Effects of the invasion of Ralstonia solanacearum on soil microbial community structure in Wuhan, China.

This study investigated the change in the microbiome of tomato rhizosphere soils after the invasion of Ralstonia solanacearum and analyzed the correlation between microbes and soil physicochemical properties. Diversity analyses of the bacteria in healthy and diseased rhizosphere soil samples (HRS and DRS) revealed that HRS had a higher species diversity and were compositionally different from DRS (P ≤ 0.05). Substantial differences in the relative abundance of Actinobacteria (37.52% vs 28.96%, P ≤ 0.05) and Proteobacteria (29.20% vs 35.59%, P ≤ 0.05) were identified in HRS and DRS, respectively. Taxonomic composition analysis showed ten differentially abundant genera, and seven of them (Gaiella, Roseisolibacter, Solirubrobacter, Kribbella, Acidibacter, Actinomarinicola, and Marmoricola) are more abundant in HRS. Soil pH and enzyme activities were negatively correlated with the abundance of R. solanacearum. The contents of total nitrogen (TN), total phosphorus (TP), total potassium (TK), alkaline nitrogen (alkaline N), available phosphorus (AP), available potassium (AK), NO3-N(NN), NH4+-N (AN), and organic matter (OM) were all significantly increased in DRS. The composition and richness of protozoa in the samples show significant differences. Cephalobus, Acrobeles, Heteromita, norank_Tylenchida, and Rotylenchulus were enriched in DRS. Microbial interaction networks revealed that the HRS networks were more complex than the DRS networks. Overall, the results of this study demonstrate that healthy soil has a more complex microbial community structure and higher enzyme activity, and the invasion of R. solanacearum damages the soil microbial system.IMPORTANCEHow does the invasion of Ralstonia solanacearum affect tomato rhizosphere bacteria and protozoa? Which microbial changes can affect the growth of R. solanacearum? To date, most research studies focus on bacteria, with little research on protozoa, and even less on the synergistic effects between protozoa and bacteria. Here, we analyzed the correlation between tomato rhizosphere bacterial and protozoan communities and soil physicochemical properties during the invasion of R. solanacearum. We found that the diversity and abundance of rhizosphere microorganisms in healthy rhizosphere soil samples (HRS) were significantly higher than those in diseased rhizosphere soil samples (DRS), and there were significant changes in soil pH and enzyme activity. Overall, in this study, the analysis of microbial changes during the invasion of R. solanacearum provides a theoretical basis for the prevention and control of bacterial wilt.

Early inoculation of an endophyte alters the assembly of bacterial communities across rice plant growth stages.

The core endophytes of plants are regarded as promising resources in future agroecosystems. How they affect the assembly of rice-related bacterial communities after early inoculation remains unclear. Here, we examined bacterial communities across 148 samples, including bulk and rhizosphere soils, sterilized roots, stems, and seeds at the seedling, tillering, booting, and maturity stages. Tissue cultured rice seedlings were inoculated with Xathomonas sacchari JR3-14, a core endophytic bacterium of rice seeds, before transplanting. The results revealed that α-diversity indices were significantly enhanced in the root and stem endosphere at the seedling stage. ß-diversity was altered at most plant developmental stages, except for the root and stem at the booting stage. Network complexity consequently increased in the root and stem across rice growth stages, other than the stem endosphere at the booting stage. Four abundant beneficial bacterial taxa, Bacillus, Azospira, Azospirillum, and Arthrobacter, were co-enriched during the early growth stage. Infer Community Assembly Mechanisms by Phylogenetic-bin-based null model analysis revealed a higher relative contribution of drift and other eco-evolutionary processes mainly in root compartments across all growth stages, but the opposite pattern was observed in stem compartments. IMPORTANCE Endophytic bacteria are regarded as promising environmentally friendly resources to promote plant growth and plant health. Some of microbes from the seed are able to be carried over to next generation, and contribute to the plant's ability to adapt to new environments. However, the effects of early inoculation with core microbes on the assembly of the plant microbiome are still unclear. In our study, we demonstrate that early inoculation of the rice seed core endophytic bacterium Xanthomonas sacchari could alter community diversity, enhance complexity degree of network structure at most the growth stages, and enrich beneficial bacteria at the seedling stage of rice. We further analyzed the evolutionary processes caused by the early inoculation. Our results highlight the new possibilities for research and application of sustainable agriculture by considering the contribution of seed endophytes in crop production and breeding.

Pseudomonas syringae Type III Secretion Protein HrpP Manipulates Plant Immunity To Promote Infection.

The bacterial plant pathogen Pseudomonas syringae deploys a type III secretion system (T3SS) to deliver effector proteins into plant cells to facilitate infection, for which many effectors have been characterized for their interactions. However, few T3SS Hrp (hypersensitive response and pathogenicity) proteins from the T3SS secretion apparatus have been studied for their direct interactions with plants. Here, we show that the P. syringae pv. tomato DC3000 T3SS protein HrpP induces host cell death, suppresses pattern-triggered immunity (PTI), and restores the effector translocation ability of the hrpP mutant. The hrpP-transgenic Arabidopsis lines exhibited decreased PTI responses to flg22 and elf18 and enhanced disease susceptibility to P. syringae pv. tomato DC3000. Transcriptome analysis reveals that HrpP sensing activates salicylic acid (SA) signaling while suppressing jasmonic acid (JA) signaling, which correlates with increased SA accumulation and decreased JA biosynthesis. Both yeast two-hybrid and bimolecular fluorescence complementation assays show that HrpP interacts with mitogen-activated protein kinase kinase 2 (MKK2) on the plant membrane and in the nucleus. The HrpP truncation HrpP1-119, rather than HrpP1-101, retains the ability to interact with MKK2 and suppress PTI in plants. In contrast, HrpP1-101 continues to cause cell death and electrolyte leakage. MKK2 silencing compromises SA signaling but has no effect on cell death caused by HrpP. Overall, our work highlights that the P. syringae T3SS protein HrpP facilitates effector translocation and manipulates plant immunity to facilitate bacterial infection. IMPORTANCE The T3SS is required for the virulence of many Gram-negative bacterial pathogens of plants and animals. This study focuses on the sensing and function of the T3SS protein HrpP during plant interactions. Our findings show that HrpP and its N-terminal truncation HrpP1-119 can interact with MKK2, promote effector translocation, and manipulate plant immunity to facilitate bacterial infection, highlighting the P. syringae T3SS component involved in the fine-tuning of plant immunity.

Delayed progressive sensorineural hearing loss due to a novel compound heterozygous PTPRQ mutation in a Chinese patient.

BACKGROUND: The Protein tyrosine phosphatase receptor Q (PTPRQ) gene encodes a member of the type III receptor-like protein tyrosine phosphatase family found in the stereocilium. Mutations in PTPRQ are mostly associated with deafness, autosomal recessive type 84 (DFNB 84), which usually results in progressive familial hearing loss. METHODS: A 25-year-old woman and her sister, both with postlingual-delayed progressive sensorineural hearing loss, were examined. They were from a nonconsanguineous marriage and had no family history of hearing loss. New compound heterozygous PTPRQ gene mutations, nonsense (c.90C > A, p.Y30X) and splice (c.5426 + 1G > A) mutations in two PTPRQ alleles, were identified in the two sisters and were presumably autosomal recessive. The c.90C > A (p.Y30X) mutation was mapped to exon 2 of PTPRQ (NM_001145026). RESULTS: The c.90C > A mutation leads to a premature stop codon and a truncated protein. The c.5426 + 1G > A mutation leads to a truncated protein lacking the extracellular domain. Hence, both mutations were predicted to be pathogenic, leading to a deficiency of the extracellular, transmembrane, and phosphatase domains because of nonsense-mediated mRNA degradation. CONCLUSIONS: This study increases the spectrum of PTPRQ gene mutations that might be involved in delayed progressive autosomal recessive non-syndromic hearing loss.

Deciphering the rhizosphere bacteriome associated with biological control of tobacco black shank disease.

Introduction: The black shank disease seriously affects the health of tobacco plants. Conventional control methods have limitations in terms of effectiveness or economic aspects and cause public health concerns. Thus, biological control methods have come into the field, and microorganisms play a key role in suppressing tobacco black shank disease. Methods: In this study, we examined the impact of soil microbial community on black shank disease basing on the structural difference of bacterial communities in rhizosphere soils. We used Illumina sequencing to compare the bacterial community diversity and structure in different rhizosphere soil samples in terms of healthy tobacco, tobacco showing typical black shank symptoms, and tobacco treated with the biocontrol agent, Bacillus velezensis S719. Results: We found that Alphaproteobacteria in the biocontrol group, accounted for 27.2% of the ASVs, was the most abundant bacterial class among three groups. Heatmap and LEfSe analyses were done to determine the distinct bacterial genera in the three sample groups. For the healthy group, Pseudomonas was the most significant genus; for the diseased group, Stenotrophomonas exhibited the strongest enrichment trend, and Sphingomonas showed the highest linear discriminant analysis score, and was even more abundant than Bacillus; for the biocontrol group, Bacillus, and Gemmatimonas were the largely distributed genus. In addition, co-occurrence network analysis confirmed the abundance of taxa, and detected a recovery trend in the network topological parameters of the biocontrol group. Further functional prediction also provided a possible explanation for the bacterial community changes with related KEGG annotation terms. Discussion: These findings will improve our knowledge of plant-microbe interactions and the application of biocontrol agents to improve plant fitness, and may contribute to the selection of biocontrol strains.

Dynamics of rice microbiomes reveal core vertically transmitted seed endophytes.

BACKGROUND: Plants and their associated microbiota constitute an assemblage of species known as holobionts. The plant seed microbiome plays an important role in nutrient uptake and stress attenuation. However, the core vertically transmitted endophytes remain largely unexplored. RESULTS: To gain valuable insights into the vertical transmission of rice seed core endophytes, we conducted a large-scale analysis of the microbiomes of two generations of six different rice varieties from five microhabitats (bulk soil, rhizosphere, root, stem, and seed) from four geographic locations. We showed that the microhabitat rather than the geographic location and rice variety was the primary driver of the rice microbiome assemblage. The diversity and network complexity of the rice-associated microbiome decreased steadily from far to near the roots, rice exterior to interior, and from belowground to aboveground niches. Remarkably, the microbiomes of the roots, stems, and seeds of the rice interior compartments were not greatly influenced by the external environment. The core bacterial endophytes of rice were primarily comprised of 14 amplicon sequence variants (ASVs), 10 of which, especially ASV_2 (Pantoea) and ASV_48 (Xanthomonas), were identified as potentially vertically transmitted taxa because they existed across generations, were rarely present in exterior rice microhabitats, and were frequently isolated from rice seeds. The genome sequences of Pantoea and Xanthomonas isolated from the parental and offspring seeds showed a high degree of average nucleotide and core protein identity, indicating vertical transmission of seed endophytes across generations. In silico prediction indicated that the seed endophytes Pantoea and Xanthomonas possessed streamlined genomes with short lengths, low-complexity metabolism, and various plant growth-promoting traits. We also found that all strains of Pantoea and Xanthomonas exhibited cellulase activity and produced indole-3-acetic acid. However, most strains exhibited insignificant antagonism to the major pathogens of rice, such as Magnaporthe oryzae and X. oryzae pv. oryzae. CONCLUSION: Overall, our study revealed that microhabitats, rather than site-specific environmental factors or host varieties, shape the rice microbiome. We discovered the vertically transmitted profiles and keystone taxa of the rice microbiome, which led to the isolation of culturable seed endophytes and investigation of their potential roles in plant-microbiome interactions. Our results provide insights on vertically transmitted microbiota and suggest new avenues for improving plant fitness via the manipulation of seed-associated microbiomes.  Video Abstract.

Safety and efficacy of meplazumab in healthy volunteers and COVID-19 patients: a randomized phase 1 and an exploratory phase 2 trial.

Recent evidence suggests that CD147 serves as a novel receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Blocking CD147 via anti-CD147 antibody could suppress the in vitro SARS-CoV-2 replication. Meplazumab is a humanized anti-CD147 IgG2 monoclonal antibody, which may effectively prevent SARS-CoV-2 infection in coronavirus disease 2019 (COVID-19) patients. Here, we conducted a randomized, double-blinded, placebo-controlled phase 1 trial to evaluate the safety, tolerability, and pharmacokinetics of meplazumab in healthy subjects, and an open-labeled, concurrent controlled add-on exploratory phase 2 study to determine the efficacy in COVID-19 patients. In phase 1 study, 59 subjects were enrolled and assigned to eight cohorts, and no serious treatment-emergent adverse event (TEAE) or TEAE grade ≥3 was observed. The serum and peripheral blood Cmax and area under the curve showed non-linear pharmacokinetic characteristics. No obvious relation between the incidence or titer of positive anti-drug antibody and dosage was observed in each cohort. The biodistribution study indicated that meplazumab reached lung tissue and maintained >14 days stable with the lung tissue/cardiac blood-pool ratio ranging from 0.41 to 0.32. In the exploratory phase 2 study, 17 COVID-19 patients were enrolled, and 11 hospitalized patients were involved as concurrent control. The meplazumab treatment significantly improved the discharged (P = 0.005) and case severity (P = 0.021), and reduced the time to virus negative (P = 0.045) in comparison to the control group. These results show a sound safety and tolerance of meplazumab in healthy volunteers and suggest that meplazumab could accelerate the recovery of patients from COVID-19 pneumonia with a favorable safety profile.

Genomic insights into a plant growth-promoting Pseudomonas koreensis strain with cyclic lipopeptide-mediated antifungal activity.

Strain S150 was isolated from the tobacco rhizosphere as a plant growth-promoting rhizobacterium. It increased plant fresh weight significantly and lateral root development, and it antagonized plant pathogenic fungi but not phytobacteria. Further tests showed that strain S150 solubilized organic phosphate and produced ammonia, siderophore, protease, amylase, and cellulase, but it did not produce indole-3-acetic acid. Using morphology, physiological characteristics, and multi-locus sequence analysis, strain S150 was identified as Pseudomonas koreensis. The complete genome of strain S150 was sequenced, and it showed a single circular chromosome of 6,304,843 bp with a 61.09% G + C content. The bacterial genome contained 5,454 predicted genes that occupied 87.7% of the genome. Venn diagrams of the identified orthologous clusters of P. koreensis S150 with the other three sequenced P. koreensis strains revealed up to 4,167 homologous gene clusters that were shared among them, and 21 orthologous clusters were only present in the genome of strain S150. Genome mining of the bacterium P. koreensis S150 showed that the strain possessed 10 biosynthetic gene clusters for secondary metabolites, which included four clusters of non-ribosomal peptide synthetases (NRPSs) involved in the biosynthesis of cyclic lipopeptides (CLPs). One of the NRPSs possibly encoded lokisin, a cyclic lipopeptide produced by fluorescent Pseudomonas. Genomic mutation of the lokA gene, which is one of the three structural NRPS genes for lokisin in strain S150, led to a deficiency in fungal antagonism that could be restored fully by gene complementation. The results suggested that P. koreensis S150 is a novel plant growth-promoting agent with specific cyclic lipopeptides and contains a lokisin-encoding gene cluster that is dominant against plant fungal pathogens.

Identification of Benzyloxy Carbonimidoyl Dicyanide Derivatives as Novel Type III Secretion System Inhibitors via High-Throughput Screening.

The type III secretion system (T3SS) in many Gram-negative bacterial pathogens is regarded as the most critical virulence determinant and an attractive target for novel anti-virulence drugs. In this study, we constructed a T3SS secretion reporter containing the ß-lactamase gene fused with a signal peptide sequence of the T3SS effector gene, and established a high-throughput screening system for T3SS inhibitors in the plant pathogenic bacterium Acidovorax citrulli. From a library of 12,000 chemical compounds, we identified a series of benzyloxy carbonimidoyl dicyanide (BCD) derivatives that effectively blocked T3SS-dependent ß-lactamase secretion. Substitution of halogens or nitro groups at the para-position on the benzene ring contributed to an increased inhibitory activity. One representative compound, BCD03 (3,4-dichloro-benzyloxy carbonimidoyl dicyanide), dramatically reduced pathogenicity of A. citrulli on melon seedlings, and attenuated hypersensitive responses in the non-host Nicotiana tabacum caused by pathogenic bacteria A. citrulli, Xanthomonas oryzae pv. oryzae and Pseudomonas syringae pv. tomato at sub-MIC concentrations. Western blotting assay further confirmed that BCD03 inhibited effector secretion from the above bacteria via T3SS in the liquid medium. Taken together, our data suggest that BCD derivatives act as novel inhibitors of T3SS in multiple plant bacterial pathogens.

Comprehensive analysis of key genes and microRNAs in radioresistant nasopharyngeal carcinoma.

BACKGROUND: Radioresistance is one of the main obstacle limiting the therapeutic efficacy and prognosis of patients, the molecular mechanisms of radioresistance is still unclear. The purpose of this study was to identify the key genes and miRNAs and to explore their potential molecular mechanisms in radioresistant nasopharyngeal carcinoma. METHODS: In this study, we analysis the differentially expressed genes and microRNA based on the database of GSE48501 and GSE48502, and then employed bioinformatics to analyze the pathways and GO terms in which DEGs and DEMS target genes are involved. Moreover, Construction of protein-protein interaction network and identification of hub genes. Finally, analyzed the biological networks for validated target gene of hub miRNAs. RESULTS: A total of 373 differentially expressed genes (DEGs) and 14 differentially expressed microRNAs (DEMs) were screened out. The up-regulated gene JUN was overlap both in DEGs and publicly available studies, which was potentially targeted by three miRNAs, including hsa-miR-203, hsa-miR-24 and hsa-miR-31. Moreover, Pathway analysis showed that both up-regulated gene and DEMs target genes were enriched in TGF-beta signaling pathway, Hepatitis B, Pathways in cancer and p53 signaling pathway. Finally, we further constructed protein-protein interaction network (PPI) of DEGs and analyzed the biological networks for above mentioned common miRNAs, the result indicated that JUN was a core hub gene in PPI network, hsa-miR-24 and its target gene were significantly enriched in P53 signaling pathway. CONCLUSIONS: These results might provide new clues to improve the radiosensitivity of Nasopharyngeal Carcinoma.

SATB2 suppresses non-small cell lung cancer invasiveness by G9a.

Special AT-rich sequence-binding protein 2 (SATB2) is a transcription factor, which plays an important role in transcriptional regulation and chromatin recombinant by combining with matrix attachment regions. More evidence shows that SATB2 is involved in progression of breast cancer, head and neck squamous cell carcinomas and osteosarcoma. However, the role of SATB2 in cancer initiation and progression is still not well understood. Our study identified that decreased expression of SATB2 was correlated with tumor progression and poor prognosis in non-small cell lung cancer (NSCLC) patients. Furthermore, SATB2 suppressed lung cancer cell invasion and metastasis and regulated the expression of EMT-related proteins and histone methylation by G9a. In summary, SATB2 may act as a tumor suppressor gene in NSCLC.

Reference Intervals of Mitochondrial DNA Copy Number in Peripheral Blood for Chinese Minors and Adults.

BACKGROUND: Mitochondrial DNA (mtDNA) content measured by different techniques cannot be compared between studies, and age- and tissue-related control values are hardly available. In the present study, we aimed to establish the normal reference range of mtDNA copy number in the Chinese population. METHODS: Two healthy cohorts of 200 Chinese minors (0.1-18.0 years) and 200 adults (18.0-88.0 years) were recruited. Then, they were further categorized into eight age groups. The absolute mtDNA copy number per cell was measured by a quantitative real-time polymerase chain reaction. We subsequently used this range to evaluate mtDNA content in four patients (0.5-4.0 years) with molecularly proven mitochondrial depletion syndromes (MDSs) and 83 cases of mitochondrial disease patients harboring the m.3243A>G mutation. RESULTS: The reference range of mtDNA copy number in peripheral blood was 175-602 copies/cell (mean: 325 copies/cell) in minors and 164-500 copies/cell (mean: 287 copies/cell) in adults. There was a decreasing trend in mtDNA copy number in blood with increasing age, especially in 0-2-year-old and >50-year-old donors. The mean mtDNA copy number level among the mitochondrial disease patients with m.3243A>G mutation was significantly higher than that of healthy controls. The mtDNA content of POLG, DGUOK, TK2, and SUCLA2 genes in blood samples from MDS patients was reduced to 25%, 38%, 32%, and 24%, respectively. CONCLUSIONS: We primarily establish the reference intervals of mtDNA copy number, which might contribute to the clinical diagnosis and monitoring of mitochondrial disease.

Involvement of microRNA-141-3p in 5-fluorouracil and oxaliplatin chemo-resistance in esophageal cancer cells via regulation of PTEN.

microRNAs (miRNAs) act as a major regulator of acquired chemo-resistance in various types of cancer therapeutics. This study investigated the contribution of miRNAs in influencing multiple drug resistance in esophageal squamous cell carcinoma (ESCC). The sensitivity of four ESCC cell lines (EC109, EC9706, TE-1 and KYSE-150) to 5-fluorouracil (5-FU) and oxaliplatin (OX) was determined by MTT assay. A 5-FU and OX-resistant subline, EC9706R, was established by continuous exposure to stepwise increasing concentration of 5-FU and OX. Microarray technology was used to compare the differential expression of miRNAs between resistant cells and parental cells. Chemo-sensitivity assay was performed to evaluate drug response in EC9706R cells transfected with miRNA mimic or inhibitor. The direct targets of miRNA were identified by employing pathway analysis and then confirmed with luciferase assay. Sixty ESCC tissue samples and their paired adjacent normal tissues were collected to validate the expression of identified miRNA. Mouse models were further utilized to investigate the function of miRNA on acquired chemo-resistance. MicroRNA panel results indicated that a total of 12 miRNAs were differentially expressed and miR-141-3p was highly over expressed in resistant cells. Inhibition of miR-141-3p reversed acquired chemo-resistance in EC9706R cells by stimulating apoptosis. The expression of miR-141-3p was significantly increased in ESCC tissue samples compared to their matched distant normal tissues. In addition, the elevated miR-141-3p expression was found to be associated with ESCC differentiation status and TNM stage. Moreover, Phosphatase and tensin homolog (PTEN) was identified as direct target of miR-141-3p. Western blot exhibited altered protein levels of PTEN, Akt, and PI3k with miR-141-3p inhibitor. An inverse correlation between PTEN expression and miR-141-3p expression was also observed in tissue samples. EC9706R xenograft mouse model became sensitized to 5-FU and OX treatment following miR-141-3p inhibitor transfection in vivo. Our study demonstrated that miR-141-3p contributed to an acquired chemo-resistance through PTEN modulation both in vitro and in vivo.

Clinical and Molecular Characteristics in 100 Chinese Pediatric Patients with m.3243A>G Mutation in Mitochondrial DNA.

BACKGROUND: Mitochondrial diseases are a group of energy metabolic disorders with multisystem involvements. Variable clinical features present a major challenge in pediatric diagnoses. We summarized the clinical spectrum of m.3243A>G mutation in Chinese pediatric patients, to define the common clinical manifestations and study the correlation between heteroplasmic degree of the mutation and clinical severity of the disease. METHODS: Clinical data of one-hundred pediatric patients with symptomatic mitochondrial disease harboring m.3243A>G mutation from 2007 to 2013 were retrospectively reviewed. Detection of m.3243A>G mutation ratio was performed by polymerase chain reaction (PCR)-restriction fragment length polymorphism. Correlation between m.3243A>G mutation ratio and age was evaluated. The differences in clinical symptom frequency of patients with low, middle, and high levels of mutation ratio were analyzed by Chi-square test. RESULTS: Sixty-six patients (66%) had suffered a delayed diagnosis for an average of 2 years. The most frequent symptoms were seizures (76%), short stature (73%), elevated plasma lactate (70%), abnormal magnetic resonance imaging/computed tomography (MRI/CT) changes (68%), vomiting (55%), decreased vision (52%), headache (50%), and muscle weakness (48%). The mutation ratio was correlated negatively with onset age (r = -0.470, P < 0.001). Myopathy was more frequent in patients with a high level of mutation ratio. However, patients with a low or middle level of m.3243A>G mutation ratio were more likely to suffer hearing loss, decreased vision, and gastrointestinal disturbance than patients with a high level of mutation ratio. CONCLUSIONS: Our study showed that half of Chinese pediatric patients with m.3243A>G mutation presented seizures, short stature, abnormal MRI/CT changes, elevated plasma lactate, vomiting, and headache. Pediatric patients with these recurrent symptoms should be considered for screening m.3243A>G mutation. Clinical manifestations and laboratory abnormalities should be carefully monitored in patients with this point mutation.

Effect of baicalein on the expression of SATB1 in human breast cancer cells.

The aim of the present study was to investigate the effects of baicalein on the protein expression of SATB1 in the MDA-MB-231 human breast cancer cell line. MDA-MB-231 cells were treated with various concentrations of baicalein (0, 10, 20, 40 µM). Following treatment, a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay and wound healing assay were used to detect the changes in cell proliferation and migration. In addition, western blot analysis was performed to detect the changes in the protein expression levels of SATB1 in the MDA-MB-231 cell line. With the prolongation of administration time and the increase in drug concentration, the inhibitory effect of baicalein on the proliferation and migration of MDA-MB-231 cells gradually increased in a time- and dose-dependent manner (P<0.05). In addition, baicalein was shown to markedly decrease the protein expression levels of SATB1 in the MDA-MB-231 cells. With increasing drug concentrations, the protein expression levels of SATB1 decreased gradually (P<0.05). Therefore, baicalein was demonstrated to inhibit the proliferation of MDA-MB-231 cells and downregulate the protein expression of SATB1, indicating that baicalein can significantly inhibit the proliferation, migration and invasiveness of MDA-MB-231 cells by downregulating the expression of SATB1.

[Expression and significance of respiratory chain enzyme of cells in urine sediment in MELAS syndrome].

OBJECTIVE: To explore the expression and significance of respiratory chain enzyme of cells in urine sediment in mitochondrial encephalopathy myopathy, lactic acidosis and stroke-like episodes (MELAS) syndrome. METHODS: Through enzyme histochemistry, the authors analyzed the changes of respiratory chain enzyme in urine sediment in 20 MELAS patients due to mitochondrial A3243G mutation (MELAS group) and 20 health peoples (control group). And the impact on the expression of protein encoded by nuclear DNA (A21347) and mitochondrial DNA (A6404) was detected by immunochemistry. Image pro Plus 6.0 software was used for analysis of absorbance (A) of staining images as staining intensity. The data were expressed as M (Q1, Q3) and analyzed through statistical software. RESULTS: The staining intensity of complexes Iin the MELAS group was lower than that in the control group (0.06(0.01, 0.12) vs 0.12(0.01, 0.62), P = 0.010). The intergroup staining intensity of complex II showed no marked difference. Increased density of blue particle and cytoplasmic gathering was found in 13 cased (65%) of the MELAS group under light microscope. The staining intensity of complexes IV was expressed at a low level in the MELAS group (0.14(0.03, 0.32) vs 0.23(0.06, 0.43), P = 0.038). The expression of protein encoded by nuclear DNA (A21347) was lower than that in the control group (0.05(0.02, 0.45) vs 0.17(0.03, 0.70), P = 0.000). The expression of protein encoded by mitochondrial DNA (A6404) was also lower than that in the control group (0.03(0.01, 0.07) vs 0.15 (0.09, 0.23), P = 0.000). CONCLUSION: Abnormal change of respiratory chain enzyme in urine sediment in MELAS due to mitochondrial A3243G mutation and a low expression of proteins encoded by two kinds of DNA in complexes IV can help to confirm the genetic diagnosis of mitochondrial encephalomyopathies so that different subtypes may be classified and its pathogenesis elucidated.

[Clinical, pathological and molecular biological characteristics of mitochondrial encephalomyopathy with lactic acidosis and stroke-like episode in children].

OBJECTIVE: To delineate the characteristics of the clinical manifestation, pathology of skeletal muscle and gene mutations of mitochondrial encephalomyopathy with lactic acidosis and stroke-like episode (MELAS) in children. METHOD: The clinical manifestation, laboratorial data, brain images, muscle pathology and mitochondrial gene mutations were analyzed in 24 patients with MELAS who were diagnosed in Department of Pediatrics, Peking University First Hospital. Their prognosis was evaluated by following up. RESULT: Symptoms of central nervous system such as stroke-like episodes, vomiting, convulsion and headache were present in all the 24 cases. Nine cases had the symptoms of myopathy. Twenty cases had developmental delay. Short stature, being thin and hairy was very common in these cases. Serum lactate level increased in all the cases, pyruvate increased in 17 cases. Elevated CSF lactate was found in 2 cases. Magnetic resonance imaging (MRI) was performed on 24 cases, out of them 23 were abnormal. The lesions mainly involved cerebral lobes. Occipital lobe was the most common site of lesions. Computed tomography (CT) was performed on 13 cases, low density lesions were present in 10 cases, basal ganglia calcifications in 5 cases. Muscle biopsy was performed on 8 cases, ragged-red fibers (RRF) were found in 4/8 cases, and abnormal accumulation of mitochondria were found in 3/8 cases. The mtDNA gene mutational analysis showed A3243G mutation in these patients. The mutation rates varied from 11.6% to 75.0%. The same mutation were found in 4/5 mothers who had the genetic tests, and the mutation rates of the mothers varied from 15.0% to 23.6%. The clinical information of 11 cases was available through recent following up. Three cases died, the others had some degrees of mental retardation. CONCLUSION: Children with MELAS had various clinical manifestations. Central nervous system and skeletal muscle were usually involved. Short stature and hypertrichosis were common signs. The prognosis of this disease was disappointing. mtDNA A3243G was the most common mutation in MELAS. Fully understanding the characteristics of its clinical manifestation, laboratory tests, brain image, muscle pathology and molecular features can be helpful to the early diagnosis and treatment.

[Audiologic features of mitochondrial DNA A3243G mutation and its correlation with mutation rate].

OBJECTIVE: To summarize the clinical audiologic features of patients with mitochondrial DNA (mtDNA) A3243G mutation and explore the lesion location of hearing loss so as to examine its correlation with the related syndrome. METHODS: A total of 44 patients with mtDNA A3243G mutation from 2009-2011 were studied. Audiological evaluations consisted of measurements of pure-tone and speech audiometry, tympanometry, distortion-product otoacoustic emissions and auditory brainstem response. We investigated a possible correlation between the degree of hearing loss and gender, age and mutation rate. RESULTS: (1) Pure tone test was performed in 41 patients and showed normal hearing or symmetrical sensorineural hearing loss. Pure tone audiogram (PTA) showed high-frequency loss and descending curve in a majority of patients. There were 75 ears with hearing loss in 82 ears (91.46%), 22 ears with abnormal speech audiometry in 26 ears, 77 ears with abnormal distortion product otoacoustic emissions (DPOAE)testing in 86 ears, including 5 ears with normal PTA, 31 ears with abnormal electrocochleography in 75 ears, 25 ears with abnormal auditory brainstem response (ABR) in 82 ears. The abnormal ABR showed elevated threshold in 10 ears, delayed interpeak latencies of wave I-V in 2 ears and disappearance of wave V before wave I in 1 ear. In addition, there were 2 ears with speech audiometry abnormal but with normal ABR. (2) The correlation between the severity of hearing and gender did not reach statistical significance, nor the severity of hearing and mutation ratio. Age could influence the hearing of A3243G-induced MELAS. CONCLUSIONS: The predominant lesions of mtDNA A3243G is at cochlea and retrocochlear sites. Significant variations in clinical manifestation of hearing are the prominent features in patient with A3243G mutation. There was no correlation between the degree of hearing loss and mutation load. However, hearing impairment is the most common symptom of A3243G mutation.