Protein Fluorescence Research Articles

Human Hemoglobin and Antipsychotics Clozapine, Ziprasidone and Sertindole: Friends or Foes?

Packed with hemoglobin, an essential protein for oxygen transport, human erythrocytes are a suitable model system for testing the pleiotropic effects of lipophilic drugs. Our study investigated the interaction between antipsychotic drugs clozapine, ziprasidone, sertindole, and human hemoglobin under simulated physiological conditions. Analysis of protein fluorescence quenching at different temperatures and data obtained from the van't Hoff diagram and molecular docking indicate that the interactions are static and that the tetrameric human hemoglobin has one binding site for all drugs in the central cavity near αβ interfaces and is dominantly mediated through hydrophobic forces. The association constants were lower-moderate strength (~104 M-1), the highest observed for clozapine (2.2 × 104 M-1 at 25 °C). The clozapine binding showed "friendly" effects: increased α-helical content, a higher melting point, and protein protection from free radical-mediated oxidation. On the other hand, bound ziprasidone and sertindole had a slightly pro-oxidative effect, increasing ferrihemoglobin content, a possible "foe". Since the interaction of proteins with drugs plays a vital role in their pharmacokinetic and pharmacodynamic properties, the physiological significance of the obtained findings is briefly discussed.

Separation and colorimetric detection of Escherichia coli by phage tail fiber protein combined with nano-magnetic beads.

A colorimetric detection method for Escherichia coli(E. coli)in water was established based on a T7 phage tail fiber protein-magnetic separation. Firstly, the tail fiber protein (TFP) was expressed and purified to specifically recognize E. coli, which was verified by using fusion protein GFP-tagged TFP (GFP-TFP) and fluorescence microscopy. Then TFP conjugated with magnetic beads were applied to capture and separate E. coli. The TFP was covalently immobilized on the surface of magnetic beads and captured E. coliasverified by scanning electron microscopy (SEM). Finally, polymyxin B was used to lyse E. coli in solution and the released intracellular β-galactosidase (β-gal) could hydrolyze the colorimetric substrate chlorophenol red-β-D-galactopyranoside (CPRG), causing color changefrom yellow to purple. The high capture efficiencies of E. coli ranged from 88.70% to 95.65% and E. coli could be detected at a concentration of 102CFU/mL by naked eyes. The specificity of the chromogenic substrate was evaluated using five different pathogen strains as competitors and tests withfour kinds of real water samples showed recoveries of86.00% to 92.25%. The colorimetric changes determined by visual inspection can be developed as an efficient platform for point-of-care detection of E. coli in resource-limited regions.

Energy dissipation efficiency in the CP43 assembly intermediate complex of photosystem II

Photosystem II in oxygenic organisms is a large membrane bound rapidly turning over pigment protein complex. During its biogenesis, multiple assembly intermediates are formed, including the CP43-preassembly complex (pCP43). To understand the energy transfer dynamics in pCP43, we first engineered a His-tagged version of the CP43 in a CP47-less strain of the cyanobacterium Synechocystis 6803. Isolated pCP43 from this engineered strain was subjected to advanced spectroscopic analysis to evaluate its excitation energy dissipation characteristics. These included measurements of steady-state absorption and fluorescence emission spectra for which correlation was tested with Stepanov relation. Comparison of fluorescence excitation and absorptance spectra determined that efficiency of energy transfer from β-carotene to chlorophyll a is 39 %. Time-resolved fluorescence images of pCP43-bound Chl a were recorded on streak camera, and fluorescence decay dynamics were evaluated with global fitting. These demonstrated that the decay kinetics strongly depends on temperature and buffer used to disperse the protein sample and fluorescence decay lifetime was estimated in 3.2–5.7 ns time range, depending on conditions. The pCP43 complex was also investigated with femtosecond and nanosecond time-resolved absorption spectroscopy upon excitation of Chl a and β-carotene to reveal pathways of singlet excitation relaxation/decay, Chl a triplet dynamics and Chl a → β-carotene triplet state sensitization process. The latter demonstrated that Chl a triplet in the pCP43 complex is not efficiently quenched by carotenoids. Finally, detailed kinetic analysis of the rise of the population of β-carotene triplets determined that the time constant of the carotenoid triplet sensitization is 40 ns.

Interactions of Different Urolithins With Bovine Serum Albumin

Backgound/Objectives Urolithins (UROs) are the metabolites derived from the gut microbial action on ellagitannins and ellagic acid-rich foods. Following their absorption in the intestine, UROs are transported through the systemic circulation to various tissues where they can express their biological function as antimicrobial, anti-inflammatory, and anticancer agents. In addition to blood plasma, where they can be found as glucuronide and sulfate conjugates, they are also found in urine. Therefore, the interactions of UROs with serum proteins are of great clinical interest. Methods A powerful technique for examining these urolithin-serum protein interactions is fluorescence spectroscopy. Bovine serum albumin (BSA) is a particularly suitable model protein because it is readily available, affordable, and similar to human serum albumin. This work aimed to study the binding of UROs (urolithin A, UROA and urolithin B, UROB) and their glucuronide conjugates (UROAG and UROBG) to BSA by quenching the intrinsic fluorescence of protein. Results The spectra obtained showed that the binding process is influenced by the polyphenol's structure and the conjugation process with the glucuronide. The calculated Stern Vollmer binding constants (Ksv): UROA and UROB Ksv were 59236 ± 5706 and 69653 ± 14922, respectively, while for UROAG and UROBG, these values were 15179 ± 2770 and 9462 ± 1955, respectively, which showed that the binding affinity decreased with glucuronidation. Molecular docking studies confirmed that all of the studied molecules will bind favorably to BSA. The preferential binding site for both UROs and UROGs is Sudlow I, while UROs will also bind to Sudlow II. URO-Gs can bind to BSA in the cleft region with lower binding scores than for the Sudlow I binding site. Conclusion The aglycone's higher hydrophobicity increases the binding affinity to BSA, thus reducing its bioavailability in the blood.

Interaction between STK33 and autophagy promoted renal cell carcinoma metastasis by regulating mTOR/ULK1 signaling pathway

The roles of STK33 in renal cell carcinoma (RCC) remain unclear. This study was designed to investigate the interaction between STK33 and the autophagy in the RCC. STK33 was knocked down in 786-O and CAKI-1 cells. Then CCK8, clony formation assay, wound healing assay and Transwell assay were performed to analyze the proliferation, migration and invasion of the cancer cells. In addition, the activation of autophagy was determined using fluorescence, followed by investigating the potential signaling pathways in this process. After STK33 knockdown, the proliferation and migration of cell lines were inhibited, and the apoptosis of renal cancer cells was promoted. Autophagy fluorescence experiment showed that after STK33 knockdown, green LC3 protein fluorescence particles could be seen in the cells. Western blot analysis showed that after STK33 knockdown, there was significant down-regulation in P62 and p-mTOR, as well as significant up-regulation of Beclin1, LC3 and p-ULK1. STK33 affected autophagy in RCC cells by activating mTOR/ ULK1pathway.

Abstract 2389: In vivo multiphoton autofluorescence imaging is sensitive to CD8 T cell and tumor cell metabolic changes during immunotherapy in a murine melanoma model

Abstract Introduction: In vivo multiphoton autofluorescence microscopy provides live, label free, single cell imaging of metabolic changes. These metabolic changes are quantified via the metabolic coenzymes NAD(P)H and FAD which are autofluorescent molecules endogenous to all cells. Metabolic reprogramming of tumor and immune cells is closely associated with cancer progression and cell phenotype. We aim to study metabolic changes during administration of an effective, triple-combination immunotherapy within murine melanoma tumors. This therapy includes external beam radiation, intratumoral hu14.18-IL2 immunocytokine (anti-GD2 mAb fused to IL2, provided by Anyxis Immuno-Oncology GmbH of Vienna, Austria), and intraperitoneal anti-CTLA-4 leading to in situ vaccination and cure of GD2+ murine tumors. Previous work has shown that a T cell response is critical to the efficacy of this therapy, so we created an mCherry-labeled T cell mouse model to study this response. Methods: We implanted syngeneic B78 (GD2+) melanoma cells into the flanks of mCherry-labeled CD8 T cell reporter mice (C57Bl/6 background) to induce tumors. Under anesthesia, skin flap surgery was performed and tumors were imaged at several time points during therapy. Multiphoton imaging was performed to collect NAD(P)H, FAD, mCherry, and collagen signal with a 40X objective. Fluorescence lifetime data was collected using time correlated single photon counting electronics. Tissues were harvested and analyzed via flow cytometry and multiplex immunofluorescence to corroborate intravital imaging findings and characterize the immune infiltrate. Results: Here we demonstrate that our in vivo imaging is sensitive to metabolic changes in both B78 melanoma and CD8 T cells from immunotherapy treated versus control tumors. These metabolic differences include changes in protein binding, redox balance, and fluorescence lifetime. We also show remodeling of collagen, a major component of the extracellular matrix, during immunotherapy that may be explained by an observed decrease in macrophages. Flow cytometry and multiplex immunofluorescence illustrate changes in the immune infiltrate composition, activation, cytotoxicity, and spatial distribution during therapy. We are also currently investigating metabolic changes in an MC38 mouse model - a hot tumor model - that we anticipate will provide excellent metabolic contrast to our B78 immunologically cold tumor model. Conclusions: These results show that in vivo metabolic imaging enables single cell quantification of metabolic changes in tumor and immune cells during therapy. Combined with other traditional assays, we can elucidate key immune cell populations and the crucial timepoints during therapy where changes are occurring. With continued efforts, this imaging platform may be leveraged to develop new combinations of immunotherapies. Citation Format: Alexa R. Heaton, Anna Hoefges, Peter R. Rehani, Nathaniel J. Burkard, Arika S. Feils, Dan V. Spiegelman, Noah W. Tsarovsky, Alina A. Hampton, Amy K. Erbe Gurel, Alexander L. Rakhmilevich, Paul M. Sondel, Melissa C. Skala. In vivo multiphoton autofluorescence imaging is sensitive to CD8 T cell and tumor cell metabolic changes during immunotherapy in a murine melanoma model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2389.

Fluorescent visualization and evaluation of NPC1L1-mediated vesicular endocytosis during intestinal cholesterol absorption in mice

Abstract Excessive cholesterol absorption from intestinal lumen contributes to the pathogenesis of hypercholesterolemia, which is an independent risk factor for atherosclerotic cardiovascular disease. Niemann-Pick C1-like 1 (NPC1L1) is a major membrane protein responsible for cholesterol absorption, in which the physiological role of vesicular endocytosis is still controversial, and it lacks a feasible tool to visualize and evaluate the endocytosis of NPC1L1 vesicles in vivo. Here, we genetically labeled endogenous NPC1L1 protein with EGFP in a knock-in mouse model, and demonstrated fluorescent visualization and evaluation of the endocytic vesicles of NPC1L1-cago during intestinal cholesterol absorption. The homozygous NPC1L1-EGFP mice have normal NPC1L1 expression pattern as well as cholesterol homeostasis on chow or high-cholesterol diets. The fluorescence of NPC1L1-EGFP fusion protein localizes at the brush border membrane of small intestine, and EGFP-positive vesicles is visualized beneath the membrane as early as 5 min post oral gavage of cholesterol. Of note, the vesicles colocalize with the early endosomal marker early endosome antigen 1 (EEA1) and the filipin-stained free cholesterol. Pretreatment with NPC1L1 inhibitor ezetimibe inhibits the formation of these cholesterol-induced endocytic vesicles. Our data support the notion that NPC1L1-mediated cholesterol absorption is a vesicular endocytic process. NPC1L1-EGFP mice are a useful model for visualizing cellular NPC1L1-cargo vesicle itineraries and for evaluating NPC1L1 activity in vivo in response to diverse pharmacological agents and nutrients.

Using AlphaFold to predict the impact of single mutations on protein stability and function.

AlphaFold changed the field of structural biology by achieving three-dimensional (3D) structure prediction from protein sequence at experimental quality. The astounding success even led to claims that the protein folding problem is "solved". However, protein folding problem is more than just structure prediction from sequence. Presently, it is unknown if the AlphaFold-triggered revolution could help to solve other problems related to protein folding. Here we assay the ability of AlphaFold to predict the impact of single mutations on protein stability (ΔΔG) and function. To study the question we extracted the pLDDT and <pLDDT> metrics from AlphaFold predictions before and after single mutation in a protein and correlated the predicted change with the experimentally known ΔΔG values. Additionally, we correlated the same AlphaFold pLDDT metrics with the impact of a single mutation on structure using a large scale dataset of single mutations in GFP with the experimentally assayed levels of fluorescence. We found a very weak or no correlation between AlphaFold output metrics and change of protein stability or fluorescence. Our results imply that AlphaFold may not be immediately applied to other problems or applications in protein folding.

Outer Membrane Integrity-Dependent Fluorescence of the Japanese Eel UnaG Protein in Live Escherichia coli Cells.

Reporter genes are important tools in many biological disciplines. The discovery of novel reporter genes is relatively rare. However, known reporter genes are constantly applied to novel applications. This study reports the performance of the bilirubin-dependent fluorescent protein UnaG from the Japanese eel Anguilla japonicas in live Escherichia coli cells in response to the disruption of outer membrane (OM) integrity at low bilirubin (BR) concentrations. Using the E. coli wild-type strain MC4100, its isogenic OM-deficient mutant strain NR698, and different OM-active compounds, we show that BR uptake and UnaG fluorescence depend on a leaky OM at concentrations of 10 µM BR and below, while fluorescence is mostly OM integrity-independent at concentrations above 50 µM BR. We suggest that these properties of the UnaG-BR couple might be applied as a biosensor as an alternative to the OM integrity assays currently in use.

Interaction mechanisms of the binding of polychlorinated biphenyls to thyroid hormone transporters revealed based on quantum chemistry and spectroscopy

Polychlorinated biphenyls (PCBs) are a kind of persistent, bioaccumulative and refractory environmental pollutants. In this work, three typical PCBs (CB54, CB125, CB152) were selected to investigate the interaction between thyroid hormone (TH) transporter and PCBs by combining spectroscopy and calculation. The fluorescence results showed that PCBs had a high binding affinity to TH transport proteins and strongly quenched the intrinsic fluorescence of TH transport proteins through a static quenching mechanism. Analysis of thermodynamic parameters revealed that hydrophobic interactions played an important role in the complex formation. Furthermore, the microenvironment and conformation of transthyretin (TTR) and thyroxine binding globulin (TBG) were altered in the presence of PCBs, which was confirmed by Fourier transform infrared (FT-IR) spectroscopy and quantum chemical analysis. Finally, the optimal binding mode of PCBs to TH transport proteins and the stability of the complexes were studied through molecular docking and molecular dynamics simulations.

Formation mechanism of salted egg yolk mudding during storage: Protein oxidation, gel structure, and conformation

The aim of this study was to investigate the formation mechanism of salted egg yolk (SEY) mudding during storage. Results showed that the soluble protein, hardness, and intrinsic fluorescence intensity of SEY decreased significantly during storage, while total volatile basic nitrogen, sulfhydryl group, dityrosine, adhesiveness, and surface hydrophobicity increased significantly, and the intrinsic fluorescence peak position red-shifted at first and then blue-shifted. In addition, from the results of infrared and microstructure analyses, there was an obvious oxidation reaction between protein and lipid in the late storage stage; the structure of SEY was destroyed, many random coils were formed, and the degree of protein–lipid binding and the crystallinity of SEY protein decreased during storage. Finally, the heatmap analysis revealed that the protein and lipid oxidation and conformational changes might be the main reasons for SEY mudding. This study can provide theoretical guidance for the control of SEY mudding.

Abstract WMP101: Plasma Proteomic Determinants Of White Matter Hyperintensities And Covert Brain Infarction In The Cardiovascular Health Study

Background: Identification of novel biomarkers of cerebral small vessel disease is critical to elucidate pathophysiology and guide therapeutic development for prevention. We evaluated plasma proteomic associations of clinically asymptomatic, covert brain infarction (cBI) and white matter hyperintensity (WMH) burden on cranial magnetic resonance imaging (MRI) in the population-based Cardiovascular Health Study (CHS). Methods: Eligible CHS participants underwent one or two cranial MRI scans (~5 years apart) and aptamer-based SOMAScan platform measurement of up to 7288 plasma proteins concurrent with the initial MRI. cBI was defined by MRI signal characteristics and size ≥ 3 mm, and WMH burden was graded (WMG) 0 (least) to 9 (most severe). The relationship between 1-standard deviation increase in each log 2 -normalized plasma protein fluorescence was modeled separately with incident cBI and WMG worsening (≥1 grade increase) using multivariable relative risk regression, adjusting for demographics, estimated glomerular filtration rate (eGFR), and stroke risk factors. In secondary analyses, multivariable linear regression evaluated the cross-sectional relationship between each plasma protein with number of cBI and WMG on initial MRI. Bonferroni correction, based on the number of principal components that explained 99% of the protein fluorescence variance in CHS participants, accounted for multiple comparisons (p&lt;2.1x10 -5 (0.05/2377)). Results: For eligible participants (n=2615), mean age was 74.4 ± 4.9 years, 61.7% were women, and 16.4% were Black. After adjustment for demographics, eGFR, and stroke risk factors, multiple circulating proteins were associated with cBI and WMG (Table). Conclusion: Multiple plasma proteins - implicated in systemic inflammation, fatty acid metabolism, neuronal adhesion/migration, and cellular cytoskeletal integrity/remodeling - were independently associated with cBI and WMG in a cohort of older adults.

The effect of isovitexin on lipopolysaccharide-induced renal injury and inflammation by induction of protective autophagy.

Chronic kidney disease (CKD) is a systemic inflammatory syndrome that includes tubulointerstitial inflammation. Lipopolysaccharide (LPS), the outer membrane of Gram-negative bacteria, can increase reactive oxygen species production (ROS) that triggers cell inflammation. Isovitexin (IV) is a flavone that has the potential for anticancer, antioxidant, and anti-inflammatory. This study aimed to hypothesize that IV inhibited LPS-induced renal injury in vitro and in vivo. In vitro study, IV prevented LPS-induced ROS production and increased cell viability on SV40-MES-13cells. Additionally, IV ameliorated mitochondrial membrane potential, downregulated inflammation and pyroptosis factors on LPS treatment. We found that LPS treatment reduced the expression of autophagy, however, this effect was reversed by IV. In vivo study, the renal injury model in C57BL/6 mice cotreatment with IV was examined. In addition, IV decreased LPS-induced glomerular atrophy and reduced inflammation-related cytokines releases. Further showed that IV could significantly reduce LPS-induced inflammation and pyroptosis factors in mice. Under the immunostaining, increased fluorescence of LC3 autophagy-related protein was recovered by IV. In summary, IV ameliorated renal injury, inflammation and increased protected autophagy by anti-ROS production, anti-inflammation, and anti-pyroptosis. In the future, the safety of isovitexin as a novel perspective for CKD patients should be evaluated in further clinical studies.

Monitoring oligomeric forms of amyloid beta in the early stages of aggregation using hybrid X-ray footprinting mass spectrometry with inline fluorescence.

The aggregation-prone Amyloid Beta (Aβ) peptide has been the target of the majority of efforts to develop Alzheimer's Disease therapeutics, most of which have failed or have shown mixed results. It is no longer widely believed that buildup of amyloid fibrils is a causal factor in cognitive decline associated with the disease, and a growing body of evidence now suggests that low molecular weight oligomeric forms of Aβ may be the culprits in disease progression. However, while numerous Aβ fibril structures have been characterized, primarily by NMR and cryo-EM, obtaining structural information on oligomeric forms of Aβ that presumably precede and/or seed fibril formation has proved challenging. These transient forms are heterogeneous, and depend heavily on experimental conditions such as buffer, temperature, concentration, and degree of quiescence during measurement. Here, we present a new approach to delineating structural features of early-stage low molecular weight Aβ oligomers using a solvent accessibility assay in conjunction with inline fluorescence measurements. In this method, inline fluorescence is collected nearly simultaneously from samples during collection of X-ray footprinting mass spectrometry (XFMS) data. In the XFMS method, X-ray irradiation dissociates solvent water to produce hydroxyl radicals, which covalently modify solvent accessible side chains. Liquid chromatography-mass spectrometry is subsequently used to analyze the covalent modifications produced, and the data provide a “water map” at the single residue level, which is used to determine protein structure. This new hybrid approach circumvents issues in batch-to-batch variability of Aβ aggregation which has made this peptide so difficult to study, and provides a method to combine residue-specific structural information nearly simultaneously with global protein structure readouts from either intrinsic protein fluorescence or fluorophores such as Thioflavin T.

Kinetics of Ca2+ Dissociation from Cod Parvalbumin Studied by Fluorescent Stopped-flow Method.

Small Ca2+-binding protein parvalbumin possesses two strong Ca2+/Mg2+- binding sites located within two EF-hand domains. Most parvalbumins have no tryptophan residues, while cod protein contains a single tryptophan residue, which fluorescence (spectrum maximum position and fluorescence quantum yield) is highly sensitive to the Ca2+ association/dissociation. Intrinsic protein fluorescence of cod parvalbumin can be used for elucidating the mechanism of Ca2+ binding to this protein. Fluorescence of the single tryptophan residue of cod parvalbumin has been used to monitor Ca2+-induced changes in the protein, both in steady-state and kinetic mode. Steady-state fluorescence spectra of cod parvalbumin were measured using Cary Eclipse spectrofluorimeter. Stopped-flow accessories in combination with a novel high-speed spectrofluorimeter were used for measurements of kinetics of Ca2+ dissociation from cod parvalbumin after fast mixing of Ca2+-loaded protein with a chelator of divalent metal cations ethylenediaminetetraacetic acid (EDTA). The fluorescent phase plots (fluorescence intensity at a fixed wavelength plotted against a fluorescence intensity at another fixed wavelength), constructed from steady state and kinetical data, shows a break at [Ca2+]/[parvalbumin] ratio close to 1. This means that the transition passes through an intermediate state, which is a protein with one bound calcium ion. These observations indicate that the binding of Ca2+ to cod parvalbumin is sequential. The results of the present spectral study showed that the binding of Ca2+ to cod parvalbumin is a sequential process. Calcium dissociation rate constants for the two binding sites of cod parvalbumin evaluated from the kinetic data are koff1 = 1.0 s-1 and koff2 = 1.5 s-1.

A generic rotamer model to explain the temperature dependence of BSA protein fluorescence

AbstractProtein fluorescence signals essential information about the conformational dynamics of proteins. Different types of intrinsic fluorophores reflect different protein local or global structural changes. Bovine Serum Albumin (BSA) is a transport protein that contains two intrinsic fluorophores: Tryptophan134 (Trp134) and Tryptophan213 (Trp213). This protein displays an interesting temperature dependence of the tryptophan fluorescence. However, the molecular mechanism of the temperature dependence is still unclear. In this work, we propose a generic rotamer model to explain this phenomenon. The model assumes the presence of rotamer‐specific fluorescence lifetimes. The fluorescence temperature dependence is caused by the population shifts between different rotamers due to thermal effects. As a proof of concept, we show that the tryptophan's two fluorescence lifetimes (𝜏1 = 0.4–0.5 ns and 𝜏2 = 2‐4 ns) are sufficient to qualitatively explain the fluorescence intensity change at different temperatures, both in buffer solution (water) and in the protein. To computationally verify our rotamer hypothesis, we use an all‐atom molecular dynamics simulation to study the effects of temperature on the two tryptophans' rotamer dynamics. The simulations show that Trp134 is more sensitive to temperature, consistent with experimental observations. Overall, the results support that the temperature dependence of fluorescence in the protein BSA is due to local conformational changes at the residue level. This work sheds light on the relationship between tryptophan's rotamer dynamics and its ability to fluorescence.

Comprehensive physicochemical characterization of a peptide-based medicine: Teduglutide (Revestive®) structural description and stress testing

Teduglutide (Revestive®) is a glucagon-like peptide-2 analogue used for the treatment of short bowel syndrome, a rare life-threatening condition in which the amount of functional gut is too short to enable proper absorption of nutrients and fluids. During handling prior to administration to the patient in hospital, it is possible that peptide-based medicines may be exposed to environmental stress conditions that could affect their quality. It is therefore essential to carry out stress testing studies to evaluate how such medicines respond to these stresses. For this reason, in this paper we present a strategy for a comprehensive analytical characterization of a peptide and a stress testing study in which it was subjected to various stress conditions: heating at 40 °C and 60 °C, light exposure and shaking. Several complementary analytical techniques were used throughout this study: Far UV circular dichroism, intrinsic protein fluorescence spectroscopy, dynamic light scattering, size-exclusion chromatography and intact and peptide mapping reverse-phase chromatography coupled to mass spectrometry. To the best of our knowledge, this is the first study to offer an in-depth description of the chemical structure of teduglutide peptide and its physicochemical characteristics after stress stimuli were applied to the reconstituted medicine Revestive®.

Cigarette smoke extract-induced inflammatory response via inhibition of the TFEB-mediated autophagy in NR8383 cells

Objective: Chronic pulmonary inflammation caused by long-term smoking is the core pathology of COPD. Alveolar macrophages (AMs) are involved in the pulmonary inflammation of COPD. The accumulation of damaged materials caused by impaired autophagy triggers inflammatory response in macrophages. As a key transcription regulator, transcription factor EB (TFEB) activates the transcription of target genes related autophagy and lysosome by binding to promoters, whereas it is unclarified for the relationship between inflammatory response induced by cigarette smoke extract (CSE) and TFEB-mediated autophagy. Thus, we investigated the role of TFEB-mediated autophagy in inflammatory response induced by CSE in NR8383 cells, and to explore its potential mechanism. Methods: Based on cell viability and autophagy, cells treated with 20% concentration of CSE for 24 h were selected for further studies. Cells were divided into control group, chloroquine (CQ, the autophagy inhibitor) group, CSE group, CSE + rapamycin (the autophagy inducer) group and CSE + fisetin (the TFEB inducer) group. The levels of tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β), and IL-6 in supernatant were detected by ELISA kits. The protein expressions were tested by western blot. The intensity of fluorescence of Lysosome-associated membrane protein 1 (LAMP1) and TFEB was detected by immunofluorescence. Lyso-Tracker Red staining was applied to detect the lysosome environment. Results: CSE inhibited the cell viability, increased the contents of TNF-α, IL-1β, IL-6, the ratio of LC3II/I, and the level of P62 protein. Besides, CSE decreased the fluorescence intensity of LAMP1 protein and Lyso-Tracker Red staining, as well as the ratio of nucleus/cytosol of TFEB protein. Activating autophagy with rapamycin alleviated CSE-induced inflammatory response. The activation of TFEB via fisetin alleviated CSE-induced autophagy impairment and lysosomal dysfunction, thus alleviated inflammatory response in NR8383 cells. Conclusion: CSE-induced inflammatory response in NR8383 cells, which may be related to the inhibition of TFEB-mediated autophagy.

Exploring the In‐Vitro Antibacterial Activity and Protein (Human Serum Albumin, Human Hemoglobin and Lysozyme) Interaction of Hexagonal Silver Nanoparticle Obtained from Wood Extract of Wild Cherry Shrub

AbstractHere, the silver nanoparticles were synthesized using aqueous wood extract of cerasus macrocarpa (wild Cherry) shrub. This green synthesis method was both eco‐friendly and straightforward. The nanoparticles were characterized using FT‐IR, zeta potential, ultraviolet‐visible, TEM, and SEM‐EDX analysis. Green synthesized AgNPs demonstrated effective antibacterial activity against gram‐positive and gram‐negative bacteria (S. aureus and E. coli). Furthermore, fluorometric, circular dichroism and UV‐vis were used to study the interaction of green synthesized AgNPs with Human serum albumin (HSA (, Human hemoglobin (HHb), and Lysozyme (Lys) in simulated physiological conditions. The studies of interaction with HSA, HHb and Lys show good binding affinity for green synthesized silver nanoparticles; the binding affinity was found in the order Lys&gt;HSA&gt;HHb at 298.15 K. The results show that the native conformation of HSA, HHb, and Lys was preserved at the secondary structure level, which is crucial in providing insights into the side effects of newly synthesized drugs on their carriers. The fluorescence data represented that green synthesized AgNPs quench the intrinsic fluorescence of proteins through a static quenching procedure.

Excessive ultraviolet C irradiation causes spore protein denaturation and prohibits the initiation of spore germination in Bacillus subtilis.

Ultraviolet (UV) -C is widely used to kill bacteria as it damages chromosomal DNA. We analyzed the denaturation of the protein function of Bacillus subtilis spores after UV-C irradiation. Almost all of the B. subtilis spores germinated in Luria-Bertani (LB) liquid medium, but the colony-forming unit (CFU) of the spores on LB agar plates decreased to approximately 1/103 by 100 mJ/cm2 of UV-C irradiation. Some of the spores germinated in LB liquid medium under phase-contrast microscopy, but almost no colonies formed on the LB agar plates after 1 J/cm2 of UV-C irradiation. The fluorescence of the green fluorescent protein (GFP) -fused spore proteins, YeeK-GFP, YeeK is a coat protein, decreased following UV-C irradiation of over 1 J/cm2, while that of SspA-GFP, SspA is a core protein, decreased following UV-C irradiation of over 2 J/ cm2, respectively. These results revealed that UV-C affected on coat proteins more than core proteins. We conclude that 25 to 100 mJ/cm2 of UV-C irradiation can cause DNA damage, and more than 1 J/cm2 of UV-C irradiation can cause the denaturation of spore proteins involved in germination. Our study would contribute to improve the technology to detect the bacterial spores, especially after UV sterilization.