Biological Characteristics of Recombinant Arthrobotrys oligospora Chitinase AO-801.

Chitinase AO-801 is a hydrolase secreted by Arthrobotrys oligospora during nematode feeding, while its role remained elusive. This study analyzed the molecular characteristics of recombinant chitinase of Arthrobotrys oligospora (reAO-801). AO-801 belongs to the typical glycoside hydrolase 18 family with conserved chitinase sequence and tertiary structure of (α/ß)8 triose-phosphate isomerase (TIM) barrel. The molecular weight of reAO-801 was 42 kDa. reAO-801 effectively degraded colloidal and powdered chitin, egg lysate, and stage I larval lysate of Caenorhabditis elegans. The activity of reAO-801 reached its peak at 40˚C and pH values between 4-7. Enzyme activity was inhibited by Zn2+, Ca2+, and Fe3+, whereas Mg2+ and K+ potentiated its activity. In addition, urea, sodium dodecyl sulfate, and 2-mercaptoethanol significantly inhibited enzyme activity. reAO-801 showed complete nematicidal activity against C. elegans stage I larvae. reAO-801 broke down the C. elegans egg shells, causing them to die or die prematurely by hatching the eggs. It also invoked degradation of Haemonchus contortus eggs, resulting in apparent changes in the morphological structure. This study demonstrated the cytotoxic effect of reAO-801, which laid the foundation for further dissecting the mechanism of nematode infestation by A. oligospora.

Overexpression of mitochondrial histidyl-tRNA synthetase restores mitochondrial dysfunction caused by a deafness-associated tRNAHis mutation.

The deafness-associated m.12201T>C mutation affects the A5-U68 base-pairing within the acceptor stem of mitochondrial tRNAHis The primary defect in this mutation is an alteration in tRNAHis aminoacylation. Here, we further investigate the molecular mechanism of the deafness-associated tRNAHis 12201T>C mutation and test whether the overexpression of the human mitochondrial histidyl-tRNA synthetase gene (HARS2) in cytoplasmic hybrid (cybrid) cells carrying the m.12201T>C mutation reverses mitochondrial dysfunctions. Using molecular dynamics simulations, we demonstrate that the m.12201T>C mutation perturbs the tRNAHis structure and function, supported by decreased melting temperature, conformational changes, and instability of mutated tRNA. We show that the m.12201T>C mutation-induced alteration of aminoacylation tRNAHis causes mitochondrial translational defects and respiratory deficiency. We found that the transfer of HARS2 into the cybrids carrying the m.12201T>C mutation raises the levels of aminoacylated tRNAHis from 56.3 to 75.0% but does not change the aminoacylation of other tRNAs. Strikingly, HARS2 overexpression increased the steady-state levels of tRNAHis and of noncognate tRNAs, including tRNAAla, tRNAGln, tRNAGlu, tRNALeu(UUR), tRNALys, and tRNAMet, in cells bearing the m.12201T>C mutation. This improved tRNA metabolism elevated the efficiency of mitochondrial translation, activities of oxidative phosphorylation complexes, and respiration capacity. Furthermore, HARS2 overexpression markedly increased mitochondrial ATP levels and membrane potential and reduced production of reactive oxygen species in cells carrying the m.12201T>C mutation. These results indicate that HARS2 overexpression corrects the mitochondrial dysfunction caused by the tRNAHis mutation. These findings provide critical insights into the pathophysiology of mitochondrial disease and represent a step toward improved therapeutic interventions for mitochondrial disorders.

Contribution of the tRNAIle 4317A→G mutation to the phenotypic manifestation of the deafness-associated mitochondrial 12S rRNA 1555A→G mutation.

The 1555A→G mutation in mitochondrial 12S rRNA has been associated with aminoglycoside-induced and non-syndromic deafness in many individuals worldwide. Mitochondrial genetic modifiers are proposed to influence the phenotypic expression of m.1555A→G mutation. Here, we report that a deafness-susceptibility allele (m.4317A→G) in the tRNAIle gene modulates the phenotype expression of m.1555A→G mutation. Strikingly, a large Han Chinese pedigree carrying both m.4317A→G and m.1555A→G mutations exhibited much higher penetrance of deafness than those carrying only the m.1555A→G mutation. The m.4317A→G mutation affected a highly conserved adenine at position 59 in the T-loop of tRNAIle We therefore hypothesized that the m.4317A→G mutation alters both structure and function of tRNAIle Using lymphoblastoid cell lines derived from members of Chinese families (three carrying both m.1555A→G and m.4317A→G mutations, three harboring only m.1555A→G mutation, and three controls lacking these mutations), we found that the cell lines bearing both m.4317A→G and m.1555A→G mutations exhibited more severe mitochondrial dysfunctions than those carrying only the m.1555A→G mutation. We also found that the m.4317A→G mutation perturbed the conformation, stability, and aminoacylation efficiency of tRNAIle These m.4317A→G mutation-induced alterations in tRNAIle structure and function aggravated the defective mitochondrial translation and respiratory phenotypes associated with the m.1555A→G mutation. Furthermore, mutant cell lines bearing both m.4317A→G and m.1555A→G mutations exhibited greater reductions in the mitochondrial ATP levels and membrane potentials and increasing production of reactive oxygen species than those carrying only the m.1555A→G mutation. Our findings provide new insights into the pathophysiology of maternally inherited deafness arising from the synergy between mitochondrial 12S rRNA and tRNA mutations.

The interaction between mitochondria and oncoviruses.

Mitochondria play important roles in multiple aspects of viral tumorigenesis. Mitochondrial genomes contribute to the host's genetic background. After viruses enter the cell, they modulate mitochondrial function and thus alter bioenergetics and retrograde signaling pathways. At the same time, mitochondria also regulate and mediate viral oncogenesis. In this context, oncogenesis by oncoviruses like Hepatitis B virus (HBV), Hepatitis C virus (HCV), Human papilloma virus (HPV), Human Immunodeficiency virus (HIV) and Epstein-Barr virus (EBV) will be discussed.

Synthesis of Isoquinolines from Benzimidates and Alkynes via Cobalt(III)-Catalyzed C-H Functionalization/Cyclization.

C-H alkenylation/annulation of benzimidates with alkynes has been realized by using a Cp*Co(III) catalyst under air. A series of substituted isoquinolines were obtained with moderate to good yields under mild reaction conditions.

A deafness-associated tRNAHis mutation alters the mitochondrial function, ROS production and membrane potential.

In this report, we investigated the molecular genetic mechanism underlying the deafness-associated mitochondrial tRNAHis 12201T>C mutation. The destabilization of a highly conserved base-pairing (5A-68U) by the m.12201T>C mutation alters structure and function of tRNAHis. Using cybrids constructed by transferring mitochondria from lymphoblastoid cell lines derived from a Chinese family into mtDNA-less (ρo) cells, we showed ∼70% decrease in the steady-state level of tRNAHis in mutant cybrids, compared with control cybrids. The mutation changed the conformation of tRNAHis, as suggested by slower electrophoretic mobility of mutated tRNA with respect to the wild-type molecule. However, ∼60% increase in aminoacylated level of tRNAHis was observed in mutant cells. The failure in tRNAHis metabolism was responsible for the variable reductions in seven mtDNA-encoded polypeptides in mutant cells, ranging from 37 to 81%, with the average of ∼46% reduction, as compared with those of control cells. The impaired mitochondrial translation caused defects in respiratory capacity in mutant cells. Furthermore, marked decreases in the levels of mitochondrial ATP and membrane potential were observed in mutant cells. These mitochondrial dysfunctions caused an increase in the production of reactive oxygen species in the mutant cells. The data provide the evidence for a mitochondrial tRNAHis mutation leading to deafness.

Expression of serine proteinase P186 of Arthrobotrys oligospora and analysis of its nematode-degrading activity.

The nematode-trapping fungi possess a unique capability of predating and invading nematodes. As a representative nematode-trapping fungus, Arthrobotrys oligospora has been widely used to study the interactions between nematode-trapping fungi and their hosts. Serine proteinase is one of the important virulence factors during process of invasion of the nematode-trapping fungi into nematodes. In this study, using reverse transcription polymerase chain reaction, we amplified the gene sequence of serine proteinase 186 from A. oligospora, cloned it into pPIC9K vector and expressed it in the yeast Pichia pastoris. The expressed recombinant serine proteinase186 (reP186) was purified via Ni-affinity chromatography. The in vitro nematode-degrading activity of reP186 was analyzed. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blot analysis revealed that reP186 with molecular weight of 33 kDa was successfully obtained. ReP186 was capable of degrading a series of protein substrates including casein, gelatin, bovine serum albumin, denatured collagen and nematode cortical layer. The reP186 exhibited the maximal activity at pH 8.0 and 55 °C and was highly sensitive to the inhibitor, phenylmethanesulfonylfluoride. Treatment of Caenorhabditis elegans and Haemonchus contortus with reP186 for 12, 24 and 36 h, respectively, resulted in 62, 88 and 100 % of killing rates for C. elegans, and 52, 65 and 84 % of killing rates for H. contortus, respectively, indicating a relatively strong nematode-degrading bioactivity of reP186.

[Characterization of two Chinese families with aminoglycoside-induced and nonsyndromic hearing loss both carrying a mitochondrial 12S rRNA 1494C>T mutation].

OBJECTIVE: To evaluate the effect of mitochondrial DNA(mtDNA) secondary mutations, haplotypes, GJB2 gene mutations on phenotype of 1494C>T mutation, and to study the molecular pathogenic mechanism of maternally transmitted aminoglycoside-induced and nonsyndromic hearing loss. METHODS: Two Chinese Han pedigrees of maternally transmitted aminoglycoside induced and nonsyndromic hearing loss were collected. The two probands and their family members underwent clinical, genetic and molecular evaluations including audiological examinations and mutational analysis of mitochondrial genome and GJB2 gene. RESULTS: Clinical evaluation revealed wide range of severity, age-at-onset and audiometric configuration of hearing impairment in matrilineal relatives in both families, for which the penetrance of hearing loss was respectively 42.9% and 28.6% when aminoglycoside-induced deafness was included. When the effect of aminoglycosides was excluded, the penetrances of hearing loss were 14.3% and 14.3%. Sequence analysis of mitochondrial genomes identified a known 12S rRNA 1494C>T mutation, in addition with distinct sets of mtDNA polymorphisms belonging to Eastern Asian haplogroups C4a1a and B4b1c, respectively. CONCLUSION: Mitochondrial 12S rRNA 1494C>T mutation probably underlie the deafness in both families. Lack of significant mutation in the GJB2 gene ruled out involvement of GJB2 in the phenotypic expression. However, aminoglycosides and other nuclear modifier genes may still modify the phenotype of the 1494C>T mutation in these families. The B4b1c is a newly identified haplogroup in aminoglycoside-induced and nonsyndromic hearing loss family carrying the 1494C>T mutation. The 1494C>T mutation seems to have occurred sporadically through evolution.

Frequency and spectrum of mitochondrial 12S rRNA variants in 440 Han Chinese hearing impaired pediatric subjects from two otology clinics.

BACKGROUND: Aminoglycoside ototoxicity is one of the common health problems. Mitochondrial 12S rRNA mutations are one of the important causes of aminoglycoside ototoxicity. However, the incidences of 12S rRNA mutations associated with aminoglycoside ototoxicity are less known. METHODS: A total of 440 Chinese pediatric hearing-impaired subjects were recruited from two otology clinics in the Ningbo and Wenzhou cities of Zhejiang Province, China. These subjects underwent clinical, genetic evaluation and molecular analysis of mitochondrial 12S rRNA. Resultant mtDNA variants were evaluated by structural and phylogenetic analysis. RESULTS: The study samples consisted of 227 males and 213 females. The age of all participants ranged from 1 years old to 18 years, with the median age of 9 years. Ninety-eight subjects (58 males and 40 females) had a history of exposure to aminoglycosides, accounting for 22.3% cases of hearing loss in this cohort. Molecular analysis of 12S rRNA gene identified 41 (39 known and 2 novel) variants. The incidences of the known deafness-associated 1555A > G, 1494C > T and 1095T > C mutations were 7.5%, 0.45% and 0.91% in this entire hearing-impaired subjects, respectively, and 21.4%, 2% and 2% among 98 subjects with aminoglycoside ototoxicity, respectively. The structural and phylogenetic evaluations showed that a novel 747A > G variant and known 839A > G, 1027A > G, 1310C > T and 1413T > C variants conferred increased sensitivity to aminoglycosides or nonsyndromic deafness as they were absent in 449 Chinese controls and localized at highly conserved nucleotides of this rRNA. However, other variants were polymorphisms. Of 44 subjects carrying one of definite or putative deafness-related 12S rRNA variants, only one subject carrying the 1413T > C variant harbored the 235DelC/299DelAT mutations in the GJB2 gene, while none of mutations in GJB2 gene was detected in other 43 subjects. CONCLUSIONS: Mutations in mitochondrial 12S rRNA accounted for ~30% cases of aminoglycoside-induced deafness in this cohort. Our data strongly support the idea that the mitochondrial 12S rRNA is the hot spot for mutations associated with aminoglycoside ototoxicity. These data have been providing valuable information and technology to predict which individuals are at risk for ototoxicity, to improve the safety of aminoglycoside antibiotic therapy, and eventually to decrease the incidence of deafness.

A literature review of quality in lower gastrointestinal endoscopy from the patient perspective.

BACKGROUND: Given the limited state of health care resources, increased demand for colorectal cancer (CRC) screening raises concerns about the quality of endoscopy services. Little is known about quality in colonoscopy and endoscopy from the patient perspective. OBJECTIVE: To systematically review the literature on quality that is relevant to patients who require colonoscopy or endoscopy services. METHODS: A systematic PubMed search was performed on articles that were published between January 2000 and February 2011. Keywords included "colonoscopy" or "sigmoidoscopy" or "endoscopy" AND "quality"; "colonoscopy" or "sigmoidoscopy" or "endoscopy" AND "patient satisfaction" or "willingness to return". The included articles were qualitative and quantitative English language studies regarding aspects of colonoscopy and/or endoscopy services that were evaluated by patients in which data were collected within one year of the colonoscopy/endoscopy procedure. RESULTS: In total, 28 quantitative studies were identified, of which eight (28.6%) met the inclusion criteria (four cross-sectional, three prospective cohort and one single-blinded controlled study). Aspects of quality included comfort, management of pain and anxiety, endoscopy unit staff manner, skills and specialty, procedure and results discussion with the doctor, physical environment, wait times for the appointment and procedure, and discharge. Qualitative studies eliciting the patient perspective on what constituted quality in colonoscopy/endoscopy were not found. CONCLUSIONS: Factors related to comfort, staff, communication and the service environment were evaluated from the patient perspective using closed-ended questions that were designed by clinicians and researchers. Future research using qualitative methodology to elicit the patient perspective on quality in colonoscopy and/or endoscopy services is needed.

[Maternally inherited aminoglycoside-induced and nonsyndromic hearing loss in five Han Chinese pedigrees].

OBJECTIVE: To study the effect of the mitochondrial 12S rRNA mutations on aminoglycoside-induced and nonsyndromic hearing loss, to carry out the clinical and molecular characterization of five Han Chinese pedigrees with maternally transmitted aminoglycoside-induced and nonsyndromic hearing loss. METHODS: Five pedigrees of maternally transmitted aminoglycoside-induced and nonsyndromic hearing loss were collected, genomic DNA was extracted, and complete mitochondrial genomes and the gap junction protein beta 2 (GJB2) gene were amplified and sequenced. RESULTS: Clinical evaluation revealed a wide range of severity, age-at-onset and audiometric configuration of hearing impairment in the matrilineal relatives in these families. The penetrance rates of hearing loss in these pedigrees were 17.6%, 50.0%, 66.7%, 31.3% and 23.1%, with an average of 37.7%, when aminoglycoside-induced deafness was included. Sequence analysis of the complete mitochondrial genomes in these pedigrees identified the known 1555A>G mutation and distinct sets of mitochondrial DNA(mtDNA) polymorphisms belonging to Eastern Asian haplogroups D4b2b, B4c1b1, F3, C1 and D5a, respectively. Of these variants, ND1 L89T and CO3 A200T mutations resided at the highly conservative regions. However, there were no functionally significant mutations in tRNAs and rRNAs or secondary known mutations. No hearing loss related GJB2 gene mutation was observed. CONCLUSION: The lack of significant mutation in the ruled out the possible involvement of GJB2 in the phenotypic expression of the 1555A>G mutation in those affected subjects. However, aminoglycosides, mtDNA variations and other nuclear modifier genes may play an important role in the phenotypic manifestation of the 1555A>G mutation in these Chinese families.

Maternally inherited hearing loss is associated with the novel mitochondrial tRNA Ser(UCN) 7505T>C mutation in a Han Chinese family.

Mutations in mitochondrial DNA (mtDNA) have been found to be one of the most important causes of sensorineural hearing loss. We report here a clinical, genetic, molecular and biochemical characterization of a Han Chinese pedigree with maternally transmitted nonsyndromic hearing impairment. Seven of nine matrilineal relatives exhibited a variable severity and age-at-onset (8 years old) of hearing loss. Mutational analysis of mtDNA identified the novel homoplasmic tRNA(Ser(UCN)) 7505T>C mutation and other 37 variants belonging to haplogroup F1. The 7505T>C mutation, which is absent in 449 Chinese controls, is located at a highly conserved base-pairing (10A-20U) of tRNA(Ser(UCN)). The abolishment of 10A-20U base-pairing likely alters the tRNA(Ser(UCN)) metabolism. Functional significant of this mutation was supported by approximately 65% reductions in the level of tRNA(Ser(UCN)) observed in the lymphoblastoid cell lines carrying the 7505T>C mutation, compared with the wild-type cell lines. This reduced tRNA level is below the proposed threshold to support a normal respiration in lymphoblastoid cells. Furthermore, the highly conserved tRNA(Ala) 5587T>C and Cytb C93Y variants may have a modifying role of deafness expression associated with the 7505T>C mutation. However, genotyping analysis of nuclear modifier gene TRMU and the prominent deafness-cause gene GJB2 failed to detect any mutations in the member of this family. These data strongly indicate that the novel tRNA(Ser(UCN)) 7505T>C mutation is involved in maternally transmitted hearing loss. However, other genetic, epigenetic or environmental factors may contribute to the phenotypic variability of this family. Our findings will be helpful for counseling families of maternally inherited hearing loss.

Novel Insights Into Triple-Negative Breast Cancer Prognosis by Comprehensive Characterization of Aberrant Alternative Splicing.

BACKGROUND: Alternative splicing (AS) is important in the regulation of gene expression and aberrant AS is emerging as a major factor in the pathogenesis of human conditions, including cancer. Triple-negative breast cancer (TNBC) is the most challenging subtype of breast cancer with strong invasion, high rate of metastasis, and poor prognosis. Here we report a systematic profiling of aberrant AS in TNBC. METHODS: The percent spliced in (PSI) values for AS events in 151 TNBC patients were obtained from The Cancer Genome Atlas (TCGA) SpliceSeq database. Univariate Cox and stepwise Multivariate Cox regression analyses were conducted to find the best prognostic AS model. Splicing regulatory networks were constructed by prognosis-related spliceosome and aberrant AS events. Additionally, pathway enrichment and gene set enrichment analysis (GSEA) were further employed to reveal the significant pathways for prognosis-related AS genes. Finally, splicing regulatory networks were constructed via Spearman's rank correlation coefficients between prognosis-related AS events and splicing factor expressions. RESULTS: A total of 1,397 prognosis-associated AS events were identified in TNBC. The majority of the parent genes of prognostic AS events exhibited direct interactions to each other in the STRING gene network. Pathways of focal adhesion (p < 0.001), RNA splicing (p = 0.007), homologous recombination (p = 0.042) and ECM-receptor interaction (p = 0.046) were found to be significantly enriched for prognosis-related AS. Additionally, the area under curve (AUC) of the best AS prognostic predictor model reached 0.949, showing a powerful capability to predict outcomes. The Exon Skip (ES) type of AS events displayed more robust and efficient capacity in predicting performance than any other specific AS events type in terms of prognosis. The ES AS signature might confer a strong oncogenic phenotype in the high-risk group with elevated activities in cell cycle and SUMOylating pathways of tumorigenesis, while programmed cell death and metabolism pathways were found to be enriched in the low-risk group of TNBC. The splicing correlation network also revealed a regulatory mode of prognostic splicing factors (SFs) in TNBC. CONCLUSION: Our analysis of AS events in TNBC could not only contribute to elucidating the tumorigenesis mechanism of AS but also provide clues to uncovering underlying prognostic biomarkers and therapeutic targets for further study.

Multi-Epitope Fusion Protein Eg mefAg-1 as a Serodiagnostic Candidate for Cystic Echinococcosis in Sheep

Cystic echinococcosis (CE) in sheep is a hazardous zoonotic parasitic disease that is caused by Echinococcus granulosus (Eg). At present, serological test is an important diagnostic method for Eg infection in domestic animals. Here, a fusion protein Eg mefAg-1 harboring 8 dominant B-cell epitopes of Eg such as antigen B, tetraspanin 1, tetraspanin 6, reticulon and Eg95 was produced in E. coli and evaluated for CE in sheep by indirect ELISA. Eg mefAg-1 showed in ELISA a high sensitivity (93.41%) and specificity (99.31%), with a coincidence rate of 97.02%. Overall, it is suggested that the Eg mefAg-1 could be a potential antigen candidate for CE serodiagnosis in sheep.

Low amounts and high thiol oxidation of peroxiredoxins in spermatozoa from infertile men.

Seminal oxidative stress occurs when there is an increased production of reactive oxygen species (ROS) and/or a decrease of antioxidant activity, promoting impaired sperm function. Peroxiredoxins (PRDX) are abundant in human semen and are important antioxidant enzymes, which act as ROS scavengers and modulators in ROS-dependent signaling. Our aim was to determine whether the levels of PRDX1 and PRDX6 and their oxidation on thiol groups are associated with a decrease in sperm motility and DNA integrity. We evaluated the sperm and seminal PRDX level in men (13 healthy controls, 15 men with clinical varicocele, and 17 men with idiopathic infertility). We assessed conventional semen parameters, sperm DNA integrity (by the sperm chromatin structure assay), lipid peroxidation in seminal plasma and spermatozoa (by the thiobarbituric acid reactive substances assay), and the amount and thiol oxidation of PRDX1 and PRDX6 (by immunoblotting). PRDXs were affected in seminal plasma (lower amounts) and in sperm samples (lower amounts and higher levels of thiol oxidation) characterized by lower sperm motility, higher lipid peroxidation, and sperm DNA damage. The thioloxidation ratio of PRDXs (thiol-oxidized PRDX/total PRDX) correlated negatively with sperm motility (total and progressive) and positively with sperm DNA damage and sperm lipid peroxidation. In conclusion, because of the lower amount of total PRDX1 and PRDX6 and the high thiol oxidation of these PRDXs, very little (less than 20%) protection due to PRDXs remains, and this is associated with impaired sperm function and poor DNA integrity and suggests an important role of PRDXs in the protection of human spermatozoa against oxidative stress.