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The Journal of Biological Chemistry May 2024Extracellular secretion is an essential mechanism for α-synuclein (α-syn) proteostasis. Although it was reported that neuronal activity affects α-syn secretion, the...
Extracellular secretion is an essential mechanism for α-synuclein (α-syn) proteostasis. Although it was reported that neuronal activity affects α-syn secretion, the underlying mechanisms remain unclear. Here, we investigated the autophagic processes that regulate the physiological release of α-syn in mouse primary cortical neurons and SH-SY5Y cells. Stimulating neuronal activity with glutamate or depolarization with high KCl enhanced α-syn secretion. This glutamate-induced α-syn secretion was blocked by a mixture of NMDA receptor antagonist AP5 and AMPA receptor antagonist NBQX, as well as by cytosolic Ca chelator BAPTA-AM. Additionally, mTOR inhibitor rapamycin increased α-syn and p62/SQSTM1 (p62) secretion, and this effect of rapamycin was reduced in primary cortical neurons deficient in the autophagy regulator beclin 1 (derived from BECN1 mice). Glutamate-induced α-syn and p62 secretion was suppressed by knockdown of ATG5, which is required for autophagosome formation. Glutamate increased LC3-II generation and decreased intracellular p62 levels, and the increase in LC3-II levels was blocked by BAPTA-AM. Moreover, glutamate promoted co-localization of α-syn with LC3-positive puncta, but not with LAMP1-positive structures in the neuronal somas. Glutamate-induced α-syn and p62 secretion was also reduced by knockdown of RAB8A, which is required for autophagosome fusion with the plasma membrane. Collectively, these findings suggest that stimulating neuronal activity mediates autophagic α-syn secretion in a cytosolic Ca-dependent manner, and autophagosomes may participate in autophagic secretion by functioning as α-syn carriers.
PubMed: 38815862
DOI: 10.1016/j.jbc.2024.107419 -
JCI Insight May 2024The non-physiological nutrient levels found in traditional culture media have been shown to affect numerous aspects of cancer cell physiology, including how cells...
The non-physiological nutrient levels found in traditional culture media have been shown to affect numerous aspects of cancer cell physiology, including how cells respond to certain therapeutic agents. Here, we comprehensively evaluated how physiological nutrient levels impact therapeutic response by performing drug screening in human plasma-like medium (HPLM). We observed dramatic nutrient-dependent changes in sensitivity to a variety of FDA-approved and clinically trialed compounds including rigosertib, an experimental cancer therapeutic that has recently failed in phase 3 clinical trials. Mechanistically, we found that the ability of rigosertib to destabilize microtubules is strongly inhibited by the purine metabolism end product uric acid, which is uniquely abundant in humans relative to traditional in vitro and in vivo cancer models. These results demonstrate the broad and dramatic effects nutrient levels can have on drug response, and how incorporation of human-specific physiological nutrient media might help to identify compounds whose efficacy could be impacted in humans.
PubMed: 38815134
DOI: 10.1172/jci.insight.174329 -
Blood Transfusion = Trasfusione Del... May 2024Several countries have recently reassessed the international risk of variant Creutzfeldt-Jakob disease (vCJD) transmission through transfusion of blood and blood...
Several countries have recently reassessed the international risk of variant Creutzfeldt-Jakob disease (vCJD) transmission through transfusion of blood and blood components (red blood cells, platelets and plasma) and relaxed donor deferrals based on geographic and transfusion exposure in countries formerly considered to be high risk, such as the UK. In this regard, the European Blood Alliance organised a consensus meeting of experts and involved professionals to discuss current knowledge, epidemiological data, prevention and various methods for assessing the risk of transfusion-transmitted vCJD, as well as to develop an appropriate position on possible approaches to address these challenges in Europe. Participants reached a consensus that the current risk of transfusion-transmitted vCJD associated with blood donors who either travelled to or received transfusions in the UK during the vCJD outbreak is minimal. In addressing such risks, it would be pragmatic that assessments and guidelines are developed by European expert bodies, rather than individual assessments by Member States. Regardless of the approach used, European or national, a qualitative risk assessment based on a review and analysis of available data, considering all the uncertainties and experiences of other countries, would provide crucial information to reassess blood donation strategies regarding the transfusion-associated vCJD risk.
PubMed: 38814884
DOI: 10.2450/BloodTransfus.778 -
Blood Transfusion = Trasfusione Del... May 2024Quality assessment of modified or processed red blood cell (RBC) components, such as pathogen-reduced RBCs, using only in vitro testing may not always be predictive of...
BACKGROUND
Quality assessment of modified or processed red blood cell (RBC) components, such as pathogen-reduced RBCs, using only in vitro testing may not always be predictive of in vivo performance. Mouse or rat in vivo models are limited by a lack of applicability to certain aspects of human RBC biology. Here, we used a guinea pig model to study the effects of riboflavin combined with UV light on the integrity of RBCs in vitro and following transfusion in vivo.
MATERIALS AND METHODS
Guinea pig RBCs were collected from whole blood (WB) treated with varying UV doses (10, 20, 40 or 80 J/mL) in the presence of riboflavin (UVR-RBCs). In vitro tests for UVR-RBCs included hemolysis, osmotic fragility, and cellular morphology by scanning electron microscopy. Guinea pigs transfused with one-day post-treatment UVR-RBCs were evaluated for plasma hemoglobin (Hb), non-transferrin bound iron (NTBI), total iron and Perls-detectable hemosiderin deposition in the spleen and kidney, and renal uptake of Hb.
RESULTS
Acute RBC injury was dose dependently accelerated after treatment with UV light in the presence of riboflavin. Aberrant RBC morphology was evident at 20, 40, and 80 J/mL, and membrane lysis with Hb release was prominent at 80 J/mL. Guinea pigs transfused with 40 and 80 J/mL UVR-RBCs showed increased plasma Hb levels, and plasma NTBI was elevated in all UVR-RBC groups (10-80 J/mL). Total iron levels and Perls-hemosiderin staining in spleen and kidney as well as Hb uptake in renal proximal tubules were increased 8 hours post-transfusion with 40 and 80 J/mL UVR-RBCs.
DISCUSSION
UVR-RBCs administered to guinea pigs increased markers of intravascular and extravascular hemolysis in a UV dose-dependent manner. This model may allow for the discrimination of RBC injury during testing of extensively processed RBCs intended for transfusion.
PubMed: 38814883
DOI: 10.2450/BloodTransfus.718 -
MSphere May 2024is the leading cause of severe mold infections in immunocompromised patients. This common fungus possesses innate attributes that allow it to evade the immune system,...
is the leading cause of severe mold infections in immunocompromised patients. This common fungus possesses innate attributes that allow it to evade the immune system, including its ability to survive the high copper (Cu) levels in phagosomes. Our previous work has revealed that under high Cu levels, the transcription factor AceA is activated, inducing the expression of the copper exporter CrpA to expel excess Cu. To identify additional elements in Cu resistance, we evolved wild-type and mutant Δ or Δ strains under increasing Cu concentrations. Sequencing of the resultant resistant strains identified both shared and unique evolutionary pathways to resistance. Reintroduction of three of the most common mutations in genes encoding Pma1 (plasma membrane H-ATPase), Gcs1 (glutamate cysteine-ligase), and Cpa1 (carbamoyl-phosphate synthetase), alone and in combination, into wild-type confirmed their additive role in conferring Cu resistance. Detailed analysis indicated that the mutation L424I preserves Pma1 H-ATPase activity under high Cu concentrations and that the mutation A37V confers a survival advantage to conidia in the presence of Cu. Interestingly, simultaneous mutations of all three genes did not alter virulence in infected mice. Our work has identified novel Cu-resistance pathways and provides an evolutionary approach for dissecting the molecular basis of adaptation to diverse environmental challenges.IMPORTANCE is the most common mold infecting patients with weakened immunity. Infection is caused by the inhalation of mold spores into the lungs and is often fatal. In healthy individuals, spores are engulfed by lung immune cells and destroyed by a combination of enzymes, oxidants, and high levels of copper. However, the mold can protect itself by pumping out excess copper with specific transporters. Here, we evolved under high copper levels and identified new genetic mutations that help it resist the toxic effects of copper. We studied how these mutations affect the mold's ability to resist copper and how they impact its ability to cause disease. This is the first such study in a pathogenic mold, and it gives us a better understanding of how it manages to bypass our body's defenses during an infection.
PubMed: 38814077
DOI: 10.1128/msphere.00253-24 -
Haematologica May 2024Multiple myeloma (MM) remains an incurable hematological malignancy. Despite tremendous advances in the treatment, about 10% of patients still have very poor outcomes...
Multiple myeloma (MM) remains an incurable hematological malignancy. Despite tremendous advances in the treatment, about 10% of patients still have very poor outcomes with median overall survival less than 24 months. Our study aimed to underscore the critical mechanisms pertaining to the rapid disease progression and provide novel therapeutic selection for these ultra-high-risk patients. We utilized single-cell transcriptomic sequencing to dissect the characteristic bone marrow niche of patients with survival of less than two years (EM24). Notably, an enrichment of LILRB4high pre-matured plasma-cell cluster was observed in the patients in EM24 compared to patients with durable remission. This cluster exhibited aggressive proliferation and drug-resistance phenotype. High-level LILRB4 promoted MM clonogenicity and progression. Clinically, high expression of LILRB4 was correlated with poor prognosis in both newly diagnosed MM patients and relapsed/refractory MM patients. The ATAC-seq analysis identified that high chromosomal accessibility caused the elevation of LILRB4 on MM cells. CRISPR-Cas9 deletion of LILRB4 alleviated the growth of MM cells, inhibited the immunosuppressive function of MDSCs, and further rescued T cell dysfunction in MM microenvironment. The more infiltration of myeloid-derived suppressive cells (MDSCs) was observed in EM24 patients as well. Therefore, we innovatively generated a TCR-based chimeric antigen receptor (CAR) T cell, LILRB4-STAR-T. Cytotoxicity experiment demonstrated that LILRB4-STAR-T cells efficaciously eliminated tumor cells and impeded MDSCs function. In conclusion, our study elucidates that LILRB4 is an ideal biomarker and promising immunotherapy target for high-risk MM. LILRB4-STAR-T cell immunotherapy is promising against tumor cells and immunosuppressive tumor microenvironment in MM.
PubMed: 38813706
DOI: 10.3324/haematol.2024.285099 -
Arteriosclerosis, Thrombosis, and... May 2024Pulmonary hypertension (PH) represents an important phenotype in heart failure with preserved ejection fraction (HFpEF). However, management of PH-HFpEF is challenging...
BACKGROUND
Pulmonary hypertension (PH) represents an important phenotype in heart failure with preserved ejection fraction (HFpEF). However, management of PH-HFpEF is challenging because mechanisms involved in the regulation of PH-HFpEF remain unclear.
METHODS
We used a mass spectrometry-based comparative plasma proteomics approach as a sensitive and comprehensive hypothesis-generating discovery technique to profile proteins in patients with PH-HFpEF and control subjects. We then validated and investigated the role of one of the identified proteins using in vitro cell cultures, in vivo animal models, and independent cohort of human samples.
RESULTS
Plasma proteomics identified high protein abundance levels of B2M (β2-microglobulin) in patients with PH-HFpEF. Interestingly, both circulating and skeletal muscle levels of B2M were increased in mice with skeletal muscle SIRT3 (sirtuin-3) deficiency or high-fat diet-induced PH-HFpEF. Plasma and muscle biopsies from a validation cohort of PH-HFpEF patients were found to have increased B2M levels, which positively correlated with disease severity, especially pulmonary capillary wedge pressure and right atrial pressure at rest. Not only did the administration of exogenous B2M promote migration/proliferation in pulmonary arterial vascular endothelial cells but it also increased PCNA (proliferating cell nuclear antigen) expression and cell proliferation in pulmonary arterial vascular smooth muscle cells. Finally, deletion improved glucose intolerance, reduced pulmonary vascular remodeling, lowered PH, and attenuated RV hypertrophy in mice with high-fat diet-induced PH-HFpEF.
CONCLUSIONS
Patients with PH-HFpEF display higher circulating and skeletal muscle expression levels of B2M, the magnitude of which correlates with disease severity. Our findings also reveal a previously unknown pathogenic role of B2M in the regulation of pulmonary vascular proliferative remodeling and PH-HFpEF. These data suggest that circulating and skeletal muscle B2M can be promising targets for the management of PH-HFpEF.
PubMed: 38813697
DOI: 10.1161/ATVBAHA.123.320270 -
Therapeutic Advances in Neurological... 2024Myasthenia gravis (MG) is an autoimmune disorder characterized by fluctuating muscle weakness. Severe patients may develop life-threatening respiratory failure and...
Myasthenia gravis (MG) is an autoimmune disorder characterized by fluctuating muscle weakness. Severe patients may develop life-threatening respiratory failure and experience crisis. Plasma exchange or intravenous immunoglobulin (IVIg) is the first-line treatment option for myasthenia crisis, but some patients still poorly respond to them. Here, we first reported a generalized MG patient from China who was in a state of impending myasthenic crisis and did not respond effectively to IVIg but was successfully rescued by add-on efgartigimod. Especially, we also detected meaningful changes in T-cell and B-cell subsets after efgartigimod, promoting a potential role of efgartigimod in re-establishing immune homeostasis.
PubMed: 38813520
DOI: 10.1177/17562864241254895 -
Turkish Journal of Medical Sciences 2023Atherosclerosis is significantly influenced by endothelial cell activation and dysfunction. Studies have demonstrated the substantial presence of fibronectin (Fn) within...
BACKGROUND/AIM
Atherosclerosis is significantly influenced by endothelial cell activation and dysfunction. Studies have demonstrated the substantial presence of fibronectin (Fn) within atherosclerotic plaques, promoting endothelial inflammation and activation. However, cellular Fn (cFn) secreted by various cell types, including endothelial cells and smooth muscle cells, and plasma Fn (pFn) produced by hepatocytes. They are distinct forms of Fn that differ in both structure and function. The specific contribution of different types of Fn in promoting endothelial cell activation and dysfunction remain uncertain. Therefore, this study aimed to investigate the respective roles of pFn and endothelial cell-derived Fn (Fn) in promoting endothelial cell activation and dysfunction.
MATERIALS AND METHODS
Initially, endothelial cell injury was induced by exposing the cells to oxidized low-density lipoprotein (ox-LDL) and subsequently we generated a mutant strain of aortic endothelial cells with Fn knockdown (Fn). The impact of the Fn arel the addition of pFn on the expression levels of inflammatory factors, vasoconstrictors, and diastolic factors were compared.
RESULTS
The results showed that the Fn significantly inhibited ox-LDL-induced intercellular adhesion molecule 1 (ICAM-1, p < 0.05), vascular cell adhesion molecule (VCAM-1, p < 0.05), and endothelin (p < 0.05) expression, and nuclear factor kappa-B (NFκB, p < 0.05) activation. These results implied that Fn inhibited both endothelial cell activation and dysfunction. Surprisingly, the addition of pFn significantly inhibited the ox-LDL-induced ICAM-1 (p < 0.05), VCAM-1 (p < 0.05), and endothelin (p < 0.05) expression and NFκB (p < 0.05) activation. Implying that pFn inhibits endothelial cell activation and dysfunction. Additionally, the study revealed that ox-LDL stimulation enhanced the production of excessive nitric oxide, leading to severe endothelial cell damage.
CONCLUSION
Aortic Fn promotes endothelial cell activation and endothelial dysfunction, whereas pFn inhibits ox-LDL-induced endothelial cell activation and endothelial dysfunction.
Topics: Fibronectins; Lipoproteins, LDL; Endothelial Cells; Humans; Vascular Cell Adhesion Molecule-1; Intercellular Adhesion Molecule-1; Endothelium, Vascular; Cells, Cultured; NF-kappa B
PubMed: 38813506
DOI: 10.55730/1300-0144.5735 -
Turkish Journal of Medical Sciences 2023This study was designed to evaluate the relationship of two new biomarkers [tribbles homolog 3 (TRB3) and sestrin 2 levels], which were previously associated with...
BACKGROUND/AIM
This study was designed to evaluate the relationship of two new biomarkers [tribbles homolog 3 (TRB3) and sestrin 2 levels], which were previously associated with obesity, with metabolic parameters in obese and nonobese women with polycystic ovary syndrome (PCOS).
MATERIALS AND METHODS
This cross-sectional case control study was conducted between September 2017 and August 2019 in the gynecology department of a tertiary referral hospital. The values of the plasma sestrin 2, TRB3, insulin, fasting plasma glucose, lipid profile, and homeostasis model assessment of insulin resistance (HOMA-IR) were compared in 90 obese women with PCOS (BMI > 30), 90 women with nonobese PCOS (BMI < 30), and 90 control patients (BMI < 30).
RESULTS
The mean age of the study group consisting of all PCOS patients (26.11 ± 4.64 years) and the mean age of the control group (26.3 ± 4.4 years) were statistically similar (p = 0.239). The serum sestrin 2 values of the obese PCOS group were found to be statistically significantly lower than the control and non-obese PCOS groups (p = 0.001, p = 0.0001), while the sestrin 2 values of the nonobese PCOS group were found to be statistically significantly lower than the control group (p = 0.0001). The TRB3 values of the control group were found to be statistically significantly lower than the obese and nonobese PCOS groups (p = 0.0001), while the TRB3 values of the nonobese PCOS group were found to be statistically significantly lower than the obese PCOS group (p = 0.0001). A negative correlation was observed between the sestrin 2 level and BMI (r = -0.272 p = 0.0001), insulin (r = -0.261 p = 0.0001), and HOMA-IR levels (r = -0.250 p = 0.0001). A positive correlation was observed between the TRB3 values and TG (r = 0.248 p = 0.0001), and LDL-C values (r = 0.235 p = 0.0001).
CONCLUSION
According to the findings in this study, low sestrin 2 and high TRB3 levels may be related to impaired metabolic status in the obese PCOS group. Thus, it may be promising for the development of treatment of PCOS and associated metabolic disorder in the future.
Topics: Humans; Female; Polycystic Ovary Syndrome; Obesity; Adult; Case-Control Studies; Cross-Sectional Studies; Protein Serine-Threonine Kinases; Biomarkers; Insulin Resistance; Nuclear Proteins; Young Adult; Blood Glucose; Sestrins; Repressor Proteins; Cell Cycle Proteins
PubMed: 38813505
DOI: 10.55730/1300-0144.5738