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.

Level of polychlorinated biphenyls in tumor and blood serum of breast cancer patients and control subjects from Punjab, Pakistan.

The current study examined the level of Polychlorinated biphenyls (PCBs) in tumor and blood serum of female breast cancer patients and control individuals recruited from Punjab, Pakistan. Breast tumor and blood serum from 40 patients and only blood serum from ten control subjects were obtained and concentration of 32 PCB congeners was analyzed through Gas chromatography coupled with Mass spectrophotometry. Sociodemographic variables of the patients along with essential clinical and haematological parameters were taken as covariates. Tumor reflects the highest median (min-max) concentration (ng g-1 lw) of Æ©PCBs at 115.94 (0.05-17.75) followed by 16.53 (0.09-2.94) and 5.24 (0.01-0.59) in blood serum of cancer patients and control group respectively. Median concentrations (ng g-1 lw) of non-dioxine like Æ©PCBs were considerably higher at 83.04, 32.89 and 4.27 compared to 13.03 and 3.50 and 0.97 for dioxin like Æ©PCBs in tumor, serum of breast cancer patients and control subjects respectively. PCB-87 was most dominant congeners in tumor followed by PCB-170 and -82 whereas PCB-28 and -52 reflected greatest contribution in serum of breast cancer patients. Blood haemoglobin, potassium and chloride ions showed significant positive whereas body mass index reflect inverse relationship when regressed with Æ©PCBs in tumor. This pioneer study depicts elevated concentrations of PCBs in patients compared to control, reflecting potential positive association of PCBs with breast cancer which need further confirmation. We concluded that chronic exposure to PCBs might be associated with an increasing number of breast cancer incidences in developing countries like Pakistan, which should be further elucidated through detail in vitro and in vivo studies.

Quantitative analysis of mRNA-lipid nanoparticle stability in human plasma and serum by size-exclusion chromatography coupled with dual-angle light scattering.

Understanding the stability of mRNA loaded lipid nanoparticles (mRNA-LNPs) is imperative for their clinical development. Herein, we propose the use of size-exclusion chromatography coupled with dual-angle light scattering (SEC-MALS) as a new approach to assessing mRNA-LNP stability in pure human serum and plasma. By applying a dual-column configuration to attenuate interference from plasma components, SEC-MALS was able to elucidate the degradation kinetics and physical property changes of mRNA-LNPs, which have not been observed accurately by conventional dynamic light scattering techniques. Interestingly, both serum and plasma had significantly different impacts on the molecular weight and radius of gyration of mRNA-LNPs, suggesting the involvement of clotting factors in desorption of lipids from mRNA-LNPs. We also discovered that a trace impurity (~1 %) in ALC-0315, identified as its O-tert-butyloxycarbonyl-protected form, greatly diminished mRNA-LNP stability in serum. These results demonstrated the potential utility of SEC-MALS for optimization and quality control of LNP formulations.

Realizing the label-free sensitive detection of carcinoembryogenic antigen (CEA) in blood serum via a MNC-decorated flexible immunosensor.

A label-free electrochemical immunosensor utilising nitrogen-rich mesoporous carbon (MNC) as the substrate material was developed for the sensitive quantification of carcinoembryonic antigen (CEA). The synergic interactions between MNC and AbCEA also eliminated the need for coupling agents such as EDC/NHS. The novel immunosensor demonstrated a wide detection range from 500 fM (9.04 pg mL-1) to 50 nM (1 µg mL-1) and a low detection limit (LOD) of 500 fM. Moreover, the immunosensor showed sensitivities of 12.27 mA nM-1 cm-2 and 0.066 mA nM-1 cm-2 for detecting CEA in the linear ranges 10 pM to 1 nM and 2 nM to 50 nM, respectively, while maintaining long-term storage stability of 6 weeks. Analysis of real serum sample analysis yielded highly accurate results with recovery rates ranging from 99.3% to 103.7%. Furthermore, the developed paper-based screen-printed electrode exhibited a similar detection range, suggesting its potential for use in point-of-care detection devices in future applications.

Serum levels of inflammatory cytokines in mercury mining workers in a precarious situation: A preliminary study.

Mercury is a ubiquitous environmental xenobiotic; the primary sources of exposure to this metal are artisanal gold mining and the direct production of mercury. In Mexico, artisanal mercury mining continues to be an important activity in different regions of the country. Exposure to mercury vapors releases can have severe health impacts, including immunotoxic effects such as alterations in cytokine profiling. Therefore, in the present work, we evaluated the inflammatory cytokines profile in the blood serum of miners exposed to mercury. A cross-sectional observational study was performed on 27 mining workers (exposed group) and 20 control subjects (nonexposed group) from central Mexico. The mercury urine concentration (U-Hg) was determined by atomic absorption spectrometry, and IL-2, IL-6, IL-8, IL-10, and TNF-α were measured using a Multiplex Assay. The results showed that the U-Hg in the miners had a median value of 552.70 µg/g creatinine. All cytokines showed a significant increase in the miner group compared with the control group, except for TNF-α. In addition, we observed a positive correlation between U-Hg concentration and cytokine levels. In conclusion, mercury exposure correlated with cytokine levels (considered acute inflammatory marker) in miners; therefore, workers exposed to this metal show an acute systemic inflammation that could lead to alterations in other organs and systems.

Polychlorinated alkanes in paired blood serum and breast milk in a Swedish cohort study: Matrix dependent partitioning differences compared to legacy POPs.

BACKGROUND: Polychlorinated alkanes (PCAs) constitute a large group of individual congeners originating from commercial chlorinated paraffin (CP) products with carbon chain lengths of PCAs-C10-13, PCAs-C14-17, and PCAs-C18-32, occasionally containing PCAs-C6-9 impurities. The extensive use of CPs has led to global environmental pollution of PCAs. This study aimed to quantify PCAs in paired serum and breast milk of lactating Swedish mothers, exploring their concentration relationship. METHODS: Twenty-five paired samples of mothers' blood serum and breast milk were analysed and concentrations were determined for PCAs C6-32 and compared to 4,4'-DDE, the PCB congener 2,2',4,4',5,5'-hexachlorobiphenyl (CB-153), and hexachlorobenzene (HCB). RESULTS: The median concentrations of PCAs-C6-9, PCAs-C10-13, PCAs-C14-17, PCAs-C18-32 and ΣPCAs in serum were 14, 790, 520, 16 and 1350 ng/g lipid weight (lw), respectively, and in breast milk 0.84, 36, 63, 6.0 and 107 ng/g lw. Levels of 4,4'-DDE, CB-153 and HCB were comparable in the two matrices, serum and breast milk at 17, 12 and 4.9 ng/g lw. The results show significant differences of PCAs-C10-13 and PCAs-C14-17 in breast milk with 22- and 6.2-times lower lw-based concentrations than those measured in serum. On wet weight the differences serum/breast milk ratios of PCAs-C6-9, PCAs-C10-13, PCAs-C14-17, PCAs-C18-32 and ΣPCAs were 1.7, 3.2, 1.0, 0.4 and 1.6, respectively, while the ratio for 4,4'-DDE, CB-153 and HCB were each close to 0.1. CONCLUSION: Swedish lactating mothers had high serum concentrations of PCAs-C10-13 and PCAs-C14-17, with the ΣPCAs median serum concentration of 1350 ng/g lw. The breast milk concentration, although considerably lower at 107 ng/g lw, still surpassed those of 4,4'-DDE, CB-153 and HCB, suggesting an exposure risk of infants to PCAs. The variation in blood and breast milk accumulation between PCAs and studied legacy POPs, is rarely discussed but warrants further studies on partitioning properties as well as associated toxicological implications.

Serum trace element levels and activity of enzymes associated with oxidative stress in endometriosis and endometrial cancer.

Endometriosis and endometrial cancer are closely related to oxidative stress. However, the direct relationship between copper and zinc levels and oxidative stress in the extracellular and intracellular space remains unclear. The presented study is focused on the determination of serum Zn and Cu levels, glutathione concentration and enzyme activity in three groups: patients diagnosed with endometrial cancer (EC), patients diagnosed with endometriosis (EM), and a healthy control group. Spectrophotometric determination of trace elements revealed that levels of zinc and copper were lower in blood plasma of patients with endometriosis as compared with the other groups; however, there were no significant differences in the Cu/Zn ratio. Furthermore, significantly increased blood serum glutathione levels were detected in both EM and EC groups compared with the control group. While the activity of superoxide dismutase (SOD) was similar across the studied groups, we observed differences in the activity of other enzymes associated with oxidative stress, including glutathione peroxidase (GPx), glutathione reductase (GR) and glutathione S-transferase (GST), between the control group and the EM and EC patients. Additionally, analysis of gene expression based on free circulating mRNA indicated significant differences in the expression of SOD isoenzymes between the patient groups and the control group; expression of GPx isoenzymes was also altered. Obtained results may have potential application in diagnostics as well as monitoring of endometriosis and endometrial cancer.

Richer than previously probed: An application of 1H NMR reveals one hundred metabolites using only fifty microliter serum.

The classical approach restricts the detection of metabolites in serum samples by using nuclear magnetic resonance (NMR) spectroscopy; however, the presence of copious proteins and lipoproteins emphasize and necessitate the development of a contemporary, high-throughput tactic. To eliminate the lipoproteins and proteins from sera to engender filtered sera (FS), the study was executed with 50 µl serum obtained from five healthy individuals with 5 years of age difference from 25 to 45 years old and the application of a unique mechanical filter with molecular weight cut-off value of 2KDa. The 10 µl FS from each individual was pooled to make 50 µl final volume filled in a co-axial tube for acquisition of a battery of 1D/2D investigations at 800 MHz NMR spectrometer and the assigned metabolites was confirmed through mass spectrometry as well as by comparing 1H NMR spectra of individual metabolites. This innovative tactic is commissioning to reveal more than 100 metabolites. In contrast to the protein precipitation method, 24 new metabolites were recognized in the present study. The present innovative approach characterizes nucleosides, nitrogenous bases, and volatile metabolites that possibly produce a landmark for the delineation of a comprehensive metabolic profile applicable for detection of the molecular cause of pathogenicity, early-stage disease detection and prognosis, inborn error of metabolism, and forensic investigations exerting the least volume of FS and NMR spectroscopy. The assignment of 100 metabolites using 1H NMR-based FS is described for the first time in the present report.

Trends in Serum Per- and Polyfluoroalkyl Substance (PFAS) Concentrations in Teenagers and Adults, 1999-2018 NHANES.

Some types of per- and poly-fluoroalkyl substances (PFAS) have been banned over the last two decades, but millions of Americans continue to have exposure to the compounds through drinking water and consumer products. Therefore, understanding the changes in serum PFAS concentrations after their limited use is necessary to protect public health. In this study, we evaluated trends of serum PFAS compounds (PFOS, PFOA, PFHxS, PFDA, and PFNA) to determine their distribution among the United States general population. We analyzed serum concentrations of PFAS measured from random subsamples of the National Health and Nutrition Examination Survey (NHANES) participants. The study results demonstrated that demographic factors such as race/ethnicity, age, and sex may influence the levels of serum PFAS over time. Adults, males, Asians, Non-Hispanic Blacks, and Non-Hispanic Whites had high risks of exposure to the selected PFAS. Overall, serum PFAS levels declined continuously in the studied population from 1999 to 2018. Among the studied population, PFOS and PFDA were the most and least prevalent PFAS in blood serum, respectively. Serum levels of PFDA, PFOA, and PFHxS showed upward trends in at least one racial/ethnic group after 2016, which underscores the need for continuous biomonitoring of PFAS levels in humans and the environment.

LC-MS/MS Quantitation of HILIC-Enriched N-glycopeptides Derived from Low-Abundance Serum Glycoproteins in Patients with Narcolepsy Type 1.

Glycoproteomic analysis is always challenging because of low abundance and complex site-specific heterogeneity. Glycoproteins are involved in various biological processes such as cell signaling, adhesion, and cell-cell communication and may serve as potential biomarkers when analyzing different diseases. Here, we investigate glycoproteins in narcolepsy type 1 (NT1) disease, a form of narcolepsy characterized by cataplexy-the sudden onset of muscle paralysis that is typically triggered by intense emotions. In this study, 27 human blood serum samples were analyzed, 16 from NT1 patients and 11 from healthy individuals serving as controls. We quantified hydrophilic interaction liquid chromatography (HILIC)-enriched glycopeptides from low-abundance serum samples of controls and NT1 patients via LC-MS/MS. Twenty-eight unique N-glycopeptides showed significant changes between the two studied groups. The sialylated N-glycopeptide structures LPTQNITFQTESSVAEQEAEFQSPK HexNAc6, Hex3, Neu5Ac2 (derived from the ITIH4 protein) and the structure IVLDPSGSMNIYLVLDGSDSIGASNFTGAK HexNAc5, Hex4, Fuc1 (derived from the CFB protein), with p values of 0.008 and 0.01, respectively, were elevated in NT1 samples compared with controls. In addition, the N-glycopeptide protein sources Ceruloplasmin, Complement factor B, and ITH4 were observed to play an important role in the complement activation and acute-phase response signaling pathways. This may explain the possible association between the biomarkers and pathophysiological effects.

In-house standards derived from doping peptides: Enzymatic and serum stability and degradation profile of GHRP and GHRH-related peptides.

Matrix effect and sample pretreatment significantly affect the percentage recovery of peptides in biological matrices, affecting the method robustness and accuracy. To counteract this effect, an internal standard (IS) is used; however, in most cases this is not available, which limits the analytical method. It is important to identify short peptides that can be used as ISs in the quantification of peptides in biological matrices. In this study, doping peptides GHRP-4, GHRP-5, GHRP-6, Sermorelin (1-11), Sermorelin (13-20) and Sermorelin (22-29) were synthesized using solid-phase peptide synthesis. Treatment with human blood, trypsin and chymotrypsin was used to determine the stability of the peptides. Products were evaluated using the high-performance liquid chromatography-diode array detector (HPLC-DAD) method. The analytical methodology and sample pretreatment were effective for the analysis of these molecules. A unique profile related to protein binding and enzymatic stability of each peptide was established. GHRP-4, GHRP-6 and Sermorelin (22-29) can be considered as in-house ISs as they were stable to enzyme and blood treatment and can be used for the quantification of peptides in biological samples. Peptides GHRP-6 and Sermorelin (22-29) were used to analyse a dimeric peptide (26 [F] LfcinB (20-30)2 ) in four different matrices to test these peptides as in-house IS.

Orbitrap Mass Spectrometry and High-Field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS) Enable the in-Depth Analysis of Human Serum Proteoforms.

Blood serum and plasma are arguably the most commonly analyzed clinical samples, with dozens of proteins serving as validated biomarkers for various human diseases. Top-down proteomics may provide additional insights into disease etiopathogenesis since this approach focuses on protein forms, or proteoforms, originally circulating in blood, potentially providing access to information about relevant post-translational modifications, truncations, single amino acid substitutions, and many other sources of protein variation. However, the vast majority of proteomic studies on serum and plasma are carried out using peptide-centric, bottom-up approaches that cannot recapitulate the original proteoform content of samples. Clinical laboratories have been slow to adopt top-down analysis, also due to higher sample handling requirements. In this study, we describe a straightforward protocol for intact proteoform sample preparation based on the depletion of albumin and immunoglobulins, followed by simplified protein fractionation via polyacrylamide gel electrophoresis. After molecular weight-based fractionation, we supplemented the traditional liquid chromatography-tandem mass spectrometry (LC-MS2) data acquisition with high-field asymmetric waveform ion mobility spectrometry (FAIMS) to further simplify serum proteoform mixtures. This LC-FAIMS-MS2 method led to the identification of over 1000 serum proteoforms < 30 kDa, outperforming traditional LC-MS2 data acquisition and more than doubling the number of proteoforms identified in previous studies.

Versatile MXene composite probe-mediated homogeneous electrochemiluminescence biosensor with integrated signal transduction and near-infrared modulation strategy for concanavalin A detection.

Based on the highly specific interaction between concanavalin A (Con A) and glucose (Glu), a competitive electrochemiluminescence (ECL) biosensor was constructed for ultrasensitive detection of Con A. Nanocomposites with excellent electrocatalytic and photothermal properties were obtained by covalently bonding zinc oxide quantum dots (ZnO QDs) to vanadium carbide MXene (V2C MXene) surfaces. The modification of ZnO QDs hinders the aggregation of V2C MXene and increases the catalytic activity of oxygen reduction reaction, thus amplifying the luminol cathodic emission. In addition, the excellent photothermal performance of the V2C MXene-ZnO QDs can convert light energy into heat energy under the irradiation of 808 nm near infrared laser, thus increasing the temperature of the reaction system and accelerating the electron transfer process to realize the synergistic amplified homogeneous ECL system. This innovative work not only enriches the fundamental research on multifunctional MXene nanomaterials for biosensing, but also provides an effective strategy for ECL signal amplification.

Detection of major psychoactive compounds (safrole, myristicin, and elemicin) of nutmeg in human serum via GC-MS/MS using MonoSpin® extraction: Application in a nutmeg poisoning case.

Nutmeg is an inexpensive, readily available spice used in a variety of recipes. However, the use of nutmeg powder as a recreational drug for its hallucinogenic effects is resulting in an increase in overdose rates. We encountered a male patient being hospitalized after ingesting 75 g of commercially available nutmeg powder with the intent of committing suicide. There are no available reports documenting the toxic or comatose-fatal blood concentrations or time-course of drug action in cases of nutmeg poisoning. Therefore, to improve patient management, we endeavored to determine the blood serum levels and time-course of the major psychoactive compounds (safrole, myristicin, and elemicin) present in nutmeg. We designed a simple and reliable method using the MonoSpin® extraction kit and gas chromatography-tandem mass spectrometry to detect the presence of these psychoactive compounds in human serum. The method had detection and quantitation limits of 0.14-0.16 and 0.5 ng/mL (lowest calibration points), respectively. The calibration curves displayed excellent linearity (0.996-0.997) for all three compounds at 0.5-300 ng/mL blood concentrations. The intra- and inter-day precision values for quality assurance were in the ranges of 2.4-11 % and 2.5-11 %, respectively; bias ranged from - 2.6 % to 2.1 %. Blood serum levels of safrole, myristicin, and elemicin were measured at admission (approximately 8 h post-ingestion) and approximately 94 h after a post-admission fluid therapy to evaluate their biological half-lives. We developed this method to obtain information on the psychoactive constituents of nutmeg and, thereby, determine the toxicokinetic parameters of nutmeg in a case of nutmeg poisoning.

A novel device for swift and efficient CD44 protein digestion of pipette tips in human serum.

For molecular diagnostics in modern biomedical research, electrospray ionisation mass spectrometry (ESI-MS) based on proteome profiling is important. Now a days, sample preparation such as proteolysis and protein extraction remain incredibly challenging and inefficient. Recent sample-preparation methods based on micro tips show promising results toward the aim "a proteome in an hour". Proteolysis at the tip, is still infrequently observed and does not represent the processing of complex bio-samples. In this study, we outline a unique technique for detecting and extracting human serum CD44 biomarkers by ligand-protein interactions. This method employs macropores silica particles (MPSP) or (MOSF) modified with hyaluronic acid (HA). In order to assist in the profile of the human serum proteome, we limitations of immunoassays for rapid and multimodal proteolysis. For effective in situ proteolysis, in micropipette tips, MPSP were designed as nanoreactors with variable pore size and surface chemistry. In MS-based bottom-up proteome analysis, the device as-built demonstrated favourable sensitivity (LOD of 0.304 ± 0.007 ng/mL and LOQ of 0.973 ± 0.054 ng/mL), selectivity, durability (at -20 °C for 2 months), reuse (at least 10 times), and minimal memory impact. In addition, we examined into specific surface chemistries of nanoparticles for the absorption of proteins in serum and profiled the HA-binding serum proteome, setting a new preliminary benchmark for future databases. Our study not only helped establish a new platform for extracting/detection of CD44 and identifying the HA-binding proteome, but it also offered design recommendations for ligand affinity-based techniques for the antibody-free study of serum biomarkers with a view towards diagnostic applications.

Diagnostic performance of the IMMY cryptococcal antigen lateral flow assay on serum and cerebrospinal fluid for diagnosis of cryptococcosis in HIV-negative patients: a systematic review.

BACKGROUND: The incidence of cryptococcosis amongst HIV-negative persons is increasing. Whilst the excellent performance of the CrAg testing in people living with HIV is well described, the diagnostic performance of the CrAg LFA has not been systematically evaluated in HIV-negative cohorts on serum or cerebrospinal fluid. METHODS: We performed a systematic review to characterise the diagnostic performance of IMMY CrAg® LFA in HIV-negative populations on serum and cerebrospinal fluid. A systematic electronic search was performed using Medline, Embase, Global Health, CENTRAL, WoS Science Citation Index, SCOPUS, Africa-Wide Information, LILACS and WHO Global Health Library. Studies were screened and data extracted from eligible studies by two independent reviewers. A fixed effect meta-analysis was used to estimate the diagnostic sensitivity and specificity. RESULTS: Of 447 records assessed for eligibility, nine studies met our inclusion criteria, including 528 participants overall. Amongst eight studies that evaluated the diagnostic performance of the IMMY CrAg® LFA on serum, the pooled median sensitivity was 96% (95% Credible Interval (CrI) 68-100%) with a pooled specificity estimate of 96% (95%CrI 84-100%). Amongst six studies which evaluated the diagnostic performance of IMMY CrAg® LFA on CSF, the pooled median sensitivity was 99% (95%CrI 95-100%) with a pooled specificity median of 99% (95%CrI 95-100%). CONCLUSIONS: This review demonstrates a high pooled sensitivity and specificity for the IMMY CrAg® LFA in HIV-negative populations, in keeping with findings in HIV-positive individuals. The review was limited by the small number of studies. Further studies using IMMY CrAg® LFA in HIV-negative populations would help to better determine the diagnostic value of this test.

Development of serum substitute medium for bone tissue engineering.

In tissue engineering, cells are grown often on scaffolds and subjected to chemical/mechanical stimuli. Most such cultures still use fetal bovine serum (FBS) despite its known disadvantages including ethical concerns, safety issues, and variability in composition, which greatly influences the experimental outcomes. To overcome the disadvantages of using FBS, chemically defined serum substitute medium needs to be developed. Development of such medium depends on cell type and application-which makes it impossible to define one universal serum substitute medium for all cells in any application. Here, we developed a serum substitute medium for bone tissue engineering (BTE) in a step-by-step process. Essential components were added to the medium while human bone marrow mesenchymal stromal cells (hBMSCs, osteoblast progenitor cells) were cultured in two-dimensional and three-dimensional substrates. In a 3-week culture, the developed serum substitute medium worked equally well as FBS containing medium in term of cell attachment to the substrate, cell survival, osteoblast differentiation, and deposition of extracellular matrix. In the next step, the use of serum substitute medium was evaluated when culturing cells under mechanical loading in the form of shear stress. The outcomes showed that the application of shear stress is essential to improve extracellular matrix formation while using serum substitute medium. The developed serum substitute medium could pave the way in replacing FBS for BTE studies eliminating the use of controversial FBS and providing a better-defined chemical environment for BTE studies.

Metabolomic differentiation of benign vs malignant pulmonary nodules with high specificity via high-resolution mass spectrometry analysis of patient sera.

Differential diagnosis of pulmonary nodules detected by computed tomography (CT) remains a challenge in clinical practice. Here, we characterize the global metabolomes of 480 serum samples including healthy controls, benign pulmonary nodules, and stage I lung adenocarcinoma. The adenocarcinoma demonstrates a distinct metabolomic signature, whereas benign nodules and healthy controls share major similarities in metabolomic profiles. A panel of 27 metabolites is identified in the discovery cohort (n = 306) to distinguish between benign and malignant nodules. The discriminant model achieves an AUC of 0.915 and 0.945 in the internal validation (n = 104) and external validation cohort (n = 111), respectively. Pathway analysis reveals elevation in glycolytic metabolites associated with decreased tryptophan in serum of lung adenocarcinoma vs benign nodules and healthy controls, and demonstrates that uptake of tryptophan promotes glycolysis in lung cancer cells. Our study highlights the value of the serum metabolite biomarkers in risk assessment of pulmonary nodules detected by CT screening.

Week-Long Operation of Electrochemical Aptamer Sensors: New Insights into Self-Assembled Monolayer Degradation Mechanisms and Solutions for Stability in Serum at Body Temperature.

Conventional wisdom suggests that widely utilized self-assembled alkylthiolate monolayers on gold are too unstable to last more than several days when exposed to complex fluids such as raw serum at body temperature. Demonstrated here is that these monolayers can not only last at least 1 week under such harsh conditions but that significant applied value can be captured for continuous electrochemical aptamer biosensors. Electrochemical aptamer biosensors provide an ideal tool to investigate monolayer degradation, as aptamer sensors require a tightly packed monolayer to preserve sensor signal vs background current and readily reveal fouling by albumin and other solutes when operating in biofluids. Week-long operation in serum at 37 °C is achieved by (1) increasing van der Waals interactions between adjacent monolayer molecules to increase the activation energy required for desorption, (2) optimizing electrochemical measurement to decrease both alkylthiolate oxidation and electric-field-induced desorption, and (3) mitigating fouling using protective zwitterionic membranes and zwitterion-based blocking layers with antifouling properties. This work further proposes origins and mechanisms of monolayer degradation in a logical stepwise manner that was previously unobservable over multiday time scales. Several of the observed results are surprising, revealing that short-term improvements to sensor longevity (i.e., hours) actually increase sensor degradation in the longer term (i.e., days). The results and underlying insights on mechanisms not only push forward fundamental understanding of stability for self-assembled monolayers but also demonstrate an important milestone for continuous electrochemical aptamer biosensors.