Visualizing DNA/RNA, Proteins, and Small Molecule Metabolites within Live Cells.

Live cell imaging is essential for obtaining spatial and temporal insights into dynamic molecular events within heterogeneous individual cells, in situ intracellular networks, and in vivo organisms. Molecular tracking in live cells is also a critical and general requirement for studying dynamic physiological processes in cell biology, cancer, developmental biology, and neuroscience. Alongside this context, this review provides a comprehensive overview of recent research progress in live-cell imaging of RNAs, DNAs, proteins, and small-molecule metabolites, as well as their applications in molecular diagnosis, immunodiagnosis, and biochemical diagnosis. A series of advanced live-cell imaging techniques have been introduced and summarized, including high-precision live-cell imaging, high-resolution imaging, low-abundance imaging, multidimensional imaging, multipath imaging, rapid imaging, and computationally driven live-cell imaging methods, all of which offer valuable insights for disease prevention, diagnosis, and treatment. This review article also addresses the current challenges, potential solutions, and future development prospects in this field.

Mass spectrometry-based metabolomics: Targeting the crosstalk between gut microbiota and brain in neurodegenerative disorders.

Metabolomics seeks to take a "snapshot" in a time of the levels, activities, regulation and interactions of all small molecule metabolites in response to a biological system with genetic or environmental changes. The emerging development in mass spectrometry technologies has shown promise in the discovery and quantitation of neuroactive small molecule metabolites associated with gut microbiota and brain. Significant progress has been made recently in the characterization of intermediate role of small molecule metabolites linked to neural development and neurodegenerative disorder, showing its potential in understanding the crosstalk between gut microbiota and the host brain. More evidence reveals that small molecule metabolites may play a critical role in mediating microbial effects on neurotransmission and disease development. Mass spectrometry-based metabolomics is uniquely suitable for obtaining the metabolic signals in bidirectional communication between gut microbiota and brain. In this review, we summarized major mass spectrometry technologies including liquid chromatography-mass spectrometry, gas chromatography-mass spectrometry, and imaging mass spectrometry for metabolomics studies of neurodegenerative disorders. We also reviewed the recent advances in the identification of new metabolites by mass spectrometry and metabolic pathways involved in the connection of intestinal microbiota and brain. These metabolic pathways allowed the microbiota to impact the regular function of the brain, which can in turn affect the composition of microbiota via the neurotransmitter substances. The dysfunctional interaction of this crosstalk connects neurodegenerative diseases, including Parkinson's disease, Alzheimer's disease and Huntington's disease. The mass spectrometry-based metabolomics analysis provides information for targeting dysfunctional pathways of small molecule metabolites in the development of the neurodegenerative diseases, which may be valuable for the investigation of underlying mechanism of therapeutic strategies.

Metabolomic profiling of the excretory-secretory products of hookworm and whipworm.

INTRODUCTION: Soil-transmitted helminths infect billions of people, livestock and companion animals worldwide, and chronic infections with these nematodes represent a major health burden in many developing countries. On the other hand, complete elimination of parasitic helminths and other infectious pathogens has been implicated with rising rates of autoimmune and allergic disorders in developed countries. Given the enormous health impact of these parasites, it is surprising how little is known about the non-protein small metabolites of the excretory-secretory products (ESP), including their composition and pharmacological properties. OBJECTIVES: We sought proof-of-concept that Nippostrongylus brasiliensis and Trichuris muris, rodent models of two of the most important human soil-transmitted helminths, secrete small metabolites and that some of these metabolites may have specific pharmacological functions. METHODS: N. brasiliensis and T. muris ESP were collected from adult worms and filtered using a 10 kDa cut-off membrane to produce excretory-secretory metabolites (ESM). The ESM were analysed using targeted gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry for polar and non-polar small metabolites. RESULTS: ESM from both N. brasiliensis and T. muris contained small molecules. A total of 54 small molecules (38 polar metabolites and 16 fatty acids) were identified, 36 known polar metabolites from N. brasiliensis and 35 from T. muris. A literature review of the identified compounds revealed that 17 of them have various demonstrated pharmacological activities. CONCLUSION: N. brasiliensis and T. muris secrete polar and non-polar small molecules with as many as 17 metabolites known to exhibit various pharmacological activities.

A surface plasmon resonance based inhibition immunoassay for measurement of steroid hormones.

Quantitative measurement of small-molecule metabolites is now emerging as an effective way to link the metabolite profile to disease state. Surface plasmon resonance (SPR) is a sensing platform that has demonstrated applicability for a large range of biomolecules. However, direct detection of small molecules with SPR challenges the refractive index based detection mechanism. Herein, we utilized an indirect detection format and developed an inhibition immunoassay for the quantitative measurement of 17ß-estradiol (E2) using SPR. One competitor, BSA-E2 conjugate, was immobilized to the SPR chip via the reaction between the primary amino group of the conjugate and the succinimide group (NHS) introduced by the formation of a thiol-NHS monolayer on gold surface. Free E2 molecules compete with BSA-E2 on chip surface for binding sites provided by a monoclonal anti-E2 antibody. It was found the binding affinity of the antibody to BSA-E2 conjugate increases with decreasing surface coverage of BSA-E2 conjugate. Under optimal conditions, a sigmoidal calibration curve with a negative slope and a dynamic range from 10 pM to 2 nM was generated. The detection limit of the immunoassay is estimated to be 0.3 pM. Moreover, the immunoassay exhibits high specificity for E2 detection using estrone (E1) as a potential interference.

Effects of ultrasound treatment on muscle structure, volatile compounds, and small molecule metabolites of salted Culter alburnus fish.

This study investigated the effects of ultrasound treatment on the quality of salted Culter alburnus fish. The results showed that with the increasing ultrasound power, the structural degradation of muscle fibers was intensified, and the conformation of myofibrillar protein was significantly changed. The high-power ultrasound treatment group (300 W) had relatively higher thiobarbiturate reactive substance content (0.37 mg malondialdehyde eq/kg) and peroxidation value (0.63 mmol/kg). A total of 66 volatile compounds were identified with obvious differences among groups. The 200 W ultrasound group exhibited fewer fishy substances (Hexanal, 1-Pentene-3-ol, and 1-Octane-3-ol). Compared with control group, ultrasound groups (200, 300 W) contained more umami taste-related amino peptides such as γ-Glu-Met, γ-Glu-Ala, and Asn-pro. In the ultrasound treatment group, L-isoleucine and L-methionine, which may be used as flavor precursors, were significantly down-regulated, while carbohydrates and its metabolites were up-regulated. Amino acid, carbohydrate, and FA (fatty acyls) metabolism products in salted fish were enriched by ultrasound treatment, and those products might ultimately be related to the taste and flavor of salted fish.

Flexible electrochemical sensors integrated with nanomaterials for in situ determination of small molecules in biological samples: A review.

Flexible biosensors have aroused research enthusiasm due to their potential for in situ quantification of chemical constituents in the human body, which perform a vital function in health monitoring and disease diagnosis. Especially, flexible electrochemical biosensors based on different advanced nanomaterials combine the merits of electrochemical analysis with unique structural/chemical properties of flexible electrode materials, and have exhibited excellent performance for in situ biomarkers detection toward different biological samples. These flexible electrochemical sensors can be integrated into implantable/wearable devices, which demonstrate great promise in invasive/noninvasive in-situ analysis. Consequently, the development of flexible electrochemical biosensors is of great significance for both scientific research and clinical application. In this review, we focus on the state-of-the-art progress in flexible electrochemical biosensors integrated with a wide spectrum of nanomaterials, which are aimed at in situ sensitive detection of small molecule metabolites in different biological specimens including live cells, tissues, body fluids (e.g., human blood, cerebrospinal fluid (CSF), and interstitial fluid (ISF)), and exudate secretion liquors (e.g., human saliva, tear, sweat, and urine). From the perspectives of flexible electrochemical biosensors toward different biological samples, we discuss their innovations in nanomaterials with diverse structures. We also introduce the research status of integrated flexible implantable and wearable electrochemical sensing devices with various types and functionalities for practical application. Furthermore, we share our opinions on the recent progress of flexible electrochemical sensors based on nanomaterials and look forward to applying flexible electrochemical sensors in medical diagnosis and healthcare.

Acetone immersion enhanced MALDI-MS imaging of small molecule metabolites in biological tissues.

Profiling the endogenous tissue metabolites with spatial features is significant for our understanding of molecular histology, and provides an insightful way to uncover the complex associations between tissue metabolic response and external stimuli. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) is an effective molecular imaging technology to illustrate the spatial locations of molecules in tissue. However, due to the limited sensitivity and the presence of multiple matrix-related ions, it is still challenging to globally image the small molecule metabolites (SMMs) using MALDI, especially for those low-content functional ones. Here, a simple acetone washing method was developed to improve the sensitivity of MALDI-MS for imaging SMMs. After immersing in acetone and shaken for 15 min, key functional SMMs were well-visualized with significantly enhanced ion intensities. In addition to lipids, more than 160 SMM ions, including polyamines, cholines, carnitines, amino acids, nitrogenous bases, nucleosides, carbohydrates, organic acids, vitamins were imaged. The acetone washes-based MALDI-MSI was then applied to profile the metabolic alternations that occurred in osteosarcoma, and the abnormally altered SMMs and lipids were clearly visualized. Moreover, with the protection of acetone against tissue antigenicity, we successfully characterized the expression of three metabolites-related enzymes, fatty acid synthase (FASN), glutaminase (GLS), and cytosolic phospholipase A2 (cPLA2) in osteosarcoma. The spatially-resolved metabolite and corresponding enzyme information reveals what occured in osteosarcoma at the molecular level, providing new insights into the understanding of tumour metabolic reprogramming.

Assessing the Pre-Analytical Stability of Small-Molecule Metabolites in Cerebrospinal Fluid Using Direct-Infusion Metabolomics.

Metabolomics studies aiming to find biomarkers frequently make use of historical or multicenter cohorts. These samples often have different pre-analytical conditions that potentially affect metabolite concentrations. We studied the effect of different storage conditions on the stability of small-molecule metabolites in cerebrospinal fluid to aid a reliable interpretation of metabolomics data. Three cerebrospinal fluid pools were prepared from surplus samples from the Amsterdam Dementia Cohort biobank. Aliquoted pools were exposed to different storage conditions to assess the temperature and freeze/thaw stability before final storage at -80 °C: storage up to four months at -20 °C and up to one week at either 5-8 °C or 18-22 °C and exposure to up to seven freeze/thaw cycles. Direct-infusion high-resolution mass spectrometry was performed, resulting in the identification of 1852 m/z peaks. To test the storage stability, principal component analyses, repeated measures analysis of variance, Kruskal‒Wallis tests, and fold change analyses were performed, all demonstrating that small-molecule metabolites in the cerebrospinal fluid (CSF) are relatively unaffected by 1‒3 freeze/thaw cycles, by storage at -20 °C up to two months, by storage at 5-8 °C for up to 72 h, or by storage at 18-22 °C for up to 8 h. This suggests that these differences do not affect the interpretation of potential small-molecule biomarkers in multicenter or historical cohorts and implies that these cohorts are suitable for biomarker studies.