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Analytical Chemistry Jun 2024Lipids are critical modulators of membrane protein structure and function. However, it is challenging to investigate the thermodynamics of protein-lipid interactions...
Lipids are critical modulators of membrane protein structure and function. However, it is challenging to investigate the thermodynamics of protein-lipid interactions because lipids can simultaneously bind membrane proteins at different sites with different specificities. Here, we developed a native mass spectrometry (MS) approach using single and double mutants to measure the relative energetic contributions of specific residues on Aquaporin Z (AqpZ) toward cardiolipin (CL) binding. We first mutated potential lipid-binding residues on AqpZ, and mixed mutant and wild-type proteins together with CL. By using native MS to simultaneously resolve lipid binding to the mutant and wild-type proteins in a single spectrum, we directly determined the relative affinities of CL binding, thereby revealing the relative Gibbs free energy change for lipid binding caused by the mutation. Comparing different mutants revealed that W14 contributes to the tightest CL binding site, with R224 contributing to a lower affinity site. Using double mutant cycling, we investigated the synergy between W14 and R224 sites on CL binding. Overall, this novel native MS approach provides unique insights into the binding of lipids to specific sites on membrane proteins.
Topics: Cardiolipins; Mass Spectrometry; Mutation; Aquaporins; Binding Sites; Protein Binding; Membrane Proteins; Thermodynamics; Models, Molecular; Escherichia coli Proteins
PubMed: 38859611
DOI: 10.1021/acs.analchem.4c01704 -
Journal of Clinical Apheresis Jun 2024Plasma exchange (PE) is considered a Category II option for the treatment of acute attacks and relapse cases of neuromyelitis optica spectrum disorder (NMOSD). However,... (Comparative Study)
Comparative Study
INTRODUCTION
Plasma exchange (PE) is considered a Category II option for the treatment of acute attacks and relapse cases of neuromyelitis optica spectrum disorder (NMOSD). However, neurologists are also considering intravenous immunoglobulins (IVIg) as an add-on therapy for this disorder.
AIMS
The aim of this study is to evaluate the efficacy of PE in acute attacks of NMOSD when compared with IVIg, in terms of improvement in the Expanded disability status scale (EDSS) and activities of daily living (ADL) scale score and levels of anti-Aquaporin P4 (AQP4) antibody in seropositive patients.
METHODS
We enrolled 43 NMOSD patients in two groups: Group 1 (n = 29) received steroids and PE, and Group 2 (n = 14) received steroids with IVIg. The baseline EDSS and ADL scores were recorded and compared with scores at the end of therapy, 4 weeks, and 3 months after. Also, anti-AQP4 antibody was measured at baseline and post-therapy in seropositive patients of both groups.
RESULTS
We observed a significant difference in EDSS (p = 0.00) and ADL score (p = 0.00) at day 10 and 3 months in both groups. However, no significant difference in EDSS, as well as ADL score from baseline (p = 0.83; p = 0.25) to 3 months (p = 0.85; p = 0.19), was observed when delta change of score at 3 months was compared across the two groups (p = 0.39; p = 0.52). We observed improved visual acuity in both groups with mild improvement in findings of magnetic resonance imaging at 3 months. We observed a significant decline in AQP4 antibody concentration (at day 10) in group 1 seropositive patients (p = 0.013) with improved EDSS (p = 0.027) and ADL scores (p = 0.026) of these patients.
CONCLUSIONS
PE should be considered as a choice of an add-on therapy in anti-AQP4 antibody-positive NMOSD patients compared with IVIg as it is more effective in reducing antibody concentrations.
Topics: Humans; Neuromyelitis Optica; Immunoglobulins, Intravenous; Plasma Exchange; Female; Adult; Male; Aquaporin 4; Middle Aged; Activities of Daily Living; Treatment Outcome; Autoantibodies
PubMed: 38850074
DOI: 10.1002/jca.22129 -
Journal of Cystic Fibrosis : Official... Jun 2024The management of cystic fibrosis (CF) requires knowledge of the patient's microbiological status. The serology of anti-Pseudomonas aeruginosa antibodies against...
BACKGROUND
The management of cystic fibrosis (CF) requires knowledge of the patient's microbiological status. The serology of anti-Pseudomonas aeruginosa antibodies against exoenzymes or water-soluble antigens has gained diagnostic value, particularly to detect the onset of colonization with P. aeruginosa. However, the diversity and variable expression of these antigens, which was unknown when the ELISAs became common diagnostic procedures at CF clinics, prohibits the quantitative evaluation of bacterial antigen load during intermittent and chronic infection.
METHODS
An ELISA was developed to measure the serum IgG antibody levels against P. aeruginosa porin OprF, a species-specific, conserved, immunogenic and constitutively expressed protein present in the outer membrane and extracellular vesicles.
RESULTS
Serial serum samples were collected from 310 people with CF (pwCF) over a period of up to 15 years. Compared to a reference of P. aeruginosa - negative CF sera set to 1, OprF antibody titers ranged from 0.3 to 13.2 (median: 1.7) in 56 intermittently colonized patients and from 0.5 to 51.2 (median: 11.8) in 176 chronically colonized pwCF showing higher anti-OprF antibody levels during chronic than during intermittent colonization with P. aeruginosa (P = 0, Z = - 21.7, effect size 0.62). Inhalation with twice daily 80 mg tobramycin decreased OprF antibody titers (P = 5 × 10), particularly during the third and fourth year of chronic colonization.
CONCLUSION
The OprF ELISA should be an appropriate tool to monitor Pseudomonas serology at all stages of infection and disease severity and to study the impact of short- and long-term therapeutic interventions.
PubMed: 38845269
DOI: 10.1016/j.jcf.2024.06.001 -
Journal of Chemical Information and... Jun 2024Cholesterol (CHL) plays an integral role in modulating the function and activity of various mammalian membrane proteins. Due to the slow dynamics of lipids, conventional...
Cholesterol (CHL) plays an integral role in modulating the function and activity of various mammalian membrane proteins. Due to the slow dynamics of lipids, conventional computational studies of protein-CHL interactions rely on either long-time scale atomistic simulations or coarse-grained approximations to sample the process. A highly mobile membrane mimetic (HMMM) has been developed to enhance lipid diffusion and thus used to facilitate the investigation of lipid interactions with peripheral membrane proteins and, with customized solvents to replace phospholipid tails, with integral membrane proteins. Here, we report an updated HMMM model that is able to include CHL, a nonphospholipid component of the membrane, henceforth called HMMM-CHL. To this end, we had to optimize the effect of the customized solvents on CHL behavior in the membrane. Furthermore, the new solvent is compatible with simulations using force-based switching protocols. In the HMMM-CHL, both improved CHL dynamics and accelerated lipid diffusion are integrated. To test the updated model, we have applied it to the characterization of protein-CHL interactions in two membrane protein systems, the human β-adrenergic receptor (βAR) and the mitochondrial voltage-dependent anion channel 1 (VDAC-1). Our HMMM-CHL simulations successfully identified CHL binding sites and captured detailed CHL interactions in excellent consistency with experimental data as well as other simulation results, indicating the utility of the improved model in applications where an enhanced sampling of protein-CHL interactions is desired.
Topics: Cholesterol; Humans; Molecular Dynamics Simulation; Receptors, Adrenergic, beta-2; Voltage-Dependent Anion Channel 1; Protein Binding; Membrane Proteins; Models, Molecular; Diffusion; Solvents
PubMed: 38844760
DOI: 10.1021/acs.jcim.4c00619 -
Biochimica Et Biophysica Acta.... Aug 2024Albumin infusions improve circulatory and renal function in patients with decompensated cirrhosis. However, there is no convincing evidence that hypoalbuminemia...
Albumin infusions improve circulatory and renal function in patients with decompensated cirrhosis. However, there is no convincing evidence that hypoalbuminemia contributes to ascites formation in liver cirrhosis. The aim of our study is to determine the exact role of hypoalbuminemia in the formation of ascites caused by liver cirrhosis and its underlying mechanism. Clinical profiles of patients with liver cirrhosis retrospectively analyzed. The details of albumin involved in ascites formation were investigated in rat model and murine model. Statistical analysis demonstrated hypoalbuminemia was an independent risk factor for ascites formation in patients with liver cirrhosis (OR = 0.722, P < 0.001). In carbon tetrachloride (CCl)-induced rat model of liver cirrhosis, a significant reduction in serum albumin was observed in rats with ascites (13.37 g/L) compared with rats without ascites (21.43 g/L, P < 0.001). In thioacetamide (TAA)-treated mice, ascites amount of heterozygous albumin (Alb) mice (112.0 mg) was larger than that of wild-type (Alb) mice (58.46 mg, P < 0.001). In CCl-induced chronic liver injury, ascites amounts of Alb or Alb mice were 80.00 mg or 48.46 mg (P = 0.001). Further study demonstrated 24-h urinary sodium excretion in Alb mice was lower than that of Alb mice in TAA/CCl-induce murine models of liver cirrhosis. Additionally, serum sodium concentration of Alb mice was lower than that of Alb mice. In cirrhotic mice, higher level of antidiuretic hormone was observed in Alb mice compared with the control; and renal aquaporin (AQP2) expression in Alb mice was significantly higher than that of WT mice. These revealed hypoalbuminemia contributed to the occurrence of ascites in liver cirrhosis through sodium and water retention.
Topics: Animals; Hypoalbuminemia; Ascites; Sodium; Mice; Male; Humans; Liver Cirrhosis; Female; Rats; Carbon Tetrachloride; Middle Aged; Aquaporin 2; Disease Models, Animal; Retrospective Studies; Serum Albumin; Thioacetamide; Water; Aged
PubMed: 38844112
DOI: 10.1016/j.bbadis.2024.167275 -
Nucleic Acids Research Jun 2024The ompD transcript, encoding an outer membrane porin in Salmonella, harbors a controlling element in its coding region that base-pairs imperfectly with a 'seed' region...
The ompD transcript, encoding an outer membrane porin in Salmonella, harbors a controlling element in its coding region that base-pairs imperfectly with a 'seed' region of the small regulatory RNA (sRNA) MicC. When tagged with the sRNA, the ompD mRNA is cleaved downstream of the pairing site by the conserved endoribonuclease RNase E, leading to transcript destruction. We observe that the sRNA-induced cleavage site is accessible to RNase E in vitro upon recruitment of ompD into the 30S translation pre-initiation complex (PIC) in the presence of the degradosome components. Evaluation of substrate accessibility suggests that the paused 30S PIC presents the mRNA for targeted recognition and degradation. Ribonuclease activity on PIC-bound ompD is critically dependent on the recruitment of RNase E into the multi-enzyme RNA degradosome, and our data suggest a process of substrate capture and handover to catalytic sites within the degradosome, in which sequential steps of seed matching and duplex remodelling contribute to cleavage efficiency. Our findings support a putative mechanism of surveillance at translation that potentially terminates gene expression efficiently and rapidly in response to signals provided by regulatory RNA.
PubMed: 38842944
DOI: 10.1093/nar/gkae455 -
Genes, Brain, and Behavior Jun 2024Duchenne muscular dystrophy is a severe neuromuscular disorder that is caused by mutations in the DMD gene, resulting in a disruption of dystrophin production. Next to...
Duchenne muscular dystrophy is a severe neuromuscular disorder that is caused by mutations in the DMD gene, resulting in a disruption of dystrophin production. Next to dystrophin expression in the muscle, different isoforms of the protein are also expressed in the brain and lack of these isoforms leads to cognitive and behavioral deficits in patients. It remains unclear how the loss of the shorter dystrophin isoform Dp140 affects these processes. Using a variety of behavioral tests, we found that mdx and mdx mice (which lack Dp427 or Dp427 + Dp140, respectively) exhibit similar deficits in working memory, movement patterns and blood-brain barrier integrity. Neither model showed deficits in spatial learning and memory, learning flexibility, anxiety or spontaneous behavior, nor did we observe differences in aquaporin 4 and glial fibrillary acidic protein. These results indicate that in contrast to Dp427, Dp140 does not play a crucial role in processes of learning, memory and spontaneous behavior.
Topics: Animals; Mice; Blood-Brain Barrier; Muscular Dystrophy, Duchenne; Dystrophin; Male; Mice, Inbred mdx; Mice, Inbred C57BL; Aquaporin 4; Memory, Short-Term; Memory
PubMed: 38837620
DOI: 10.1111/gbb.12895 -
PLoS Genetics Jun 2024The cell envelope fortifies bacterial cells against antibiotics and other insults. Species in the Mycobacteriales order have a complex envelope that includes an outer...
The cell envelope fortifies bacterial cells against antibiotics and other insults. Species in the Mycobacteriales order have a complex envelope that includes an outer layer of mycolic acids called the mycomembrane (MM) and a cell wall composed of peptidoglycan and arabinogalactan. This envelope architecture is unique among bacteria and contributes significantly to the virulence of pathogenic Mycobacteriales like Mycobacterium tuberculosis. Characterization of pathways that govern envelope biogenesis in these organisms is therefore critical in understanding their biology and for identifying new antibiotic targets. To better understand MM biogenesis, we developed a cell sorting-based screen for mutants defective in the surface exposure of a porin normally embedded in the MM of the model organism Corynebacterium glutamicum. The results revealed a requirement for the conserved σD envelope stress response in porin export and identified MarP as the site-1 protease, respectively, that activate the response by cleaving the membrane-embedded anti-sigma factor. A reporter system revealed that the σD pathway responds to defects in mycolic acid and arabinogalactan biosynthesis, suggesting that the stress response has the unusual property of being induced by activating signals that arise from defects in the assembly of two distinct envelope layers. Our results thus provide new insights into how C. glutamicum and related bacteria monitor envelope integrity and suggest a potential role for members of the σD regulon in protein export to the MM.
Topics: Cell Wall; Corynebacterium glutamicum; Mycolic Acids; Sigma Factor; Cell Membrane; Stress, Physiological; Porins; Bacterial Proteins; Galactans; Gene Expression Regulation, Bacterial; Peptidoglycan
PubMed: 38829907
DOI: 10.1371/journal.pgen.1011127 -
NeuroImage Jul 2024Understanding the physiological processes in aging and how neurodegenerative disorders affect cognitive function is a high priority for advancing human health. One...
Understanding the physiological processes in aging and how neurodegenerative disorders affect cognitive function is a high priority for advancing human health. One specific area of recently enabled research is the in vivo biomechanical state of the brain. This study utilized reverberant optical coherence elastography, a high-resolution elasticity imaging method, to investigate stiffness changes during the sleep/wake cycle, aging, and Alzheimer's disease in murine models. Four-dimensional scans of 44 wildtype mice, 13 mice with deletion of aquaporin-4 water channel, and 12 mice with Alzheimer-related pathology (APP/PS1) demonstrated that (1) cortical tissue became softer (on the order of a 10% decrease in shear wave speed) when young wildtype mice transitioned from wake to anesthetized, yet this effect was lost in aging and with mice overexpressing amyloid-β or lacking the water channel AQP4. (2) Cortical stiffness increased with age in all mice lines, but wildtype mice exhibited the most prominent changes as a function of aging. The study provides novel insight into the brain's biomechanics, the constraints of fluid flow, and how the state of brain activity affects basic properties of cortical tissues.
Topics: Animals; Alzheimer Disease; Aging; Elasticity Imaging Techniques; Mice; Brain; Sleep; Wakefulness; Mice, Transgenic; Aquaporin 4; Male; Mice, Inbred C57BL
PubMed: 38823503
DOI: 10.1016/j.neuroimage.2024.120662 -
Microbial Pathogenesis Jul 2024Pseudomonas aeruginosa is a significant pathogen responsible for severe multisite infections with high morbidity and mortality rates. This study analyzed...
Pseudomonas aeruginosa is a significant pathogen responsible for severe multisite infections with high morbidity and mortality rates. This study analyzed carbapenem-resistant Pseudomonas aeruginosa (CRPA) at a tertiary hospital in Shandong, China, using whole-genome sequencing (WGS). The objective was to explore the mechanisms and molecular characteristics of carbapenem resistance. A retrospective analysis of 91 isolates from January 2022 to March 2023 was performed, which included strain identification and antimicrobial susceptibility testing. WGS was utilized to determine the genome sequences of these CRPA strains, and the species were precisely identified using average nucleotide identification (ANI), with further analysis on multilocus sequence typing and strain relatedness. Some strains were found to carry the ampD and oprD genes, while only a few harbored carbapenemase genes or related genes. Notably, all strains possessed the mexA, mexE, and mexX genes. The major lineage identified was ST244, followed by ST235. The study revealed a diverse array of carbapenem resistance mechanisms among hospital isolates, differing from previous studies in mainland China. It highlighted that carbapenem resistance is not due to a single mechanism but rather a combination of enzyme-mediated resistance, AmpC overexpression, OprD dysfunction, and efflux pump overexpression. This research provides valuable insights into the evolutionary mechanisms and molecular features of CRPA resistance in this region, aiding in the national prevention and control of CRPA, and offering references for targeting and developing new drugs.
Topics: Pseudomonas aeruginosa; Multilocus Sequence Typing; Whole Genome Sequencing; China; Carbapenems; Humans; Anti-Bacterial Agents; Bacterial Proteins; Microbial Sensitivity Tests; Pseudomonas Infections; Retrospective Studies; beta-Lactamases; Porins; Genome, Bacterial; Membrane Transport Proteins; Tertiary Care Centers; Bacterial Outer Membrane Proteins
PubMed: 38815778
DOI: 10.1016/j.micpath.2024.106720