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Biomedicines Dec 2023Platelet lipid rafts are critical membrane domains for adhesion, aggregation, and clot retraction. Lipid rafts are isolated as a detergent-resistant membrane fraction...
Platelet lipid rafts are critical membrane domains for adhesion, aggregation, and clot retraction. Lipid rafts are isolated as a detergent-resistant membrane fraction via sucrose density gradient centrifugation. The platelet detergent-resistant membrane shifted to a higher density on the sucrose density gradient upon thrombin stimulation. The shift peaked at 1 min and returned to the control level at 60 min. During this time, platelets underwent clot retraction and spreading on a fibronectin-coated glass strip. Thrombin induced the transient tyrosine phosphorylation of several proteins in the detergent-resistant membrane raft fraction and the transient translocation of fibrin and myosin to the detergent-resistant membrane raft fraction. The level of phosphatidylserine (36:1) was increased and the level of phosphatidylserine (38:4) was decreased in the detergent-resistant membrane raft fraction via the thrombin stimulation. Furthermore, Glanzmann's thrombasthenia integrin αIIbβ3-deficient platelets underwent no detergent-resistant membrane shift to a higher density upon thrombin stimulation. As the phosphorylation of the myosin regulatory light chain on Ser19 was at a high level in Glanzmann's thrombasthenia resting platelets, thrombin caused no further phosphorylation of the myosin regulatory light chain on Ser19 or clot retraction. These observations suggest that the fibrin-integrin αIIbβ3-myosin axis and compositional change of phosphatidylserine species may be required for the platelet detergent-resistant membrane shift to a higher density upon stimulation with thrombin.
PubMed: 38255176
DOI: 10.3390/biomedicines12010069 -
Frontiers in Bioengineering and... 2023Limited availability of the organs donors has facilitated the establishment of xenogeneic organ sources for transplantation. Numerous studies have decellularized several...
Limited availability of the organs donors has facilitated the establishment of xenogeneic organ sources for transplantation. Numerous studies have decellularized several organs and assessed their implantability in order to provide such organs. Among all the decellularized organs studies for xenotransplantation, the pancreas has garnered very limited amount of research. The presently offered alternatives for pancreas transplantation are unable to liberate patients from donor dependence. The rat and mice pancreas are not of an accurate size for transplantation but can only be used for studies mimicking immune response in humans, while the porcine pancreas can cause zoonotic diseases as it carries porcine endogenous retrovirus (PERV- A/B/C). Therefore, we propose caprine pancreas as a substitute for these organs, which not only reduces donor dependence but also poses no risk of zoonosis. Upon decellularization the extracellular matrix (ECM) of different tissues responds differently to the detergents used for decellularization at physical and physiological level; this necessitates a comprehensive analysis of each tissue independently. This study investigates the impact of decellularization by ionic (SDS and SDC), non-ionic (Triton X-100 and Tween-20), and zwitterionic detergents (CHAPS). All these five detergents have been used to decellularize caprine pancreas via immersion (ID) and perfusion (PD) set-up. In this study, an extensive comparison of these two configurations (ID and PD) with regard to each detergent has been conducted. The final obtained scaffold with each set-up has been evaluated for the left-over cytosolic content, ECM components like sGAG, collagen, and fibronectin were estimated via Prussian blue and Immunohistochemical staining respectively, and finally for the tensile strength and antimicrobial activity. All the detergents performed consistently superior in PD than in ID. Conclusively, PD with SDS, SDC, and TX-100 successfully decellularizes caprine pancreatic tissue while retaining ECM architecture and mechanical properties. This research demonstrates the viability of caprine pancreatic tissue as a substitute scaffold for porcine organs and provides optimal decellularization protocol for this xenogeneic tissue. This research aims to establish a foundation for further investigations into potential regenerative strategies using this ECM in combination with other factors.
PubMed: 37790257
DOI: 10.3389/fbioe.2023.1253804 -
Frontiers in Microbiology 2023Maize () is one of the most widely cultivated crops used as energy feeds. The aim of this study was to evaluate the effects of two lactic acid bacteria additives on the...
Maize () is one of the most widely cultivated crops used as energy feeds. The aim of this study was to evaluate the effects of two lactic acid bacteria additives on the fermentation quality and bacterial community of high moisture ear corn (HMEC) silage at different moisture levels. The study utilized corn kernels and cobs harvested at the stage of complete ripeness as the primary material. The cob was crushed and divided into three treatment groups: an untreated control group (CK), a group treated with a mixture of and (TQ), or a group treated with a mixture of and (KT). Moisture contents were adjusted to 37.5% (L), 42.5% (M) or 47.5% (H) and then silaged for 180 days. Compared to CK, TQ, and KT elevated the dry matter, crude protein, starch, lactic and acetic acid content of HMEC and reduced the pH, neutral detergent fiber, acid detergent fiber and ammonia nitrogen content ( < 0.05). Even though both additives improved the bacterial community structure after fermentation, KT experienced the greater enhancement. At a phylum and genus level, KT had the higher relative abundance of and , respectively. Compared with the group of 37.5% (L) moisture content, the 42.5% (M) and 47.5% moisture content (H) group increased lactic acid, acetic acid and ammonia nitrogen concentrations and reduced the pH value ( < 0.05). In conclusion, the addition of TQ and KT at the appropriate moisture content might be helpful for producing high-quality HMEC. Among the three moisture contents, 42.5% (M) moisture content provides the best silage qualities.
PubMed: 38094628
DOI: 10.3389/fmicb.2023.1251946 -
Biomolecules Jan 2024The purpose of this review is to succinctly examine the methodologies used in lipid raft research in the brain and to highlight the drawbacks of some investigative... (Review)
Review
The purpose of this review is to succinctly examine the methodologies used in lipid raft research in the brain and to highlight the drawbacks of some investigative approaches. Lipid rafts are biochemically and biophysically different from the bulk membrane. A specific lipid environment within membrane domains provides a harbor for distinct raftophilic proteins, all of which in concert create a specialized platform orchestrating various cellular processes. Studying lipid rafts has proved to be arduous due to their elusive nature, mobility, and constant dynamic reorganization to meet the cellular needs. Studying neuronal lipid rafts is particularly cumbersome due to the immensely complex regional molecular architecture of the central nervous system. Biochemical fractionation, performed with or without detergents, is still the most widely used method to isolate lipid rafts. However, the differences in solubilization when various detergents are used has exposed a dire need to find more reliable methods to study particular rafts. Biochemical methods need to be complemented with other approaches such as live-cell microscopy, imaging mass spectrometry, and the development of specific non-invasive fluorescent probes to obtain a more complete image of raft dynamics and to study the spatio-temporal expression of rafts in live cells.
Topics: Detergents; Membrane Microdomains; Brain
PubMed: 38397393
DOI: 10.3390/biom14020156 -
Nanotechnology Aug 2023imaging of protein complexes is a powerful method for understanding the underlying biological function of these key biomolecules. Though the engineering of small, high...
imaging of protein complexes is a powerful method for understanding the underlying biological function of these key biomolecules. Though the engineering of small, high affinity nanobodies have become more prevalent, the off-rates of these tags may result in incomplete or partial labeling of proteins in live cells. The SpyCatcher003 and SpyTag split protein system allow for irreversible, covalent binding to a short target peptide unlike nanobody-affinity based probes. However, delivering these tags into a cell without disrupting its normal function is a key challenge. Cell penetrating peptides (CPPs) are short peptide sequences that facilitate the transduction of otherwise membrane-impermeable 'cargo' , such as proteins, into cells. Here we report on our efforts to engineer and characterize CPP-SpyCatcher003 fusions as modular imaging probes. We selected three CPPs, CUPID, Pentratin, and pVEC, to engineer fusion protein probes for superresolution microscopy, with the aim to eliminate prior permeabilization treatments that could introduce imaging artifacts. We find that fusing the CPP sequences to SpyCatcher003 resulted in dimer and multimer formation as determined by size exclusion chromatography, dynamic light scattering, and SDS resistant dimers on SDS-PAGE gels. By isolating and labeling the monomeric forms of the engineered protein, we show these constructs retained their ability to bind SpyTag and all three CPP sequences remain membrane active, as assessed by CD spectroscopy in the presence of SDS detergent. Using fluorescence and super resolution Lattice structured illumination microscopy (Lattice SIM) imaging we show that the CPPs did not enhance uptake of SpyCatcher byhowever withcells, we show that Penetratin, and to a lesser degree CUPID, does enhance uptake. Our results demonstrate the ability of the CPP-SpyCatcher003 to label targets within living cells, providing the groundwork for using split protein systems for targetedimaging.
Topics: Cell-Penetrating Peptides; Proteins; Biological Transport
PubMed: 37336203
DOI: 10.1088/1361-6528/acdf65 -
Analytical Biochemistry Jan 2024Because of the heterogeneity among seedlings of outbreeding species, the use of seedling tissues as a source of DNA is unsuitable for the genomic characterization of...
Because of the heterogeneity among seedlings of outbreeding species, the use of seedling tissues as a source of DNA is unsuitable for the genomic characterization of elite germplasms. High-quality DNA, free of RNA, proteins, polysaccharides, secondary metabolites, and shearing, is mandatory for downstream molecular biology applications, especially for next-generation genome sequencing and pangenome analysis aiming to capture the complete genetic diversity within a species. The study aimed to accomplish an efficient protocol for the extraction of high-quality DNA suitable for diverse plant species/tissues. We describe a reliable, and consistent protocol suitable for the extraction of DNA from 42 difficult-to-extract plant species belonging to 33 angiosperm (monocot and dicot) families, including tissues such as seeds, roots, endosperm, and flower/fruit tissues. The protocol was first optimized for the outbreeding recalcitrant trees viz., Prosopis cineraria, Conocarpus erectus, and Phoenix dactylifera, which are rich in proteins, polysaccharides, and secondary metabolites, and the quality of the extracted DNA was confirmed by downstream applications. Nine procedures were attempted to extract high-quality, impurities-free DNA from these three plant species. Extraction of the ethanol-precipitated DNA from cetyltrimethylammonium bromide (CTAB) protocol using sodium dodecyl sulfate (SDS) buffer, i.e., the extraction using a cationic (CTAB) detergent followed by an anionic (SDS) detergent was the key for high yield and high purity (1.75-1.85 against A and an A ratio of >2) DNA. A vice versa extraction procedure, i.e., SDS buffer followed by CTAB buffer, and also CTAB buffer followed by CTAB, did not yield good-quality DNA. PCR (using different primers) and restriction endonuclease digestion of the DNA extracted from these three plants validated the protocol. The accomplishment of the genome of P. cineraria using the DNA extracted using the modified protocol confirmed its applicability to genomic studies. The optimized protocol successful in extracting high-quality DNA from diverse plant species/tissues extends its applicability and is useful for accomplishing genome sequences of elite germplasm of recalcitrant plant species with quality reads.
Topics: Humans; Cetrimonium; Detergents; DNA; Plants; Genomics; Polysaccharides; DNA, Plant
PubMed: 37940013
DOI: 10.1016/j.ab.2023.115372 -
Cell Reports. Medicine Mar 2024Selenoprotein N (SEPN1) is a protein of the endoplasmic reticulum (ER) whose inherited defects originate SEPN1-related myopathy (SEPN1-RM). Here, we identify an...
Selenoprotein N (SEPN1) is a protein of the endoplasmic reticulum (ER) whose inherited defects originate SEPN1-related myopathy (SEPN1-RM). Here, we identify an interaction between SEPN1 and the ER-stress-induced oxidoreductase ERO1A. SEPN1 and ERO1A, both enriched in mitochondria-associated membranes (MAMs), are involved in the redox regulation of proteins. ERO1A depletion in SEPN1 knockout cells restores ER redox, re-equilibrates short-range MAMs, and rescues mitochondrial bioenergetics. ERO1A knockout in a mouse background of SEPN1 loss blunts ER stress and improves multiple MAM functions, including Ca levels and bioenergetics, thus reversing diaphragmatic weakness. The treatment of SEPN1 knockout mice with the ER stress inhibitor tauroursodeoxycholic acid (TUDCA) mirrors the results of ERO1A loss. Importantly, muscle biopsies from patients with SEPN1-RM exhibit ERO1A overexpression, and TUDCA-treated SEPN1-RM patient-derived primary myoblasts show improvement in bioenergetics. These findings point to ERO1A as a biomarker and a viable target for intervention and to TUDCA as a pharmacological treatment for SEPN1-RM.
Topics: Humans; Mice; Animals; Muscle Proteins; Muscular Diseases; Taurochenodeoxycholic Acid; Oxidoreductases; Mice, Knockout
PubMed: 38402623
DOI: 10.1016/j.xcrm.2024.101439 -
Bioscience Reports Aug 2023Lipid transporters play a crucial role in supporting essential cellular processes such as organelle assembly, vesicular trafficking, and lipid homeostasis by driving... (Review)
Review
Lipid transporters play a crucial role in supporting essential cellular processes such as organelle assembly, vesicular trafficking, and lipid homeostasis by driving lipid transport across membranes. Cryo-electron microscopy has recently resolved the structures of several ATP-dependent lipid transporters, but functional characterization remains a major challenge. Although studies of detergent-purified proteins have advanced our understanding of these transporters, in vitro evidence for lipid transport is still limited to a few ATP-dependent lipid transporters. Reconstitution into model membranes, such as liposomes, is a suitable approach to study lipid transporters in vitro and to investigate their key molecular features. In this review, we discuss the current approaches for reconstituting ATP-driven lipid transporters into large liposomes and common techniques used to study lipid transport in proteoliposomes. We also highlight the existing knowledge on the regulatory mechanisms that modulate the activity of lipid transporters, and finally, we address the limitations of the current approaches and future perspectives in this field.
Topics: Liposomes; ATP-Binding Cassette Transporters; Cryoelectron Microscopy; Membrane Transport Proteins; Adenosine Triphosphate
PubMed: 37417269
DOI: 10.1042/BSR20221268 -
Antimicrobial Resistance and Infection... Oct 2023Coagulase-Negative Staphylococci (CoNS) are opportunistic and nosocomial pathogens. The excessive use of antimicrobial agents, including antiseptics, represents one of...
BACKGROUND
Coagulase-Negative Staphylococci (CoNS) are opportunistic and nosocomial pathogens. The excessive use of antimicrobial agents, including antiseptics, represents one of the world's major public health problems. This study aimed to test the susceptibility of CoNS to antiseptics.
METHODS
Out of 250 specimens collected from different sections of the hospital, 55 samples were identified as CoNS, categorized into three groups based on their sources: environmental samples (n = 32), healthcare worker carriers samples (n = 14), and clinical infection samples (n = 9). Isolates were examined for susceptibility to antibiotics and antiseptics, such as benzalkonium chloride (BC), cetyltrimethylammonium bromide (CTAB), and chlorhexidine digluconate (CHDG). Mupirocin and antiseptic resistance genes, as well as the mecA gene, were detected using polymerase chain reaction. CoNS isolates with notable resistance to antiseptics and antibiotics were identified using the API-Staph system.
RESULTS
A high frequency of multidrug resistance among CoNS clinical infection isolates was observed. Approximately half of the CoNS isolates from healthcare workers were susceptible to CHDG, but 93% were resistant to BC and CTAB. The frequency of antiseptics and antibiotics resistance genes in CoNS isolates was as follows: qacA/B (51/55; 92.7%), smr (22/55; 40.0%), qacG (1/55; 1.8%), qacH (6/55; 10.9%), qacJ (4/55; 7.3%), mecA (35/55; 63.6%), mupB (10/55; 18.2%), and mupA (7/55; 12.7%). A significant difference in the prevalence of smr gene and qacJ genes between CoNS isolates from healthcare workers and other isolates was reported (P value = 0.032 and ˂0.001, respectively). Four different CoNS species; S. epidermidis, S. chromogene, S. haemolyticus, and S. hominis, were identified by API.
CONCLUSIONS
CoNS isolates colonizing healthcare workers showed a high prevalence of antiseptic resistance genes, while clinical infection samples were more resistant to antibiotics. CHDG demonstrated greater efficacy than BC and CTAB in our hospital.
Topics: Humans; Anti-Infective Agents, Local; Mupirocin; Coagulase; Cetrimonium; Staphylococcal Infections; Bacterial Proteins; Staphylococcus; Anti-Bacterial Agents; Staphylococcus epidermidis; Benzalkonium Compounds
PubMed: 37794413
DOI: 10.1186/s13756-023-01310-3 -
Animals : An Open Access Journal From... Sep 2023This study aimed to evaluate (1) the net energy for lactation of broken rice in dairy cows and (2) the effects of broken rice substituting in diets on feed intake,...
This study aimed to evaluate (1) the net energy for lactation of broken rice in dairy cows and (2) the effects of broken rice substituting in diets on feed intake, nutrient energy utilization, and milk production. An energy metabolism experiment was conducted using a respiration chamber system in four multiparous Holstein crossbred cows (88.6% Holstein × 11.4% Native Thai; body weight of 438 ± 16.0 kg; 70 ± 31 days in milk) according to a 4 × 4 Latin square design with four 21-d periods. The four dietary treatments included a basal diet substitution with broken rice at 0%, 12%, 24%, and 36%. Increasing the substitution rate of broken rice in the diet resulted in unaffected feed intake, milk yield and composition, and energy balance ( > 0.05); however, a linear increase in the digestibility of dry matter, organic matter, and neutral detergent fiber ( < 0.05). The estimated net energy for lactation of broken rice was 8.68 MJ/kg. The net energy requirement for maintenance was estimated at 504 kJ/kg of metabolic body weight. Our results indicated that broken rice is a good energy-feed resource and that increasing the proportion in the diet up to 36% had no adverse effect on dairy cows' production performance.
PubMed: 37835648
DOI: 10.3390/ani13193042