The gene spectrum of thalassemia in Yangjiang of western Guangdong Province.

Background: Thalassemia presents a higher incidence in southern China. The objective of this study is to analyze the genotype distribution of thalassemia in Yangjiang, a western city of Guangdong Province in China. Methods: The genotypes of suspected cases with thalassemia were tested by PCR and reverse dot blot (RDB). Unidentified rare thalassemia genotypes of the samples were further ascertained by PCR and direct DNA sequencing. Results: Among 22467 suspected cases with thalassemia, 7658 cases were found with thalassemia genotypes using our PCR-RDB kit. Among these 7658 cases, 5313 cases were found with α-thalassemia (α-thal) alone, --SEA/αα was the most common genotype, accounting for 61.75% of α-thal genotypes, and the following mutations were found: α3.7/αα, -α4.2/αα, αCSα/αα, αWSα/αα, and αQSα/αα. A total of 2032 cases were found with ß-thalassemia (ß-thal) alone. ßCD41-42/ßN, ßIVS-II-654/ßN, and ß-28/ßN accounted for 80.9% of all ß-thal genotypes, and the following genotypes were found: ßCD17/ßN, ßCD71-72/ßN, and ßE/ßN. Compound heterozygotes of ß-thal and ß-thalassemia homozygotes were identified in 11 and five cases, respectively, in this study. α-thal combined with ß-thal was identified in 313 cases, showing 57 genotype combinations of the coincidence of both Hb disorders; one extreme patient had a genotype of --SEA/αWSα and ßCD41-42/ß-28. In addition, four rare α-mutations (--THAI, HKαα, Hb Q-Thailand, and CD31 AGG>AAG) and six rare ß-mutations (CD39 CAG>TAG, IVS-Ⅱ-2 (-T), -90(C>T), Chinese Gγ+(Aγδß)0, CD104 (-G), and CD19 A>G) were also found in this study population. Conclusion: This study provided detailed genotypes of thalassemia in Yangjiang of western Guangdong Province in China and reflected the complexity of genotypes in this high-prevalence region, and this would be valuable for diagnosis and counseling for thalassemia in this area.

Identification of three potential novel biomarkers for early diagnosis of acute ischemic stroke via plasma lipidomics.

INTRODUCTION: Acute ischemic stroke (AIS) accounts for the majority of all stroke, globally the second leading cause of death. Due to its rapid development after onset, its early diagnosis is crucial. OBJECTIVES: We aim to identify potential highly reliable blood-based biomarkers for early diagnosis of AIS using quantitative plasma lipid profiling via a machine learning approach. METHODS: Lipidomics was used for quantitative plasma lipid profiling, based on ultra-performance liquid chromatography tandem mass spectrometry. Our samples were divided into a discovery and a validation set, each containing 30 AIS patients and 30 health controls (HC). Differentially expressed lipid metabolites were screened based on the criteria VIP > 1, p < 0.05, and fold change > 1.5 or < 0.67. The least absolute shrinkage and selection operator (LASSO) and random forest algorithms in machine learning were used to select differential lipid metabolites as potential biomarkers. RESULTS: Three key differential lipid metabolites, CarnitineC10:1, CarnitineC10:1-OH and Cer(d18:0/16:0), were identified as potential biomarkers for early diagnosis of AIS. The former two, associated with thermogenesis, were down-regulated, whereas the latter, associated with necroptosis and sphingolipd metabolism, was upregulated. Univariate and multivariate logistic regressions showed that these three lipid metabolites and the resulting diagnostic model exhibited a strong ability in discriminating between AIS patients and HCs in both the discovery and validation sets, with an area under the curve above 0.9. CONCLUSIONS: Our work provides valuable information on the pathophysiology of AIS and constitutes an important step toward clinical application of blood-based biomarkers for diagnosing AIS.

Molecular epidemiological investigation of G6PD deficiency in Yangjiang region, western Guangdong province.

Objectives: The prevalence of G6PD deficiency has not been reported in Yangjiang, a western city in Guangdong province. This study aims to investigate the molecular characteristics of G6PD deficiency in this region. Methods: Blood samples were collected from adults at a local hospital to screen for G6PD deficiency. The deficient samples were subjected to further analysis using PCR and reverse dot blot to determine the specific G6PD variants. Results: Among the 3314 male subjects, 250 cases of G6PD deficiency were found using the G6PD enzyme quantitative assay, resulting in a prevalence of 7.54% (250/3314) in the Yangjiang region. The prevalence of G6PD deficiency in females was 3.42% (176/5145). Out of the 268 cases of G6PD deficiency tested for G6PD mutations, reverse dot blot identified 20 different G6PD variants. The most common G6PD variant was c.1388G>A (81/268), followed by c.1376G>T (48/268), c.95A>G (32/268), c.1024C>T (9/268), c.392G>T (7/268), and c.871G>A/c.1311C>T (6/268). It was observed that c.871G>A was always linked to the polymorphism of c.1311C>T in this population. Conclusion: This investigation into G6PD deficiency in this area is expected to significantly improve our understanding of the prevalence and molecular characterization of this condition.

ALB, HP, OAF and RBP4 as novel protein biomarkers for identifying cured patients with pulmonary tuberculosis by DIA.

BACKGROUND: Pulmonary tuberculosis (TB) is a serious infectious disease that lacks robust blood-based biomarkers to identify cured TB. Some discharged patients are not fully cured and may relapse or even develop multidrug-resistant TB. This study is committed to finding proteomic-based plasma biomarkers to support establishing laboratory standards for clinical TB cure. METHODS: Data-independent acquisition (DIA) was used to obtain the plasma protein expression profiles of TB patients at different treatment stages compared with healthy controls. Multivariate statistical methods and bioinformatics were used to analyze the data. RESULTS: Bioinformatic analysis suggests coagulation dysfunction and vitamin and lipid metabolism disturbances in TB. Albumin (ALB), haptoglobin (HP), out at first protein homolog (OAF), and retinol-binding protein 4 (RBP4) can be used to establish a diagnostic model for the efficacy evaluation of TB with an area under the curve of 0.963, which could effectively distinguish untreated TB patients from cured patients. CONCLUSIONS: Our research demonstrated that ALB, HP, OAF and RBP4 can be potential biomarkers for evaluating the efficacy of TB. These findings may provide experimental data for establishing the laboratory indicators of clinical TB cure and providing clinicians with new targets for exploring the underlying mechanisms of TB pathogenesis.

Glucose-6-phosphate dehydrogenase deficiency screening and gene analysis in blood donors of Guangdong province.

OBJECTIVES: The characteristic of glucose-6-phosphate dehydrogenase (G6PD) deficiency is red blood cell (RBC) destruction in response to oxidative stress. Patients requiring RBC transfusions may simultaneously receive oxidative medications or have concurrent infections, both of which can induce hemolysis in G6PD-deficient RBCs. We intend to investigate the incidence of G6PD deficiency in voluntary blood donors and to evaluate the transfusion risk associated with G6PD deficiency in Guangdong province. METHODS: G6PD enzyme was analyzed in 3042 donors and gene mutations were genotyped in G6PD-deficient samples. RESULTS: The G6PD-deficient prevalence of voluntary blood donors was 6.97% (212/3042), 55.19% blood donors with G6PD deficiency donated blood more than twice. Eighty-five cases of G6PD deficiency were genotyped, and the common types of G6PD mutations were c.1376 G > T, c.1388 G > A, c.95 A > G, c.1024 C > T, and c.871 G > A. CONCLUSIONS: Due to the high prevalence of G6PD deficiency in Foshan area, we recommended that the screening of G6PD deficiency should be carried out for the regular blood donors to ensure the safety of blood users.

Clinical validation of a single-tube PCR and reverse dot blot assay for detection of common α-thalassaemia and ß-thalassaemia in Chinese.

OBJECTIVE: To evaluate a novel reverse dot blot assay for the simultaneous detection six types of common α-thalassaemia alleles (three deletional and three common non-deletional mutations) and 19 types of common ß-thalassaemia alleles in a Chinese population. METHODS: Genomic DNA samples were collected from three hospitals in southern China. The novel thalassaemia gene assay involved one multiplex polymerase chain reaction amplification system and one round of hybridization. Each of the clinically validated DNA samples was re-tested using the new multiplex polymerase chain reaction/reverse dot blot assay II (M-PCR/RDB II) assay in a double-blind manner. RESULTS: A total of 1060 unrelated study participants, including 829 patients with thalassaemia and 231 healthy control subjects, were analysed. The whole PCR and RDB procedures were completed in 260 min. All the samples, including heterozygous thalassaemia, homozygous thalassaemia and compound heterozygous thalassaemia, were correctly genotyped, yielding 100% concordance with the reference assays. HKαα/--SEA and HKαα/-α4.2, which were not included in the detection panel, yielded a contradictory result with this new assay. CONCLUSION: The novel M-PCR/RDB II assay was simple, rapid and accurate, suggesting that it could be used for the genetic screening and clinical diagnosis of common α-thalassaemia and ß-thalassaemia variants in Chinese populations.

Postpartum Depression: Current Status and Possible Identification Using Biomarkers.

Postpartum depression (PPD) is a serious health issue that can affect about 15% of the female population within after giving birth. It often conveys significant negative consequences to the offsprings. The symptoms and risk factors are somewhat similar to those found in non-postpartum depression. The main difference resides in the fact that PPD is triggered by postpartum specific factors, including especially biological changes in the hormone levels. Patients are usually diagnosed using a questionnaire onsite or in a clinic. Treatment of PPD often involves psychotherapy and antidepressant medications. In recent years, there have been more researches on the identification of biological markers for PPD. In this review, we will focus on the current research status of PPD, with an emphasis on the recent progress made on the identification of PPD biomarkers.

YY1/LncRNA GAS5 complex aggravates cerebral ischemia/reperfusion injury through enhancing neuronal glycolysis.

Yin-Yang 1 (YY1) has been identified as playing critical roles in multiple diseases. However, little is known regarding its roles and mechanisms in cerebral ischemia/reperfusion (I/R) injury. This study is aimed to explore the roles of YY1 in regulating neuronal apoptosis in cerebral I/R injury and its underlying mechanisms. Primary mouse cerebral cortical neurons were isolated and subjected to OGD/R to mimic cerebral I/R injury in vitro. The roles of YY1 on OGD/R-induced neuronal injury were investigated by performing western blotting, quantitative real-time polymerase chain reaction, TUNEL, RNA-binding protein immunoprecipitation, chromatin immunoprecipitation, chromatin isolation by RNA purification assay, glucose uptake assay, lactate production assay, and extracellular acidification rate assay. YY1-binding long non-coding RNAs (LncRNAs) in neurons subjected to OGD/R were identified by RIP and RNA sequencing. The roles of YY1 on cerebral I/R in vivo were detected by assessing neuronbehaviour, infarct size, and neuronal apoptosis. We found that YY1 expression is downregulated, and LncRNA GAS5 is upregulated in neurons subjected to OGD/R. OGD/R treatment promotes YY1 interacting with GAS5 in neurons, and YY1 negatively regulates GAS5 expression by binding to GAS5 promoter to repress its transcription. Besides, YY1 and GAS5 bind to the same region of PFKFB3 promoter to promote PFKFB3 expression and strengthen neuronal glycolysis, resulting in aggravating OGD/R-induced neuronal apoptosis. Knockdown of YY1 or GAS5 protects against I/R-induced ischemic brain damage and improves overall neurological functions in vivo. Overall, YY1 interacts with LncRNA GAS5 to promote PFKFB3 transcription to enhance neuronal glycolysis, resulting in aggravating cerebral I/R injury.

Effects and mechanism of action of Huang-Lian-Jie-Du-Tang in atopic dermatitis-like skin dysfunction in vivo and in vitro.

ETHNOPHARMACOLOGICAL RELEVANCE: Atopic dermatitis (AD), a disorder prevalent during childhood and adulthood, seriously affects the patient's quality of life. Although Huang-Lian-Jie-Du-Tang (HLJDT) has shown anti-inflammatory effects in previous studies, its effects and mechanism of action underlying AD disorder are still largely unknown. OBJECTIVE: This study explored the anti-inflammatory and immunomodulatory effects of HLJDT on the AD-like dermal disorder, induced in vitro by lipopolysaccharide (LPS)-triggered inflammation, and in vivo by 2,4-dinitrochlorobenzene (DNCB). MATERIALS AND METHODS: In vivo HLJDT effects were investigated by determining the severity of dermatitis, which consisted of observing signs of skin lesions, visually and through haematoxylin and eosin (HE) staining, in mouse ears and dorsal skin, measuring serum levels of interleukin (IL)-1α, IL-1ß, IL-2, IL-4, IL-5, IL-6, interferon (IFN)-γ, the tumour necrosis factor (TNF)-α, and determining the splenic index, number of splenic CD4+/CD8+ T-lymphocytes, as well as the phosphorylation levels of mitogen-activated protein kinases (including MAPKs-p38, ERK, and JNK), IκB-α, and nuclear factor kappa B (NF-κB) (p65) within dermal lesions. Morphological changes in LPS-induced inflammation were observed under a microscope, and ELISA and qPCR assays were used to measure IL-1α, IL-1ß, IL-6, and TNF-α expression levels. The protein expression levels of P-ERK/ERK, P-p38/p38, P-JNK/JNK, P-IKß-α, and P-p65 were measured through western blotting. Additionally, p65 expression was assessed by immunofluorescence, and LPS binding to RAW264.7 cell membrane was studied with laser confocal microscopy. RESULTS: HLJDT could remarkably mitigate DNCB-induced AD-like lesion symptoms, alleviating inflammatory mediator infiltration in mouse ears and dorsal skin tissue, down-regulating serum expression levels of IL-1α, IL-1ß, IL-2, IL-4, IL-5, IL-6, IFN-γ, and TNF-α, normalising the splenic CD4+/CD8+ T-lymphocyte ratio, and inactivating MAPKs (including p38, ERK, and JNK), IκB-α, and NF-κB (p65) in dorsal skin. Furthermore, HLJDT inhibited LPS-induced differentiation of RAW264.7 cells, as evidenced by the decreased protein and mRNA expression of IL-1α, IL-1ß, IL-6, and TNF-α. Additionally, it decreased ERK, p38, JNK, IKß-α, and p65 phosphorylation levels in the MAPKs/NF-κB pathway, inhibited p65 nuclear translocation, and reduced LPS binding to the RAW264.7 cell membrane. CONCLUSIONS: HLJDT significantly improved AD-like symptoms via inhibition of the MAPKs/NF-κB pathway. Therefore, administration of HLJDT might be a potential treatment for AD in the clinical setting.

YY1 promotes IL-6 expression in LPS-stimulated BV2 microglial cells by interacting with p65 to promote transcriptional activation of IL-6.

Neuroinflammation plays a critical role in the process of neurodegenerative disorders, during which microglia, the principal resident immune cells in the central nervous system, are activated and produce proinflammatory mediators. Yin-Yang 1 (YY1), a multi-functional transcription factor, is widely expressed in cells of the immune system and participate in various cellular processes. However, whether YY1 is involved in the process of neuroinflammation is still unknown. In the present study, we found that YY1 was progressively up-regulated in BV2 microglial cells stimulated with lipopolysaccharide (LPS), which was dependent on the transactivation function of nuclear factor kappa B (NF-κB). Furthermore, YY1 knockdown notably inhibited LPS-induced the activation of NF-κB signaling and interleukin-6 (IL-6) expression in BV-2 cells, but not mitogen-activated protein kinase (MAPK) signaling. Moreover, YY1 strengthened p65 binding to IL-6 promoter by interacting with p65 but decreased H3K27ac modification on IL-6 promoter, eventually increasing IL-6 transcription. Taken together, these results for the first time uncover the regulatory mechanism of YY1 on IL-6 expression during neuroinflammation responses and provide new lights into neuroinflammation.

Proteomic analysis of chondromodulin-I-induced differentiation of mesenchymal stem cells into chondrocytes.

To identify novel proteins that might help clarify the molecular mechanisms underlying chondromodulin-I (ChM-I) induction of mesenchymal stem cells (MSCs) differentiate into chondrocytes. MSCs are triggered to differentiate into chondrocytes, which are recognized as important factors in cartilage tissue engineering. ChM-I is a glycoprotein that stimulates the growth of chondrocytes and inhibits angiogenesis in vitro. In this study, the proteomic approach was used to evaluate protein changes between undifferentiated MSCs and ChM-I-transfected MSCs. The expression of the protein spots was analyzed using two-dimensional gel electrophoresis. Then, 14 protein spots were identified between MSCs and ChM-I-transfected MSCs. 309 proteins were identified using mass spectrometry (MS). The differentially regulated proteins were categorized and annotated using Protein Analysis Through Evolutionary Relationships (PANTHER) analysis with the aid of the Database for Annotation, Visualization and Integrated Discovery (DAVID) tool. These proteins are included in a variety of metabolic pathways and signal transduction pathways, such as focal adhesion, glycolysis, actin cytoskeleton regulation, and ribosome. These results demonstrate novel information about the molecular mechanism by which ChM-I induce MSCs to differentiate into chondrocytes. These results also provide a solid foundation for the development of tissue-engineered cartilage.

[Pathogenesis of Parkinson's Disease Viewing from Modern Chinese Medicine].

Modern pathological studies of Parkinson's disease. (PD) reveal that 70% -80% sub- stantia nigra striatum nerve endings degenerate before -motor symptoms occur. But till now, Chinese medicine (CM) infers its pathogeneses still at the theoretical level. Braak H, et al. raised six pathological stages of PD. They also beleived that symptoms of PD occurred one by one acocrding to staging sequence. All these indicated important evidence for the developing process of PD's pathogenesis. Authors expounded modern CM pathogenesis of PD.