Functional Expression Research Articles

Distinct actions of testicular endocrine and lumicrine signaling on the proximal epididymal transcriptome.

The epididymal function and gene expression in mammals are under the control of the testis. Sex steroids are secreted from the testis and act on the epididymis in an endocrine manner. There is another, non-sex steroidal secreted signaling, named lumicrine signaling, in which testis-derived secreted proteins go through the male reproductive tract and act on the epididymis. The effects of such multiple regulations on the epididymis by the testis have been investigated for many genes. The recent development of high-throughput next-generation sequencing now enables us a further comparative survey of endocrine and lumicrine action-dependent gene expression. In the present study, testis-derived endocrine and lumicrine actions on epididymal gene expression were comparatively investigated by RNA-seq transcriptomic analyses. This investigation utilized experimental animal models in which testis-derived endocrine and/or lumicrine actions were interfered with, such as unilateral or bilateral orchidectomy. By bilateral orchidectomy, which interferes with both endocrine and lumicrine actions, 431 genes were downregulated. By unilateral orchidectomy, which also interferes with endocrine and lumicrine actions by the unilateral testis, but the endocrine action was compensated by the contralateral testis, 283 genes were downregulated. The content of such genes downregulated by unilateral orchidectomy was like those of lumicrine action-interfered efferent duct-ligation, W/Wv, and Nell2-/- mice. When genes affected by unilateral and bilateral orchidectomy were compared, 154 genes were commonly downregulated, whereas 217 genes were specifically downregulated only by bilateral orchidectomy, indicating the distinction between endocrine and lumicrine actions on the proximal epididymal transcriptome. Comparative transcriptome analyses also showed that the expressions of genes emerging since Amniota were notably impacted by bilateral orchidectomy, unilateral orchidectomy, and lumicrine action-interfering treatments; the degree of influence from these treatments varied based on the evolutionary stage beyond Amniota. These findings unveil an evolutional transition of regulated gene expression in the proximal epididymis by two different testis-derived signaling mechanisms.

CGAS suppresses hepatocellular carcinoma independent of its cGAMP synthase activity.

Cyclic GMP-AMP synthase (cGAS) is a key innate immune sensor that recognizes cytosolic DNA to induce immune responses against invading pathogens. The role of cGAS is conventionally recognized as a nucleotidyltransferase to catalyze the synthesis of cGAMP upon recognition of cytosolic DNA, which leads to the activation of STING and production of type I/III interferon to fight against the pathogen. However, given that hepatocytes are lack of functional STING expression, it is intriguing to define the role of cGAS in hepatocellular carcinoma (HCC), the liver parenchymal cells derived malignancy. In this study, we revealed that cGAS was significantly downregulated in clinical HCC tissues, and its dysregulation contributed to the progression of HCC. We further identified cGAS as an immune tyrosine inhibitory motif (ITIM) containing protein, and demonstrated that cGAS inhibited the progression of HCC and increased the response of HCC to sorafenib treatment by suppressing PI3K/AKT/mTORC1 pathway in cellular and animal models. Mechanistically, cGAS recruits SH2-containing tyrosine phosphatase 1 (SHP1) via ITIM, and dephosphorylates p85 in phosphatidylinositol 3-kinase (PI3K), which leads to the suppression of AKT-mTORC1 pathway. Thus, cGAS is identified as a novel tumor suppressor in HCC via its function independent of its conventional role as cGAMP synthase, which indicates a novel therapeutic strategy for advanced HCC by modulating cGAS signaling.

Niche-dependent sponge hologenome expression profiles and the host-microbes interplay: a case of the hawaiian demosponge Mycale Grandis.

Most researches on sponge holobionts focus primarily on symbiotic microbes, yet data at the level of the sponge hologenome are still relatively scarce. Understanding of the sponge host and its microbial gene expression profiles and the host-microbes interplay in different niches represents a key aspect of sponge hologenome. Using the Hawaiian demosponge Mycale grandis in different niches as a model, i.e. on rocks, on the surface of coral Porites compressa, under alga Gracilaria salicornia, we compared the bacterial and fungal community structure, functional gene diversity, expression pattern and the host transcriptome by integrating open-format (deep sequencing) and closed-format (GeoChip microarray) high-throughput techniques. Little inter-niche variation in bacterial and fungal phylogenetic diversity was detected for M. grandis in different niches, but a clear niche-dependent variability in the functional gene diversity and expression pattern of M. grandis host and its symbiotic microbiota was uncovered by GeoChip microarray and transcriptome analyses. Particularly, sponge host genes related to innate immunity and microbial recognition showed a strong correlation with the microbial symbionts' functional gene diversity and transcriptional richness in different niches. The cross-niche variability with respect to the symbiont functional gene diversity and the transcriptional richness of M. grandis holobiont putatively reflects the interplay of niche-specific selective pressure and the symbiont functional diversity. Niche-dependent gene expression profiles of M. grandis hologenome and the host-microbes interplay were suggested though little inter-niche variation in bacterial and fungal diversity was detected, particularly the sponge innate immunity was found to be closely related to the symbiotic microbes. Altogether, these findings provide novel insights into the black box of one sponge holobiont in different niches at the hologenome level.

Thermosensitive TRP Channels Are Functionally Expressed and Influence the Lipogenesis in Human Meibomian Gland Cells.

While the involvement of thermosensitive transient receptor potential channels (TRPs) in dry eye disease (DED) has been known for years, their expression in the meibomian gland (MG) has never been investigated. This study aims to show their expression and involvement in the lipogenesis of the MG, providing a possible new drug target in the treatment of DED. Our RT-PCR, Western blot and immunofluorescence analysis showed the expression of TRPV1, TRPV3, TRPV4 and TRPM8 in the MG at the gene and the protein level. RT-PCR also showed gene expression of TRPV2 but not TRPA1. Calcium imaging and planar patch-clamping performed on an immortalized human meibomian gland epithelial cell line (hMGECs) demonstrated increasing whole-cell currents after the application of capsaicin (TRPV1) or icilin (TRPM8). Decreasing whole-cell currents could be registered after the application of AMG9810 (TRPV1) or AMTB (TRPM8). Oil red O staining on hMGECs showed an increase in lipid expression after TRPV1 activation and a decrease after TRPM8 activation. We conclude that thermo-TRPs are expressed at the gene and the protein level in MGs. Moreover, TRPV1 and TRPM8's functional expression and their contribution to their lipid expression could be demonstrated. Therefore, TRPs are potential drug targets and their clinical relevance in the therapy of meibomian gland dysfunction requires further investigation.

Photophysical image analysis: Unsupervised probabilistic thresholding for images from electron-multiplying charge-coupled devices.

We introduce the concept photophysical image analysis (PIA) and an associated pipeline for unsupervised probabilistic image thresholding for images recorded by electron-multiplying charge-coupled device (EMCCD) cameras. We base our approach on a closed-form analytic expression for the characteristic function (Fourier-transform of the probability mass function) for the image counts recorded in an EMCCD camera, which takes into account both stochasticity in the arrival of photons at the imaging camera and subsequent noise induced by the detection system of the camera. The only assumption in our method is that the background photon arrival to the imaging system is described by a stationary Poisson process (we make no assumption about the photon statistics for the signal). We estimate the background photon statistics parameter, λbg, from an image which contains both background and signal pixels by use of a novel truncated fit procedure with an automatically determined image count threshold. Prior to this, the camera noise model parameters are estimated using a calibration step. Utilizing the estimates for the camera parameters and λbg, we then introduce a probabilistic thresholding method, where, for the first time, the fraction of misclassified pixels can be determined a priori for a general image in an unsupervised way. We use synthetic images to validate our a priori estimates and to benchmark against the Otsu method, which is a popular unsupervised non-probabilistic image thresholding method (no a priori estimates for the error rates are provided). For completeness, we lastly present a simple heuristic general-purpose segmentation method based on the thresholding results, which we apply to segmentation of synthetic images and experimental images of fluorescent beads and lung cell nuclei. Our publicly available software opens up for fully automated, unsupervised, probabilistic photophysical image analysis.

Abstract LB068: Repurposing chimeric antigen receptor regulatory T cells for treating cancer

Abstract Solid tumors are the deadliest form of cancer. Over the past decade, chimeric antigen receptor (CAR) T-cell therapy has led to remarkable regression rates in leukemia and lymphoma. However, solid tumors have remained refractory to CAR T-cell therapy, with CAR T cells either failing to penetrate the solid tumor microenvironment (TME) or becoming exhausted once in it. Mechanisms behind these properties of the TME include nutrient scarcity, aberrant vascularization, stromal cell deposition, expression of inhibitory immune receptors by cancer cells, and the presence of immunosuppressive cells. Interestingly, there is a subset of T cells found in the TME - regulatory T cells (Tregs). Dedicated to suppressing immune responses, Tregs constitute a barrier to anticancer immunity by migrating to and heavily accumulating in solid tumors, inhibiting local immunity. Tregs can be redirected towards any cell surface antigen using a CAR and have been shown to be cytotoxic towards target antigen presenting cells. Can Tregs be converted from tumor protectors to anti-tumor effector cells? To test the anti-tumor potential of CAR Tregs, we modified human Tregs with an anti-CD19 CD28-CD3z CAR. Tregs activated via the CAR or endogenous TCR/CD28 were evaluated at the functional and gene expression levels. CAR and endogenous TCR/CD28 activation resulted in similar cell expansion and Treg stability (FOXP3 and HELIOS expression). However, CAR-mediated activation markedly increased Treg proinflammatory cytokine production. Moreover, compared to their TCR/CD28-activated counterparts, CAR Tregs displayed increased cytotoxic activity towards tumor cells and a diminished capacity to modulate antigen-presenting cells and suppress T cell proliferation, suggesting a global shift from suppression to inflammation. Strikingly, CAR Tregs suppressed tumor growth in immunodeficient mice inoculated with antigen-expressing B-cell leukemia, myeloid leukemia, and lung epithelial carcinoma. Single-cell RNA sequencing identified a cell cluster enriched in CAR Tregs characterized by high expression of effector T cell genes, such as IFNG, GZMB, and CD40LG, revealing that CAR activation creates a highly active population of proinflammatory Tregs. Multiplex cytokine analysis, whole-transcriptome RNA-seq, and intracellular cytokine staining confirmed that IFNG production by FOXP3+ Tregs is induced specifically by CAR activation. Of note, murine CAR Tregs are also cytotoxic towards tumor cells. Current experiments are focused on pinpointing and validating transcription factors and surface markers characteristic of proinflammatory CAR Tregs and further assessing CAR Treg antitumor efficacy in vivo. In conclusion, CAR-mediated activation induces a novel proinflammatory Treg phenotype that can be exploited in the next generation of engineered immune cell therapies against solid tumors. Citation Format: Leonardo M. Ferreira, Russell W. Cochrane, Rob A. Robino, Bryan Granger, Eva Allen, Vishnu Gudivada, Aguirre A. de Cubas, Stefano Berto. Repurposing chimeric antigen receptor regulatory T cells for treating cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB068.

Proteasome-Associated Syndromes: Updates on Genetics, Clinical Manifestations, Pathogenesis, and Treatment.

The ubiquitin-proteasome system (UPS) has a critical role in post-translational protein modification that is essential for the maintenance of all cellular functions, including immune responses. The proteasome complex is ubiquitously expressed and is responsible for degradation of short-lived structurally abnormal, misfolded and not-needed proteins that are targeted for degradation via ubiquitin conjugation. Over the last 14years, an increasing number of human diseases have been linked to pathogenic variants in proteasome subunits and UPS regulators. Defects of the proteasome complex or its chaperons - which have a regulatory role in the assembly of the proteasome - disrupt protein clearance and cellular homeostasis, leading to immune dysregulation, severe inflammation, and neurodevelopmental disorders in humans. Proteasome-associated diseases have complex inheritance, including monogenic, digenic and oligogenic disorders and can be dominantly or recessively inherited. In this review, we summarize the current known genetic causes of proteasomal disease, and discuss the molecular pathogenesis of these conditions based on the function and cellular expression of mutated proteins in the proteasome complex.

Low mutation rate of spontaneous mutants enables detection of causative genes by comparing whole genome sequences.

In the early 1900s, mutation breeding to select varieties with desirable traits using spontaneous mutation was actively conducted around the world, including Japan. In rice, the number of fixed mutations per generation was estimated to be 1.38-2.25. Although this low mutation rate was a major problem for breeding in those days, in the modern era with the development of next-generation sequencing (NGS) technology, it was conversely considered to be an advantage for efficient gene identification. In this paper, we proposed an in silico approach using NGS to compare the whole genome sequence of a spontaneous mutant with that of a closely related strain with a nearly identical genome, to find polymorphisms that differ between them, and to identify the causal gene by predicting the functional variation of the gene caused by the polymorphism. Using this approach, we found four causal genes for the dwarf mutation, the round shape grain mutation and the awnless mutation. Three of these genes were the same as those previously reported, but one was a novel gene involved in awn formation. The novel gene was isolated from Bozu-Aikoku, a mutant of Aikoku with the awnless trait, in which nine polymorphisms were predicted to alter gene function by their whole-genome comparison. Based on the information on gene function and tissue-specific expression patterns of these candidate genes, Os03g0115700/LOC_Os03g02460, annotated as a short-chain dehydrogenase/reductase SDR family protein, is most likely to be involved in the awnless mutation. Indeed, complementation tests by transformation showed that it is involved in awn formation. Thus, this method is an effective way to accelerate genome breeding of various crop species by enabling the identification of useful genes that can be used for crop breeding with minimal effort for NGS analysis.

Cloning, functional expression, and pharmacological characterization of inwardly rectifying potassium channels (Kir) from Apis mellifera

Potassium channels belong to the super family of ion channels and play a fundamental role in cell excitability. Kir channels are potassium channels with an inwardly rectifying property. They play a role in setting the resting membrane potential of many excitable cells including neurons. Although putative Kir channel family genes can be found in the Apis mellifera genome, their functional expression, biophysical properties, and sensitivity to small molecules with insecticidal activity remain to be investigated. We cloned six Kir channel isoforms from Apis mellifera that derive from two Kir genes, AmKir1 and AmKir2, which are present in the Apis mellifera genome. We studied the tissue distribution, the electrophysiological and pharmacological characteristics of three isoforms that expressed functional currents (AmKir1.1, AmKir2.2, and AmKir2.3). AmKir1.1, AmKir2.2, and AmKir2.3 isoforms exhibited distinct characteristics when expressed in Xenopus oocytes. AmKir1.1 exhibited the largest potassium currents and was impermeable to cesium whereas AmKir2.2 and AmKir2.3 exhibited smaller currents but allowed cesium to permeate. AmKir1 exhibited faster opening kinetics than AmKir2. Pharmacological experiments revealed that both AmKir1.1 and AmKir2.2 are blocked by the divalent ion barium, with IC50 values of 10−5 and 10−6 M, respectively. The concentrations of VU041, a small molecule with insecticidal properties required to achieve a 50% current blockade for all three channels were higher than those needed to block Kir channels in other arthropods, such as the aphid Aphis gossypii and the mosquito Aedes aegypti. From this, we conclude that Apis mellifera AmKir channels exhibit lower sensitivity to VU041.

Combined sensory, volatilome and transcriptome analyses identify a limonene terpene synthase as a major contributor to the characteristic aroma of a Coffea arabica L. specialty coffee

BackgroundThe fruity aromatic bouquet of coffee has attracted recent interest to differentiate high value market produce as specialty coffee. Although the volatile compounds present in green and roasted coffee beans have been extensively described, no study has yet linked varietal molecular differences to the greater abundance of specific substances and support the aroma specificity of specialty coffees.ResultsThis study compared four Arabica genotypes including one, Geisha Especial, suggested to generate specialty coffee. Formal sensory evaluations of coffee beverages stressed the importance of coffee genotype in aroma perception and that Geisha Especial-made coffee stood out by having fine fruity, and floral, aromas and a more balanced acidity. Comparative SPME–GC–MS analyses of green and roasted bean volatile compounds indicated that those of Geisha Especial differed by having greater amounts of limonene and 3-methylbutanoic acid in agreement with the coffee cup aroma perception. A search for gene ontology differences of ripening beans transcriptomes of the four varieties revealed that they differed by metabolic processes linked to terpene biosynthesis due to the greater gene expression of prenyl-pyrophosphate biosynthetic genes and terpene synthases. Only one terpene synthase (CaTPS10-like) had an expression pattern that paralleled limonene loss during the final stage of berry ripening and limonene content in the studied four varieties beans. Its functional expression in tobacco leaves confirmed its functioning as a limonene synthase.ConclusionsTaken together, these data indicate that coffee variety genotypic specificities may influence ripe berry chemotype and final coffee aroma unicity. For the specialty coffee variety Geisha Especial, greater expression of terpene biosynthetic genes including CaTPS10-like, a limonene synthase, resulted in the greater abundance of limonene in green beans, roasted beans and a unique citrus note of the coffee drink.

CDK inhibition results in pharmacologic BRCAness increasing sensitivity to olaparib in BRCA1-WT and olaparib resistant in Triple Negative Breast Cancer

One in three Triple Negative Breast Cancer (TNBC) is Homologous Recombination Deficient (HRD) and susceptible to respond to PARP inhibitor (PARPi), however, resistance resulting from functional HR restoration is frequent. Thus, pharmacologic approaches that induce HRD are of interest. We investigated the effectiveness of CDK-inhibition to induce HRD and increase PARPi sensitivity of TNBC cell lines and PDX models. Two CDK-inhibitors (CDKi), the broad range dinaciclib and the CDK12-specific SR-4835, strongly reduced the expression of key HR genes and impaired HR functionality, as illustrated by BRCA1 and RAD51 nuclear foci obliteration. Consequently, both CDKis showed synergism with olaparib, as well as with cisplatin and gemcitabine, in a range of TNBC cell lines and particularly in olaparib-resistant models. In vivo assays on PDX validated the efficacy of dinaciclib which increased the sensitivity to olaparib of 5/6 models, including two olaparib-resistant and one BRCA1-WT model. However, no olaparib response improvement was observed in vivo with SR-4835. These data support that the implementation of CDK-inhibitors could be effective to sensitize TNBC to olaparib as well as possibly to cisplatin or gemcitabine.

Adiponectin overexpression improves metabolic abnormalities caused by acid ceramidase deficiency but does not prolong lifespan in a mouse model of Farber Disease

Farber Disease is a debilitating and lethal childhood disease of ceramide accumulation caused by acid ceramidase deficiency. The potent induction of a ligand-gated neutral ceramidase activity promoted by adiponectin may provide sufficient lowering of ceramides to allow for the treatment of Farber Disease. In vitro, adiponectin or adiponectin receptor agonist treatments lowered total ceramide concentrations in human fibroblasts from a patient with Farber Disease. However, adiponectin overexpression in a Farber Disease mouse model did not improve lifespan or immune infiltration. Intriguingly, mice heterozygous for the Farber Disease mutation were more prone to glucose intolerance and insulin resistance when fed a high-fat diet, and adiponectin overexpression protected from these metabolic perturbations. These studies suggest that adiponectin evokes a ceramidase activity that is not reliant on the functional expression of acid ceramidase, but indicates that additional strategies are required to ameliorate outcomes of Farber Disease.

Identification of two critical amino acid residues in short-chain aldehyde-responsive odorant receptors.

Mammalian odorant receptors (ORs) are crucial for detecting a broad spectrum of odorants, yet their functional expression poses a significant challenge, often requiring Receptor-transporting proteins (RTPs). This study examines mouse Olfr733 and Olfr732, which, despite high homology, show different functional expression profiles in heterologous cell systems. Our research aimed to identify key amino acids impacting Olfr733's functional expression. We discovered that G112FBW3.40 and L148PBW4.49 (Ballesteros-Weinstein numbering in superscript) substitutions in Olfr732 markedly enhance its RTP-independent expression and ligand responsiveness, mirroring Olfr733. These substitutions, particularly Phe112 and Leu148, are crucial for aldehyde recognition and membrane localization in Olfr733, respectively. While Olfr732-type ORs are conserved across species, Olfr733-types, unique to specific rodents, appear to have evolved from Olfr732, with Pro148 enhancing membrane expression and aldehyde sensitivity. Mouse ORs with ProBW4.49 tend to exhibit improved membrane expression compared to their paralogs, especially when co-expressed with RTP1S. This study concludes that the Pro residue in the fourth transmembrane domain significantly contributes to the structural stability of certain olfactory receptors, highlighting the intricate molecular mechanisms underlying OR functionality and evolution.

An Explicit Form of Ramp Function

In this paper, an analytical exact form of the ramp function is presented. This seminal function constitutes a fundamental concept of the digital signal processing theory and is also involved in many other areas of applied sciences and engineering. In particular, the ramp function is performed in a simple manner as the pointwise limit of a sequence of real and continuous functions with pointwise convergence. This limit is zero for strictly negative values of the real variable x, whereas it coincides with the independent variable x for strictly positive values of the variable x. Here, one may elucidate beforehand that the pointwise limit of a sequence of continuous functions can constitute a discontinuous function, on the condition that the convergence is not uniform. The novelty of this work, when compared to other research studies concerning analytical expressions of the ramp function, is that the proposed formula is not exhibited in terms of miscellaneous special functions, e.g., gamma function, biexponential function, or any other special functions, such as error function, hyperbolic function, orthogonal polynomials, etc. Hence, this formula may be much more practical, flexible, and useful in the computational procedures, which are inserted into digital signal processing techniques and other engineering practices.

Sequential estimation for the multiple linear regression models with balanced loss functions

Sequential analysis (SA) as a sampling technique has notable advantages like smaller average sample size and reduced value of risk compared to similarly comparable fixed-sample techniques. In this study, we first propose a few models for the estimation of the regression parameters or functions of parameters under the multiple linear regression (MLR) setup using a balanced loss function (BLF). Thereafter, we obtain the expressions of risk functions and optimal fixed sample sizes for the proposed models based on the bounded risk criteria. We establish that no fixed-sample procedures can tackle these estimation problems. Hence, we propose different multistage sampling methodologies, viz. (i) two-stage sampling, (ii) three-stage sampling, (iii) purely sequential sampling, and (iv) batch sequential sampling, and corroborate the same with the detailed simulation and real data analyses.

Molecular and Cellular Characterization of Primary Endothelial Cells from a Familial Cavernomatosis Patient.

Cerebral cavernous malformation (CCM) or familial cavernomatosis is a rare, autosomal dominant, inherited disease characterized by the presence of vascular malformations consisting of blood vessels with an abnormal structure in the form of clusters. Based on the altered gene (CCM1/Krit1, CCM2, CCM3) and its origin (spontaneous or familial), different types of this disease can be found. In this work we have isolated and cultivated primary endothelial cells (ECs) from peripheral blood of a type 1 CCM patient. Differential functional and gene expression profiles of these cells were analyzed and compared to primary ECs from a healthy donor. The mutation of the familial index case consisted of a heterozygous point mutation in the position +1 splicing consensus between exons 15 and 16, causing failure in RNA processing and in the final protein. Furthermore, gene expression analysis by quantitative PCR revealed a decreased expression of genes involved in intercellular junction formation, angiogenesis, and vascular homeostasis. Cell biology analysis showed that CCM1 ECs were impaired in angiogenesis and cell migration. Taken together, the results obtained suggest that the alterations found in CCM1 ECs are already present in the heterozygous condition, suffering from vascular impairment and somewhat predisposed to vascular damage.

Cellular plasticity in the breast cancer ecosystem.

The complex interplay between genetically diverse tumor cells and their microenvironment significantly influences cancer progression and therapeutic responses. This review highlights recent findings on cellular plasticity and heterogeneity within the breast cancer ecosystem, focusing on the roles of cancer-associated fibroblasts (CAFs) and tumor-associated macrophages (TAMs). We discuss evidence suggesting that breast cancer cells exhibit phenotypic plasticity driven by both intrinsic genetic factors and external microenvironmental cues, impacting treatment responses and disease recurrence. Moreover, single-cell RNA sequencing studies reveal diverse subtypes of CAFs and TAMs, each with distinct functional gene expression programs and spatial organization within the tumor microenvironment. Understanding the hierarchical relationships and niche cues governing cellular phenotypes offers new opportunities for targeted therapeutic interventions. By elucidating the organizational principles of the tumor ecosystem, future therapies may target phenotypic states or entire cellular niches, advancing precision medicine approaches in breast cancer treatment.

Performance analysis of energy harvesting‐enabled relay networks in κ‐μ fading channels

AbstractIn this paper, we investigate the performance of an energy harvesting (EH)‐enabled multiple relay network operating over generalized ‐ fading channels. Our approach involves utilizing a partial relay selection strategy and employing an amplify and forward relay protocol for efficient EH from RF signals. Additionally, we integrate a selection combining scheme at the receiver to combine both direct and relaying path signals. In our research, we first obtain the exact closed‐form expression of the cumulative distribution function (CDF) for the system under investigation. Subsequently, we derive expressions for the outage probability (OP) and the moment generating function (MGF) using the derived CDF expression. Furthermore, with the assistance of a CDF‐based approach, we derive closed‐form expressions for the average symbol error rate (ASER) for coherent quadrature amplitude modulation (QAM) schemes, including rectangular QAM (RQAM) and hexagonal QAM (HQAM). We also analyze the ASER expression for non‐coherent frequency shift keying (NCFSK), leveraging the derived MGF expression. To gain valuable insights into the performance of EH‐enabled multiple relay networks, we assess the asymptotic expression of the OP. The outcomes indicate how the behavior of the system is affected by various factors such as time switching ratio, channel fading components, number of relays, and the distance between nodes. To corroborate the accuracy of the analytical findings, Monte Carlo simulations are executed.

Evolutionary origins of bitter taste receptors in jawed vertebrates

Taste is a sense that detects information about nutrients and toxins in foods. Of the five basic taste qualities, bitterness is associated with the detection of potentially harmful substances like plant alkaloids. In bony vertebrates, type 2 taste receptors (T2Rs), which are G-protein-coupled receptors (GPCRs), act as bitter taste receptors1,2. In vertebrates, six GPCR gene families are described as chemosensory receptor genes, encoding taste receptor families (T1Rs and T2Rs) and olfactory receptor families (ORs, V1Rs, V2Rs, and TAARs). These families of receptors have been found in all major jawed vertebrate lineages, except for the T2Rs, which are confined to bony vertebrates3. Therefore, T2Rs are believed to have emerged later than the other chemosensory receptor genes in the bony vertebrate lineage. So far, only the genomes of two cartilaginous fish species have been mined for TAS2R genes, which encode T2Rs4. Here, we identified novel T2Rs in elasmobranchs, namely selachimorphs (sharks) and batoids (rays, skates, and their close relatives) by an exhaustive search covering diverse cartilaginous fishes. Using functional and mRNA expression analyses, we demonstrate that their T2Rs are expressed in the oral taste buds and contribute to the detection of bitter compounds. This finding indicates the early origin of T2Rs in the common ancestor of jawed vertebrates.

358 In Vitro Uptake of Harmful Algal Bloom Toxin Microcystin-LR in Human Placental Cells

OBJECTIVES/GOALS: Harmful algal blooms (HABs) are increasing in both frequency and intensity due to climate change. HABs release the toxin microcystin-LR (MC-LR) which enters cells via organic anion-transporting polypeptides (OATPs). In this study, we sought to assess the ability of MC-LR to accumulate in trophoblasts, potentially disrupting placental functions. METHODS/STUDY POPULATION: Intracellular accumulation of MC-LR at exposure concentrations of 0.1, 1, and 10 µM over 6 hrs was evaluated in immortalized JAR placental cytotrophoblasts. Western blotting was used to evaluate protein-bound MC-LR accumulation in JAR cells. The function of OATP transporters in JAR cells was determined by pre-incubating cells with 10µM cyclosporin A, a general OATP inhibitor for 1 hr, and then incubated with 1µM OATP substrate fluorescein for up to 40 min. Fluorescence of fluorescein was measured at Ex/Em: 494nm/515nm by spectrophotometry. RESULTS/ANTICIPATED RESULTS: A concentration-dependent increase of MC-LR bound proteins in JAR cells was observed at 6 hrs with the greatest intracellular accumulation of MC-LR at 10µM. In the transporter experiments, a significant decrease of fluorescein uptake by up to 45% into JAR cells was observed following cyclosporin A inhibition of OATPs. These findings are consistent with the functional expression of OATP transporters in JAR placenta cells. Ongoing studies are evaluating whether the cyclosporin A-mediated inhibition of OATPs also inhibits the uptake of MC-LR. DISCUSSION/SIGNIFICANCE: Although MC-LR is well-known for its hepatotoxic and neurotoxic effects, there is growing interest in examining its potential adverse impacts on female reproductive health, particularly during pregnancy. Active uptake of MC-LR into the placenta could interfere with placental and fetal development.