Low Molecular Weight Proteins Research Articles

Trypsin Encapsulation in the Zeolitic Imidazolate Framework for Low-Molecular Weight Protein Analysis with High Selectivity and Efficiency.

Low-molecular weight proteins (LWPs) are important sources of biological information in biomarkers, signaling molecules, and pathology. However, the separation and analysis of LWPs in complex biological samples are challenging, mainly due to their low abundance and the complex sample pretreatment procedure. Herein, trypsin modified by poly(acrylic acid) (PAA) was encapsulated by a zeolitic imidazolate framework (ZIF-L). Mesopores were formed on the ZIF-L with the introduction of PAA. An alternative strategy for separation and pretreatment of LWPs was developed based on the prepared ZIF-L-encapsulated trypsin with adjustable pore size. The mesoporous structure of the prepared materials selectively excluded high-molecular weight proteins from the reaction system, allowing LWPs to enter the pores and react with the internal trypsin, resulting in an improved separation efficiency. The hydrophobicity of the ZIF-L simplified the digestion process by inducing significant structural changes in substrate proteins. In addition, the enzymatic activity was significantly enhanced by the developed encapsulation method that maintained the enzyme conformation, allowed low mass transfer resistance, and possessed a high enzyme-to-substrate ratio. As a result, the ZIF-L-encapsulated trypsin can achieve highly selective separation, valid denaturation, and efficient digestion of LWPs in a short time by simply mixing with substrate proteins, greatly simplifying the separation and pretreatment process of the traditional hydrolysis method. The prepared materials and the developed strategy demonstrated an excellent size-selective assay performance in model protein mixtures, showing great potential in the application of proteomics analysis.

Small peptide CSF fingerprint of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by abnormal protein aggregation in the motor neurons. Present and earlier proteomic studies to characterize peptides in cerebrospinal fluid (CSF) associated with motoneuron pathology did not target low molecular weight proteins and peptides. We hypothesized that specific changes in CSF peptides or low molecular weight proteins are significantly altered in ALS, and that these changes may support deciphering molecular pathophysiology and even guide approaches towards therapeutic interventions. Cerebrospinal fluid (CSF) from 50 ALS patients and 50 non-ALS controls was collected, centrifuged immediately after collection, aliquoted into polypropylene test tubes, frozen within 30-40 min after the puncture, and stored at -80°C until use. Peptides were sequenced using capillary electrophoresis or liquid chromatography/mass spectrometry (CE-MS/MS or LC-MS/MS). In the CSF of 50 patients and 50 non-ALS controls 33 peptides were found, of which 14 could be sequenced using a non-lytic single-pot proteomic detection method, CE/MS. ALS deregulated peptides vs. controls included Integral membrane protein 2B, Neurosecretory protein VGF, Osteopontin, Neuroendocrine protein 7B2 (Secretogranin-V), EGF-containing fibulin-like extracellular matrix protein 1, Xylosyltransferase 1 XT-1, Chromogranin-A, Superoxide dismutase SOD-1, Secretogranin-1 (Chromogranin B), NR2F2 Nuclear Receptor Subfamily 2 Group F Member 2 and Collagen alpha-1(VII) chain. Most striking deregulations in CSF from ALS patients were found in VGF, Osteopontin, SOD-1 and EFEMP1 peptides. No associations of disease severity, duration and region of onset with sequenced peptides were found.

Bio-adhesives derived from sewage sludge via hydrothermal carbonization: Influence of aqueous phase recycling

This paper describes the preparation of formaldehyde-free plywood bio-adhesive using hydrochar from sewage sludge in the presence (and absence) of aqueous phase containing a high content of inorganic matter. Aqueous phase recycling was observed to enhance the removal of low molecular weight proteins and the retention of large molecular weight proteins in hydrochar. The hydrochar-based bio-adhesive, incorporating the aqueous phase, exhibits excellent wet shear strength, meeting the Chinese national standard GB/T 9846-2015 (≥0.7 MPa). The wet shear strength of the bio-adhesive with tap water surpasses that of the bio-adhesive with the aqueous phase. During hot-pressing, both low molecular weight proteins in the aqueous phase and large molecular weight proteins in hydrochar participate in existing reactions including those between inter-components in bio-adhesives and bio-adhesives/plywood. The presence of low molecular weight proteins in the aqueous phase negatively affects wet shear strength and water resistance. The formation of covalent bonds, such as C–N and C–O–C, emerges as the key factor to enhance the wet shear strength of bio-adhesives. This study presents a novel approach for the valorization of sewage sludge, particularly the aqueous phase.

Сytokines as promising diagnostic and prognostic biomarkers of microvascular complications of diabetes

Classical knowledge about the pathogenesis of diabetes and its complications in recent years has been supplemented by ideas about the role of chronic inflammation. It has been established that inflammatory reactions play a role in the beta cell dysfunction, the formation of insulin resistance and remodeling of the vascular wall. Cytokines, soluble low molecular weight proteins and peptides that perform informational and regulatory functions, play central role in the development of inflammation. A wide range of biological activity and involvement in many aspects of pathogenesis make it possible to consider cytokines as promising molecules for diagnosing and predicting the complications. In this review, we summarize data from studies that assessed the role of cytokines as diagnostic and prognostic markers for the development of microvascular diabetic complications. Current data indicate that angiogenic and pro-inflammatory cytokines (VEGF, TNF-α, IL-6, IL-8, IL-15, IL-17, MCP-1, IP-10, INF-γ, PEDF, etc.) are promising biomarkers for proliferative diabetic retinopathy, especially when their local production is assessed (in vitreous, aqueous humor and tears). The role of these molecules as biomarkers of non-proliferative diabetic retinopathy and diabetic macular edema needs further research. Serum proinflammatory and fibrogenic cytokines (primarily MCP-1, IL-6, TNF-α, YKL-40, TGF-β and bFGF) and cytokine receptors (sTNFR1, sTNFR2) are considered as promising diagnostic and prognostic markers of diabetic kidney disease. Urinary excretion of IL-6 and MCP-1 turned out to be a predictor of the progression of diabetic nephropathy. Multi-bead assay and mass spectrometry make it possible to study cytokine panels in small samples of biological material. Combined biomarkers, including several cytokines, may increase the reliability of the prognosis of diabetic complications.

Effects of Grinding Methods of Tartary Buckwheat Leaf Powder on the Characteristics and Micromorphology of Wheat Dough.

The functional components in tartary buckwheat leaf powder can give flour products higher nutritional value. To comprehensively realize the high-value utilization of tartary buckwheat and its by-products, electric stone mill powder (EMP), ultra-fine mill powder (UMP), steel mill powder (SMP), and grain mill powder (GMP) from tartary buckwheat leaves were used in the preparation of wheat dough, and this was used to explore their effects on dough properties and protein microstructure. With an increase in tartary buckwheat leaf powder, the hydration characteristics, protein weakening rate, and starch gelatinization characteristics of the dough changed, and the water holding capacity and swelling capacity decreased. The retrogradation value increased, which could prolong the shelf life of related products. The water solubility of the dough showed an upward trend and was the lowest at 10% UMP. The addition of UMP produced a more uniform dough stability time and the lowest degree of protein weakening, which made the dough more resistant to kneading. An increasing amount of tartary buckwheat leaf powder augmented the free sulfhydryl content of the dough and decreased the disulfide bond content. The disulfide bond content of the dough containing UMP was higher than that of the other doughs, and the stability of the dough was better. The peaks of the infrared spectrum of the dough changed after adding 10% UMP and 20% EMP. The content of α-helical structures was the highest at 10% UMP, and the content of ordered structures was enhanced. The polymerization of low molecular weight proteins to form macromolecular polymers led to a reduction in surface hydrophobic regions and the aggregation of hydrophobic groups. The SEM results also demonstrated that at 10% tartary buckwheat leaf powder, the addition of UMP was significantly different from that of the other three leaf powders, and at 20%, the addition of EMP substantially altered the structure of the dough proteins. Considering the effects of different milling methods and different added amounts of tartary buckwheat leaf powder on various characteristics of dough, 10% UMP is the most suitable amount to add to the dough.

Unraveling the impacts of fermentation methods on the protein structural, foaming and air-water interfacial properties of buckwheat steamed bread dough liquor

Liquid lamella has crucial impacts on stabilizing gas cells and buckwheat steamed bread (BSB) quality due to the high fiber content and lack of gluten protein in buckwheat. The liquid lamella was collected by ultracentrifugation as the dough liquor (DL). This study explored the composition, foaming and interfacial properties of DL from lactic acid bacteria (LAB), yeast, LAB and yeast cooperate (LAB-yeast), and chemical acidified and yeast (CA-yeast) fermented buckwheat dough, as well as the unfermented buckwheat dough. Compared with the unfermented dough, the DL yield of LAB-yeast, yeast, and CA-yeast fermented dough decreased by 4.09%, 3.93%, and 7.67%, respectively. The protein molecular weight and surface hydrophobicity of DL were dramatically reduced by LAB, LAB-yeast, and CA-yeast fermentation as compared to unfermented DL. Among all DL samples, the unfermented and yeast DL showed the highest protein surface hydrophobicity and surface activity, resulting in the greatest foam stability. The protein in CA-yeast DL displayed the fastest interface diffusion because its DL had the highest amount of low molecular weight protein, the lowest viscosity and protein content. Compared with the unfermented dough, the LAB and LAB-yeast fermentation decreased, while the CA-yeast fermentation increased the strain hardening index (SHI) of the dough. This enhanced strain hardening behavior contributed to a decrease in the average cell area and an increase in cell density, making the crumb structure of CA-yeast fermented BSB homogeneous and dense.

Effect of thermal and defatting treatments on the composition, protein profile and structure of house cricket (Acheta domesticus) protein extracts

This study investigated the impact of blanching (100 °C, 40 s), defatting method (maceration, Soxhlet) and solvent polarity (hexane, ethanol) on the profile, structure and solubility of house cricket protein extracts. Blanching and Soxhlet using ethanol impacted the protein profile, with a lower content of myosin heavy chain and a higher abundance of low molecular weight proteins (<25 kDa). Moreover, ethanol induced aggregation of non-blanched cricket proteins, with a 13–72% reduction in protein recovery yield. The protein secondary structure of non-blanched extracts was also affected by ethanol with 18% more β-sheets. Furthermore, blanching resulted in a lower protein surface hydrophobicity by a factor of 3 to 7, with no impact of solvent polarity. Finally, the solubility of protein extracts remained >75%, regardless of the blanching and defatting methods. These findings, combined with the evaluation of techno-functional properties, could be used for the development of cricket-based protein ingredients for food formulations.

Physicochemical and emulsification properties of okara-derived soluble dietary fiber modified by steam explosion

The effective utilization of by-products still poses a challenge in the food industry. In this work, soluble dietary fiber (SDF) extracted from okara was modified by steam explosion (SE) via adjusting steam pressure (1.0, 1.5, and 2.0 MPa) and SE duration (60 and 120 s). The structural and emulsification properties of modified SDF were studied. The untreated SDF exhibited a low yield (5.17%) and poor emulsification properties due to its large molecular weight (162.67 kDa), compact structure, and high interfacial tension (14.27 mN/m). SE increased SDF content by a maximum of 30.31%, 4.86 times higher than that of untreated SDF. Under mild SE conditions (1.0 MPa for 60 s), SDF displayed a loose structure with abundant side chains and a higher glyoxylate content (26.77%). Its near-neutral contact angle (82.0°) and lower interfacial tension (12.69 mN/m) endowed it with higher emulsification capacity, smaller emulsion droplet size, and enhanced emulsifying stability. The molecular weight of SDF decreased and the specific surface area enlarged with increasing SE intensity. Notably, SDF exhibited the best interfacial adsorption characteristics under SE of 2.0 MPa for 120 s, attributed to its low molecular weight (46.99 kDa) and higher protein content (7.58%). Overall, the improved emulsification properties of SDF were attributed to the synergistic effects of amphiphilic equilibrium, electrostatic repulsion, spatial resistance, and enhanced droplet-to-droplet interactions.

Assessment of IL-8 Levels in Saliva of Healthy and Chronic Periodontitis Individuals

ABSTRACT Inflammation of the gums and other tissues supporting the teeth, as well as gradual loss of attachment and bone, are the results of chronic periodontitis, an infectious illness. During inflammation, a group of low molecular weight proteins called cytokines facilitate a complex interaction between inflammatory cells (such neutrophils) and other cellular components in connective tissue. The cytokine interleukin-8 (IL-8) is a potent neutrophil chemoattractant. Therefore, it is possible that IL-8 is crucial to the development of periodontitis’s pathology. Objectives: 1) To estimate concentration of IL-8 levels in healthy individuals and chronic periodontitis individuals. 2) To compare IL-8 levels in healthy and chronic periodontitis individuals Materials and Methods: Participants in this research will be recruited from among those who visit the outpatient department (OPD) at the NGH Institute of Dental Sciences and Research Centre, Belagavi, run by the Maratha Mandal. Control Group: Subjects with no clinical attachment loss (CAL) and a probing depth of 3.0 mm are considered to be periodontally healthy. Those in Group 2 (chronic periodontitis) have a chronic form of the disease, as shown by a probing pocket depth (PPD) of less than 5 mm and CAL of less than 2 mm. Unstimulated saliva sample will be collected in a 5 mL wide-mouthed sterile container by spitting method. Samples collected will be centrifuged. The supernatant is collected and stored at –80°C and then assayed for IL-8 concentration by using the standardized IL-8 ELISA kit.

Simultaneous determination of multiple urine biomarkers for kidney injury using SPE combined with LC-MS/MS

Background and ObjectivesUrinary biomarkers such as low molecular weight proteins and small molecular weight metabolites are crucial in the diagnosis of kidney injury. The objective of this study was to develop and preliminarily validate a sensitive and specific method using solid-phase extraction (SPE) in conjunction with liquid chromatography–tandem mass spectrometry (LC-MS/MS) for the simultaneous measurement of these biomarkers in human urine. MethodThis study presents the development of a solid-phase extraction method integrated with LC-MS/MS analyzing biomarkers including creatinine, urea, β2-microglobulin, α1-microglobulin, and cystatin C in human urine. An enhanced solid-phase cartridge technique was employed for peptide purification and dilution of small molecule metabolites during sample preparation. ResultsThe developed LC-MS/MS method achieved satisfactory separation of the five analytes within 15 min. Accuracy levels ranged from −8.6% to 13.6%. Both intra-assay and inter-assay imprecision rates were maintained below 7.9% for all analytes. ConclusionsThe established LC-MS/MS method effectively quantifies creatinine, urea, β2-microglobulin, α1-microglobulin and cystatin C concurrently. This offers a viable alternative for the detection of kidney injury biomarkers in human urine, demonstrating potential for clinical application in kidney injury diagnosis.

Uncovering the antimicrobial activity of G-type lysozyme 2 derived from Cyprinus carpio mucus against bacterial and fungal pathogens

This study aimed to explore the antimicrobic potential of mucus samples collected from Cyprinus carpio and identify the specific antimicrobial peptides responsible for its activity. The crude extract was tested against various bacterial and fungal pathogens, and its protein content and profile were analyzed. Purification steps, including gel filtration chromatography, were employed to isolate the most active fraction (peak IV), which was further identified via liquid chromatography and mass spectroscopy. The results revealed varying degrees of antimicrobial activity of the crude extract against different bacterial and fungal strains, with Leclercia adecarboxylata, Candida glabrata, and Candida parapsilosis showing the highest susceptibility. SDS-PAGE analysis demonstrated the existence of multiple low molecular weight protein bands in the crude extract, while fraction IV obtained from gel filtration chromatography exhibited the strongest antimicrobial activity. Peak IV displayed a range of minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and minimum fungicidal concentration (MFC) values against the tested pathogens, spanning from 0.038 to 4.960 mg/mL. Further investigation identified the purified peptide derived from peak IV as G-type lysozyme 2, characterized by a molecular weight of 21 kDa. These findings shed light on the existence of a highly effective antimicrobial peptide, G-type lysozyme 2, within the mucus of Cyprinus carpio. This peptide demonstrates notable activity against diverse bacterial and fungal pathogens. The insights from this study enhance our understanding of the fish's antimicrobial defense mechanisms and hold promise for developing novel antimicrobial agents.

Expression of concern: Microchip-based structure determination of low-molecular weight proteins using cryo-electron microscopy.

Expression of concern for 'Microchip-based structure determination of low-molecular weight proteins using cryo-electron microscopy' by Michael A. Casasanta et al., Nanoscale, 2021, 13, 7285-7293, https://doi.org/10.1039/D1NR00388G.

Comprehensive bioinformatics-based annotation and functional characterization of bovine chymosin protein revealed novel biological insights

Chymosin, an aspartic protease present in the stomachs of young ruminants like cows (bovine), causes milk coagulation and cheese production through the breakdown of κ-casein peptide bonds at the Met105-Phe106 site. Bovine chymosin is first synthesized as a pre-prochymosin that is cleaved to produce the mature chymosin protein. Despite significant strides in research, our understanding of this crucial enzyme remains incomplete. The purpose of this work was to perform in silico evolutionary and functional analysis and to gain unique insights into the structure of this protein. For this, the sequence of Bos taurus chymosin from UniProt database was subjected to various bioinformatics analyses. We found that bovine chymosin is a low molecular weight and hydrophilic protein that has homologs in other Bovidae species. Two active sites of aspartic peptidases, along with a functional domain, were identified. Gene Ontology analysis further confirmed chymosin's involvement in proteolysis and aspartic endopeptidase activity. Potential disordered residues and post-translational modification sites were also uncovered. It was revealed that the secondary structure of bovine chymosin is comprised of beta strands (44.27%), coils (43.65%), and alpha helices (12.07%). A highly optimized 3D structure was also obtained. Moreover, crucial protein–protein interactions were unveiled. Altogether, these findings provide valuable insights that could guide future research on bovine chymosin and its biological roles.

A common edible insect (Antheraea assamensis) protein hydrolysate regulates LPS-induced oxidative stress and inflammation by modulating the TLR4/NF- κ β Signaling Pathway

Abstract Dietary intake of antioxidant and anti-inflammatory-rich foods is a growing approach for fighting inflammation and its associated disorders. Edible insects are gaining popularity as a food source; however, their therapeutic properties, anti-inflammatory activity and their mechanism of action remain largely unexplored. Herein, we evaluate the effectiveness of Antheraea assamensis pupae hydrolysates against oxidative stress and inflammation. The proteins isolated from the pupae were digested with pepsin or pancreatin, or pepsin + pancreatin and passed through a 30 kDa molecular cut-off membrane. The low molecular weight protein hydrolysates were characterised using RP-HPLC, HRMS, XRD, and FTIR-ATR. Hydrolysate from pepsin + pancreatin digestion showed significantly higher DPPH (77.13±2.57%), superoxide (72.08±1.15%) and hydroxyl (56.32±0.90%) radical scavenging activity in a cell-free system. Further, it reduced intercellular ROS production and suppressed the lipopolysaccharide-induced protein expression of TLR4, p-IKKβ, p-NF-, IL-1β, and COX2 in RAW 264.7 macrophages. Furthermore, it inhibited the secretion of pro-inflammatory mediators, including IL-6, IL-1β, and MIP-2. These findings suggest that A. assamensis pupae hydrolysates can be a potential source of bioactive peptides for managing oxidative stress and inflammation.

In Situ ATR Spectroscopy Study of the Interaction of Acacia senegal Gum with Gold Nanoparticles Films at the Solid-Liquid Interface.

The adsorption process of Acacia gum (A. senegal), a complex heteropolysaccharide, was followed by using a spectroscopic method to unravel the relative contribution of the protein moieties and the carbohydrate blocks on the adsorption process. In situ ATR-FTIR was used to investigate the kinetics and conformational changes associated with the adsorption of A. senegal gum on gold nanoparticle films (Au-NPs) at different pHs. The results of this thorough study highlighted the adsorption of A. senegal gum through its protein moieties, in particular, AGPs of low molecular weight and high protein content, close to the Au-NPs surface. Isotherm experiments, by gradually increasing the concentration, showed that the gum adsorption was heterogeneous and followed the Freundlich model for the amide part, while the polysaccharide part followed the Langmuir model. In addition, the hydration and structural organization of the gum layer depended on the gum concentration. A. senegal gum adsorbed irreversibly on Au-NPs whatever the pHs, but the adsorbed layer presented a different behavior depending on pH. A more aggregated and less hydrated structure was observed at acidic pH, while a very hydrated and continuous layer was detected at higher pH. The secondary structure analysis through amide III band revealed a change in the gum secondary structure at high pH with the increase in β-turn while random coil decreased.

Detection of IgG Anti-Giardia duodenalis Antibodies in Sera by Indirect Immunofluorescence and Western Blotting Assays.

Serological assays are alternative laboratory tools for the diagnosis of parasitic infections. The aim of this work was to evaluate the performance of the indirect fluorescent antibody test (IFAT) and Western blotting (WB) for the detection of IgG anti-Giardia antibodies in human sera. Sera from individuals infected with Giardia duodenalis, other parasites or non-parasitized were selected for serological assays. Ninety-seven sera were tested by IFAT at 1:20 and 1:40 dilutions and of these, 40 samples were also analyzed by WB. The sensitivity and specificity of the IFAT was 97% and 46.9% at 1:20 sera dilution, and 39.4% and 59.4% at 1:40 sera dilution. The low molecular weight polypeptides fractions of 25kDa, 27-31kDa and 45-55kDa were the most frequently identified by the sera of individuals infected with G. duodenalis, along with low cross-reactivity, presenting an individual sensitivity of 42.8%, 50.0% and 57.1%, and specificity of 83.3%, 83.3% and 91.7%, respectively. The highest overall sensitivity of WB (85.7%) was based on the immunoreactivity of sera with at least one of those proteins. The concordance between the detection of G. duodenalis in feces by microscopy and the WB results was considered substantial (Kappa = 0.61). Constant exposure to Giardia infection throughout a lifetime can maintain high levels of specific antibodies in serum, even without active infection. Moreover, proteins found in intestinal amoebas may hinder the serological diagnosis of giardiasis in endemic areas due to cross-reactivity, which can be partially solved using Giardia low molecular weight proteins.

Elucidating structural variability in p53 conformers using combinatorial refinement strategies and molecular dynamics

ABSTRACT Low molecular weight proteins and protein assemblies can now be investigated using cryo-electron microscopy (EM) as a complement to traditional structural biology techniques. It is important, however, to not lose sight of the dynamic information inherent in macromolecules that give rise to their exquisite functionality. As computational methods continue to advance the field of biomedical imaging, so must strategies to resolve the minute details of disease-related entities. Here, we employed combinatorial modeling approaches to assess flexible properties among low molecular weight proteins (~100 kDa or less). Through a blend of rigid body refinement and simulated annealing, we determined new hidden conformations for wild type p53 monomer and dimer forms. Structures for both states converged to yield new conformers, each revealing good stereochemistry and dynamic information about the protein. Based on these insights, we identified fluid parts of p53 that complement the stable central core of the protein responsible for engaging DNA. Molecular dynamics simulations corroborated the modeling results and helped pinpoint the more flexible residues in wild type p53. Overall, the new computational methods may be used to shed light on other small protein features in a vast ensemble of structural data that cannot be easily delineated by other algorithms.

Preclinical Studies Demonstrating Efficacy of Tasquinimod in Models of Advanced Myeloproliferative Neoplasm (MPN) in Blastic Phase

The alarmins, S100A8 (A8) and S100A9 (A9), are low molecular weight proteins belonging to the S100 protein family. Also called calprotectins, A8 and A9 can form homodimers or heterodimers and bind Ca++. A8 and A9 are secreted into the extracellular space and plasma, where they interact with TLR4 (Toll like receptor 4), RAGE (Receptor for Advanced Glycation End products) and CD33. Intracellularly, they act as Ca++ biosensors and activate NADPH oxidase, stimulating ROS production, which activates NLRP3 inflammasome. A8 and A9 also amplify the inflammatory response through TLR4, and induce inflammatory cytokines, including TNFα and IL-6, through activation of NFkB and MAP kinase pathways. A8 and A9 are highly expressed in AML, especially myelomonocytic and monocytic AML, and are associated with poor prognosis. Through scRNA-Seq analysis, A8/A9 heterocomplex was shown to be highly expressed in myeloid and stromal cells and marked progression of bone marrow fibrosis in PMF (Primary Myelofibrosis). In the murine JAK2-V617F-driven PMF model, Tasquinimod (TM, Active Biotech), an orally active, quinoline-3-carboxamide linomide analogue immune-modulatory agent was shown to bind A9, inhibiting its interaction with TLR and RAGE receptors. Notably, TM treatment reduced myeloproliferation, splenomegaly and MF in JAK2-V617F mice. Therefore, based on these findings, in present studies, we determined in vitro and in vivo efficacy of TM against cellular models of advanced MPN cell lines and patient-derived (PD) CD34+ blastic phase (BP, &amp;gt; 5% blasts in PB) MPN cells. Treatment with TM (10 to 30 µM for 96 hours) induced loss of viability in the MPN-AML SET2, HEL92.1.7 cell lines and PD MPN-BP cells, but not in normal CD34+ progenitor cells. RNA-Seq analysis showed that exposure to TM (20 µM for 16 hours) led to negative enrichment of the gene-sets of MYC and E2F targets, of mTORC1 and IL6-JAK-STAT3 signaling, and of inflammatory response in PD MPN-AML cells. This was associated with log2 fold-decline in MYC, BCL2, CDK6, MPL, CCND1 and myeloperoxidase (MPO), but upregulation of mRNA of GFI1 and p21. CyTOF analysis revealed that TM treatment significantly reduced expressions of A8, A9, p-ERK, MPO, CXCR4, Cyclin D1, PU.1 and Ki67, but increased protein levels of GFI1, p21 and cleaved PARP in phenotypically defined PD, CD34+ MPN-BP cells expressing high levels of CLEC12A, CD99, CD123 but low expression of CD11b. Additionally, our findings showed that co-treatment with TM (5 to 30 µM) and ruxolitinib (250 to 1000 nM), BET inhibitor OTX015 (50 to 250 nM) or pelabresib (CPI-0610) (100 to 500 nM), or BCL2/Bcl-xL inhibitor navitoclax induced synergistic lethality in advanced MPN-BP cells exhibiting delta synergy scores of &amp;gt;1.0 (by the ZIP method). Notably, our findings also demonstrated that treatment with TM (10 or 30 mg/kg/day, by oral gavage) for 8-weeks, significantly improved survival without inducing any toxicity in immune-depleted NSG mice engrafted with PDX cells of MPN-AML with mutations in CALR and TERT. In a separate experiment on the same PDX model, treatment with TM (30 mg/kg/day) also induced significantly greater survival advantage than treatment with ruxolitinib (30 mg/kg/day) or OTX015 (30 mg/kg/day) by oral gavage. These findings clearly demonstrate preclinical efficacy of TM in advanced MPN-BP cellular models and create the rationale to further interrogate the efficacy of TM alone and in combinations with current, front-line therapies for advanced MPN with excess blasts.

Identified Small Open Reading Frame-Encoded Peptides in Human Serum with Nanoparticle Protein Coronas.

The low-molecular-weight proteins (LMWP) in serum and plasma are related to various human diseases and can be valuable biomarkers. A small open reading frame-encoded peptide (SEP) is one kind of LMWP, which has been found to function in many bioprocesses and has also been found in human blood, making it a potential biomarker. The detection of LMWP by a mass spectrometry (MS)-based proteomic assay is often inhibited by the wide dynamic range of serum/plasma protein abundance. Nanoparticle protein coronas are a newly emerging protein enrichment method. To analyze SEPs in human serum, we have developed a protocol integrated with nanoparticle protein coronas and liquid chromatography (LC)/MS/MS. With three nanoparticles, TiO2, Fe3O4@SiO2, and Fe3O4@SiO2@TiO2, we identified 164 new SEPs in the human serum sample. Fe3O4@SiO2 and a nanoparticle mixture obtained the maximum number and the largest proportion of identified SEPs, respectively. Compared with acetonitrile-based extraction, nanoparticle protein coronas can cover more small proteins and SEPs. The magnetic nanoparticle is also fit for high-throughput parallel protein separation before LC/MS. This method is fast, efficient, reproducible, and easy to operate in 96-well plates and centrifuge tubes, which will benefit the research on SEPs and biomarkers.

Determination and Kinetic Characterization of a New Potential Inhibitor for AmsI Protein Tyrosine Phosphatase from the Apple Pathogen Erwinia amylovora

Erwinia amylovora is a Gram-negative bacterium, responsible for the fire blight disease in Rosaceae plants. Its virulence is correlated with the production of an exopolysaccharide (EPS) called amylovoran, which protects the bacterium from the surrounding environment and helps its diffusion inside the host. Amylovoran biosynthesis relies on the expression of twelve genes clustered in the ams operon. One of these genes, amsI, encodes for a Low Molecular Weight Protein Tyrosine Phosphatase (LMW-PTP) called EaAmsI, which plays a key role in the regulation of the EPS production pathway. For this reason, EaAmsI was chosen in this work as a target for the development of new antibacterial agents against E. amylovora. To achieve this aim, a set of programs (DOCK6, OpenEye FRED) was selected to perform a virtual screening using a database of ca. 700 molecules. The six best-scoring compounds identified were tested in in vitro assays. A complete inhibition kinetic characterization carried out on the most promising molecule (n-Heptyl β-D-glucopyranoside, N7G) showed an inhibition constant of 7.8 ± 0.6 µM. This study represents an initial step towards the development of new EaAmsI inhibitors able to act as antibacterial agents against E. amylovora infections.