Proteomic and metabolomic advances uncover biomarkers of mitochondrial disease pathophysiology and severity

Advancing proteomic and metabolomic technologies that integrate curated omic databases have crossed a threshold to enable their clinical utility. In this issue of the JCI, Sharma et al. exploit emerging technologies to evaluate whether biomarkers identified in the mitochondrial encephalomyopathy lactic acidosis and stroke-like episodes (MELAS) syndrome could refine disease characterization, uncover pathways to monitor therapeutic efficacy, and/or delineate disease-modifying targets. The authors analyzed blood and urine samples from patients with this genetic mitochondrial disease and elucidated proteins and metabolites related to NADH-reductive stress. These circulating biomarkers have intriguing clinical potential that implicate disease pathophysiology and may prove important biomarkers for the future management of MELAS.     

Islet-Specific T-Cell Responses and Proinflammatory Monocytes Define Subtypes of Autoantibody-Negative Ketosis-Prone Diabetes

OBJECTIVE

Ketosis-prone diabetes (KPD) is characterized by diabetic ketoacidosis (DKA) in patients lacking typical features of type 1 diabetes. A validated classification scheme for KPD includes two autoantibody-negative (“A−”) phenotypic forms: “A−β−” (lean, early onset, lacking β-cell functional reserve) and “A−β+” (obese, late onset, with substantial β-cell functional reserve after the index episode of DKA). Recent longitudinal analysis of a large KPD cohort revealed that the A−β+ phenotype includes two distinct subtypes distinguished by the index DKA episode having a defined precipitant (“provoked,” with progressive β-cell function loss over time) or no precipitant (“unprovoked,” with sustained β-cell functional reserve). These three A− KPD subtypes are characterized by absence of humoral islet autoimmune markers, but a role for cellular islet autoimmunity is unknown.

RESEARCH DESIGN AND METHODS

Islet-specific T-cell responses and the percentage of proinflammatory (CD14+CD16+) blood monocytes were measured in A−β− (n = 7), provoked A−β+ (n = 15), and unprovoked A−β+ (n = 13) KPD patients. Genotyping was performed for type 1 diabetes–associated HLA class II alleles.

RESULTS

Provoked A−β+ and A−β− KPD patients manifested stronger islet-specific T-cell responses (P < 0.03) and higher percentages of proinflammatory CD14+CD16+ monocytes (P < 0.01) than unprovoked A−β+ KPD patients. A significant relationship between type 1 diabetes HLA class II protective alleles and negative T-cell responses was observed.

CONCLUSIONS

Provoked A−β+ KPD and A−β− KPD are associated with a high frequency of cellular islet autoimmunity and proinflammatory monocyte populations. In contrast, unprovoked A−β+ KPD lacks both humoral and cellular islet autoimmunity.Ketosis-prone diabetes (KPD), characterized by presentation with diabetic ketoacidosis (DKA) in patients lacking the typical features of autoimmune type 1 diabetes, is a heterogeneous syndrome (1,2). A validated classification scheme for KPD, based on the presence or absence of β-cell autoantibodies (“A+” or “A−”) and presence or absence of β-cell functional reserve (“β+” or “β−”) (3) includes two autoantibody-negative A− phenotypic forms: “A−β−” (lean, early onset, lacking β-cell functional reserve) and “A−β+” (obese, late onset, with substantial β-cell functional reserve after the index episode of DKA). Long-term longitudinal follow-up of a large cohort of A−β+ KPD patients has revealed that this phenotype comprises two distinct subtypes distinguished by whether the index DKA episode had a defined precipitant (“provoked” A−β+) or no precipitant (“unprovoked” A−β+) (4). Provoked A−β+ KPD patients have progressive loss of β-cell function after initial recovery from the DKA episode, with relapse to insulin dependence, no sex predominance, and an increased frequency of the HLA class II alleles DQB1*0302 and DRB1*04 associated with susceptibility to autoimmune type 1 diabetes. In contrast, unprovoked A−β+ KPD patients have sustained preservation of β-cell function after recovery from the DKA episode, prolonged insulin independence, male predominance, and an increased frequency of the protective allele DQB1*0602 (4). The unique clinical features and natural histories of these two subtypes of A−β+ KPD patients suggest distinctive underlying pathophysiologic processes for each. Although an underlying “occult” autoimmune element is suggested in the provoked A−β+ subtype by progressive β-cell loss and the presence of type 1 diabetes–associated HLA susceptibility alleles, the unprovoked A−β+ subtype could represent a truly nonautoimmune syndrome of KPD.We have previously shown that the T cells of a significant proportion of individuals with an apparent phenotype of type 2 diabetes react strongly to islet antigens, despite lacking β-cell autoantibodies, and that this reactivity is associated with low C-peptide levels, indicating underlying cellular immune damage to β-cells (5,6). This finding expands the range of diabetic phenotypes—including those labeled as having “type 2” diabetes—with a potential pathophysiologic basis in islet autoimmunity. In the current study, we extended these findings to the unique, emerging forms of A− KPD. Specifically, we hypothesized that differences in cellular immune responses might distinguish the three A− KPD subtypes (A−β−, unprovoked A−β+, and provoked A−β+) with regard to a cellular autoimmune etiology. To test this hypothesis, we measured islet-specific T-cell responses using the validated cellular immunoblotting assay and islet autoantibody responses to determine the presence of islet autoimmunity in patients carefully phenotyped for these three KPD subtypes. We further assessed the percentage of proinflammatory (CD14+CD16+) monocytes in the peripheral blood of the three KPD subtypes.In healthy subjects, 90–95% of classical monocytes express high levels of the cell surface marker CD14 (CD14+), without expression of CD16 (CD16−). Inflammation and infection are associated with the emergence of a distinct monocyte population characterized by coexpression of CD14 and CD16 (CD14+CD16+). CD14+CD16+ monocytes secrete high levels of proinflammatory tumor necrosis factor-α and low levels of anti-inflammatory interleukin-10, leading to their designation as proinflammatory monocytes (7).Our results demonstrate that 1) provoked A−β+ KPD, associated with progressive loss of β-cell function, is associated with a high frequency of cellular islet autoimmunity and proinflammatory monocyte populations; 2) unprovoked A− KPD is a distinct syndrome of reversible β-cell dysfunction lacking evidence of humoral or cellular islet autoimmunity; and 3) a substantial proportion of A−β− KPD patients, who resemble patients with type 1 diabetes but lack autoantibodies, have evidence of cellular islet autoimmunity.     

Global Urine Metabolomics in Patients Treated with First-Line Tuberculosis Drugs and Identification of a Novel Metabolite of Ethambutol

Population level variation of drug metabolism phenotype (DMP) has great implications in treatment outcome, drug-related side effects, and resistance development. In this study, we used a gas chromatography-time of flight-mass spectrometry (GC-TOF-MS)-based untargeted urine metabolomics approach to understand the DMP of a tuberculosis (TB) patient cohort (n = 20) from Tripura, a state in the northeastern part of India. Urine samples collected at different postdose time points (2 h, 6 h, 12 h, 24 h, 36 h, and 48 h) from these newly diagnosed TB patients receiving first-line anti-TB drugs were analyzed, and we have successfully detected three of the four first-line drugs, viz., isoniazid (INH), ethambutol (ETB), and pyrazinamide (PZA). The majority of their known metabolites, acetyl-isoniazid (AcINH), isonicotinic acid (INA), isonicotinuric acid (INTA), 2,2′-(ethylenediimino)-dibutyric acid (EDBA), 5-hydroxypyrazinamide (5OH-PZA), pyrazinoic acid (POA), and 5-hydroxypyrazinoic acid (5OH-POA), were also detected. Analyzing the variation in abundances of drugs and their known metabolites and calculating the metabolic ratios in these samples, we offer comprehensive DMP information on this small patient cohort that represents Tripura, India. The majority (75%) of these patients are found to be slow acetylators of INH. The average metabolic ratios of POA/PZA and 5OH-POA/POA are 3.16 ± 3.03 and 6.09 ± 6.15, respectively. Employing correlation analysis of the metabolomics metadata and a manual prediction of drug catabolism, we have proposed 2-aminobutyric acid (AABA) as a novel metabolite of ETB. These observations indicate the usefulness of GC-MS-based metabolomics to characterize the DMP at a population level and also to identify novel drug metabolites.     

Preclinical validation of a potent γ-secretase modulator for Alzheimer’s disease prevention

A potent γ-secretase modulator (GSM) has been developed to circumvent problems associated with γ-secretase inhibitors (GSIs) and to potentially enable use in primary prevention of early-onset familial Alzheimer’s disease (EOFAD). Unlike GSIs, GSMs do not inhibit γ-secretase activity but rather allosterically modulate γ-secretase, reducing the net production of Aβ42 and to a lesser extent Aβ40, while concomitantly augmenting production of Aβ38 and Aβ37. This GSM demonstrated robust time- and dose-dependent efficacy in acute, subchronic, and chronic studies across multiple species, including primary and secondary prevention studies in a transgenic mouse model. The GSM displayed a >40-fold safety margin in rats based on a comparison of the systemic exposure (AUC) at the no observed adverse effect level (NOAEL) to the 50% effective AUC or AUC_effective, the systemic exposure required for reducing levels of Aβ42 in rat brain by 50%.     

Metabolites of Cerebellar Neurons and Hippocampal Neurons Play Opposite Roles in Pathogenesis of Alzheimer's Disease

Metabolites of neural cells, is known to have a significant effect on the normal physiology and function of neurons in brain. However, whether they play a role in pathogenesis of neurodegenerative diseases is unknown. Here, we show that metabolites of neurons play essential role in the pathogenesis of Alzheimer's disease (AD). Firstly, in vivo and in vitro metabolites of cerebellar neurons both significantly induced the expression of Aβ-degrading enzymes in the hippocampus and cerebral cortex and promoted Aβ clearance. Moreover, metabolites of cerebellar neurons significantly reduced brain Aβ levels and reversed cognitive impairments and other AD-like phenotypes of APP/PS1 transgenic mice, in both early and late stages of AD pathology. On the other hand, metabolites of hippocampal neurons reduced the expression of Aβ-degrading enzymes in the cerebellum and caused cerebellar neurodegeneration in APP/PS1 transgenic mice. Thus, we report, for the first time, that metabolites of neurons not only are required for maintaining the normal physiology of neurons but also play essential role in the pathogenesis of AD and may be responsible for the regional-specificity of Aβ deposition and AD pathology.     

Adult-onset of Mitochondrial Myopathy,Encephalopathy, Lactic Acidosis,and Stroke-Like Episodes (MELAS) Syndrome Presenting as Acute Meningoencephalitis: A Case Report

Background: Mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome is a rare mitochondrial disorder with a wide range of multisystemic symptoms. Epileptic seizures are common features of both MELAS and meningoencephalitis and are typically treated with anticonvulsants. Objectives: To provide the reader with a better understanding of MELAS and the adverse effects of valproic acid. Case Report: A 47-year-old man with a history of diabetes, hearing loss, sinusitis, and otitis media was brought to our emergency department due to acute onset of fever, headache, generalized seizure, and agitation. Because acute meningoencephalitis was suspected, the patient was treated with antibiotics on an empirical basis. The seizure activity was aggravated by valproic acid and abated after its discontinuation. MELAS was suspected and the diagnosis was confirmed by the presence of a nucleotide 3243 A→G mutation in the mitochondrial DNA. Conclusion: Detailed history-taking and systematic review help emergency physicians differentiate MELAS from meningoencephalitis in patients with the common presentation of epileptic seizures. Use of valproic acid to treat epilepsy in patients suspected of having mitochondrial disease should be avoided. Underlying mitochondrial disease should be suspected if seizure activity worsens with valproic acid therapy.     

MELAS with multiple stroke‐like episodes due to the variant m.13513G>A in MT‐ND5

Mitochondrial encephalopathy, lactic acidosis, and stroke‐like episodes (MELAS) is characterized by metabolic stroke, seizures, cognitive decline, lactic acidosis, ragged‐red fibers, headache, and vomiting, and in 80% of cases due to the mtDNA variant m.3243A>G. We report the case of a MELAS patient carrying a variant in subunit‐5 of the respiratory chain (MT‐ND5), rarely reported in MELAS. The patient is a 33‐year‐old male, who experienced a series of stroke‐like episodes (StLEs) since age 23 years, which manifested clinically as seizures transient sensory disturbances, weakness, and visual or cognitive impairment. Over 9 years, these StLEs were misinterpreted as ischemic strokes, respectively, as cerebral vasculitis. He presented with mild, recurrent elevations of the creatine kinase. Initially, anti‐seizure drugs and steroids appeared to be beneficial. Despite good recovery of each single StLE, the patient experienced a progressive decline of cognitive functions and activities of daily living. Cerebral imaging showed corresponding stroke‐like lesions in changing locations. At age 32y, genetic work‐up revealed the variant m.13513G>A in MT‐ND5. The patient profited significantly from a cocktail with anti‐oxidants/cofactors. This case shows that the variant m.13513G>A in MT‐ND5 can manifest as MELAS that StLEs recover spontaneously and that the course of MELAS is slowly progressive.     

Branched-Chain Amino Acids and Insulin Metabolism: The Insulin Resistance Atherosclerosis Study (IRAS)

OBJECTIVE

Recent studies using untargeted metabolomics approaches have suggested that plasma branched-chain amino acids (BCAAs) are associated with incident diabetes. However, little is known about the role of plasma BCAAs in metabolic abnormalities underlying diabetes and whether these relationships are consistent across ethnic populations at high risk for diabetes. We investigated the associations of BCAAs with insulin sensitivity (S_I), acute insulin response (AIR), and metabolic clearance of insulin (MCRI) in a multiethnic cohort.

RESEARCH DESIGN AND METHODS

In 685 participants without diabetes of the Insulin Resistance Atherosclerosis Study (IRAS) (290 Caucasians, 165 African Americans, and 230 Hispanics), we measured plasma BCAAs (sum of valine, leucine, and isoleucine) by mass spectrometry and S_I, AIR, and MCRI by frequently sampled intravenous glucose tolerance tests.

RESULTS

Elevated plasma BCAAs were inversely associated with S_I and MCRI and positively associated with fasting insulin in regression models adjusted for potential confounders (β = −0.0012 [95% CI −0.0018, −0.00059], P < 0.001 for S_I; β = −0.0013 [95% CI −0.0018, −0.00082], P < 0.001 for MCRI; and β = 0.0015 [95% CI 0.0008, 0.0023], P < 0.001 for fasting insulin). The association of BCAA with S_I was significantly modified by ethnicity, with the association only being significant in Caucasians and Hispanics. Elevated plasma BCAAs were associated with incident diabetes in Caucasians and Hispanics (multivariable-adjusted odds ratio per 1-SD increase in plasma BCAAs: 1.67 [95% CI 1.21, 2.29], P = 0.002) but not in African Americans. Plasma BCAAs were not associated with S_I-adjusted AIR.

CONCLUSIONS

Plasma BCAAs are associated with incident diabetes and underlying metabolic abnormalities, although the associations were generally stronger in Caucasians and Hispanics.     

Proteomics and metabolomics analysis reveal potential mechanism of extended-spectrum β-lactamase production in Escherichia coli

In this study, ten clinical susceptible strains and ten clinical ESBL-EC (extended-spectrum β-lactamase-producing Escherichia coli) were screened and obtained by microbial identification using ITEK® 2 Compact. TMT (Tandem Mass Tag) proteomics analysis discovered 1553 DEPs (differentially expressed proteins) between ESBL-EC and non-ESBL-EC. In addition, an untargeted metabolomics assay by using UHPLC-MS (ultra-high-performance liquid chromatography-mass spectrometry) was applied to compare the differential profiles of metabolites between β-lactam antibiotic-sensitive E. coli and multidrug-resistant ESBL-producing E. coli strains. The PCA (principal component analysis) score plots and OPLS-DA (orthogonal projections to latent structures discriminant analysis) plots clearly discriminated ESBL-EC and non-ESBL-EC, and volcano analysis presented 606 and 459 altered metabolites between ESBL-EC vs. non-ESBL-EC in positive and negative ion modes, respectively. Interestingly, the bioinformatics analysis demonstrated that the purine metabolism pathway was enriched in ESBL-EC. These results suggest that the existence of extended-spectrum β-lactamase affects the metabolite and protein profiles of E. coli. The correlation analysis of metabolomics and proteomics data established a correlation between DEPs and differential metabolites in the purine metabolism pathway. Moreover, three metabolite candidates in the purine metabolism pathway were validated by the UPLC-MRM-MS (ultra-performance liquid chromatography multiple reaction monitoring mass spectrometry) method. Our data suggest that these DEPs and differential metabolites may play important roles in the antibiotic resistance of ESBL-EC. Our study can provide scientific data for the mechanism study of antibiotic resistance of ESBL-EC at the metabolite and protein levels and targeting modulators to these pathways may be effective for treatment of ESBL-EC strains.

Proteomic and metabolomics revealed the underlying mechanism of extended-spectrum β-lactamase production in Escherichia coli.     

Abeta42-Induced Neurodegeneration via an Age-Dependent Autophagic-Lysosomal Injury in Drosophila

The mechanism of widespread neuronal death occurring in Alzheimer's disease (AD) remains enigmatic even after extensive investigation during the last two decades. Amyloid beta 42 peptide (Aβ_1–42) is believed to play a causative role in the development of AD. Here we expressed human Aβ_1–42 and amyloid beta 40 (Aβ_1–40) in Drosophila neurons. Aβ_1–42 but not Aβ_1–40 causes an extensive accumulation of autophagic vesicles that become increasingly dysfunctional with age. Aβ_1–42-induced impairment of the degradative function, as well as the structural integrity, of post-lysosomal autophagic vesicles triggers a neurodegenerative cascade that can be enhanced by autophagy activation or partially rescued by autophagy inhibition. Compromise and leakage from post-lysosomal vesicles result in cytosolic acidification, additional damage to membranes and organelles, and erosive destruction of cytoplasm leading to eventual neuron death. Neuronal autophagy initially appears to play a pro-survival role that changes in an age-dependent way to a pro-death role in the context of Aβ_1–42 expression. Our in vivo observations provide a mechanistic understanding for the differential neurotoxicity of Aβ_1–42 and Aβ_1–40, and reveal an Aβ_1–42-induced death execution pathway mediated by an age-dependent autophagic-lysosomal injury.     

Prevalence and genetic analysis of thalassemia in childbearing age population of Hainan,The Free Trade Island in Southern China

BackgroundHainan has one of the high incidences of thalassemia in China, but the epidemiological data in the whole province has not been reported yet. The objective of our study was to reveal the true prevalence and molecular mutation spectrum of thalassemia in the population of Hainan who are of childbearing age.MethodsWe screened 166,936 individuals from 19 cities and counties in Hainan by hematological parameters analysis, and further conducted genetic analysis for individuals whose MCV was less than 82fL.ResultsIn total, 21,619 (12.95%) subjects were diagnosed as thalassemia carriers or patients. The overall prevalence of α‐thalassemia, β‐thalassemia, and α+β‐thalassemia were 10.39%, 1.38%, and 1.18%, respectively. Eleven α‐thalassemia mutations and sixteen β‐thalassemia mutations were identified. The high‐frequent genotypes of α‐thalassemia were ‐α^3.7/αα (19.70%), ‐α^4.2/αα (19.39%), αα/‐‐^SEA (15.60%), α^WSα/αα (9.24%), and ‐α^3.7/‐α^4.2 (8.90%), and those of β‐thalassemia were β^{CD41/42(−TTCT)}/β^N (58.92%), β^−28(A>G)/β^N (16.05%), β^{IVS−Ⅱ−654(C>T)}/β^N (8.42%), β^CD71/72(+A)/β^N (6.03%), β^CD17(A>T)/β^N (5.47%), and β^{CD26 (GAG>AAG)}/β^N (2.69%). In addition, the frequencies and hematological profiles of many rare mutations of α‐ [Fusion, HKαα, αααanti^4.2, IVS‐II‐55 (T>G), IVS‐II‐119 (−G,+CTCGGCCC)] and β‐globin genes [−50 (G>A), IVS‐Ⅱ‐81 (C>T)] in Hainan were reported for the first time.ConclusionOur study revealed the high prevalence and extensive molecular spectrum of thalassemia in childbearing age population of Hainan, suggesting thalassemia in Hainan ranks second in prevalence among all regions in China. The findings will be useful for genetic counseling and prevention of thalassemia.     

Investigation of plasma metabolomics and neurotransmitter dysfunction in the process of Alzheimer's disease rat induced by amyloid beta 25-35

Alzheimer''s disease (AD) has become one of the major diseases endangering the health of the elderly. Clarifying the features of each AD animal model is valuable for understanding the onset and progression of diseases and developing potential treatments in the pharmaceutical industry. In this study, we aimed to clarify plasma metabolomics and neurotransmitter dysfunction in the process of AD model rat induced by amyloid beta 25-35 (Aβ 25-35). Firstly, Morris Water Maze (MWM) test was used to investigate cognitive impairment in AD rat after 2, 4 and 8 weeks of modelling. Based on this, the effects on levels of AD-related enzymes and eight neurotransmitters were analyzed. And plasma metabolomics analysis based on ultra high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS) was used to research the metabolic disturbances in the process of AD rat. The results shown the injury on the spatial learning ability of AD rats was gradually aggravated within 4 weeks, reached the maximum at 4 weeks and then was stable until 8 weeks. During 8 weeks of modeling, the levels of enzymes including β-secretase, γ-secretase, glycogen synthase kinase-3β (GSK-3β), acetyl cholinesterase (AchE) and nitric oxide synthase (NOS) were significant increased in the plasma of AD rats. The neurotransmitter dysfunction was mainly involved in γ-aminobutyric acid (GABA), acetyl choline (Ach), glutamic acid (Glu), 5-hydroxytryptamine (5-HT), dopamine (DA) and norepinephrine (NE). 17 endogenous metabolites correlated with AD were successfully detected in the metabolomics analysis. These metabolites were mainly involved in fatty acids, sphingolipids, and sterols metabolisms, vitamin metabolism, and amino acid metabolism. These metabolites might be the potential biomarkers that correctly mark different stages of AD. The study on peripheral plasma indices reflecting the process of AD laid the foundation for understand the pathophysiology of AD and find an effective and radical cure. And the rules of endogenous metabolic disorder in AD rats also have a certain guiding significance for the future study of food–drug interactions at different stages of AD.

The cognitive impairment, Alzheimer''s disease (AD) related enzymes, neurotransmitters and endogenous metabolites shown a dynamic change in AD model rat induced by amyloid beta 25-35.     

Molecular analysis of alpha‐ and beta‐thalassemia in Meizhou region and comparison of gene mutation spectrum with different regions of southern China

BackgroundThalassemia is a group of inherited autosomal recessive hemolytic anemia disease caused by reduced or absent synthesis of globin chain/chains of hemoglobin. Only few studies showed the molecular characterization of α‐ and β‐thalassemia in Meizhou city of China.MethodsA total of 22,401 individuals were collected; hematological and hemoglobin electrophoresis analysis and thalassemia genetic testing were performed.ResultsEleven thousand and thirty (49.24%) cases with microcytosis (mean corpuscular volume (MCV) < 82 fl), 11,074 (49.44%) cases with hypochromia (mean corpuscular Hb (MCH) < 27 pg) in 22,401 subjects, 11,085 cases with abnormal hemoglobin results were identified in subjects aged ≥6 months. 7,322 (32.69%) subjects harbored thalassemia mutations, including 4,841 (21.61%) subjects with α‐thalassemia, 2,237 (9.99%) with β‐thalassemia, and 244 (1.09%) with α‐thalassemia combined β‐thalassemia. 18 genotypes of α‐thalassemia mutations and 27 genotypes of β‐thalassemia mutations were characterized. The most frequent α gene mutation was ‐‐^SEA (64.69%), followed by ‐α^3.7 (19.93%), ‐α^4.2 (7.73%), α^CSα (3.97%), and α^WSα (2.83%). The six most common β‐thalassemia mutations were IVS‐II‐654 (C>T) (39.79%), CD41‐42 (‐TCTT) (33.02%), −28 (A>G) (10.38%), CD17 (A>T) (9.08%), CD27‐28 (+C) (2.14%), and CD26 (G>A) (2.02%). In addition, MCV and MCH were sensitive markers for α‐ and β‐thalassemia except for ‐α^3.7/αα, ‐α^4.2/αα, α^CSα/αα, α^WSα/αα, and β^Cap+40−43/β^N.ConclusionsThe ‐‐^SEA, ‐α^3.7, and ‐α^4.2 deletions were the main mutations of α‐thalassemia, while IVS‐II‐654 (C>T), CD41‐42 (‐TCTT), −28 (A>G), and CD17 (A>T) mutations of β‐thalassemia in Meizhou. There were some differences in thalassemia mutation frequencies in Meizhou city from other populations in China.     

FcγRIIb mediates amyloid-β neurotoxicity and memory impairment in Alzheimer’s disease

Amyloid-β (Aβ) induces neuronal loss and cognitive deficits and is believed to be a prominent cause of Alzheimer’s disease (AD); however, the cellular pathology of the disease is not fully understood. Here, we report that IgG Fcγ receptor II-b (FcγRIIb) mediates Aβ neurotoxicity and neurodegeneration. We found that FcγRIIb is significantly upregulated in the hippocampus of AD brains and neuronal cells exposed to synthetic Aβ. Neuronal FcγRIIb activated ER stress and caspase-12, and Fcgr2b KO primary neurons were resistant to synthetic Aβ-induced cell death in vitro. Fcgr2b deficiency ameliorated Aβ-induced inhibition of long-term potentiation and inhibited the reduction of synaptic density by naturally secreted Aβ. Moreover, genetic depletion of Fcgr2b rescued memory impairments in an AD mouse model. To determine the mechanism of action of FcγRIIb in Aβ neurotoxicity, we demonstrated that soluble Aβ oligomers interact with FcγRIIb in vitro and in AD brains, and that inhibition of their interaction blocks synthetic Aβ neurotoxicity. We conclude that FcγRIIb has an aberrant, but essential, role in Aβ-mediated neuronal dysfunction.     

Diagnostic value of HSP90α and related markers in lung cancer

PurposeTo investigate the expression of heat shock protein 90α (HSP90α) in patients with lung cancer (LC) and the clinical value of HSP90α and other related markers in the diagnosis of LC.MethodsOf 335 patients enrolled in the study cohort, 175 were screened for LC and 160 were healthy (HC). The plasma levels of HSP90α and related markers (CEA, NSE, CYFRA21‐1 and ProGRP) were detected in all individuals in the cohort by enzyme‐linked immunosorbent assay (ELISA). Groups were divided according to gender (male/female), age (≤60 years/>60 years), types of LC (small‐cell carcinoma, squamous carcinoma and adenocarcinoma), staging (I, II, III and IV) and metastasis (metastasis and non‐metastasis) separately. Wilcoxon Mann–Whitney test and Kruskal–Wallis test were used to compare statistical differences between two groups/among the multiple groups for each factor of HSP90α. The r‐value and Kappa were used to compare HSP90α with related markers, and the receiver operating curve (ROC) was used to evaluate the efficacy of plasma HSP90α in predicting LC.ResultsNo statistical difference was found in the plasma level of HSP90α among different age and gender groups (p > 0.05). In the group divided by LC type, staging and metastasis status, there were statistical differences among different groups in HSP90α level (p < 0.05). The levels of HSP90α, CEA, NSE, CYFRA21‐1 and ProGRP in LC groups were significantly higher than HC (p < 0.001). R values of HSP90α correlated with other related markers in the diagnosis of LC (p < 0.05). Although HSP90α and other related markers did not fit the satisfactory conformance, in terms of the positive rate of diagnosis, it was statistically differences in the diagnostic positive rate between HSP90α and each marker (p < 0.01). ROC analysis showed that a plasma HSP90α cut‐off point of 50.02 ng/ml had an optimal predictive value for LC.ConclusionsHSP90α has significant clinical value in early screening and diagnosis of LC. The combined application of HSP90α and related markers can improve the positive rate of early diagnosis of LC effectively.     

Monascus pigment rubropunctatin derivative FZU-H reduces Aβ(1-42)-induced neurotoxicity in Neuro-2A cells

Alzheimer''s disease (AD) is an extremely complex disease, characterized by several pathological features including oxidative stress and amyloid-β (Aβ) aggregation. Blockage of Aβ-induced injury has emerged as a potential therapeutic approach for AD. Our previous efforts resulted in the discovery of Monascus pigment rubropunctatin derivative FZU-H with potential neuroprotective effects. This novel lead compound significantly diminishes toxicity induced by Aβ(1-42) in Neuro-2A cells. Our further mechanism investigation revealed that FZU-H inhibited Aβ(1-42)-induced caspase-3 protein activation and the loss of mitochondrial membrane potential. In addition, treatment of FZU-H was proven to attenuate Aβ(1-42)-induced cell redox imbalance and Tau hyperphosphorylation which caused by okadaic acid in Neuro-2A cells. These results indicated that FZU-H shows promising neuroprotective effects for AD.

Monascus pigment rubropunctatin derivative FZU-H shows promising neuroprotective effects for AD.     

Preparation and photo-induced activities of water-soluble amyloid β-C60 complexes

We have shown that fullerene (C₆₀) becomes soluble in water by mixing fullerene and amyloid β peptide (Aβ40) whose fibril structures are considered to be associated with Alzheimer''s disease. The water-solubility of fullerene arises from the generation of a nanosized complex between fullerene and the monomer species of Aβ40 (Aβ40-C₆₀). The prepared Aβ40-C₆₀ exhibits photo-induced activity with visible light to induce the inhibition of Aβ40 fibrillation and the cytotoxicity for cultured HeLa cells. The observed photo-induced phenomena result from the generation of singlet oxygen via photoexcitation, inducing oxidative damage to Aβ40 and HeLa cells. The oxidized Aβ40 following photoexcitation of Aβ40-C₆₀ was confirmed by mass spectrometry.

We have shown that fullerene (C₆₀) becomes soluble in water by mixing fullerene and amyloid β peptide (Aβ40) whose fibril structures are considered to be associated with Alzheimer''s disease.     

Aβ Mediated Diminution of MTT Reduction—An Artefact of Single Cell Culture?

The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide (MTT) reduction assay is a frequently used and easily reproducible method to measure beta-amyloid (Aβ) toxicity in different types of single cell culture. To our knowledge, the influence of Aβ on MTT reduction has never been tested in more complex tissue. Initially, we reproduced the disturbed MTT reduction in neuron and astroglia primary cell cultures from rats as well as in the BV2 microglia cell line, utilizing four different Aβ species, namely freshly dissolved Aβ (25-35), fibrillar Aβ (1-40), oligomeric Aβ (1-42) and oligomeric Aβ (1-40). In contrast to the findings in single cell cultures, none of these Aβ species altered MTT reduction in rat organotypic hippocampal slice cultures (OHC). Moreover, application of Aβ to acutely isolated hippocampal slices from adult rats and in vivo intracerebroventricular injection of Aβ also did not influence the MTT reduction in the respective tissue. Failure of Aβ penetration into the tissue cannot explain the differences between single cells and the more complex brain tissue. Thus electrophysiological investigations disclosed an impairment of long-term potentiation (LTP) in the CA1 region of hippocampal slices from rat by application of oligomeric Aβ (1-40), but not by freshly dissolved Aβ (25-35) or fibrillar Aβ (1-40). In conclusion, the experiments revealed a glaring discrepancy between single cell cultures and complex brain tissue regarding the effect of different Aβ species on MTT reduction. Particularly, the differential effect of oligomeric versus other Aβ forms on LTP was not reflected in the MTT reduction assay. This may indicate that the Aβ oligomer effect on synaptic function reflected by LTP impairment precedes changes in formazane formation rate or that cells embedded in a more natural environment in the tissue are less susceptible to damage by Aβ, raising cautions against the consideration of single cell MTT reduction activity as a reliable assay in Alzheimer's drug discovery studies.