Leigh Syndrome Due to mtDNA Pathogenic Variants

Abstract Leigh syndrome is a devastating neurodegenerative disease, typically manifesting in infancy or early childhood. Hallmarks of the disease are symmetrical lesions in the basal ganglia or brain stem on MRI, and a clinical course with rapid deterioration of cognitive and motor functions. It is genetically heterogeneous, causative mutations have been disclosed in mitochondrial DNA and nuclear genes involved in the process of energy production in the mitochondria .We investigated the whole mitochondrial DNA in three Brazilian patients with LS, based on their clinical and biochemical data, with the aim to identify the disease-causing mutations. In two of the patients, with complex I deficiency, a novel heteroplasmic variant m.4142G>T (p.R279L) in MT-ND1 and a recurrent homoplasmic mutation m.10197G>A (p.A47T) in MT-ND3 were identified. In the remaining patient, with complex IV deficiency, a de novo heteroplasmic variant in MT-CO1 m.6547T>C (p.L215P) was found. The molecular investigation in mitochondrial diseases have shifted their focus from mitochondrial DNA to nuclear DNA, however, mtDNA protein-coding genes are one of the important genetic causes of mitochondrial disorders for Leigh syndrome. This study expands the molecular and clinical spectrum associated with this disease.

Molecular Characterization of Hard Ticks by Cytochrome c Oxidase Subunit 1 Sequences

Although widely studied, the natural diversity of the hard tick is not well known. In this study, we collected 194 sequences from 67 species, covering 7 genera of hard tick. The 5′ region of the mitochondrial cytochrome c oxidase subunit 1 region (586 bp) has been used to investigate intra- and inter-species variation and the phylogenetic tree of neighbor joining method has been used for assessment. As a result, by comparing the K2P-distance of intra- and interspecies, 30 samples (15.2%) shown that interspecies distance was larger than the minimum interspecfic distance. From the phylogenetic analysis, 86.8% (49) of the species were identified correctly at the genus level. On deeper analysis on these species suggested the possibility of presence cryptic species. Therefore, further work is required to delineate species boundaries and to develop a more complete understanding of hard tick diversity over larger scale.

Genetic Diversity of Echinococcus granulosus Genotype G1 in Xinjiang, Northwest of China

Cystic echinococcosis (CE) caused by E. granulosus is a serious helminthic zoonosis in humans, livestock and wildlife. Xinjiang is one of high endemic province for CE in China. A total of 55 sheep and cattle livers containing echinococcal cysts were collected from slaughterhouses in Changji and Yining City, northern region of Xinjiang. PCR was employed for cloning 2 gene fragments, 12S rRNA and CO1 for analysis of phylogenetic diversity of E. granulosus. The results showed that all the samples collected were identified as G1 genotype of E. granulosus. Interestingly, YL5 and CJ75 strains were the older branches compared to those strains from France, Argentina, Australia. CO1 gene fragment showed 20 new genotype haploids and 5 new genotype haplogroups (H1-H5) by the analysis of Network 5.0 software, and the YLY17 strain was identified as the most ancestral haplotype. The major haplotypes, such as CJ75 and YL5 strains, showed identical to the isolates from Middle East. The international and domestic trade of livestock might contribute to the dispersal of different haplotypes for E. granulosus evolution.

Expression of 5-Fluorouracil metabolic enZyme in colorectal cancer and its clinical signifi-cance / 临床与实验病理学杂志

Purpose To ana1yze the expression features of 5-F1uorouraci1(5-FU)metabo1ic enzyme thymidy1ate synthetase(TS),thy-midine phosphory1ase( TP),dihydropyrimidine dehydrogenase( DPD)and its re1ationship with c1inicopatho1ogica1 factors and progno-sis in co1orecta1 cancer,in order to further exp1ore its potentia1 significance in guiding co1orecta1 cancer chemotherapy. Methods Es-tab1ishment of a tissue microarray containing 72 patients with co1orecta1 cancer,and 56 norma1 tissue( dista1 cut edge tissue near carci-noma)was used to detect TS,TP,and DPD by immunohistochemistry,and to ana1yze its re1ationship with c1inicopatho1ogica1 factors and prognosis of co1orecta1 cancer through statistica1 method. Results The expression of TS in co1orecta1 cancer was 1ower than that in norma1 tissue(P=0. 876),which was associated with TNM(P=0. 043)and positive1y corre1ated with patients’overa11 surviva1(P=0. 027),the expression of TP in co1orecta1 cancer was higher than that in norma1 tissue(P=0. 315)that was associated with 1ymph node metastasis(P=0. 009)and negative1y corre1ated with the prognosis of patients(P=0. 040),DPD expression in co1orecta1 canc-er was higher than that in norma1 tissue(P=0. 071),which was re1ated to the histo1ogic type(P=0. 029). Overa11 surviva1 was sig-nificant1y shortened in co1orecta1 cancer with DPD high expression( P=0. 011). Conclusions TS,TP and DPD might be app1ied as important index of prognosis in co1orecta1 cancer patients using 5-FU adjuvant chemotherapy. The expression of TS re1ated c1ose1y with the c1inica1 stage is a bio1ogica1 marker of tumor progression. TP expression is c1ose1y re1ated to 1ymph nodes metastasis and recur-rence,which is an important factor of postoperative recurrence and metastasis.

Expression of IGF-Ⅱ and IGFbP-6 in colorectal cancer and their significance / 临床与实验病理学杂志

Purpose To investigate the expression of IGF-Ⅱand IGFBP-6 in co1orecta1 cancer,and to exp1ore the c1inica1 significance in co1orecta1 cancer. Methods IGF-Ⅱand IGFBP-6 were detected by immunohistochemistry and RT-PCR in 50 cases of co1orecta1 cancer(experimenta1 group)and 50 cases of the adjacent mucosa(contro1 group). Results (1)In the experimenta1 group,the ex-pression 1eve1 of IGF-Ⅱprotein and mRNA was significant1y higher than the contro1 group. The expression 1eve1s of IGF-Ⅱprotein and mRNA in co1orecta1 cancer were significant1y 1ower than the contro1 group.(2)The expression 1eve1s of IGF-Ⅱand IGFBP-6 were sig-nificant1y different between different tumor infi1tration depth,1ymph node metastasis,invasion depth and Duke’s stages( P0. 05). Conclusions There is a obvious corre1a-tion between of IGF-Ⅱ and IGFBP-6 in c1inica1 patho1ogica1 parameters in co1orecta1 cancer. Combined detection of the two markers may be the bio1ogica1 indicators of occurrence and prognosis of co1orecta1 cancer,and provide a new scheme for diagnosis and treatment of co1orecta1 cancer.

Expression of EGFR and hER-2 in colorectal carcinoma and their clinical significance / 临床与实验病理学杂志

Purpose To exp1ore the expression and c1inica1 significance of epiderma1 growth factor receptor( EGFR)and human epi-derma1 receptor-2(HER-2)in co1orecta1 carcinoma. Methods Immunohistochemistry(IHC)PV-9000 was used to detect the expres-sion of EGFR and HER-2 in 78 cases of co1orecta1 carcinoma. Si1ver in situ hybridization( SISH)was used to detect HER-2 amp1ifica-tion in co1orecta1 carcinoma. Results The positive rates of EGFR in 78 co1orecta1 carcinomas were 69. 23%( 54/78 ). The differ-ences of the expression of EGFR between different depth of invasion group and 1ymph node metastasis group were statistica11y signifi-cant. The differences of the expression of EGFR between different aging group,gender group,tumor size,different histo1ogica1 grading group and Dukes stage were insignificant. The positive rates of HER-2 in 78 cases of co1orecta1 carcinoma were 25. 64%(20/78). The differences of the expression of HER-2 between different depth of invasion group and 1ymph node metastasis group were statistica11y sig-nificant. The differences of the expression of HER-2 between different aging group,gender group,tumor size,histo1ogica1 grading group and Dukes stage were insignificant. The expression of EGFR and HER-2 was positive1y corre1ated. Among the 20 cases of HER-2 protein overexpression,10 cases showed HER-2 gene amp1ification,15 cases showed HER-2 protein overexpression(~)by IHC and 10 cases showed HER-2 gene amp1ification by SISH. The rate of HER-2 gene amp1ification was 66. 67%. 5 cases with 1ow HER-2 protein overexpression( +)and no case showed HER-2 gene amp1ification. Conclusions EGFR and HER-2 are high1y ex-pressed in co1orecta1 carcinoma. EGFR and HER-2 expression is connected to invasion and metastasis process of co1orecta1 carcinoma. The both may be synergy. The corre1ation between HER-2 protein overexpression(~)and HER-2 gene amp1ification is statisti-ca11y significant. Therefore,immunohistochemistry can be regarded as an initia1 screening method for HER-2 gene amp1ification,and then SISH testing shou1d be done as we11 to confirm the resu1t. It is usefu1 for mo1ecu1ar target therapy to detect HER-2 status in co1or-ecta1 carcinoma.

Gene sequence analysis on CO1 and ND1 of Echinococcus g ranulosus / 中国人兽共患病学报

The purpose of this study was to clarify the strains of E .granulosus from sheep of Xiwuqi and people of Xil-inhot in Inner Mongolia region and its genotypes .CO1 and ND1 of mitochondria gene were cloned and sequenced ,and then they were analyzed by MegAlign of DNAStar5 .0 .Results showed that the length of CO1 and ND1 gene of E .granulosus ,which were from sheep of Xiwuqi or people of Xilinhot ,were 936 bp and 895 bp ,respectively .The homology of CO1 gene sequences of E .granulosus strains from Xiwuqi and Xinjiang was 99 .3% ,while the homology of the corresponding gene sequences of E .granulosus from man of Xilinhot City and Xinjiang were 98 .6% .ND1 gene of E .granulosus of sheep from Xiwuqi and hu-man from Xilinhot were identical to ND1 gene of G1 type .All these indicated that the homology of E .granulosus from the two regions were high and the genotype were G1 type ,which provided an important basis for the determination of strains ,and it al-so had a great significance to prevent and control the disease .

Phylogenetic Relationships of 3 Korean Neodiplostomum Species (Digenea: Neodiplostomidae) Based on Partial CO1 Gene

The phylogenetic relationships of the 3 Neodiplostomum spp. (Digenea: Neodiplostomidae) occurring in Korea (N. seoulense, N. leei, and N. boryongense) were analyzed using the partial mitochondrial cytochrome c oxidase subunit 1 (CO1) gene. The adult flukes were recovered from Sprague-Dawley rats (N. seoulense) and newborn chicks (N. leei and N. boryongense) experimentally infected with the neodiplostomula from the grass snake, Rhabdophis tigrinus tigrinus. The genomic DNA was amplified using specific primers, and the sequence of CO1 was obtained. According to the results, the pairwise similarity was 96.1% between N. boryongense and N. seoulense, but was 95.0% between N. boryongense and N. leei and 94.2% between N. leei and N. seoulense. The results demonstrated a closer phylogenetic relationship between N. seoulense and N. boryongense. This high relationship of N. seoulense and N. boryongense may be related to their similar morphologic features including the limited distribution of vitellaria and the presence of a genital cone. N. leei is distinct on the other hand with an extensive distribution of vitellaria and the absence of a genital cone.

Evidence of geographical structuring in the Malaysian Snakehead, Channa striata based on partial segment of the CO1 gene

Channa striata, locally known as "haruan", is economically important in fisheries and aquaculture industries in several Asian countries. DNA sequencing, based on a partial segment of the Cytochrome oxidase c subunit 1 (CO1) gene, was used to determine genetic variation in C. striata samples from four different populations on the west coast of Peninsular Malaysia. The highest nucleotide and haplotype diversities were observed in the Linggi population (π = 0.0067, h = 0.835), and the lowest in the Timah Tasoh population (π = 0.0008, h = 0.286). Apart from Kajang-Linggi, which was insignificant, F ST values were significant (p < 0.05) in all pairwise-population comparisons. Consequently, it is inferred that genetic structuring C. striata populations in this region was largely shaped by a common origin, with secondary influences from geographical factors and isolation.

The Experimantal Studey of Delayed Wound Healing on Full-Thickness Skin Defects in Diabetic Rats

Wound healing was a natural process proceeded by connective tissue deposition, epithelialization, and wound contracture. Streptozotocin-induced diabetes mellitus was known to impair wound healing. However, the extend of delayed wound healing was not evaluate objectively in the diabetic rats. Therefore, we studied the extend of delayed wound healing(epithelialization and wound contracture) and histologic difference between diabetic and control rats. Twenty adult Sprague-Dawley rats(200-250 gm) were used as experimental animals(Diabetes: 10 rats, control: 10 rats). The wounds(2 x 2 cm, sized) were made on the back of the rats by excision through the panniculus carnosus. The areas of both wounds in relation to original wound areas (operative day) were serially measured at 0, 1,2, 3, 4, 5 weeks postoperatively. In addition, we performed histological examination of biopsy taken at 0, 1,2, 3, 4, 5weeks postoperatively. The difference in the mean area ratio between two groups was then compared using Kruskal Wallis test(SAS Program). Results were as follows: At postoperative 1 week, there was a significant difference(p < 0.05) in degree of epithelialization between the two groups, 1) After postoperative 2 weeks, there was a significant difference(p<0.05) in degree of wound contracture between the two groups. 2) In the diabetic group, the collagen fibers were smaller and poor organized than control group. Conclusion was that delay of epithelialization early and delay of wound contracture late in wound healing, were important roles in diabetic wound problem.

Genetic Variation of Two Mitochondrial DNA Molecules from Three Isolates of Oncomelania hupensis / 中国寄生虫学与寄生虫病杂志

Objective To study the genetic variation of two mitochondrial DNA molecules (CO1 and Cytb gene) of Oncomelania hupensis isolated from different areas. Methods Snails were collected from Jingxi of Guangxi,Yueyang of Hunan and Eryuan of Yunnan. Genomic DNA was extracted from the snails,Co1 and Cytb gene fragments were amplified by PCR,then purified and sequenced. Sequences of each isolates were edited by using Clustal W(1.82) software,and the nucleotide composition,transition and transversion were accounted by using MEGA(3.1) software. The genetic distances were computed with Kimura method and phylogenetic trees were constructed with UPGMA and MP method respectively. Results CO1 and Cytb gene fragments were about 700 bp and 600 bp(including 2 primers) respectively. A total of 106 mutation spots (15.9%) were tested in CO1 homological nucleotides,and 165 mutation spots (28.5%) were tested in Cytb homological nucleotides. The distance matrix between Guangxi isolate and Hunan isolate was 0.051 and 0.031 for CO1 gene and Cytb gene respectively;while that between Guangxi and Yunnan isolates was 0.158 and 0.405 respectively. Phylogenetic trees constructed by UPGMA and MP took on the similar topo-structure:isolates of Guangxi and Hunan clustered into one group,while the Yunnan isolate exhibited as another group. Conclusion Oncomelania hupensis in Guangxi,Hunan and Yunnan are of relatively rich polymorphism in CO1 and Cytb genes in general.

Study on Molecular Phylogeny of Schistosoma sinensium Based on Mitochondrial Genes / 中国寄生虫学与寄生虫病杂志

Objective To determine the phylogenetic position of Schistosoma sinensium in the genus Schistosoma using mitochondrial cytochrome C oxidase 1 (CO1) and NADH dehydrogenase 1(ND1) as molecular markers. Methods The genomic DNA of adult worms were extracted by the GNT\|K method. The target regions were amplified by PCR using specific primers. The PCR products were purified before ligation into the plasmid Zero\|Blunt. Recombinant plasmids were amplified in E.coli, extracted and purified using routine methods and then sequenced using M13 primers (F/R) on a Licor long\|read auto\|sequencer. Sequences of related schistosomes were retrieved from GenBank and aligned with our data in the sequence editor ESEE. Gene trees were constructed in PHYLIP and MEGA using both maximum parsimony and neighbor\|joining methods. For parsimony analysis, all characters were treated as unordered and with equal weights. At least \{3 000\} cycles of bootstrapping were carried out. For analysis in MEGA, all gap columns were deleted. The third position of codon was included. Results The nucleotide and amino acid sequences of CO1 and ND1 of S.sinensium were obtained. Conclusion The phylogenetic trees from these molecular data suggested that S.sinensium belongs to the Asian schistosome group, and the results coincided with the previous rDNA (ITS2 & LSU) analysis results.