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Microbiology Spectrum Aug 2023Many studies have suggested that gut microbiota dysbiosis may be one of the pathogenesis factors of diabetes mellitus (DM), while it is not clear whether it is involved...
Many studies have suggested that gut microbiota dysbiosis may be one of the pathogenesis factors of diabetes mellitus (DM), while it is not clear whether it is involved in the development of diabetic kidney diseases (DKD). The objective of this study was to determine bacterial taxa biomarkers during the progression of DKD by investigating bacterial compositional changes in early and late DKD. 16S rRNA gene sequencing was performed on fecal samples, including the diabetes mellitus (DM), DNa (early DKD), and DNb (late DKD) groups. Taxonomic annotation of microbial composition was performed. Samples were sequenced on the Illumina NovaSeq platform. At the genus level, we found counts of , , and were significantly elevated both in the DNa group (0.0001, 0.0007, and 0.0174, respectively) and the DNb group (0.0001, 0.0012, and 0.0003, respectively) compared with those in the DM group. Only the level of was significantly decreased in the DNa group than the DM group and in the DNb group than the DNa group. Counts of , were significantly decreased in the DNa group compared with those in the DM group (0.001 and 0.006, respectively) and in the DNb group compared with those in the DM group (0.0001 and 0.003, respectively). Levels of , and were positively correlated with an estimated glomerular filtration rate (eGFR), but negatively correlated with microalbuminuria (MAU), 24 h urinary protein quantity (24hUP), and serum creatinine (Scr). Moreover, the areas under the curve (AUCs) of and were 83.33% and 80.77%, respectively, for the DM and DNa cohorts, respectively. Notably, the largest AUC for DNa and DNb cohorts was also that of at 83.60%. Gut microbiota dysbiosis was found in the early and late stages of DKD, especially in the early stage. may be the most promising intestinal bacteria biomarker that can help distinguish different stages of DKD. It is not clear as to whether gut microbiota dysbiosis is involved in the progression of DKD. This study may be the first to explore gut microbiota compositional changes in diabetes, early-DKD, and late DKD. We identify different gut microbial characteristics during different stages of DKD. Gut microbiota dysbiosis is found in the early and late stages of DKD. may be the most promising intestinal bacteria biomarker that can help distinguish different stages of DKD, although further studies are warranted to illustrate these mechanisms.
Topics: Diabetic Nephropathies; Humans; Male; Female; Middle Aged; Gastrointestinal Microbiome; Clostridiales; Biomarkers; Diabetes Mellitus; Bacteria; Feces; Kidney Failure, Chronic
PubMed: 37341590
DOI: 10.1128/spectrum.00382-23 -
Molecular Neurodegeneration Sep 2023The accumulation of amyloid beta (Aβ) peptides in fibrils is prerequisite for Alzheimer's disease (AD). Our understanding of the proteins that promote Aβ fibril...
BACKGROUND
The accumulation of amyloid beta (Aβ) peptides in fibrils is prerequisite for Alzheimer's disease (AD). Our understanding of the proteins that promote Aβ fibril formation and mediate neurotoxicity has been limited due to technical challenges in isolating pure amyloid fibrils from brain extracts.
METHODS
To investigate how amyloid fibrils form and cause neurotoxicity in AD brain, we developed a robust biochemical strategy. We benchmarked the success of our purifications using electron microscopy, amyloid dyes, and a large panel of Aβ immunoassays. Tandem mass-spectrometry based proteomic analysis workflows provided quantitative measures of the amyloid fibril proteome. These methods allowed us to compare amyloid fibril composition from human AD brains, three amyloid mouse models, transgenic Aβ42 flies, and Aβ42 seeded cultured neurons.
RESULTS
Amyloid fibrils are primarily composed by Aβ42 and unexpectedly harbor Aβ38 but generally lack Aβ40 peptides. Multidimensional quantitative proteomics allowed us to redefine the fibril proteome by identifying 20 new amyloid-associated proteins. Notably, we confirmed 57 previously reported plaque-associated proteins. We validated a panel of these proteins as bona fide amyloid-interacting proteins using antibodies and orthogonal proteomic analysis. One metal-binding chaperone metallothionein-3 is tightly associated with amyloid fibrils and modulates fibril formation in vitro. Lastly, we used a transgenic Aβ42 fly model to test if knock down or over-expression of fibril-interacting gene homologues modifies neurotoxicity. Here, we could functionally validate 20 genes as modifiers of Aβ42 toxicity in vivo.
CONCLUSIONS
These discoveries and subsequent confirmation indicate that fibril-associated proteins play a key role in amyloid formation and AD pathology.
Topics: Humans; Animals; Mice; Amyloid; Alzheimer Disease; Amyloid beta-Peptides; Proteome; Proteomics; Amyloidogenic Proteins; Brain
PubMed: 37710351
DOI: 10.1186/s13024-023-00654-z -
European Journal of Cell Biology Dec 2023In vitro reconstitution assays using purified actin have greatly improved our understanding of cytoskeletal dynamics and their regulation by actin-binding proteins.... (Review)
Review
In vitro reconstitution assays using purified actin have greatly improved our understanding of cytoskeletal dynamics and their regulation by actin-binding proteins. However, early purification methods consisted of harsh conditions to obtain pure actin and often did not include correct maturation and obligate modification of the isolated actin monomers. Novel insights into the folding requirements and N-terminal processing of actin as well as a better understanding of the interaction of actin with monomer sequestering proteins such as DNaseI, profilin and gelsolin, led to the development of more gentle approaches to obtain pure recombinant actin isoforms with known obligate modifications. This review summarizes the approaches that can be employed to isolate natively folded endogenous and recombinant actin from tissues and cells. We further emphasize the use and limitations of each method and describe how these methods can be implemented to study actin PTMs, disease-related actin mutations and novel actin-like proteins.
Topics: Animals; Actins; Microfilament Proteins; Profilins; Protein Isoforms; Mammals; Gelsolin
PubMed: 37778219
DOI: 10.1016/j.ejcb.2023.151363 -
The Journal of Biological Chemistry Feb 2024Mammalian F-ATP synthase is central to mitochondrial bioenergetics and is present in the inner mitochondrial membrane in a dynamic oligomeric state of higher oligomers,...
Mammalian F-ATP synthase is central to mitochondrial bioenergetics and is present in the inner mitochondrial membrane in a dynamic oligomeric state of higher oligomers, tetramers, dimers, and monomers. In vitro investigations of mammalian F-ATP synthase are often limited by the ability to purify the oligomeric forms present in vivo at a quantity, stability, and purity that meets the demand of the planned experiment. We developed a purification approach for the isolation of bovine F-ATP synthase from heart muscle mitochondria that uses a combination of buffer conditions favoring inhibitor factor 1 binding and sucrose density gradient ultracentrifugation to yield stable complexes at high purity in the milligram range. By tuning the glyco-diosgenin to lauryl maltose neopentyl glycol ratio in a final gradient, fractions that are either enriched in tetrameric or monomeric F-ATP synthase can be obtained. It is expected that this large-scale column-free purification strategy broadens the spectrum of in vitro investigation on mammalian F-ATP synthase.
Topics: Animals; Cattle; Adenosine Triphosphate; Dimerization; Mitochondria, Heart; Mitochondrial Membranes; Mitochondrial Proton-Translocating ATPases; Centrifugation, Density Gradient
PubMed: 38159856
DOI: 10.1016/j.jbc.2023.105603 -
Cell Reports. Medicine May 2024Natural history and mechanisms for persistent cognitive symptoms ("brain fog") following acute and often mild COVID-19 are unknown. In a large prospective cohort of...
Natural history and mechanisms for persistent cognitive symptoms ("brain fog") following acute and often mild COVID-19 are unknown. In a large prospective cohort of people who underwent testing a median of 9 months after acute COVID-19 in the New York City/New Jersey area, we found that cognitive dysfunction is common; is not influenced by mood, fatigue, or sleepiness; and is correlated with MRI changes in very few people. In a subgroup that underwent cerebrospinal fluid analysis, there are no changes related to Alzheimer's disease or neurodegeneration. Single-cell gene expression analysis in the cerebrospinal fluid shows findings consistent with monocyte recruitment, chemokine signaling, cellular stress, and suppressed interferon response-especially in myeloid cells. Longitudinal analysis shows slow recovery accompanied by key alterations in inflammatory genes and increased protein levels of CXCL8, CCL3L1, and sTREM2. These findings suggest that the prognosis for brain fog following COVID-19 correlates with myeloid-related chemokine and interferon-responsive genes.
Topics: Humans; COVID-19; Male; Single-Cell Analysis; Female; Cognitive Dysfunction; Middle Aged; SARS-CoV-2; Aged; Receptors, Immunologic; Prospective Studies; Adult; Magnetic Resonance Imaging; Membrane Glycoproteins; Interleukin-8
PubMed: 38744274
DOI: 10.1016/j.xcrm.2024.101561 -
The Journal of General Virology Apr 2024is a family for negative-sense RNA viruses with genomes of about 17.2-21.1 kb. These viruses are maintained in and/or transmitted by arthropods among birds, reptiles...
is a family for negative-sense RNA viruses with genomes of about 17.2-21.1 kb. These viruses are maintained in and/or transmitted by arthropods among birds, reptiles and mammals. Norwaviruses and orthonairoviruses can cause febrile illness in humans. Several orthonairoviruses can infect mammals, causing mild, severe and sometimes, fatal diseases. Nairovirids produce enveloped virions containing two or three single-stranded RNA segments with open reading frames that encode a nucleoprotein (N), sometimes a glycoprotein precursor (GPC), and a large (L) protein containing an RNA-directed RNA polymerase (RdRP) domain. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) report on the family , which is available at www.ictv.global/report/nairoviridae.
Topics: Animals; Genome, Viral; Humans; Open Reading Frames; Viral Proteins; Nairovirus; RNA, Viral; Phylogeny; Virion; RNA-Dependent RNA Polymerase
PubMed: 38687001
DOI: 10.1099/jgv.0.001974 -
Polish Journal of Microbiology Jun 2024Proteases derived from demonstrate numerous commendable properties, rendering it extensively applicable in biotechnology and various industrial sectors. This study...
Proteases derived from demonstrate numerous commendable properties, rendering it extensively applicable in biotechnology and various industrial sectors. This study focused on the purification and characterization of the thermostable protease obtained from sp. CNXK100. The purified protease exhibited an estimated molecular weight of 27 kDa, with optimal activity at 75°C and pH 8.0. Notably, the enzyme remained active even without any metal ions and fully active in the presence of Na, K, Mg, and Cumetal ions. The kinetic parameters were determined with a value of 3.13 mg/ml and a value of 3.28 × 10 U/mg. Furthermore, the protease has demonstrated notable stability when subjected to a treatment temperature of up to 65°C for 60 minutes, and across a broad pH range extending from 5.0 to 10.0. This protease also demonstrated resilience against a spectrum of harsh conditions, including exposure to organic solvents, surfactants, bleaching agents, and proteolytic enzymes. Additionally, the enzyme maintained its activity following treatment with commercial detergents, accomplishing complete thrombus lysis at a concentration of 2.50 mg/ml within 4 hours. Remarkably, the protease exhibited stability in terms of activity and protein concentration for 70 days at 4°C. These findings underscore the potential industrial applications of the thermostable protease from sp. CNXK100.
Topics: Streptomyces; Enzyme Stability; Hydrogen-Ion Concentration; Kinetics; Temperature; Bacterial Proteins; Peptide Hydrolases; Molecular Weight; Metals
PubMed: 38678439
DOI: 10.33073/pjm-2024-014 -
Protein Expression and Purification Nov 2023High yield purification of Ulp1 is required during the isolation and purification of SUMO-tagged recombinant proteins. However, when expressed as a soluble protein, Ulp1...
High yield purification of Ulp1 is required during the isolation and purification of SUMO-tagged recombinant proteins. However, when expressed as a soluble protein, Ulp1 is toxic to E. coli host cells and most of the protein forms inclusion bodies. The extraction of insoluble Ulp1 followed by its purification and refolding into its active form is a lengthy and costly procedure. In our present study, we developed a simple, cost effective procedure for the large scale production of active Ulp1 that can be used for industrial scale requirements.
Topics: Recombinant Fusion Proteins; Peptide Hydrolases; Escherichia coli; Cysteine Endopeptidases; Small Ubiquitin-Related Modifier Proteins; Inclusion Bodies
PubMed: 37392905
DOI: 10.1016/j.pep.2023.106328 -
Journal of Chromatography. A Dec 2023Field-flow fractionation (FFF) with its several variants, has developed into a mature methodology. The scope of the FFF investigations has expanded, covering both a wide... (Review)
Review
Field-flow fractionation (FFF) with its several variants, has developed into a mature methodology. The scope of the FFF investigations has expanded, covering both a wide range of basic studies and especially a wide range of analytical applications. Special attention of this review is given to the achievements of FFF with reference to recent applications in the fractionation, isolation, and purification of biomacromolecules, and from which especially those of (in alphabetical order) bacteria, cells, extracellular vesicles, liposomes, lipoproteins, nucleic acids, and viruses and virus-like particles. In evaluating the major approaches and trends demonstrated since 2012, the most significant biomacromolecule applications are compiled in tables. It is also evident that asymmetrical flow field-flow fractionation is by far the most dominant technique in the studies. The industry has also shown current interest in FFF and adopted it in some sophisticated fields. FFF, in combination with appropriate detectors, handles biomacromolecules in open channel in a gentle way due to the lack of shear forces and unwanted interactions caused by the stationary phase present in chromatography. In addition, in isolation and purification of biomacromolecules quite high yields can be achieved under optimal conditions.
Topics: Chemical Fractionation; Fractionation, Field Flow; Lipoproteins; Chromatography; Liposomes
PubMed: 37944435
DOI: 10.1016/j.chroma.2023.464492 -
The Journal of Biological Chemistry Jan 2024Calreticulin (CRT) was originally identified as a key calcium-binding protein of the endoplasmic reticulum. Subsequently, CRT was shown to possess multiple intracellular...
Calreticulin (CRT) was originally identified as a key calcium-binding protein of the endoplasmic reticulum. Subsequently, CRT was shown to possess multiple intracellular functions, including roles in calcium homeostasis and protein folding. Recently, several extracellular functions have been identified for CRT, including roles in cancer cell invasion and phagocytosis of apoptotic and cancer cells by macrophages. In the current report, we uncover a novel function for extracellular CRT and report that CRT functions as a plasminogen-binding receptor that regulates the conversion of plasminogen to plasmin. We show that human recombinant or bovine tissue-derived CRT dramatically stimulated the conversion of plasminogen to plasmin by tissue plasminogen activator or urokinase-type plasminogen activator. Surface plasmon resonance analysis revealed that CRT-bound plasminogen (K = 1.8 μM) with moderate affinity. Plasminogen binding and activation by CRT were inhibited by ε-aminocaproic acid, suggesting that an internal lysine residue of CRT interacts with plasminogen. We subsequently show that clinically relevant CRT variants (lacking four or eight lysines in carboxyl-terminal region) exhibited decreased plasminogen activation. Furthermore, CRT-deficient fibroblasts generated 90% less plasmin and CRT-depleted MDA MB 231 cells also demonstrated a significant reduction in plasmin generation. Moreover, treatment of fibroblasts with mitoxantrone dramatically stimulated plasmin generation by WT but not CRT-deficient fibroblasts. Our results suggest that CRT is an important cellular plasminogen regulatory protein. Given that CRT can empower cells with plasmin proteolytic activity, this discovery may provide new mechanistic insight into the established role of CRT in cancer.
Topics: Animals; Cattle; Humans; Calreticulin; Fibrinolysin; Plasminogen; Tissue Plasminogen Activator; Urokinase-Type Plasminogen Activator; Protein Domains; Mutation; Recombinant Proteins; Gene Knockout Techniques; Cell Line, Tumor; Neoplasms
PubMed: 37979915
DOI: 10.1016/j.jbc.2023.105465