Dissecting the role of cannabinoids in vascular health and disease.

Cannabis, often recognized as the most widely used illegal psychoactive substance globally, has seen a shift in its legal status in several countries and regions for both recreational and medicinal uses. This change has brought to light new evidence linking cannabis consumption to various vascular conditions. Specifically, there is an association between cannabis use and atherosclerosis, along with conditions such as arteritis, reversible vasospasm, and incidents of aortic aneurysm or dissection. Recent research has started to reveal the mechanisms connecting cannabinoid compounds to atherosclerosis development. It is well known that the primary biological roles of cannabinoids operate through the activation of cannabinoid receptor types 1 and 2. Manipulation of the endocannabinoid system, either genetically or pharmacologically, is emerging as a promising approach to address metabolic dysfunctions related to obesity. Additionally, numerous studies have demonstrated the vasorelaxant properties and potential atheroprotective benefits of cannabinoids. In preclinical trials, cannabidiol is being explored as a treatment option for monocrotaline-induced pulmonary arterial hypertension. Although existing literature suggests a direct role of cannabinoids in the pathogenesis of atherosclerosis, the correlation between cannabinoids and other vascular diseases was only reported in some case series or observational studies, and its role and precise mechanisms remain unclear. Therefore, it is necessary to summarize and update previously published studies. This review article aims to summarize the latest clinical and experimental research findings on the relationship between cannabis use and vascular diseases. It also seeks to shed light on the potential mechanisms underlying these associations, offering a comprehensive view of current knowledge in this evolving field of study.

QTL Mapping and Genome-Wide Association Study Reveal Genetic Loci and Candidate Genes Related to Soluble Solids Content in Melon.

Melon (Cucumis melo L.) is an economically important Cucurbitaceae crop grown around the globe. The sweetness of melon is a significant factor in fruit quality and consumer appeal, and the soluble solids content (SSC) is a key index of melon sweetness. In this study, 146 recombinant inbred lines (RILs) derived from two oriental melon materials with different levels of sweetness containing 1427 bin markers, and 213 melon accessions containing 1,681,775 single nucleotide polymorphism (SNP) markers were used to identify genomic regions influencing SSC. Linkage mapping detected 10 quantitative trait loci (QTLs) distributed on six chromosomes, seven of which were overlapped with the reported QTLs. A total of 211 significant SNPs were identified by genome-wide association study (GWAS), 138 of which overlapped with the reported QTLs. Two new stable, co-localized regions on chromosome 3 were identified by QTL mapping and GWAS across multiple environments, which explained large phenotypic variance. Five candidate genes related to SSC were identified by QTL mapping, GWAS, and qRT-PCR, two of which were involved in hydrolysis of raffinose and sucrose located in the new stable loci. The other three candidate genes were involved in raffinose synthesis, sugar transport, and production of substrate for sugar synthesis. The genomic regions and candidate genes will be helpful for molecular breeding programs and elucidating the mechanisms of sugar accumulation.

LRP5 Promotes Gastric Cancer via Activating Canonical Wnt/ß-Catenin and Glycolysis Pathways.

The overactivation of canonical Wnt/ß-catenin pathway is one of the main cascades for the initiation, progression, and recurrence of most human malignancies. As an indispensable coreceptor for the signaling transduction of the canonical Wnt/ß-catenin pathway, LRP5 is up-regulated and exerts a carcinogenic role in most types of cancer. However, its expression level and role in gastric cancer (GC) has not been clearly elucidated. The current work showed that LRP5 was overexpressed in GC tissues and the expression of LRP5 was positively associated with the advanced clinical stages and poor prognosis. Ectopic expression of LRP5 enhanced the proliferation, invasiveness, and drug resistance of GC cells in vitro, and accelerated the tumor growth in nude mice, through activating the canonical Wnt/ß-catenin signaling pathway and up-regulating aerobic glycolysis, thus increasing the energy supply for GC cells. Additionally, the expression of LRP5 and glycolysis-related genes showed an obviously positive correlation in GC tissues. By contrast, the exact opposite results were observed when the endogenous LRP5 was silenced in GC cells. Collectively, these results not only reveal the carcinogenic role of LRP5 during GC development through activating the canonical Wnt/ß-catenin and glycolysis pathways, but also provide a valuable candidate for the diagnosis and treatment of human GC.

LRP5 promotes cancer stem cell traits and chemoresistance in colorectal cancer.

The overactivation of canonical Wnt/ß-catenin pathway and the maintenance of cancer stem cells (CSCs) are essential for the onset and malignant progression of most human cancers. However, their regulatory mechanism in colorectal cancer (CRC) has not yet been well demonstrated. Low-density lipoprotein receptor-related protein 5 (LRP5) has been identified as an indispensable co-receptor with frizzled family members for the canonical Wnt/ß-catenin signal transduction. Herein, we show that activation of LRP5 gene promotes CSCs-like phenotypes, including tumorigenicity and drug resistance in CRC cells, through activating the canonical Wnt/ß-catenin and IL-6/STAT3 signalling pathways. Clinically, the expression of LRP5 is upregulated in human CRC tissues and closely associated with clinical stages of patients with CRC. Further analysis showed silencing of endogenous LRP5 gene is sufficient to suppress the CSCs-like phenotypes of CRC through inhibiting these two pathways. In conclusion, our findings not only reveal a regulatory cross-talk between canonical Wnt/ß-catenin signalling pathway, IL-6/STAT3 signalling pathway and CD133-related stemness that promote the malignant behaviour of CRC, but also provide a valuable target for the diagnosis and treatment of CRC.

Carbon Dot Blinking Fingerprint Uncovers Native Membrane Receptor Organizations via Deep Learning.

Oligomeric organization of G protein-coupled receptors is proposed to regulate receptor signaling and function, yet rapid and precise identification of the oligomeric status especially for native receptors on a cell membrane remains an outstanding challenge. By using blinking carbon dots (CDs), we now develop a deep learning (DL)-based blinking fingerprint recognition method, named deep-blinking fingerprint recognition (BFR), which allows automatic classification of CD-labeled receptor organizations on a cell membrane. This DL model integrates convolutional layers, long-short-term memory, and fully connected layers to extract time-dependent blinking features of CDs and is trained to a high accuracy (∼95%) for identifying receptor organizations. Using deep blinking fingerprint recognition, we found that CXCR4 mainly exists as 87.3% monomers, 12.4% dimers, and <1% higher-order oligomers on a HeLa cell membrane. We further demonstrate that the heterogeneous organizations can be regulated by various stimuli at different degrees. The receptor-binding ligands, agonist SDF-1α and antagonist AMD3100, can induce the dimerization of CXCR4 to 33.1 and 20.3%, respectively. In addition, cytochalasin D, which inhibits actin polymerization, similarly prompts significant dimerization of CXCR4 to 30.9%. The multi-pathway organization regulation will provide an insight for understanding the oligomerization mechanism of CXCR4 as well as for elucidating their physiological functions.

The complex role of Wnt ligands in type 2 diabetes mellitus and related complications.

Type 2 diabetes mellitus (T2DM) is one of the major chronic diseases, whose prevalence is increasing dramatically worldwide and can lead to a range of serious complications. Wnt ligands (Wnts) and their activating Wnt signalling pathways are closely involved in the regulation of various processes that are important for the occurrence and progression of T2DM and related complications. However, our understanding of their roles in these diseases is quite rudimentary due to the numerous family members of Wnts and conflicting effects via activating the canonical and/or non-canonical Wnt signalling pathways. In this review, we summarize the current findings on the expression pattern and exact role of each human Wnt in T2DM and related complications, including Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, Wnt10a, Wnt10b, Wnt11 and Wnt16. Moreover, the role of main antagonists (sFRPs and WIF-1) and coreceptor (LRP6) of Wnts in T2DM and related complications and main challenges in designing Wnt-based therapeutic approaches for these diseases are discussed. We hope a deep understanding of the mechanistic links between Wnt signalling pathways and diabetic-related diseases will ultimately result in a better management of these diseases.

Enhanced Isolation of Fetal Nucleated Red Blood Cells by Enythrocyte-Leukocyte Hybrid Membrane-Coated Magnetic Nanoparticles for Noninvasive Pregnant Diagnostics.

Fetal nucleated red blood cells (fNRBCs) in maternal peripheral blood containing the whole genetic information of the fetus may serve for noninvasive pregnant diagnostics (NIPD). However, the fetal cell-based NIPD is seriously limited by the poor purity of the isolated fNRBCs. Recently, the biomimetic cell membrane-camouflaged nanoparticles containing outstanding features have been widely used to detect and isolate rare cells from the peripheral blood samples. In this work, enythrocyte (RBC) and leukocyte (WBC) membranes are fused and coated onto magnet nanoparticles and then modified with anti-CD147 to isolate fNRBCs from the maternal peripheral blood with significant efficiency (∼90%) and purity (∼87%) in simulated spiked blood samples. Further, fNRBCs were isolated and identified from a series of maternal peripheral blood samples coming from pregnant women of 11-13 gestational weeks, and different chromosomal aneuploidies were diagnosed using fNRBCs isolated from maternal blood in early pregnancy. Our strategy may offer additional opportunity to overcome the limitations of current cell-based NIPD platforms.

Silica microbeads capture fetal nucleated red blood cells for noninvasive prenatal testing of fetal ABO genotype.

ABO hemolytic disease of the newborn (ABO-HDN), which may cause neonatal jaundice and polycythemia, or even stillbirth or neonatal death, is widespread in China. Prenatal testing for the fetal ABO blood group can reduce unnecessary concerns or ensure prompt treatment. Herein, we presented a method to employ high-density silica microbeads (SiO2 MBs) for capturing fetal nucleated red blood cells (fnRBCs) in maternal peripheral blood, and we detected the ABO genotype of the fetus using these captured cells. We evaluated 52 patients using the SiO2 MBs. Among 26 pregnant women with type O blood, 8 (30.8%) of the fetuses had type A blood, 5 (19.2%) had type B blood, and 13 (50%) had type O blood. SRY genes were detected in all 27 male fetuses. This study represents a simple and effective method for noninvasive prenatal detection of the fetal ABO genotype. We believe that this method has great potential for noninvasive prenatal testing of the fetal Rh blood group and other fetal diseases as well.

High-throughput isolation of fetal nucleated red blood cells by multifunctional microsphere-assisted inertial microfluidics.

Being easy, safe and reliable, non-invasive prenatal diagnosis (NIPD) has been greatly pursued in recent years. Holding the complete genetic information of the fetus, fetal nucleated red blood cells (fNRBCs) are viewed as a suitable target for NIPD application. However, effective separating fNRBCs from maternal peripheral blood for clinic use still faces great challenges, given that fNRBCs are extremely rare in maternal blood circulation. Here, by combining the high-throughput inertial microfluidic chip with multifunctional microspheres as size amplification, we develop a novel method to isolate fNRBCs with high performance. To enlarge the size difference between fNRBCs and normal blood cells, we use the gelatin coated microspheres to capture fNRBCs with anti-CD147 as specific recognizer at first. The size difference between fNRBCs captured by the microspheres and normal blood cells makes it easy to purify the captured fNRBCs through the spiral microfluidic chip. Finally, the purified fNRBCs are mildly released from the microspheres by enzymatically degrading the gelatin coating. Cell capture efficiency about 81%, high purity of 83%, as well as cell release viability over 80% were achieved using spiked samples by this approach. Additionally, fNRBCs were successfully detected from peripheral blood of pregnant women with an average of 24 fNRBCs per mL, suggesting the great potential of this method for clinical non-invasive prenatal diagnosis.

A valve-based microfluidic device for on-chip single cell treatments.

Assays toward single-cell analysis have attracted the attention in biological and biomedical researches to reveal cellular mechanisms as well as heterogeneity. Yet nowadays microfluidic devices for single-cell analysis have several drawbacks: some would cause cell damage due to the hydraulic forces directly acting on cells, while others could not implement biological assays since they could not immobilize cells while manipulating the reagents at the same time. In this work, we presented a two-layer pneumatic valve-based platform to implement cell immobilization and treatment on-chip simultaneously, and cells after treatment could be collected non-destructively for further analysis. Target cells could be encapsulated in sodium alginate droplets which solidified into hydrogel when reacted with Ca2+ . The size of hydrogel beads could be precisely controlled by modulating flow rates of continuous/disperse phases. While regulating fluid resistance between the main channel and passages by the integrated pneumatic valves, on-chip capture and release of hydrogel beads was implemented. As a proof of concept for on-chip single-cell treatments, we showed cellular live/dead staining based on our devices. This method would have potential in single cell manipulation for biochemical cellular assays.

TiO2 nanopillar arrays coated with gelatin film for efficient capture and undamaged release of circulating tumor cells.

Circulating tumor cells (CTCs) are important for the detection and treatment of cancer. Nevertheless, a low density of circulating tumor cells makes the capture and release of CTCs an obstacle. In this work, TiO2 nanopillar arrays coated with gelatin film were synthesized for efficient capture and undamaged release of circulating tumor cells. The scanning electron microscope and atomic force microscope images demonstrate that the substrate has a certain roughness. The interaction between the cell membrane and the nanostructure substrate contributes to the efficient capture of CTC (capture efficiency up to 94.98%). The gelatin layer has excellent biocompatibility and can be rapidly digested by matrix metalloproteinase (MMP9), which realizes the non-destructive release of CTCs (0.1 mg ml-1, 5 min, nearly 100% release efficiency, activity 100%). Therefore, by our strategy, the CTCs can be efficiently captured and released undamaged, which is important for subsequent analysis.

Macrophage membrane-coated iron oxide nanoparticles for enhanced photothermal tumor therapy.

Nanotechnology possesses the potential to revolutionize the diagnosis and treatment of tumors. The ideal nanoparticles used for in vivo cancer therapy should have long blood circulation times and active cancer targeting. Additionally, they should be harmless and invisible to the immune system. Here, we developed a biomimetic nanoplatform with the above properties for cancer therapy. Macrophage membranes were reconstructed into vesicles and then coated onto magnetic iron oxide nanoparticles (Fe3O4 NPs). Inherited from the Fe3O4 core and the macrophage membrane shell, the resulting Fe3O4@MM NPs exhibited good biocompatibility, immune evasion, cancer targeting and light-to-heat conversion capabilities. Due to the favorable in vitro and in vivo properties, biomimetic Fe3O4@MM NPs were further used for highly effective photothermal therapy of breast cancer in nude mice. Surface modification of synthetic nanomaterials with biomimetic cell membranes exemplifies a novel strategy for designing an ideal nanoplatform for translational medicine.

Highly sensitive and rapid isolation of fetal nucleated red blood cells with microbead-based selective sedimentation for non-invasive prenatal diagnostics.

Non-invasive prenatal diagnostics (NIPD) has been an emerging field for prenatal diagnosis research. Carrying the whole genome coding of the fetus, fetal nucleated red blood cells (FNRBCs) have been pursued as a surrogate biomarker traveling around in maternal blood. Here, by combining a unique microbead-based centrifugal separation and enzymatic release, we demonstrated a novel method for FNRBC isolation from the blood samples. First, the gelatin-coated silica microbeads were modified with FNRBC-specific antibody (anti-CD147) to capture the target cells in the blood samples. Then, the density difference between microbead-bound FNRBCs and normal blood cells enables the purification of FNRBCs via an improved high-density percoll-based separation. The non-invasive release of FNRBCs can then be achieved by enzymatically degrading the gelatin film on the surface of the microbeads, allowing a gentle release of the captured target cells with as high as 84% efficiency and ∼80% purity. We further applied it to isolate fetal cells from maternal peripheral blood. The released cells were analyzed by real-time polymerase chain reaction to verify their fetal origin and fluorescent in situ hybridization to detect fetal chromosome disorders. This straightforward and reliable alternative platform for FNRBC detection may have the potential for realizing facile NIPD.

Zwitterionic Surfactant Modified Acetylene Black Paste Electrode for Highly Facile and Sensitive Determination of Tetrabromobisphenol A.

A electrochemical sensor for the highly sensitive detection of tetrabromobisphenol A (TBBPA) was fabricated based on acetylene black paste electrode (ABPE) modified with 3-(N,N-Dimethylpalmitylammonio) propanesulfonate (SB3-16) in this study. The peak current of TBBPA was significantly enhanced at SB3-16/ABPE compared with unmodified electrodes. To further improve the electrochemical performance of the modified electrode, corresponding experimental parameters such as the length of hydrophobic chains of zwitterionic surfactant, the concentration of SB3-16, pH value, and accumulation time were examined. The peak currents of TBBPA were found to be linearly correlated with its concentrations in the range of 1 nM to 1 µM, with a detection limit of 0.4 nM. Besides, a possible mechanism was also discussed, and the hydrophobic interaction between TBBPA and the surfactants was suggested to take a leading role in enhancing the responses. Finally, this sensor was successfully employed to detect TBBPA in water samples.

High sensitive and selective sensing of hydrogen peroxide released from pheochromocytoma cells based on Pt-Au bimetallic nanoparticles electrodeposited on reduced graphene sheets.

In this study, a high sensitive and selective hydrogen peroxide (H2O2) sensor was successfully constructed with Pt-Au bimetallic nanoparticles (Pt-Au NPs)/reduced graphene sheets (rGSs) hybrid films. Various molar ratios of Au to Pt and different electrodeposition conditions were evaluated to control the morphology and electrocatalytic activity of the Pt-Au bimetallic nanoparticles. Upon optimal conditions, wide linear ranges from 1 µM to 1.78 mM and 1.78 mM to 16.8 mM were obtained, with a detection limit as low as 0.31 µM. Besides, due to the synergetic effects of the bimetallic NPs and rGSs, the amperometric H2O2 sensor could operate at a low potential of 0 V. Under this potential, not only common anodic interferences induced from ascorbic acid, uric acid and dopamine, but also the cathodic interference induced from endogenous O2 could be effectively avoided. Furthermore, with rat pheochromocytoma cells (PC 12) as model, the proposed sensor had been successfully used in the detection of H2O2 released from the cancer cells. This method with wide linear ranges and excellent selectivity can provide a promising alternative for H2O2 monitoring in vivo in the fields of physiology, pathology and diagnosis.

Massive fabrication of functional hepatic cancer spheroids by micropatterned GelMA hydrogel chip for drug screening.

Since hepatic cancer incidence and mortality continue to grow worldwide, it is necessary to develop the biomimetic tumor models for drug development and tumor therapeutics. Cellular spheroids as an excellent simple 3D model can bridge the gap between 2D cell culture and live tissue. In this study, we proposed a biological methacrylated gelatin (GelMA) hydrogel-based microplatform for the massive generation of hepatocellular spheroids and downstream investigation of drug resistance. Micropatterned GelMA hydrogel microwell chip (GHM-chip) with tunable array was easily achieved in standard 24-culture well plates through the micro-molding fabrication strategy. The fabricated GHM-chip induced multicellular self-assembly behavior within the defined topography and further formed spheroidal structure. By regulating cell seeding density and designing microwell size, uniform hepatic cancer spheroids with tunable diameters were obtained in a simplicity, stability and controllable manner. In addition, the screening chemotherapy study of anti-cancer drug was completed through non-destructive recovery of spheroids from the GHM-chip. Beyond that, the recovered functional spheroids have potential application value in various biomedical fields such as tumor biology, pharmacology, and tissue microengineering. Finally, the proposed GHM-chip incorporated into standard cell culture plates with easy to manufacture and operate properties, may be an efficient culture microplatform for cancer research applications.

Predictive Value of Plasma PCSK9 Levels for Degree of Atherosclerosis and Major Adverse Cardiovascular and Cerebrovascular Events in Older Adult Patients with Non-Alcoholic Fatty Liver Disease.

Purpose: This study investigated the influence of plasma proprotein convertase subtilisin/kexin 9 (PCSK9) levels on the degree of atherosclerosis and major adverse cardiovascular and cerebrovascular events (MACCE) in older adults with non-alcoholic fatty liver disease. Methods: The degree of atherosclerosis severity was assessed by the standard Gensini score quartile method. According to the degree of atherosclerosis, patients were divided into mild (0-24 points; n=84), moderate (25-53 points; n=86), and severe groups (≥54 points; n=84) and then categorized as MACCE (n=30) or non-MACCE (n=224) according to 6-month follow-up data. The patients' age, sex, smoking history, medical history, and early morning fasting venous blood, for measuring biochemical indexes, were collected. Clinical data were compared between groups and the relationship between Gensini scores and PCSK9 was evaluated. Results: Compared with the mild group, the moderate and severe groups had higher high-sensitivity C-reactive protein(hs-CRP), PCSK9, triglycerides(TG), low-density lipoprotein cholesterol (LDL-C), and lipoprotein(a)[Lp(a)] levels and lower high-density lipoprotein cholesterol(HDL-C) levels (all P<0.05). Moreover, PCSK9 positively correlated with Gensini scores (r=0.657, P<0.01). The MACCE and non-MACCE groups had significantly different ages, statin use, Gensini scores, PCSK9, and LDL-C (all P<0.05). Multi-factorial Cox risk regression analysis showed the Gensini score (HR=1.018, 95% CI: 1.006~1.029) and PCSK9 (HR=1.147, 95% CI: 1.038~1.287) were independent risk factors for MACCE. Conclusion: The Gensini score and PCSK9 levels can be used as predictive indicators for the degree of illness and occurrence of MACCE in older NAFLD patients.

Embedded bioprinted multicellular spheroids modeling pancreatic cancer bioarchitecture towards advanced drug therapy.

The desmoplastic bioarchitecture and microenvironment caused by fibroblasts have been confirmed to be closely related to the drug response behavior of pancreatic ductal adenocarcinoma (PDAC). Despite the extensive progress in developing PDAC models as in vitro drug screening platforms, developing efficient and controllable approaches for the construction of physiologically relevant models remains challenging. In the current study, multicellular spheroid models that emulate pancreatic cancer bioarchitecture and the desmoplastic microenvironment are bioengineered. An extrusion-based embedded dot bioprinting strategy was established to fabricate PDAC spheroids in a one-step process. Cell-laden hydrogel beads were directly deposited into a methacrylated gelatin (GelMA) suspension bath to generate spherical multicellular aggregates (SMAs), which further progressed into dense spheroids through in situ self assembly. By modulating the printing parameters, SMAs, even from multiple cell components, could be manipulated with tunable size and flexible location, achieving tunable spheroid patterns within the hydrogel bath with reproducible morphological features. To demonstrate the feasibility of this printing strategy, we fabricated desmoplastic PDAC spheroids by printing SMAs consisting of tumor cells and fibroblasts within the GelMA matrix bath. The produced hybrid spheroids were further exposed to different concentrations of the drug gemcitabine to verify their potential for use in cell therapy. Beyond providing a robust and facile bioprinting system that enables desmoplastic PDAC bioarchitecture bioengineering, this work introduces an approach for the scalable, flexible and rapid fabrication of cell spheroids or multi-cell-type spheroid patterns as platforms for advanced drug therapy or disease mechanism exploration.

Novel cytochrome P450s for various hydroxylation of steroids from filamentous fungi.

Hydroxylated steroids are value-added products with diverse biological activities mediated by cytochrome P450 enzymes, however, few has been thoroughly characterized in fungi. This study introduces a rapid identification strategy for filamentous fungi P450 enzymes through transcriptome and bioinformatics analysis. Five novel enzymes (CYP68J5, CYP68L10, CYP68J3, CYP68N1 and CYP68N3) were identified and characterized in Saccharomyces cerevisiae or Aspergillus oryzae. Molecular docking and dynamics simulations were employed to elucidate hydroxylation preferences of CYP68J5 (11α, 7α bihydroxylase) and CYP68N1 (11α hydroxylase). Additionally, redox partners (cytochrome P450 reductase and cytochrome b5) and ABC transporter were co-expressed with CYP68N1 to enhance 11α-OH-androstenedione (11α-OH-4AD) production. The engineered cell factory, co-expressing CPR1 and CYP68N1, achieved a significant increase of 11α-OH-4AD production, reaching 0.845 g·L-1, which increased by 14 times compared to the original strain. This study provides a comprehensive approach for identifying and implementing novel cytochrome P450 enzymes, paving the way for sustainable production of steroidal products.

Tracking the disulfide rearrangement of heated lactoglobulin by matrix-assisted laser desorption/ionization-in-source decay top-down analysis.

Disulfide bond rearrangement is a common occurrence during protein analysis or treatment. A convenient and rapid method has been developed to investigate heat-induced disulfide rearrangement of lactoglobulin using matrix-assisted laser desorption/ionization-in-source decay (MALDI-ISD) technology. By analyzing heated lactoglobulin in reflectron and linear mode, we demonstrated that cysteines C66 and C160 exist as free residues other than linked ones in some protein isomers. This method provides a straightforward and expeditious way to assess the cysteine status and structural changes of proteins under heat stress.