Selenium Discrepancies in Fetal Bovine Serum: Impact on Cellular Selenoprotein Expression.

Selenium is an essential trace element in our diet, crucial for the composition of human selenoproteins, which include 25 genes such as glutathione peroxidases and thioredoxin reductases. The regulation of the selenoproteome primarily hinges on the bioavailability of selenium, either from dietary sources or cell culture media. This selenium-dependent control follows a specific hierarchy, with "housekeeping" selenoproteins maintaining constant expression while "stress-regulated" counterparts respond to selenium level fluctuations. This study investigates the variability in fetal bovine serum (FBS) selenium concentrations among commercial batches and its effects on the expression of specific stress-related cellular selenoproteins. Despite the limitations of our study, which exclusively used HEK293 cells and focused on a subset of selenoproteins, our findings highlight the substantial impact of serum selenium levels on selenoprotein expression, particularly for GPX1 and GPX4. The luciferase reporter assay emerged as a sensitive and precise method for evaluating selenium levels in cell culture environments. While not exhaustive, this analysis provides valuable insights into selenium-mediated selenoprotein regulation, emphasizing the importance of serum composition in cellular responses and offering guidance for researchers in the selenoprotein field.

Generation of Rapid and High-Quality Serum by Recombinant Prothrombin Activator Ecarin (RAPClot™).

We recently reported the potential application of recombinant prothrombin activator ecarin (RAPClot™) in blood diagnostics. In a new study, we describe RAPClot™ as an additive to develop a novel blood collection prototype tube that produces the highest quality serum for accurate biochemical analyte determination. The drying process of the RAPClot™ tube generated minimal effect on the enzymatic activity of the prothrombin activator. According to the bioassays of thrombin activity and plasma clotting, γ-radiation (>25 kGy) resulted in a 30-40% loss of the enzymatic activity of the RAPClot™ tubes. However, a visual blood clotting assay revealed that the γ-radiation-sterilized RAPClot™ tubes showed a high capacity for clotting high-dose heparinized blood (8 U/mL) within 5 min. This was confirmed using Thrombelastography (TEG), indicating full clotting efficiency under anticoagulant conditions. The storage of the RAPClot™ tubes at room temperature (RT) for greater than 12 months resulted in the retention of efficient and effective clotting activity for heparinized blood in 342 s. Furthermore, the enzymatic activity of the RAPClot™ tubes sterilized with an electron-beam (EB) was significantly greater than that with γ-radiation. The EB-sterilized RAPClot™ tubes stored at RT for 251 days retained over 70% enzyme activity and clotted the heparinized blood in 340 s after 682 days. Preliminary clinical studies revealed in the two trials that 5 common analytes (K, Glu, lactate dehydrogenase (LD), Fe, and Phos) or 33 analytes determined in the second study in the γ-sterilized RAPClot™ tubes were similar to those in commercial tubes. In conclusion, the findings indicate that the novel RAPClot™ blood collection prototype tube has a significant advantage over current serum or lithium heparin plasma tubes for routine use in measuring biochemical analytes, confirming a promising application of RAPClot™ in clinical medicine.

FBS-based cryoprotective compositions for effective cryopreservation of gut microbiota and key intestinal microorganisms.

OBJECTIVE: The need for innovative techniques to preserve microbiota for extended periods, while maintaining the species composition and quantitative balance of the bacterial community, is becoming increasingly important. To address this need, we propose an efficient approach to cryopreserve human gut microbiota using a two-component cryoprotective composition comprising fetal bovine serum (FBS) and 5% dimethyl sulfoxide (DMSO). Fetal serum is a commonly utilized component in the freezing media for eukaryotic cells, however, its effects on prokaryotic cells have not been extensively researched. RESULTS: In our study, we demonstrated the high efficiency of using a two-component cryoprotective medium, FBS + 5% DMSO, for cryopreservation of human gut microbiota using three different methods. According to the obtained results, the intact donor microbiota was preserved at a level of 85 ± 4% of the initial composition based on fluorescent analysis using the LIVE/DEAD test. No differences in survival were observed when comparing with pure DMSO and FBS media. The photometric measurement method for growth of aerobic bacteria (A. johnsoni), facultative anaerobes (E. coli, E. faecalis), microaerophilic (L. plantarum), and obligate anaerobic bacterial cultures (E. barkeri, B. breve) also demonstrated high viability rates of 94-98% in the two-component protective medium, reaching intact control levels. However, for anaerobic microflora representatives, serum proved to be a more suitable cryoprotectant. Also, we demonstrated that using cryoprotective media with 50-75% FBS content is enough to preserve a significant level of bacterial cell viability, from an economic standpoint.

Role of chemokines CXCL9, CXCL10, CXCL11, and CXCR3 in the serum and minor salivary gland tissues of patients with Sjögren's syndrome.

This study aimed to investigate the serum and expression levels of C-X-C motif chemokine ligand 9 (CXCL9), CXCL10, CXCL11, and CXC receptor 3 (CXCR3) in minor salivary glands (MSGs) of patients with primary Sjögren's syndrome (pSS), and to explore their correlations with clinical parameters. Serum samples from 49 patients diagnosed with pSS, 33 patients with rheumatoid arthritis (RA), and 30 healthy controls (HCs) were collected for measurements of CXCL9, CXCL10, CXCL11, and CXCR3. Additionally, CXCL levels in the MSG tissues were measured in 41 patients who underwent MSG biopsy. Correlations between CXCL and CXCL/CXCR levels in serum/MSG tissues and clinical factors/salivary scintigraphy parameters were analyzed. Serum CXCL11 and CXCR3 showed statistically significant differences among patients with pSS and RA and HCs (serum CXCL11, pSS:RA:HC = 235.6 ± 500.1 pg/mL:90.0 ± 200.3 pg/mL:45.9 ± 53.6 pg/mL; p = 0.041, serum CXCR3, pSS:RA:HC = 3.27 ± 1.32 ng/mL:3.29 ± 1.17 ng/mL:2.00 ± 1.12 ng/mL; p < 0.001). Serum CXCL10 showed a statistically significant difference between pSS (64.5 ± 54.2 pg/mL) and HCs (18.6 ± 18.1 pg/mL, p < 0.001), while serum CXCL9 did not exhibit a significant difference among the groups. Correlation analysis of clinical factors revealed that serum CXCL10 and CXCL11 levels positively correlated with erythrocyte sedimentation rate (r = 0.524, p < 0.001 and r = 0.707, p < 0.001, respectively), total protein (r = 0.375, p = 0.008 and r = 0.535, p < 0.001, respectively), globulin (r = 0.539, p < 0.001 and r = 0.639, p < 0.001, respectively), and European Alliance of Associations for Rheumatology SS Disease Activity Index (r = 0.305, p = 0.033 and r = 0.321, p = 0.025). Additionally, serum CXCL10 negatively correlated with the Schirmer test score (r = - 0.354, p = 0.05), while serum CXCL11 positively correlated with the biopsy focus score (r = 0.612, p = 0.02). In the MSG tissue, the percentage of infiltrating CXCL9-positive cells was highest (75.5%), followed by CXCL10 (29.1%) and CXCL11 (27.9%). In the correlation analysis, CXCL11-expressing cells were inversely related to the mean washout percentage on salivary gland scintigraphy (r = - 0.448, p = 0.007). Our study highlights distinct serum and tissue chemokine patterns in pSS, emphasizing CXCL9's potential for early diagnosis. This suggests that CXCL10 and CXCL11 are indicators of disease progression, warranting further investigation into their roles in autoimmune disorders beyond pSS.

mTORC1 and BMP-Smad1/5 regulation of serum-stimulated myotube hypertrophy: a role for autophagy.

Protein synthesis regulation is critical for skeletal muscle hypertrophy, yet other established cellular processes are necessary for growth-related cellular remodeling. Autophagy has a well-acknowledged role in muscle quality control, but evidence for its role in myofiber hypertrophy remains equivocal. Both mammalian target of rapamycin complex I (mTORC1) and bone morphogenetic protein (BMP)-Smad1/5 (Sma and Mad proteins from Caenorhabditis elegans and Drosophila, respectively) signaling are reported regulators of myofiber hypertrophy; however, gaps remain in our understanding of how this regulation is integrated with growth processes and autophagy regulation. Therefore, we investigated the mTORC1 and Smad1/5 regulation of protein synthesis and autophagy flux during serum-stimulated myotube growth. Chronic serum stimulation experiments were performed on day 5 differentiated C2C12 myotubes incubated in differentiation medium [2% horse serum (HS)] or growth medium [5% fetal bovine serum (FBS)] for 48 h. Rapamycin or LDN193189 was dosed for 48 h to inhibit mTORC1 and BMP-Smad1/5 signaling, respectively. Acute serum stimulation was examined in day 7 differentiated myotubes. Protein synthesis was measured by puromycin incorporation. Bafilomycin A1 and immunoblotting for LC3B were used to assess autophagy flux. Chronic serum stimulation increased myotube diameter 22%, total protein 21%, total RNA 100%, and Smad1/5 phosphorylation 404% and suppressed autophagy flux. Rapamycin, but not LDN193189, blocked serum-induced myotube hypertrophy and the increase in total RNA. Acute serum stimulation increased protein synthesis 111%, Smad1/5 phosphorylation 559%, and rpS6 phosphorylation 117% and suppressed autophagy flux. Rapamycin increased autophagy flux during acute serum stimulation. These results provide evidence for mTORC1, but not BMP-Smad1/5, signaling being required for serum-induced myotube hypertrophy and autophagy flux by measuring LC3BII/I expression. Further investigation is warranted to examine the role of autophagy flux in myotube hypertrophy.NEW & NOTEWORTHY The present study demonstrates that myotube hypertrophy caused by chronic serum stimulation requires mammalian target of rapamycin complex 1 (mTORC1) signaling but not bone morphogenetic protein (BMP)-Smad1/5 signaling. The suppression of autophagy flux was associated with serum-induced myotube hypertrophy and mTORC1 regulation of autophagy flux by measuring LC3BII/I expression. Rapamycin is widely investigated for beneficial effects in aging skeletal muscle and sarcopenia; our results provide evidence that rapamycin can regulate autophagy-related signaling during myotube growth, which could benefit skeletal muscle functional and metabolic health.

Bacterial vampirism mediated through taxis to serum.

Bacteria of the family Enterobacteriaceae are associated with gastrointestinal (GI) bleeding and bacteremia and are a leading cause of death, from sepsis, for individuals with inflammatory bowel diseases. The bacterial behaviors and mechanisms underlying why these bacteria are prone to bloodstream entry remain poorly understood. Herein, we report that clinical isolates of non-typhoidal Salmonella enterica serovars, Escherichia coli, and Citrobacter koseri are rapidly attracted toward sources of human serum. To simulate GI bleeding, we utilized an injection-based microfluidics device and found that femtoliter volumes of human serum are sufficient to induce bacterial attraction to the serum source. This response is orchestrated through chemotaxis and the chemoattractant L-serine, an amino acid abundant in serum that is recognized through direct binding by the chemoreceptor Tsr. We report the first crystal structures of Salmonella Typhimurium Tsr in complex with L-serine and identify a conserved amino acid recognition motif for L-serine shared among Tsr orthologues. We find Tsr to be widely conserved among Enterobacteriaceae and numerous World Health Organization priority pathogens associated with bloodstream infections. Lastly, we find that Enterobacteriaceae use human serum as a source of nutrients for growth and that chemotaxis and the chemoreceptor Tsr provide a competitive advantage for migration into enterohemorrhagic lesions. We define this bacterial behavior of taxis toward serum, colonization of hemorrhagic lesions, and the consumption of serum nutrients as 'bacterial vampirism', which may relate to the proclivity of Enterobacteriaceae for bloodstream infections.

Sepsis is the leading cause of death in patients with inflammatory bowel disease. Individuals with this condition can experience recurrent episodes of intestinal bleeding, giving intestinal (or enteric) bacteria an entry point into the bloodstream. This puts patients at risk of developing fatal infections ­ particularly from infections caused by bacteria belonging to the Enterobacteriaceae family. However, it is not well understood why this family of bacteria are particularly prone to entering the bloodstream. Enteric bacteria commonly respond to chemicals (or chemical stimuli) in their environment. This process, known as chemotaxis, helps bacteria with a variety of tasks, such as monitoring their environment, moving to different areas within their environment or colonizing their host. Chemical stimuli are classed as 'attractants' or 'repellents', with attractants luring the bacteria to an area and repellents discouraging the bacteria from being in a specific place. Intestinal bleeds will release serum (the liquid part of blood) into the gut, which could serve as a source of chemical stimuli to attract Enterobacteriaceae into the bloodstream. To find out more, Glen, Gentry-Lear et al. first used a microfluidic device to simulate an intestinal bleed and tested the response of Enterobacteriaceae bacteria to serum. Using chemotaxis, bacteria were found to be attracted to the amino acid L-serine in the serum to which they were able to attach through a receptor called Tsr. They also consumed nutrients present in the human serum to help them grow. Experiments with intestinal tissue showed that chemotaxis attracted bacteria to bleeding blood vessels and the Tsr receptor helped them to infiltrate the blood vessels. Glen et al. termed this attraction to and feeding upon blood serum as 'bacterial vampirism'. These findings suggest that chemotaxis of Enterobacteriaceae towards L-serine in serum may be linked to their tendency to enter the bloodstream. Developing therapies that target chemotaxis in Enterobacteriaceae may provide a method for managing bloodstream infections.

Direct comparison of different protocols to obtain surface enhanced Raman spectra of human serum.

Label-free Surface Enhanced Raman Spectroscopy (SERS) is a rapid technique that has been extensively applied in clinical diagnosis and biomedicine for the analysis of biofluids. The purpose of this approach relies on the ability to detect specific "metabolic fingerprints" of complex biological samples, but the full potential of this technique in diagnostics is yet to be exploited, mainly because of the lack of common analytical protocols for sample preparation and analysis. Variation of experimental parameters, such as substrate type, laser wavelength and sample processing can greatly influence spectral patterns, making results from different research groups difficult to compare. This study aims at making a step toward a standardization of the protocols in the analysis of human serum samples with Ag nanoparticles, by directly comparing the SERS spectra obtained from five different methods in which parameters like laser power, nanoparticle concentration, incubation/deproteinization steps and type of substrate used vary. Two protocols are the most used in the literature, and the other three are "in-house" protocols proposed by our group; all of them are employed to analyze the same human serum sample. The experimental results show that all protocols yield spectra that share the same overall spectral pattern, conveying the same biochemical information, but they significantly differ in terms of overall spectral intensity, repeatability, and preparation steps of the sample. A Principal Component Analysis (PCA) was performed revealing that protocol 3 and protocol 1 have the least variability in the dataset, while protocol 2 and 4 are the least repeatable.

Bone Regenerative Effect of Injectable Hypoxia Preconditioned Serum-Fibrin (HPS-F) in an Ex Vivo Bone Defect Model.

Biofunctionalized hydrogels are widely used in tissue engineering for bone repair. This study examines the bone regenerative effect of the blood-derived growth factor preparation of Hypoxia Preconditioned Serum (HPS) and its fibrin-hydrogel formulation (HPS-F) on drilled defects in embryonic day 19 chick femurs. Measurements of bone-related growth factors in HPS reveal significant elevations of Osteopontin, Osteoprotegerin, and soluble-RANKL compared with normal serum (NS) but no detection of BMP-2/7 or Osteocalcin. Growth factor releases from HPS-F are measurable for at least 7 days. Culturing drilled femurs organotypically on a liquid/gas interface with HPS media supplementation for 10 days demonstrates a 34.6% increase in bone volume and a 52.02% increase in bone mineral density (BMD) within the defect area, which are significantly higher than NS and a basal-media-control, as determined by microcomputed tomography. HPS-F-injected femur defects implanted on a chorioallantoic membrane (CAM) for 7 days exhibit an increase in bone mass of 123.5% and an increase in BMD of 215.2%, which are significantly higher than normal-serum-fibrin (NS-F) and no treatment. Histology reveals calcification, proteoglycan, and collagen fiber deposition in the defect area of HPS-F-treated femurs. Therefore, HPS-F may offer a promising and accessible therapeutic approach to accelerating bone regeneration by a single injection into the bone defect site.

Blood-Derived Eye Drops for the Treatment of Corneal Neuropathic Pain.

Blood-derived preparations, including autologous or allogenic serum, umbilical cord serum/plasma, and platelet-rich plasma eye drops, contain various growth factors, cytokines, and immunoglobulins that resemble natural tears. These components play important roles in corneal cell migration, proliferation, and wound healing. Blood-derived eye drops have demonstrated clinical effectiveness across a spectrum of ocular surface conditions, encompassing dry eye disease, Sjögren's syndrome, graft-versus-host disease, and neuropathic corneal pain (NCP). Currently, management of NCP remains challenging. The emergence of blood-derived eye drops represents a promising therapeutic approach. In this review, we discuss the benefits and limitations of different blood-derived eye drops, their mechanisms of action, and treatment efficacy in patients with NCP. Several studies have demonstrated the clinical efficacy of autologous serum eye drops in relieving pain and pain-like symptoms, such as allodynia and photoallodynia. Corneal nerve parameters were also significantly improved, as evidenced by increased nerve fiber density, length, nerve reflectivity, and tortuosity, as well as a decreased occurrence of beading and neuromas after the treatment. The extent of nerve regeneration correlated with improvement in patient-reported photoallodynia. Cord plasma eye drops also show potential for symptom alleviation and corneal nerve regeneration. Future directions for clinical practice and research involve standardizing preparation protocols, establishing treatment guidelines, elucidating underlying mechanisms, conducting long-term clinical trials, and implementing cost-effective measures such as scaling up manufacturing. With ongoing advancements, blood-derived eye drops hold promise as a valuable therapeutic option for patients suffering from NCP.

Dynamics of serum anion gaps with in-hospital mortality: Analysis of the multi-open databases.

BACKGROUND: Few studies have investigated the relationship between the anion gap, including the corrected anion gap, and patient mortality in intensive care units (ICUs) without restricting the analysis to specific diseases or medical specialties. Our primary objective was to investigate the association between the anion gap and ICU mortality using multiple open-access databases. METHODS: We identified 4229 subjects from the Medical Information Mart for Intensive Care IV (MIMIC-IV) database, whose entries were from between 2008 and 2019. For each patient, the anion gap and corrected anion gap were calculated, and the study sample was divided into tertile groups (T) according to these levels. The association between the anion gap and in-hospital mortality was assessed using hazard ratios (HRs) and 95% confidence intervals (CIs) derived from a multivariable-adjusted Cox proportional hazards model. Besides MIMIC-IV, we also incorporated study samples from two other databases (MIMIC-III and electronic ICU) to calculate summary HRs using a random-effects meta-analysis. RESULTS: Within MIMIC-IV, 1015 patients (24%) died during an average follow-up period of 15.5 days. The fully adjusted HRs and 95% CIs for T2 and T3, relative to T1, were 1.31 (95% CI 1.08-1.58) and 1.54 (95% CI 1.24-1.90), respectively. When grouped by corrected anion gap, the results remained statistically significant. In the meta-analysis, the summary HRs and 95% CIs for T2 and T3 were 1.24 (95% CI 1.08-1.43) and 1.55 (95% CI 1.33-1.82), respectively. CONCLUSIONS: Both the anion gap and corrected anion gap were associated with in-hospital mortality regardless of specific diseases or medical specialties.

Molecular detection of exosomal miRNAs of blood serum for prognosis of colorectal cancer.

Colorectal cancer (CRC) is the third most common cancer affecting people. The discovery of new, non-invasive, specific, and sensitive molecular biomarkers for CRC may assist in the diagnosis and support therapeutic decision making. Exosomal miRNAs have been demonstrated in carcinogenesis and CRC development, which makes these miRNAs strong biomarkers for CRC. Deep sequencing allows a robust high-throughput informatics investigation of the types and abundance of exosomal miRNAs. Thus, exosomal miRNAs can be efficiently examined as diagnostic biomarkers for disease screening. In the present study, a number of 660 mature miRNAs were detected in patients diagnosed with CRC at different stages. Of which, 29 miRNAs were differentially expressed in CRC patients compared with healthy controls. Twenty-nine miRNAs with high abundance levels were further selected for subsequent analysis. These miRNAs were either highly up-regulated (e.g., let-7a-5p, let-7c-5p, let-7f-5p, let-7d-3p, miR-423-5p, miR-3184-5p, and miR-584) or down-regulated (e.g., miR-30a-5p, miR-99-5p, miR-150-5p, miR-26-5p and miR-204-5p). These miRNAs influence critical genes in CRC, leading to either tumor growth or suppression. Most of the reported diagnostic exosomal miRNAs were shown to be circulating in blood serum. The latter is a novel miRNA that was found in exosomal profile of blood serum. Some of the predicted target genes of highly expressed miRNAs participate in several cancer pathways, including CRC pathway. These target genes include tumor suppressor genes, oncogenes and DNA repair genes. Main focus was given to multiple critical signaling cross-talking pathways including transforming growth factor ß (TGFß) signaling pathways that are directly linked to CRC. In conclusion, we recommend further analysis in order to experimentally confirm exact relationships between selected differentially expressed miRNAs and their predicted target genes and downstream functional consequences.

Five-Year Risk and Safety Profile of Autologous Serum Eye Drop Therapy.

PURPOSE: To evaluate the risk and safety profile of autologous serum eye drop therapy in clinical routine over a period of 5 years. METHODS: This retrospective study involved all patients treated with autologous serum between July 2014 and December 2019 at a tertiary ophthalmic referral center. The electronic patient record system was searched for all patients with autologous serum eye drop therapy at any time point. These records were subsequently searched for keywords such as infectious keratitis, corneal ulcer, conjunctivitis, or endophthalmitis at any recorded contact. The probability of an association between the therapy with autologous serum eye drops and infectious complications was investigated independently by three corneal specialists and rated as likely, potential, or unlikely. RESULTS: In total, 752 patients were treated with autologous serum eye drops between July 2014 and December 2019. There were 5 384 batches with a total of 107 680 bottles of serum eye drops that had been produced and dispensed for these patients during this period. The records of 291 patient showed a combination of autologous serum therapy and at least one keyword for infectious diseases. In 288 patients, individual case analyses revealed an unlikely association between the therapy and infection, as their infectious episodes occurred either before the start of the therapy, more than 1 month after the therapy ceased, or in the contralateral untreated eye in the case of unilateral therapy. Three cases of infectious keratitis were classified as potentially associated with autologous serum therapy. However, all three patients suffered from chronic anterior eye diseases with a high risk of spontaneous infectious complications independent of therapy with autologous eye drops. None of the infectious events was rated as being likely due to the serum eye drops. CONCLUSIONS: Serum eye drops are often used in patients with severe or chronic anterior eye diseases with an intrinsic risk of infectious diseases. Despite these preexisting risk factors, autologous serum eye drops can be considered safe, even in patients with a compromised ocular surface.

A Novel Technique of Aseptic Manufacture of Autologous Serum Eye Drops (ASEDs) and Sterility Analysis of the Bottled Ophtioles.

PURPOSE: To introduce a novel technique of the aseptic manufacture of autologous serum eye drops (ASEDs) with a prefiltered closed system and to analyze the sterility of the produced ophtioles between 2018 and 2022. METHODS: This is a prospective single-center study conducted at the Department of Ophthalmology at a Swiss University Hospital between 2018 and 2022. For regulatory reasons, closed systems for manufacturing ASEDs are strongly recommended. We attached an upstream sterile filter (Sterivex PES0.22 µm Burlington, USA) to a commercially available closed system (COL System Modena, Italy) for manufacturing ASEDs. The goal of this novel approach was to reduce the microbiological contamination of the donated autologous blood. Using the presented manufacturing method, we are able to produce, on average, 56 ophtioles per batch, containing either 1.45 mL or 2.5 mL of autologous serum per ophtiole. For each batch of ASEDs, we performed a microbiological analysis by automated blood culture testing (BACTEC). This system examines the presence of bacteria and fungi. RESULTS: We analyzed all manufactured batches between 2018 and 2022. None of the 2297 batches and the resulting 129 060 ophtioles showed bacterial or mycotic contamination. During the analyzed period, two batches were discarded: one due to fibrin-lipid aggregations, further microbiological and histological work-up excluded any contamination; another due to false-positive HIV in serological testing. Overall, the contamination rate was 0%, and the batch discharge rate was 0.09%. CONCLUSIONS: The combination of upstream sterile filtration with a commercial closed system for manufacturing ASEDs proved to be effective in ensuring sterility without any contamination over the past 4 years. This is becoming crucial, as the demand for autologous blood products for treating ocular surface disorders, such as refractory dry eyes or nonhealing defects of the corneal epithelium, is on the rise.

Serum Induces the Subunit-Specific Activation of NF-κB in Proliferating Human Cardiac Stem Cells.

Cardiovascular diseases (CVDs) are often linked to ageing and are the major cause of death worldwide. The declined proliferation of adult stem cells in the heart often impedes its regenerative potential. Thus, an investigation of the proliferative potential of adult human cardiac stem cells (hCSCs) might be of great interest for improving cell-based treatments of cardiovascular diseases. The application of human blood serum was already shown to enhance hCSC proliferation and reduce senescence. Here, the underlying signalling pathways of serum-mediated hCSC proliferation were studied. We are the first to demonstrate the involvement of the transcription factor NF-κB in the serum-mediated proliferative response of hCSCs by utilizing the NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC). RNA-Sequencing (RNA-Seq) revealed ATF6B, COX5B, and TNFRSF14 as potential targets of NF-κB that are involved in serum-induced hCSC proliferation.

Investigation of long non-coding RNAs in extracellular vesicles from low-volume blood serum specimens of colorectal cancer patients.

Colorectal cancer (CRC) is the second most prevalent cancer type worldwide, which highlights the urgent need for non-invasive biomarkers for its early detection and improved prognosis. We aimed to investigate the patterns of long non-coding RNAs (lncRNAs) in small extracellular vesicles (sEVs) collected from low-volume blood serum specimens of CRC patients, focusing on their potential as diagnostic biomarkers. Our research comprised two phases: an initial exploratory phase involving RNA sequencing of sEVs from 76 CRC patients and 29 healthy controls, and a subsequent validation phase with a larger cohort of 159 CRC patients and 138 healthy controls. Techniques such as dynamic light scattering, transmission electron microscopy, and Western blotting were utilized for sEV characterization. Optimized protocol for sEV purification, RNA isolation and preamplification was applied to successfully sequence the RNA content of sEVs and validate the results by RT-qPCR. We successfully isolated sEVs from blood serum and prepared sequencing libraries from a low amount of RNA. High-throughput sequencing identified differential levels of 460 transcripts between CRC patients and healthy controls, including mRNAs, lncRNAs, and pseudogenes, with approximately 20% being lncRNAs, highlighting several tumor-specific lncRNAs that have not been associated with CRC development and progression. The validation phase confirmed the upregulation of three lncRNAs (NALT1, AL096828, and LINC01637) in blood serum of CRC patients. This study not only identified lncRNA profiles in a population of sEVs from low-volume blood serum specimens of CRC patients but also highlights the value of innovative techniques in biomolecular research, particularly for the detection and analysis of low-abundance biomolecules in clinical samples. The identification of specific lncRNAs associated with CRC provides a foundation for future research into their functional roles in cancer development and potential clinical applications.

Human Blood Serum Counteracts EGFR/HER2-Targeted Drug Lapatinib Impact on Squamous Carcinoma SK-BR-3 Cell Growth and Gene Expression.

Lapatinib is a targeted therapeutic inhibiting HER2 and EGFR proteins. It is used for the therapy of HER2-positive breast cancer, although not all the patients respond to it. Using human blood serum samples from 14 female donors (separately taken or combined), we found that human blood serum dramatically abolishes the lapatinib-mediated inhibition of growth of the human breast squamous carcinoma SK-BR-3 cell line. This antagonism between lapatinib and human serum was associated with cancelation of the drug induced G1/S cell cycle transition arrest. RNA sequencing revealed 308 differentially expressed genes in the presence of lapatinib. Remarkably, when combined with lapatinib, human blood serum showed the capacity of restoring both the rate of cell growth, and the expression of 96.1% of the genes expression of which were altered by the lapatinib treatment alone. Co-administration of EGF with lapatinib also restores the cell growth and cancels alteration of expression of 95.8% of the genes specific to lapatinib treatment of SK-BR-3 cells. Differential gene expression analysis also showed that in the presence of human serum or EGF, lapatinib was unable to inhibit the Toll-Like Receptor signaling pathway and alter expression of genes linked to the Gene Ontology term of Focal adhesion.

N-terminal modification of an LAH4-derived peptide increases mRNA delivery in the presence of serum.

The recently developed mRNA-based coronavirus SARS-CoV-2 vaccines highlighted the great therapeutic potential of the mRNA technology. Although the lipid nanoparticles used for the delivery of the mRNA are very efficient, they showed, in some cases, the induction of side effects as well as the production of antibodies directed against particle components. Thus, the development of alternative delivery systems is of great interest in the pursuit of more effective mRNA treatments. In the present work, we evaluated the mRNA transfection capacities of a series of cationic histidine-rich amphipathic peptides derived from LAH4. We found that while the LAH4-A1 peptide was an efficient carrier for mRNA, its activity was highly serum sensitive. Interestingly, modification of this cell penetrating peptide at the N-terminus with two tyrosines or with salicylic acid allowed to confer serum resistance to the carrier.

The stability of 65 biochemistry analytes in plasma, serum, and whole blood.

OBJECTIVES: The pre-analytical stability of various biochemical analytes requires careful consideration, as it can lead to the release of erroneous laboratory results. There is currently significant variability in the literature regarding the pre-analytical stability of various analytes. The aim of this study was to determine the pre-analytical stability of 65 analytes in whole blood, serum and plasma using a standardized approach. METHODS: Blood samples were collected from 30 healthy volunteers (10 volunteers per analyte) into five vacutainers; either SST, Li-heparin, K2-EDTA, or Na-fluoride/K-oxalate. Several conditions were tested, including delayed centrifugation with storage of whole blood at room temperature (RT) for 8 h, delayed centrifugation with storage of whole blood at RT or 4 °C for 24 h, and immediate centrifugation with storage of plasma or serum at RT for 24 h. Percent deviation (% PD) from baseline was calculated for each analyte and compared to the maximum permissible instability (MPI) derived from intra- and inter-individual biological variation. RESULTS: The majority of the analytes evaluated remained stable across all vacutainer types, temperatures, and timepoints tested. Glucose, potassium, and aspartate aminotransferase, among others, were significantly impacted by delayed centrifugation, having been found to be unstable in whole blood specimens stored at room temperature for 8 h. CONCLUSIONS: The data presented provides insight into the pre-analytical variables that impact the stability of routine biochemical analytes. This study may help to reduce the frequency of erroneous laboratory results released due to exceeded stability and reduce unnecessary repeat phlebotomy for analytes that remain stable despite delayed processing.