Complex Biological Fluids Research Articles

Discovery of Mycobacterium avium subsp. paratuberculosis Lytic Phages with Extensive Host Range Across Rapid- and Slow-Growing Pathogenic Mycobacterial Species

Background/Objectives: Developing interventions for Johne’s disease, which focuses on controlling Mycobacterium avium subsp. paratuberculosis (MAP) in contaminated environments by treating infected cows and preventing transmission from diseased animals, is a critical priority. Bacteriophage (phage) therapy, an emerging biological intervention, offers a promising alternative for the treatment and management of MAP infections. Methods: In this study, we generated an MAP-specific lytic phage library aimed at characterizing the therapeutic potential of phages under environmental and biological conditions that mimic those encountered in infected cattle such as ruminal fluid, milk, colostrum, and the bovine intestinal epithelium, a key site of MAP colonization and, later, transmission. Results: Our library contains a diverse collection of phages that have demonstrated robust lytic activity against MAP. The host range of these phages was thoroughly assessed, revealing that several isolates produce clear plaques on a range of MAP strains, as well as other pathogenic non-tuberculous mycobacterial (NTM) species and M. tuberculosis strains. This broad host range expands the therapeutic potential of the phage collection, positioning it as a potential cross-species antimicrobial tool. In vitro tests under conditions replicating the rumen, milk, and colostrum environments show that selected phages maintain stability and lytic efficacy, even in the presence of complex biological fluids. Furthermore, a subset of these phages was capable of preventing MAP colonization and invasion in cultured bovine epithelial cells, suggesting their potential for direct prophylactic application in cattle. Conclusions. Our collection of MAP phages represents a valuable source that can be developed into probiotic-like preparations, offering a cost-effective solution for prophylaxis and control of Johne’s disease.

Unveiling Milk Safety in Chennai: A Comprehensive Evaluation of Adulteration Across Diverse Milk Sources

Introduction: Milk is a complex biological fluid made up mostly of water, carbohydrates, lipids, proteins, and many bioactive compounds. This extremely nutritious food contains vital components like energy-giving lactose, fat, bone-forming calcium, minerals, body-building proteins, and health-promoting vitamins necessary for all age groups. To increase the protein content, SNF, fat %, adulterants are added to milk, which unnaturally raising the milk's quality. Regretfully, there are adulterants that can have serious long-term health effects. Infants may experience renal failure and even die if they consume melamine at levels higher than what is considered safe. Objectives: This study aims to systematically evaluate the extent and nature of all milk adulteration (breast, branded vegan, unbranded vegan milk, direct cow milk and packaged cow milk) in the Chennai city, Tamil Nadu, India. Methods: The study was conducted for a period of 3 months from May 2024 to July 2024. The collected milk samples were analysed using Delstrips paper strips. Results: The findings reveal a widespread presence of adulterants, with starch being the most prevalent across nearly all milk types. There were no statistically significant differences seen between government milk, private branded cow milk, and private direct milk in adulterant's sucrose and neutralizers. In contrast, almond milk offers a viable alternative for those seeking a vegan or lactose-free option, particularly for adults and older children. Conclusion: Milk Adulteration poses a significant threat to public health, economies and the diary industry worldwide. The prevalence of adulteration, particularly in developing countries, highlights the need for improved regulatory frameworks, enhanced testing methods, and vigilant monitoring. This study has demonstrated the severity of milk adulteration, its consequences, and the imperative for collective action. The current research demonstrates that both breast milk and almond milk, when used appropriately, are safe options within their respective contexts.

Affinity peptide-based electrochemical biosensor with 2D-2D nanoarchitecture of nickel–chromium-layered double hydroxide and graphene oxide nanosheets for chirality detection of symmetric dimethylarginine

The accurate assessment of kidney dysfunction is crucial in clinical practice, necessitating the exploration of reliable biomarkers. However, current methods for measuring SDMA often fall short in terms of sensitivity and specificity. In this study, we employed phage display technology to identify high affinity peptides that specifically bind to SDMA. The selected peptide was subsequently integrated into a novel Ni-Cr layered double hydroxide-graphene oxide (NCL-GO) nanoarchitecture. We characterized the electrochemical properties of the biosensor using cyclic voltammetry, electrochemical impedance spectroscopy and differential pulse voltammetry, systematically evaluating critical parameters such as limit of detection (LOD), reproducibility, and performance in complex biological matrices including urine. The NCL-GO architecture not only enhances the surface area available for electrochemical reactions but also facilitates rapid electron transfer kinetics which are essential for the accurate quantification of small molecule, SDMA. The electrochemical biosensor exhibited an outstanding limit of detection of 0.1 ng/mL in the 0–1 ng/mL range and 7.2 ng/mL in the 1–100 ng/mL range, demonstrating exceptional sensitivity and specificity for SDMA. Furthermore, the biosensor displayed excellent reproducibility with a relative standard deviation of 4.9%. Notably, it maintained robust chirality sensing capabilities, even in complex biological fluids. These findings suggest that this biosensor could play a pivotal role in early disease diagnosis and therapeutic monitoring, ultimately improving clinical outcomes and advancing biomedical research.

Human Breast Milk Exosomes: Affecting Factors, Their Possible Health Outcomes, and Future Directions in Dietetics

Background: Human breast milk is a complex biological fluid containing multifaceted biological compounds that boost immune and metabolic system development that support the short- and long-term health of newborns. Recent literature suggests that human breast milk is a substantial source of nutrients, bioactive molecules, and exosomes. Objectives: This review examines the factors influencing exosomes noted in human milk and the impacts of exosomes on infant health. Furthermore, it discusses potential future prospects for exosome research in dietetics. Methods: Through a narrative review of the existing literature, we focused on exosomes in breast milk, exosome components and their potential impact on exosome health. Results: Exosomes are single-membrane extracellular vesicles of endosomal origin, with an approximate radius of 20–200 nm. They are natural messengers that cells secrete to transport a wide range of diverse cargoes, including deoxyribonucleic acid, ribonucleic acid, proteins, and lipids between various cells. Some studies have reported that the components noted in exosomes in human breast milk could be transferred to the infant and cause epigenetic changes. Thus, it can affect gene expression and cellular event regulation in several tissues. Conclusions: In this manner, exosomes are associated with several pathways, including the immune system, oxidative stress, and cell cycle, and they can affect the short- and long-term health of infants. However, there is still much to learn about the functions, effectiveness, and certain impacts on the health of human breast milk exosomes.

Paper immobilized BSA-decorated gold nanoclusters for single-step optical sensing of glucose and cholesterol without cross-reactivity

Minimally invasive methods for detecting glucose, cholesterol and hydrogen peroxide are crucial for monitoring the nutritional and health status of humans and animals. The peroxidase mimetic activity by nanozymes is one of the versatile methods for detecting glucose, cholesterol, hydrogen peroxide, and other biomolecules. However, the strict requirement of acidic pH limits their sensing and interfacing ability with natural enzymes. The present study developed bovine serum albumin (BSA) coated gold nanoclusters (AuNC) immobilized on paper fabric to enable single-step visual detection of glucose, cholesterol and hydrogen peroxide in complex biological fluids like serum and milk. The BSA-AuNC suspension and immobilized paper fabric synergistically interface with the natural oxidative enzymes, glucose oxidase or cholesterol oxidase, at physiological pH. The concomitant loss in the fluorescent intensity of BSA-AuNC-loaded paper fabric exposed to the generated hydrogen peroxide (glucose/glucose oxidase or cholesterol/cholesterol oxidase) was directly proportional to the concentration of glucose or cholesterol. These reactions enabled simple visual detection as well as quantification of hydrogen peroxide, glucose and cholesterol using Image-J software and common smartphone-based mobile applications. The detection ability of BSA-AuNC-embedded paper fabric is specific and remains unaltered in the presence of similar oxidase enzymes or similar substrate analogues. With these unique features, the BSA-AuNC embedded paper fabric stands out as a prominent analytical device with enormous potential as a simple, user-friendly detection tool for monitoring biomolecules that are important to health, nutrition, and environmental safeguarding.

Dual-functionalized architecture enables stable and tumor cell-specific SiO2NPs in complex biological fluids.

Most commercial anticancer nanomedicines are administered intravenously. This route is fast and precise as the drug enters directly into the systemic circulation, without undergoing absorption processes. When nanoparticles come into direct contact with the blood, however, they interact with physiological components that can induce colloidal destabilization and/or changes in their original biochemical identity, compromising their ability to selectively accumulate at target sites. In this way, these systems usually lack active targeting, offering limited therapeutic effectiveness. In the literature, there is a paucity of in-depth studies in complex environments to evaluate nanoparticle stability, protein corona formation, hemolytic activity, and targeting capabilities. To address this issue, fluorescent silica nanoparticles (SiO2NPs) are here functionalized with zwitterionic (kinetic stabilizer) and folate groups (targeting agent) to provide selective interaction with tumor cell lines in biological media. The stability of these dually functionalized SiO2NPs is preserved in unprocessed human plasma while yielding a decrease in the number of adsorbed proteins. Experiments in murine blood further proved that these nanoparticles are not hemolytic. Remarkably, the functionalized SiO2NPs are more internalized by tumor cells than their healthy counterparts. Investigations of this nature play a crucial role in garnering results with greater reliability, allowing the development of nanoparticle-based pharmaceutical drugs that exhibit heightened efficacy and reduced toxicity for medical purposes.

Fluorescence Label‐Free Doxorubicin Sensor Using Polystyrene Sulfonate as a Synthetic Receptor in Whispering Gallery Mode Microresonators

AbstractThe conventional quantification of Doxorubicin (DXR), a crucial anticancer drug, typically relies on complex and expensive techniques, limiting measurements beyond hospital settings and at different time intervals. Here, a label‐free fluorescence sensor is presented for DXR utilizing Whispering Gallery Mode (WGM) resonators. The resonators are based on fluorescent microparticles coated with a nanometer‐thick polystyrene sulfonate (PSS) layer serving as a synthetic receptor. The sensor demonstrates linear response for both position and width of the resonance peaks within the concentration range of 1–10 µg mL−1 of DXR of clinical interest. It also exhibits good selectivity against other chemotherapy drugs and can operate in complex biological fluids. The findings underscore the potential of WGM resonators as versatile multiparameter sensors for the sensitive and selective detection of small molecules, also in complex biological media.

Multiparametric profiling of HER2-enriched extracellular vesicles in breast cancer using Single Extracellular VEsicle Nanoscopy

BackgroundPatients with HER2-positive breast cancer can significantly benefit from HER2-directed therapy – such as the monoclonal antibody trastuzumab. However, some patients can develop therapy resistance or change HER2 status. Thus, we urgently need new, noninvasive strategies to monitor patients frequently. Extracellular vesicles (EVs) secreted from tumor cells are emerging as potential biomarker candidates. These membrane-delimited nanoparticles harbor molecular signatures of their origin cells; report rapidly on changes to cellular status; and can be frequently sampled from accessible biofluids.ResultsUsing Single Extracellular VEsicle Nanoscopy (SEVEN) platform that combines affinity isolation of EVs with super-resolution microscopy, here we provide multiparametric characterization of EVs with ~ 8 nm precision and molecular sensitivity. We first interrogated cell culture EVs affinity-enriched in tetraspanins CD9, CD63, and CD81; these transmembrane proteins are commonly found on EV membranes. SEVEN robustly provided critical parameters of individual, tetraspanin-enriched EVs: concentration, size, shape, molecular cargo content, and heterogeneity. Trastuzumab-resistant cells (vs. trastuzumab-sensitive) secreted more EVs. Additionally, EVs from trastuzumab-resistant cells had lower tetraspanin density and higher HER2 density. We also evaluated EVs affinity-enriched in HER2; we found that these EVs (vs. tetraspanin-enriched) were larger and more elongated. We further optimized analytical sample processing to assess a rare population of HER2-enriched EVs from patient plasma. In breast cancer patients with elevated HER2 protein expression (vs. controls), HER2-enriched EVs had distinct characteristics including typically increased number of tetraspanin molecules and larger size. Importantly, these EVs were on average 25-fold more abundant compared to no cancer controls.ConclusionsSEVEN revealed unique characteristics of HER2-enriched EVs in cultured cells and complex biological fluid. In combination with current clinical approaches, this method is well poised to support precise therapeutic decisions.Graphical abstract

Rapid and unbiased enrichment of extracellular vesicles via a meticulously engineered peptide

Extracellular vesicles (EVs) have garnered significant attention in biomedical applications. However, the rapid, efficient, and unbiased separation of EVs from complex biological fluids remains a challenge due to their heterogeneity and low abundance in biofluids. Herein, we report a novel approach to reconfigure and modify an artificial insertion peptide for the unbiased and rapid isolation of EVs in 20 min with ∼80% recovery in neutral conditions. Moreover, the approach demonstrates exceptional anti-interference capability and achieves a high purity of EVs comparable to standard ultracentrifugation and other methods. Importantly, the isolated EVs could be directly applied for downstream protein and nucleic acid analyses, including proteomics analysis, exome sequencing analysis, as well as the detection of both epidermal growth factor receptor (EGFR) and V-Ki-ras2 Kirsten Rat Sarcoma Viral Oncogene Homologue (KRAS) gene mutation in clinical plasma samples. Our approach offers great possibilities for utilizing EVs in liquid biopsy, as well as in various other biomedical applications.

Recovery and Analysis of Bacterial Membrane Vesicle Nanoparticles from Human Plasma Using Dielectrophoresis.

Bacterial membrane vesicle (BMV) nanoparticles are secreted naturally by bacteria throughout their lifecycle and are a rich source of biomarkers from the parent bacteria, but they are currently underutilized for clinical diagnostic applications, such as pathogen identification, due to the time-consuming and low-yield nature of traditional recovery methods required for analysis. The recovery of BMVs is particularly difficult from complex biological fluids. Here, we demonstrate a recovery method that uses dielectrophoretic (DEP) forces generated on electrokinetic microfluidic chips to isolate and analyze BMVs from human plasma. DEP takes advantage of the natural difference in dielectric properties between the BMVs and the surrounding plasma fluid to quickly and consistently collect these particles from as little as 25 µL of plasma. Using DEP and immunofluorescence staining of the LPS biomarker carried on BMVs, we have demonstrated a lower limit of detection of 4.31 × 109 BMVs/mL. The successful isolation of BMVs from human plasma using DEP, and subsequent on-chip immunostaining for biomarkers, enables the development of future assays to identify the presence of specific bacterial species by analyzing BMVs from small amounts of complex body fluid.

Design and optimization of an electrochemical sensor based on carbon nanotubes for the reliable voltammetric detection of serotonin in complex biological fluids

Serotonin concentration in peripherical biological fluids is correlated with the insurgence of several pathologies and represents valuable information on the human physical and mental state of health. Rapid, portable, and reliable electrochemical sensors can mine this useful information in real-time and in situ. However, the main challenge that hampers their implementation is the poor operational stability in biological fluids rich in bio-macromolecules, which can induce the fouling of the electrode altering or invalidating the analytical performance of the sensor. Herein, we propose the design of a reliable and robust voltammetric sensor for detecting serotonin in plasma. The sensor is based on multiwall carbon nanotubes modified with ligand-free gold nanoparticles obtained by metal vapor synthesis, which can efficiently catalyze the oxidation of serotonin. A thin layer of molecular imprinted polymer is added to provide selectivity and antifouling properties to the sensor. By applying a design of the experiment, to optimize the experimental condition of the differential pulsed voltammetry, and adsorptive stripping to selectively pre-accumulate serotonin in the artificial receptor, we achieved serotonin detection in plasma with sensitivity (6.7 μA μmol L−1 cm−2) and limits of detection (1.0 μmol L−1) comparable to those registered in phosphate buffer solution.

Optical Halo: A Proof of Concept for a New Broadband Microrheology Tool.

Microrheology, the study of material flow at micron scales, has advanced significantly since Robert Brown's discovery of Brownian motion in 1827. Mason and Weitz's seminal work in 1995 established the foundation for microrheology techniques, enabling the measurement of viscoelastic properties of complex fluids using light-scattering particles. However, existing techniques face limitations in exploring very slow dynamics, crucial for understanding biological systems. Here, we present a proof of concept for a novel microrheology technique called "Optical Halo", which utilises a ring-shaped Bessel beam created by optical tweezers to overcome existing limitations. Through numerical simulations and theoretical analysis, we demonstrate the efficacy of the Optical Halo in probing viscoelastic properties across a wide frequency range, including low-frequency regimes inaccessible to conventional methods. This innovative approach holds promise for elucidating the mechanical behaviour of complex biological fluids.

Effects of multiple relaxation times in the annular flow of pulsatile electro-osmotic flow of a complex biological fluid: blood with low and high cholesterol

This study investigates the electro-osmotic flow of a biological fluid (blood with varying cholesterol levels) in annular flow to simulate a first approximation to arterial occlusion. The fluid´s rheology is characterized by a multi-modal convected Maxwell model equation. The charge density follows the Boltzmann distribution, governing the electrical field. Mathematically, this scenario can be modeled by the Poisson–Boltzmann partial differential equation. Assuming a small zeta potential (less than 25 mV) using the Debye–Huckel approximation and considering a pulsatile electrical field, analytical solutions are derived using the Fourier transform formalism. These solutions, expressed in terms of the modified Bessel function, provide transfer functions for axial velocity and volumetric flow as functions of material parameters represented by characteristic dimensionless numbers. This study further analyzes thermal, electric, inertial, viscoelastic, and various interactions within the plasma, hematocrit, hematocrit–cholesterol, and cholesterol–cholesterol as well as weight concentration through numerical simulations. Finally, the flow and rheology predictions are validated using experimental data on human blood with varying cholesterol levels. The obtained transfer functions reveal that the electric–thermal–viscoelastic effects and the multiple geometric relationships contribute to the dynamic response of the interactions between the input electrical field and output volumetric flow and shear stress functions, leading to and evolution of resonance curves. It is noteworthy that electro-osmotic flow in blood with pathologies associated with low and high cholesterol has been scarcely reported in the literature on rheology. Thus, this work represents a significant contribution to the field.

A Conjugated Polymer-Based Portable Smartphone Platform for Sensitive and Point-Of-Care Detection of Monoamine Neurotransmitter.

The precise and effective detection of neurotransmitters (NTs) is crucial for clinical investigation of neuronal processes, and timely monitoring of NT-related chronic diseases. However, sensitive detection of specific NT with unprecedented selectivity is highly challenging due to similarities in chemical and electronic structures of various interfering neurochemicals. Herein, an anionic conjugated polyelectrolyte Poly[(9,9-bis(4'-sulfonatobutyl)fluorene-co-alt-1,4-phenylene) sodium], PFPS was rationally designed and synthesized for amplified detection and point-of-care (PoC) determination of monoamine neurotransmitter, serotonin (5-Hydroxy tryptamine or 5-HT, also diagnostic biomarker of carcinoid tumor) in human blood plasma. The PFPS displayed a remarkable sensing response with an exceptionally high fluorescence quenching constant of 1.14×105 M-1 and an ultralow detection limit of 0.67 μM or 0.142 ppm, much below the clinical range. Furthermore, a smartphone-enabled portable platform was constructed for real-time onsite detection of 5-HT by quantification of visual fluorescence response of PFPS into RGB values using a color recognizer android application. The smartphone platform could be readily applied for convenient, non-invasive PoC testing of 5-HT levels in complex biological fluids accurately and is expected to revolutionize clinical diagnosis and personalized health care devices.

Extralong hot-spots sensor for SERS sensitive detection of phthalate plasticizers in biological tear and serum fluids.

Phthalate plasticizers (PAEs) illegally used in food pose a great threat to human health. A new and efficient sensing platform for the sensitive detection of the PAE residues in biological fluids needs to be designed and developed. Here, we report a simple and reliable surface-enhanced Raman spectroscopy (SERS) active platform with extralong hot spots of Au nanobipyramids@Ag nanorods (Au NBPs@Ag NRs) for the rapid and sensitive detection of PAEs in biological fluids. To achieve high activity, Au NBPs@Ag NRs with different shell lengths were fabricated by controlling the synthesis conditions, and the corresponding SERS properties were investigated by using crystal violet (CryV) and butyl benzyl phthalate (BBP). The experimental results showed that a longer shell length correlated to greater Raman activity, which was confirmed by finite-difference time-domain (FDTD) electromagnetic simulation. More importantly, the extralong hot spots of the Au NBPs@Ag NR SERS-active substrate showed excellent homogeneity and reproducibility for the CryV probe molecules (6.21%), and the detection limit was 10-9M for both BBP and diethylhexyl phthalate (DEHP). Furthermore, through the standard addition method, an extralong hot spots SERS substrate could achieve highly sensitive detection of BBP and DEHP in serum and tears fluids, and the detection limit was as low as 3.52 10-8M and 2.82 10-8M. Therefore, the Au NBPs@Ag NR substrate with an extraordinarily long surface is efficient and versatile, and can potentially be used for high-efficiency sensing analysis in complex biological fluids.

SERSomes for metabolic phenotyping and prostate cancer diagnosis

Molecular phenotypic variations in metabolites offer the promise of rapid profiling of physiological and pathological states for diagnosis, monitoring, and prognosis. Since present methods are expensive, time-consuming, and still not sensitive enough, there is an urgent need for approaches that can interrogate complex biological fluids at a system-wide level. Here, we introduce hyperspectral surface-enhanced Raman spectroscopy (SERS) to profile microliters of biofluidic metabolite extraction in 15min with a spectral set, SERSome, that can be used to describe the structures and functions of various molecules produced in the biofluid at a specific time via SERS characteristics. The metabolite differences of various biofluids, including cell culture medium and human serum, are successfully profiled, showing a diagnosis accuracy of 80.8% on the internal test set and 73% on the external validation set for prostate cancer, discovering potential biomarkers, and predicting the tissue-level pathological aggressiveness. SERSomes offer a promising methodology for metabolic phenotyping.

Redox-Modified Nanostructured Electrochemical Surfaces for Continuous Glucose Monitoring in Complex Biological Fluids.

Continuous glucose monitoring is valuable for people with diabetes but faces limitations due to enzyme-electrode interactions and biofouling from biological samples that reduce sensor sensitivity and the monitoring performance. We created an enzyme-based electrochemical system with a unique nanocomposite coating that incorporates the redox molecule, aminoferrocene (NH2-Fc). This coating enhances stability via electroactivity and reduces nonspecific binding, as demonstrated through cyclic voltammetry. Our approach enables real-time glucose detection via chronoamperometry with a calculated linear range of 0.5 to 20 mM and a 1 mM detection limit. Validated with plasma and saliva, this platform shows promise for robust metabolite detection in clinical and research contexts. This versatile platform can be applied to accurately monitor a wide range of metabolites in various biological matrices, improving patient outcomes.

Abstract PO1-24-06: Prolactin Hormone-Induced Mammary Differentiation and Anti-tumorigenic Role in Breast Cancer

Abstract The premise of differentiation therapy in cancer is a strategy that aims at engaging-forward differentiation and cellular reprograming restricting the proliferative, tumor repopulation, stemness, EMT and metastatic capacities of tumor cells leading to the cessation of the aggressive tumor phenotype and offering the cancer patients improved survival for decades. Therefore, deciphering molecular mechanisms deriving differentiation in normal mammary and breast cancer cells may allow the discovery of innovative differentiation-based biomarkers and therapeutics in breast cancer. Lactation is an intricate process that results in the ability of the breast cells to produce a complex nutritious biological fluid to nurse the new-born. While the beneficial effects of breastfeeding to the infant is irrefutable, its effects on maternal health are less investigated especially in relation to breast cancer that still affects 1 in 8 women in high income countries and 1 in 20 globally. Importantly, epidemiological studies have linked breastfeeding to reduced risk of breast cancer by promoting terminal differentiation of the breast epithelial cells. While breastfeeding process is regulated by multiple hormones, growth factors, and transcriptional regulators, the lactation hormone prolactin (PRL) is known to be directly implicated in the hormonal control of breastfeeding. Extensive research including our own work has shown that PRL/PRL receptor (PRLR) pathway derives mammary gland development and importantly mammary epithelial cell terminal differentiation allowing successful lactation. In contrast the role of PRL in breast cancer is still to be fully defined. Importantly, we have accumulated compelling evidence using in-silico publicly available patient data sets and molecular data implicating this pathway as a clinically relevant pro-differentiation pathway driving differentiation and cellular reprograming suppressing stemness, EMT, metastasis and tumorigenesis in breast cancer. To better characterize PRL signaling mediating its pro-differentiation effects, we used large-scale unbiased proteomics analysis of PRLR/interacting proteins in breast cancer cells. Our analyses revealed several novel PRLR-interactors. We have further confirmed these interactions through co-immunoprecipitations/western blotting analyses in breast cancer cells representative the different breast cancer subtypes. We also examined their functional contributions to mammary differentiation and tumorigenesis using in vitro and in vivo assays. Moreover, we defined their potential clinical relevance using bioinformatics in silico databases of breast cancer. Together our study delineates novel PRL/PRLR-downstream signaling mediators important for mammary differentiation and tumorigenesis and may thus be useful as biomarkers and/or therapeutic targets in breast cancer. Citation Format: Dana Hamam, Suhad Ali. Prolactin Hormone-Induced Mammary Differentiation and Anti-tumorigenic Role in Breast Cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-24-06.

Evolutionarily conserved ovarian fluid proteins are responsible for extending egg viability in salmonid fish

In contrast to most fishes, salmonids exhibit the unique ability to hold their eggs for several days after ovulation without significant loss of viability. During this period, eggs are held in the body cavity in a biological fluid, the coelomic fluid (CF) that is responsible for preserving egg viability. To identify CF proteins responsible for preserving egg viability, a proteomic comparison was performed using 3 salmonid species and 3 non-salmonid species to identify salmonid-specific highly abundant proteins. In parallel, rainbow trout CF fractions were purified and used in a biological test to estimate their egg viability preservation potential. The most biologically active CF fractions were then subjected to mass spectrometry analysis. We identified 50 proteins overabundant in salmonids and present in analytical fractions with high egg viability preservation potential. The identity of these proteins illuminates the biological processes participating in egg viability preservation. Among identified proteins of interest, the ovarian-specific expression and abundance in CF at ovulation of N-acetylneuraminic acid synthase a (Nansa) suggest a previously unsuspected role. We show that salmonid CF is a complex biological fluid containing a diversity of proteins related to immunity, calcium binding, lipid metabolism, proteolysis, extracellular matrix and sialic acid metabolic pathway that are collectively responsible for preserving egg viability.

Unraveling the Intraday Variations in the Tear Fluid Proteome.

Tear fluid is a complex and dynamic biological fluid that plays essential roles in maintaining ocular homeostasis and protecting against the external environment. Owing to the small sample volume, studying the tear proteome is challenging. However, advances in high-resolution mass spectrometry have expanded tear proteome profiling, revealing >500 unique proteins. Tears are emerging as a noninvasive source of biomarkers for both ocular and systemic diseases; nevertheless, intraday variability of proteins in tear fluid remains questionable. This study investigates intraday variations in the tear fluid proteome to identify stable proteins that could act as candidate biomarkers. Tear samples from 15 individuals at four time points (10 am, 12 pm, 2 pm, and 4 pm) were analyzed using mass spectrometry to evaluate protein variation during these intervals. Technical variation was assessed by analyzing pooled samples and was subtracted from the total variation to isolate biological variability. Owing to high technical variation, low-abundant proteins were filtered, and only 115 proteins met the criteria for further analysis. These criteria include being detected at all four time points in at least eight subjects, having a mean peptide-spectrum match count greater than 5, and having a technical variation less than 0.10. Lactotransferrin, lipocalin-1, and several immunoglobulins were among the 51 stable proteins (mean biological coefficient of variation < 0.10). Additionally, 43 proteins displayed significant slopes across the 4 time points, with 17 increasing and 26 decreasing over time. These findings contribute to the understanding of tear fluid dynamics and further expand our knowledge of the tear proteome.