male gender, lymphovascular invasion, nodal metastasis and distant metastasis (P = 0.013, 0.006, 0.006 and 0.000 respectively) were significantly statistically associated with poor overall survival. The presence of tumor grade III and high tumor budding (P = 0.035 and 0.007 respectively) were significantly statistically associated with shorter disease free survival. Our results showed that HER2 IHC 3+ staining is highly predictive of HER2 gene amplification in colorectal carcinomas. There is a tendency towards poorer prognosis in amplified HER2 CRC cases.

Expression of mitochondrial oxidative stress response genes in muscle is associated with mitochondrial respiration, physical performance, and muscle mass in the Study of Muscle, Mobility, and Aging.

Gene expression in skeletal muscle of older individuals may reflect compensatory adaptations in response to oxidative damage that preserve tissue integrity and maintain function. Identifying associations between oxidative stress response gene expression patterns and mitochondrial function, physical performance, and muscle mass in older individuals would further our knowledge of mechanisms related to managing molecular damage that may be targeted to preserve physical resilience. To characterize expression patterns of genes responsible for the oxidative stress response, RNA was extracted and sequenced from skeletal muscle biopsies collected from 575 participants (≥70 years old) from the Study of Muscle, Mobility, and Aging. Expression levels of 21 protein-coding RNAs related to the oxidative stress response were analyzed in relation to six phenotypic measures, including maximal mitochondrial respiration from muscle biopsies (Max OXPHOS), physical performance (VO2 peak, 400-m walking speed, and leg strength), and muscle size (thigh muscle volume and whole-body D3Cr muscle mass). The mRNA level of the oxidative stress response genes most consistently associated across outcomes are preferentially expressed within the mitochondria. Higher expression of mRNAs that encode generally mitochondria located proteins SOD2, TRX2, PRX3, PRX5, and GRX2 were associated with higher levels of mitochondrial respiration and VO2 peak. In addition, greater SOD2, PRX3, and GRX2 expression was associated with higher physical performance and muscle size. Identifying specific mechanisms associated with high functioning across multiple performance and physical domains may lead to targeted antioxidant interventions with greater impacts on mobility and independence.

Identification and characterization of circular RNAs in the skin of rainbow trout (Oncorhynchus mykiss) infected with infectious hematopoietic necrosis virus

Rainbow trout (Oncorhynchus mykiss) is an economically significant freshwater-farmed fish worldwide, and the frequent outbreaks of infectious hematopoietic necrosis (IHN) in recent years have gravely compromised the healthy growth of the rainbow trout aquaculture business. Fish skin is an essential immune barrier against the invasion of external pathogens, but it is poorly known about the role of circRNAs in rainbow trout skin. Therefore, we examined the expression profiles of circRNAs in rainbow trout skin following IHNV infection using RNA-seq. A total of 6607 circRNAs were identified, of which 34 circRNAs were differentially expressed (DE) and these DE circRNA source genes were related to immune-related pathways such as Toll-like receptor signaling pathway, NOD-like receptor signaling pathway, Cytokine-cytokine receptor interaction, ubiquitin mediated proteolysis, and ferroptosis. We used qRT-PCR, Sanger sequencing, and subcellular localization to validate the chosen DE circRNAs, confirming their localization and expression patterns in rainbow trout skin. Further, 12 DE circRNAs were selected to construct the circRNA-miRNA-mRNA regulatory network, finding one miRNA could connect one or more circRNAs and mRNAs, and some miRNAs were reported to be associated with antiviral immunity. The functional prediction findings revealed that novel_circ_002779 and novel_circ_004118 may act as sponges for miR-205-z and miR-155-y to regulate the expression of target genes TLR8 and PIK3R1, respectively, thus promoting antiviral immune responses in rainbow trout. These results shed light on the immunological mechanism of circRNAs in rainbow trout skin and offer fundamental information for further research on the innate immune system and breeding rainbow trout resistant to illness.

A β-catenin chromobody-based probe highlights endothelial maturation during vascular morphogenesis in vivo.

Visualization of protein dynamics is a crucial step in understanding cellular processes. Chromobodies, fluorescently labelled single-domain antibodies, have emerged as versatile probes for live cell imaging of endogenous proteins. However, how these chromobodies behave in vivo and how accurately they monitor tissue changes remain poorly explored. Here, we generated an endothelial-specific β-catenin chromobody-derived probe and analyzed its expression pattern during cardiovascular development in zebrafish. Using high-resolution confocal imaging, we show that the chromobody signal correlates with the localization of β-catenin in the nucleus and at cell-cell junctions, and thereby can be used to assess endothelial maturation. Loss of Cadherin 5 strongly affects the localization of the chromobody at the cell membrane, confirming the cadherin-based adherens junction role of β-catenin. Furthermore, using a genetic model to block blood flow, we observed that cell junctions are compromised in most endothelial cells but not in the endocardium, highlighting the heterogeneous response of the endothelium to the lack of blood flow. Overall, our data further expand the use of chromobodies for in vivo applications and illustrate their potential to monitor tissue morphogenesis at high resolution.

Prognostic significance of CHCHD2P9 and ZNF204P in breast cancer: exploring their expression patterns and associations with malignancy-related genes.

Non-coding RNAs (ncRNAs) have a crucial impact on diverse cellular processes, influencing the progression of breast cancer (BC). The objective of this study was to identify novel ncRNAs in BC with potential effects on patient survival and disease progression. We utilized the cancer genome atlas data to identify ncRNAs associated with BC pathogenesis. We explored the association between these ncRNA expressions and survival rates. A risk model was developed using candidate ncRNA expression and beta coefficients obtained from a multivariate Cox regression analysis. Co-expression networks were constructed to determine potential relationships between these ncRNAs and molecular pathways. For validation, we employed BC samples and the RT-qPCR method. Our findings revealed a noteworthy increase in the expression of AC093850.2 and CHCHD2P9 in BC, which was correlated with a poor prognosis. In contrast, ADAMTS9-AS1 and ZNF204P displayed significant downregulation and were associated with a favorable prognosis. The risk model, incorporating these four ncRNAs, robustly predicted patient survival. The co-expression network showed an effective association between levels of AC093850.2, CHCHD2P9, ADAMTS9-AS1, and ZNF204P and genes involved in pathways like metastasis, angiogenesis, metabolism, and DNA repair. The RT-qPCR results verified notable alterations in the expression of CHCHD2P9 and ZNF204P in BC samples. Pan-cancer analyses revealed alterations in the expression of these two ncRNAs across various cancer types. This study presents a groundbreaking discovery, highlighting the substantial dysregulation of CHCHD2P9 and ZNF204P in BC and other cancers, with implications for patient survival.

Comprehensive analysis of TROP2, PD-L1, stromal (s)TILs, and HER2 expression patterns in patients (pts) with metastatic HR+HER2- breast cancer.

1062 Background: Immune checkpoint inhibitors (ICI) have shown limited efficacy in hormone receptor positive (HR+) metastatic breast cancer (mBC) and the optimal ICI-based combination is unknown. The TROP2 antibody-drug conjugate sacituzumab govitecan (SG) demonstrated antitumor activity with a 5.5-month median PFS in endocrine refractory HR+ mBC. Given SG's potential for anticancer immunity, we sought to evaluate expression patterns of biomarkers of interest in HR+ mBC. Methods: This retrospective study included pts with HR+HER2- mBC identified at DFCI between 10/2020 and 12/2021 who received 0-1 prior lines of chemotherapy in the metastatic setting and consented to analysis of archival FFPE tissue samples. We aimed to 1) characterize expression patterns of TROP2 (SP295 H-score), PD-L1 (22C3 CPS), sTILs and HER2 (per central pathology review) in the most recent available sample; 2) assess the prognostic value of selected biomarker combinations for overall survival (OS) defined as the time from consent to either death due to any cause or last follow-up (Cox proportional hazard models; p-value <0.05 considered significant). Results: Among 142 pts included, median age at diagnosis was 46 yrs (IQR: 40–56). Baseline characteristics are shown (Table). At least one biomarker was available for 123 subjects (86.6%). TROP2 expression (n=61) was Medium/High (M-H: 101-300) in 43 pts (70.5%) and Low (L: 0-100) in 18 (29.5%). PD-L1 central assessment (n=120) revealed CPS ≥1 (PD-L1+) in 32 pts (26.7%) and <1 (PD-L1-) in 88 (73.3%); only 7 (5.8%) pts had CPS ≥10. sTILs (n=87) were ≥10% in 14 pts (16.1%) and <10% in 73 (83.9%). When analyzing TROP2 and PD-L1 (n=61), 15 pts (24.6%) were TROP2-M-H/PD-L1+, 28 (45.9%) were TROP2-M-H/PD-L1-, 5 (8.2%) were TROP2-L/PD-L1+, and 13 (21.3%) were TROP2-L/PD-L1-. When analyzing TROP2 and sTILs (n=54), 6 pts (11.1%) were TROP2-M-H/sTILs ≥10%, 32 (59.3%) were TROP2-M-H/sTILs <10%, 2 (3.7%) were TROP2-L/sTILs ≥10% and 14 (25.9%) were TROP2-L/sTILs <10%. For TROP2 and HER2 (n=46), 26 pts (56.5%) had TROP2-M-H/HER2-low tumors, 7 (15.2%) TROP2-M-H/HER2-0, 3 (6.5%) TROP2-L/HER2-low, and 10 (21.7%) TROP2-Low/HER2-0. Median OS (n=138) was 43.8 months; stratification for selected biomarker combinations will be presented although no statistical significance was found. Correlations with NGS data and HER2DX are ongoing and will be presented. Conclusions: Analyzing biomarkers of interest in pts with HR+ mBC unveiled diverse patterns, without associations with OS. Patient characteristics. [Table: see text]

Identification of effector candidates in Bipolaris sorokiniana and their expression profile analysis during pathogen-wheat interactions

Spot blotch disease, caused by the fungal pathogen Bipolaris sorokiniana, poses a significant threat to global production particularly wheat, and barley due to substantial yield losses. Similar to many fungal pathogens, the infection of host plants heavily depends on the pathogen's ability to secrete effector proteins, which manipulate host defenses and aid disease progression. However, a critical knowledge gap exists in the comprehensive identification and characterization of effector candidates (ECs) in B. sorokiniana, requiring further research efforts. Therefore, study aimed to systematically identify and characterize ECs in B. sorokiniana by conducting pathogenicity tests to confirm disease symptoms on wheat leaves, utilizing a rigorous bioinformatics approach to predict ECs through sequence analysis and structural similarities, and validating effector expression profiles during infection using RNA sequencing (RNA-Seq) and quantitative real-time polymerase chain reaction (qRT-PCR). Pathogenicity testing confirmed the typical symptoms of spot blotch disease upon inoculation with B. sorokiniana. Through bioinformatics analysis, 81 ECs were identified, showing dynamic expression patterns during infection stages. Among these ECs, genes such as Cocsa1|129517, Cocsa1|141231, and Cocsa1|193443 stood out due to their different expression patterns and structural similarities, indicating their potential roles as effectors. This study offers new novel insights into the effector repertoire of B. sorokiniana and its implications for spot blotch disease management. The identified ECs present promising targets for further investigation to clarify their specific roles in fungal virulence and host immune modulation.

Unraveling the role of lipid droplets and perilipin 2 in bovine luteal cells.

Steroidogenic tissues contain cytosolic lipid droplets that are important for steroidogenesis. Perilipin 2 (PLIN2), a structural coat protein located on the surface of lipid droplets in mammalian cells, plays a crucial role in regulating lipid droplet formation and contributing to various cellular processes such as lipid storage and energy homeostasis. Herein, we examine the role that PLIN2 plays in regulating progesterone synthesis in the bovine corpus luteum. Utilizing gene array databases and Western blotting, we have delineated the expression pattern of PLIN2 throughout the follicular to luteal transition. Our findings reveal the presence of PLIN2 in both ovarian follicular and steroidogenic luteal cells, demonstrating an increase in its levels as follicular cells transition into the luteal phase. Moreover, the depletion of PLIN2 via siRNA enhanced progesterone production in small luteal cells, whereas adenovirus-mediated overexpression of both PLIN2 and Perilipin 3 (PLIN3) induced an increase in cytosolic lipid droplet accumulation and decreased hormone-induced progesterone synthesis in these cells. Lastly, invivo administration of the luteolytic hormone prostaglandin F2α resulted in an upregulation of PLIN2 mRNA and protein expression, accompanied by a decline in serum progesterone. Our findings highlight the pivotal role of PLIN2 in regulating progesterone synthesis in the bovine corpus luteum, as supported by its dynamic expression pattern during the follicular to luteal transition and its responsiveness to luteotropic and luteolytic hormones. We suggest PLIN2 as a potential therapeutic target for modulating luteal function.

Genome-scale identification, expression and evolution analysis of B-box members in Dendrobium huoshanense

B-box (BBX) proteins have been recognized as vital determinants in plant development, morphogenesis, and adaptive responses to a myriad of environmental stresses. These zinc-finger proteins play a pivotal role in various biological processes. Their influence spans photomorphogenesis, the regulation of flowering, and imparting resilience to a wide array of challenges, encompassing both biotic and abiotic factors. Chromosome localization, gene structure and conserved motifs, phylogenetic analysis, collinearity analysis, expression profiling, fluorescence quantitative analysis, and tobacco transient transformation methods were used for functional localization and expression pattern analysis of the DhBBX gene. A total of 23 DhBBX members were identified from Dendrobium huoshanense. Subsequent phylogenetic evaluations effectively segregated these genes into five discrete evolutionary subsets. The predictions of subcellular localizations revealed that all these proteins were localized in the nucleus. The genetic composition and patterns showed that the majority of these genes consisted of several exons, with a few variations that could be attributed to transposon insertion. A comprehensive analysis using qRT-PCR was conducted to unravel the expression patterns of these genes in D. huoshanense, with a specific concentration on their responses to various hormone treatments and cold stress. Subcellular localization reveals that DhBBX21 and DhBBX9 are located in the nucleus. Our results provide a deep comprehension of the complex regulatory mechanisms of BBXs in response to various environmental and hormonal stimuli. These discoveries encourage further detailed and focused investigations into the operational dynamics of the BBX gene family in a wider range of plant species.

Closing classification gaps in luminal breast cancer with single-cell RNA-seq insights from normal breast lineages.

e13168 Background: Almost two decades ago, invasive breast cancer was classified into five subgroups based on expression patterns, each with distinct biological and prognostic characteristics. Advances in single-cell RNA sequencing offers insights into the expression profiles of normal luminal epithelial cells. Here, we investigate the connection between breast cancer (BC) classification and luminal expression profiles. Methods: We applied single-cell data from recent studies (Table) to compile gene signatures corresponding to expression profiles of luminal epithelial cell clusters in normal breast tissue. Normalized enrichment scores (ESs) of these signatures were calculated for bulk RNA-seq gene expression data from open source cohorts (TCGA-BRCA, SCAN-B, METABRIC). The BC molecular subtype of each tumor sample was predicted based on a modified PAM50 classification using gene expression profiling (1); LumA and LumB subtypes were combined into Luminal. ESs of signatures were compared between the BC subtypes (Table). Results: The ESs of all signatures differed significantly across BC subtypes (p < 0.001, Kruskal-Wallis test, Table 1). The ESs of signatures for LumSec-major and Luminal progenitor clusters are the highest in subtypes HER2-low and Basal-like (p < 0.001, Dwass-Steel-Critchlow-Fligner test [DSCF]). Basal-like also had the highest enrichment of the ductal signature (p < 0.001, DSCF). Luminal subtype showed the highest ESs of signatures corresponding to HR+ clusters of mature luminal cells and of the TDLU signature (p < 0.001, DSCF). HER2-low subtype showed the highest ES of the LumSec-lac signature (p < 0.001, DSCF). Conclusions: Our analyses challenge traditional classifications by showing that luminal breast cancers are less similar to most luminal epithelial clusters in normal breast tissue than other breast cancer subtypes. These findings suggest traditional expression classification may not fully capture the complexity of this largest subset of luminal-type breast cancers. 1. Turova et al., SABCS, 2023. [Table: see text]

Unlocking the Potential: Biomarkers of Response to Antibody-Drug Conjugates.

Antibody-drug conjugates (ADCs) have reshaped the cancer treatment landscape across a variety of different tumor types. ADCs' peculiar pharmacologic design combines the cytotoxic properties of chemotherapeutic agents with the selectivity of targeted therapies. At present, the approval of many ADCs used in clinical practice has not always been biomarker-driven. Indeed, predicting ADCs' activity and toxicity through the demonstration of specific biomarkers is still a great unmet need, and the identification of patients who can derive significant benefit from treatment with ADCs may often be uncertain. With the lack of robust predictive biomarkers to anticipate primary, intrinsic resistance to ADCs and no consolidated biomarkers to aid in the early identification of treatment resistance (ie, acquired resistance), the determination of precise biologic mechanisms of ADC activity and safety becomes priority in the quest for better patient-centric outcomes. Of great relevance, whether the target antigen expression is a determinant of ADCs' primary activity is still to be clarified, and available data remain quite controversial. Antigen expression assessment is typically performed on tissue biopsy, hence only providing information on a specific tumor site, therefore unable to capture heterogeneous patterns of tumor antigen expression. Quantifying the expression of the target antigen across all tumor sites would help better understand tumor heterogeneity, whereas molecularly characterizing tumor-intrinsic features over time might provide information on resistance mechanisms. In addition, toxicity can represent a critical concern, since most ADCs have a safety profile that resembles that of chemotherapies, with often unique adverse events requiring special management, possibly because of the differential in pharmacokinetics between the small-molecule agent versus payload of a similar class (eg, deruxtecan conjugate-related interstitial lung disease). As such, the identification of robust predictive biomarkers of safety and activity of ADCs has the potential to improve patient selection and enrich the population of patients most likely to derive a substantial clinical benefit, especially in those disease settings where different ADCs happen to be approved in competing clinical indications, with undefined biomarkers to make precise decision making and unclear data on how to sequence ADCs. At this point, the identification of clinically actionable biomarkers in the space of ADCs remains a top research priority.

Haplotype-resolved T2T genome assemblies and pangenome graph of pear reveal diverse patterns of allele-specific expression and genomic basis of fruit quality traits

Hybrid crops often exhibit increased yield and greater resilience, yet the genomic mechanism(s) underlying hybrid vigor or heterosis remain unclear, hindering our ability to predict the expression of phenotypic traits in hybrid breeding. Here, we generated haplotype-resolved T2T genome assemblies of two pear hybrid varieties ‘Yuluxiangli’ (YLX) and ‘Hongxiangsu’ (HXS) that share the same maternal parent, but differ in their paternal parents. We then used these assemblies to explore genome-scale landscape of allele-specific expression and create a pangenome graph for pear. Allele specific expression (ASE) was observed for close to 6000 genes in both hybrid cultivars. A subset of ASEGs related to fruit quality including sugar, organic acid and cuticular wax were identified, suggesting their important contributions to heterosis. Specifically, Ma1, a gene regulating fruit acidity, was absent in the paternal haplotypes of HXS and YLX. Further, a pangenome graph was built based on our assemblies and eight published pear genomes. Resequencing data for 139 cultivated pear genotypes (including 97 genotypes sequenced here) were subsequently aligned to the pangenome graph, revealing numerous SV hotspots and selective sweeps during pear diversification. As predicted, the Ma1 allele was found to be absent in varieties with low organic acid content, an association that was functionally validated by Ma1 over-expression in pear fruit and calli. Overall, the results unraveled contributions of allele-specific expression to heterosis involving fruit quality and provided a robust pangenome reference for high resolution allele discovery and association mapping.

Investigating the potential role of α-SNAP in preventing chemotherapy-induced ovarian dysfunction: Insights from cellular and animal models

BackgroundThe phosphoinositide 3-kinase/Akt/mammalian target of rapamycin complex 1 (PI3K/Akt/mTORC1) pathway plays a crucial role in the activation of primordial follicles. However, excessive activation and the loss of primordial follicles can lead to ovarian dysfunction. The alpha-soluble N-ethylmaleimide sensitive factor attachment protein (α-SNAP) protein has been implicated in PI3K/Akt/mTORCl signaling, suggesting its potential involvement in follicle activation. Thus, this study aimed to explore the role of α-SNAP in the activation of the PI3K/Akt/mTORC1 signaling pathway and its ability to mitigate the effects of cisplatin on ovarian function, using both in vitro and in vivo models. MethodsWe transfected KGN human ovarian granulosa cells (GCs) with small interfering RNA (siRNA) targeting α-SNAP to investigate the effects of α-SNAP inhibition on GC proliferation and apoptosis, as well as on the activity of the PI3K/Akt/mTORC1 pathway. In a mouse model, α-SNAP siRNA was delivered via an adeno-associated virus before treatment with cisplatin to assess its effects on follicle activation and ovarian function. Follicle counts at various growth stages, western blotting, and immunohistochemistry analyses were conducted to detect the expression of cleaved caspase-3, Ki67, α-SNAP, and p-mTOR. Additionally, the serum concentrations of anti-Müllerian hormone (AMH) were measured through an enzyme-linked immunosorbent assay. ResultsIn vitro, α-SNAP depletion prevented GC proliferation by inhibiting the PI3K/Akt/mTORC1 pathway, thereby indicating its role in the regulation of cell growth. In vivo, α-SNAP knockdown attenuated the cisplatin-induced overactivation of primordial follicles by suppressing the PI3K/Akt/mTORC1 signaling pathway and partially restoring AMH levels. In addition, the expression and distribution patterns of cleaved caspase-3, Ki67, α-SNAP, and p-mTOR varied across different follicular growth stages, suggesting a protective effect against chemotherapy-induced ovarian damage. ConclusionsInhibiting α-SNAP may attenuate GC proliferation by suppressing the PI3K/Akt/mTORC1 pathway, thereby mitigating the overactivation and loss of primordial follicles induced by cisplatin. Targeting α-SNAP may emerge as a novel strategy to prevent ovarian damage resulting from chemotherapy. However, these conclusions warrant repeated testing, and the mechanistic underpinnings of α-SNAP must be further elucidated in the future.

Dual analysis of IL-8 in non-small cell lung cancer: A clinical perspective on protein and RNA evaluation via imaging mass cytometry.

e15068 Background: The cytokine Interleukin-8 (IL-8) plays a pivotal role in the tumorigenesis and progression of non-small cell lung cancer (NSCLC), influencing both the tumor microenvironment and immune escape mechanisms. Given its significance, accurately assessing IL-8 levels at the protein and RNA levels could provide valuable insights into its prognostic value and inform therapeutic targeting strategies. This study utilizes imaging mass cytometry (IMC) to conduct a comprehensive evaluation of IL-8 expression in NSCLC, integrating both protein presence and gene expression analysis within the spatial context of the tumor microenvironment. Methods: Our approach leverages the capabilities of IMC to simultaneously detect protein and RNA levels of IL-8 in NSCLC tissue samples. For protein analysis, antibodies tagged with heavy metal isotopes specifically target IL-8, allowing for precise quantification and spatial localization within the tumor. Concurrently, RNA detection is achieved through hybridization probes designed for IL-8 mRNA, enabling the direct observation of gene expression patterns. This method provides a unique opportunity to correlate IL-8's protein abundance with its mRNA expression, offering insights into the regulatory mechanisms at play and their impact on the tumor's behavior and patient outcomes. Results: Preliminary data demonstrate a marked heterogeneity in IL-8 distribution at both the protein and RNA levels, with distinct patterns emerging that relate to tumor aggressiveness and immune infiltration. High IL-8 expression, identified through our integrated IMC approach, correlates with poor prognosis and reduced response to standard therapies, underscoring its potential as a biomarker for NSCLC management. Furthermore, the spatial analysis afforded by IMC highlights the microenvironmental niches where IL-8-mediated interactions could drive tumor progression and immune evasion. Conclusions: By employing IMC for the dual analysis of IL-8 protein and RNA in NSCLC, our study provides a comprehensive view of this cytokine's role in lung cancer pathology. The ability to visualize and quantify IL-8 expression within the spatial context of the tumor microenvironment opens new avenues for understanding its biological impact and for developing targeted therapies. Our findings advocate for the inclusion of IL-8 in the biomarker panel for NSCLC, with potential implications for patient stratification and personalized treatment planning. Future research will focus on expanding these observations to a larger cohort and exploring the therapeutic modulation of IL-8 as a strategy for improving NSCLC outcomes.

Molecular characterization, expression and immune functional analysis of cystatin 10 in turbot.

Cystatin is a protease inhibitor that also regulates genes expression linked to inflammation and plays a role in defense and regulation. Cystatin 10 (Smcys10) was cloned from Scophthalmus maximus and encodes a 145 amino acid polypeptide. The results of qRT-PCR showed that Smcys10 exhibited tissue-specific expression patterns, and its expression was significantly higher in the skin than in other tissues. The expression level of Smcys10 was significantly different in the skin, gill, head kidney, spleen and macrophages after Vibrio anguillarum infection, indicating that Smcys10 may play an important role in resistance to V. anguillarum infection. The recombinant Smcys10 protein showed binding and agglutinating activity in a Ca2+-dependent manner against bacteria. rSmcys10 treatment upregulated the expression of IL-10, TNF-α and TGF-β in macrophages of turbot and hindered the release of lactate dehydrogenase (LDH) from macrophages after V. anguillarum infection, which confirmed that rSmcys10 reduced the damage to macrophages by V. anguillarum. The NF-κB pathway was suppressed by Smcys10, as demonstrated by dual-luciferase analysis. These results indicated that Smcys10 is involved in the host antibacterial immune response.

Optimising promoters and subcellular localisation for constitutive transgene expression in Marchantia polymorpha.

Marchantia polymorpha has become an important model system for comparative studies and synthetic biology. The systematic characterisation of genetic elements would make heterologous gene expression more predictable in this testbed for gene circuit assembly and bioproduction. Yet, the toolbox of genetic parts for Marchantia includes only a few constitutive promoters that need benchmarking to assess their utility. We compared the expression patterns of previously characterized and new constitutive promoters. We found that driving expression with the double enhancer version of the cauliflower mosaic virus 35S promoter (pro35S×2) provided the highest yield of proteins although it also inhibits the growth of transformants. In contrast, promoters derived from the Marchantia ETHYLENE RESPONSE FACTOR 1 (MpERF1) and the CLASS II HOMEODOMAIN-LEUCINE ZIPPER (MpC2HDZ) genes drove expression to higher levels across all tissues without growth penalty and can provide intermediate levels of gene expression. In addition, we showed that the cytosol is the best subcellular compartment to target heterologous proteins for higher levels of expression without a significant growth burden. To demonstrate the potential of these promoters in Marchantia, we expressed the polycistronic RUBY betalain synthesis cassette to demonstrate coordinated expression of metabolic enzymes. A heat-shock inducible promoter was used to further mitigate growth burdens associated with high amounts of betalain accumulation. We have expanded the existing toolkit for gene expression in Marchantia and provide new resources for the Marchantia research community.