[An exploratory clinical study of the efficacy and safety of tumor-infiltrating lymphocytes in the treatment of metastatic osteosarcoma].

Objective: To explore the safety and efficacy of tumor-infiltrating lymphocytes (TILs) extracted from tumor tissue in patients with pulmonary metastasis of osteosarcoma, the TILs were amplified in vitro to reach clinical dosage and reinfused to the patients combined with high-dose interleukin 2 (IL-2). Methods: Twelve subjects with pathologically diagnosed osteosarcoma were enrolled from December 2019 to June 20, 2021 in Shanghai General Hospital. All subjects progressed with metastasis after standard chemotherapy and failed multiple lines of treatments. Fresh tumor tissue was obtained from the metastatic site and extracted and amplified by Good Manufacturing Practice (GMP) workshop to produce TILs to clinical treatment dosage (109-1011). High-dose IL-2 (100 000-200 000 U/kg) was administered immediately after autogenous TILs infusion to promote the activation, proliferation and antitumor cytolytic activity in vivo. Adverse events (AE) were graded according to Common Terminology Criteria for Adverse Events (CTCAE) standard and tumor response was assessed according to Response Evaluation Criteria in Solid Tumors (RECIST) 1.1. Results: One patient did not receive treatment due to failure in isolating TILs, total of 11 patients received a single re-infusion of autologous TILs. There were 10 males and 1 female with a median age of 19.9 years (12-33 years). Six of these patients received higher dose levels of 1.0×1010 TILs. The 11 patients were followed-up for 1 to 13 months and tolerated well. The most common adverse events reported were fever (10/11), constipation (3/11) and elevated gamma-glutamyl transferase (GGT) (3/11). The high incidence of fever was due to the IL-2 infusion. All patients experienced a transient drop in lymphocyte count and leukopenia leading to non-myeloid ablative lymphocyte clearance. The AE included grade 4 hematologic toxicity, including 8 cases of lymphocytopenia, 2 cases of neutropenia and 1 case of thrombocytopenia. No AE of neurotoxicity occurred. Of all the 11 patients, 9 patients got stable disease (SD) and 2 patients had progressive disease (PD). The disease control rate was 9/11. The median duration of SD was more than 4 months, and the maximum tumor volume decreased by close to 20%. Patient number 9 had sustained SD status for more than 6 months. Conclusions: TILs with in vitro expansion ability could be isolated from tumor tissues of advanced osteosarcoma patients. TILs amplified and reinfused in vitro have anti-osteosarcoma activity.

[Analysis on disease burden of stroke in China and the regions with different sociodemographic index from 1990 to 2017].

Objective: To examine the trend of stroke disease burden and its main risk-attributable factors in China and regions with different Socio-Demographic Index (SDI) from 1990 to 2017. Methods: With 2017 Global Burden of Disease (GBD) data, years lived with disability (YLDs), years of life lost (YLLs) and disability-adjusted of life years (DALYs) were applied to describe the disease burden and major risk factors of stroke in China and different SDI regions from 1990 to 2017, and to analyze the changing trend of the disease burden and major risk factors of stroke. Results: From 1990 to 2017, the YLD crude rate, YLL crude rate and DALY crude rate for stroke in China showed an increasing trend and the rate of change was 126.5%, 14.6%, and 24.4%, respectively. In 2017, the YLD crude rate, YLL crude rate and DALY crude rate for stroke in China were 502.6 per 100 000, 2 633.1 per 100 000 and 3 135.7 per 100 000, respectively. Among them, the YLD crude rate, YLL crude rate, and DALY crude rate of stroke were the highest in the ≥70 age group, which were 2 617.2 per 100 000, 16 789.4 per 100 000 and 19 406.6 per 100 000, respectively. The YLD crude rate in male was 475.5 per 100 000, which was slightly lower than that of female (530.9 per 100 000), while the DALY crude rate and YLL crude rate for stroke were 3 657.1 per 100 000 and 3 181.7 per 100 000, respectively, which were significantly higher than that of female (2 591.8 per 100 000 and 2 060.9 per 100 000). Compared with regions with different SDI, the age standardized YLD rate, the age standardized YLL rate, the age standardized DALY rate in China were all at a high level. Among them, the age-standardized YLD rate increased from 286.2 per 100 000 to 374.5 per 100 000, with a rate of change of 30.9%; the age-standardized YLL rate decreased from 3 215.6 per 100 000 to 1 967.8 per 100 000, with a rate of change of -38.8%; the age-standardized DALY rate increased from 3 501.8 per 100 000 to 2 342.3 per 100 000, with a rate of change of -33.1%. The top five risk factors for stroke in China were hypertension, excessive sodium intake, insufficient fruit intake, insufficient cereal intake, and smoking in 1990 and 2017. High Body-Mass Index and Alcohol Use's rankings rose from the 9th and 10th in 1990 to the 6th and 7th in 2017, respectively. Conclusion: The burden of stroke disease in China is at a high level, and hypertension is the primary risk factor.

Characterization of the complete mitochondrial genome sequence of Tracheophilus cymbius (Digenea), the first representative from the family Cyclocoelidae.

Tracheophilus cymbius (Trematoda: Cyclocoelidae) is a common tracheal fluke of waterfowl, causing serious loss in the poultry industry. However, taxonomic identification of T. cymbius remains controversial and confused. Mitochondrial (mt) genomes can provide genetic markers for the identification of closely related species. We determined the mt genome of T. cymbius and reconstructed phylogenies with other trematodes. The T. cymbius mt genome is 13,760 bp in size, and contains 12 protein-coding genes (cox 1-3, nad 1-6, nad 4L, cyt b and atp 6), 22 transfer RNA (tRNA) genes, two ribosomal RNA genes and one non-coding region. All are transcribed in the same direction. The A + T content is 62.82%. ATG and TAG are the most common initiation and termination codons, respectively. Phylogenetic analyses of concatenated nucleotide sequences show T. cymbius grouping in suborder Echinostomata, and clustering together, with high statistical support, as a sister taxon with Echinochasmus japonicus (Echinochasmidae), the two forming a distinct branch rooted to the ancestor of all Echinostomatidae and Fasciolidae species. This is the first report of the T. cymbius mt genome, and the first reported mt genome within the family Cyclocoelidae. These data will provide a significant resource of molecular markers for studying the taxonomy, population genetics and systematics of trematodes.

According to mitochondrial DNA evidence, Parascaris equorum and Parascaris univalens may represent the same species.

Parascarosis is caused mainly by parasitic infections with Parascaris equorum and Parascaris univalens, the most common ascarid nematodes, in the small intestine of equines. Parascarosis often causes severe illness and even death in foals and yearlings. In this study, we obtained the complete sequence of the P. equorum mitochondrial (mt) genome and compared its organization and structure with that of P. equorum Japan isolate (nearly complete), and the complete mtDNA sequences of P. univalens Switzerland and USA isolates. The complete mtDNA genome of P. equorum China isolate is 13,899 base pairs (bp), making it the smallest of the four genomes. All four Parascaris mt genomes are circular, and all genes are transcribed in the same direction. The P. equorum mtDNA genome consists of 12 protein-coding genes, two ribosomal RNA genes, 22 transfer (t) RNA genes and one non-coding region, which is consistent with P. equorum Japan isolate and P. univalens Switzerland isolate but distinct from P. univalens USA isolate, which has 20 tRNA genes. Differences in nucleotide sequences of the four entire mt genomes range from 0.1-0.9%, and differences in total amino acid sequences of protein-coding genes are 0.2-2.1%. Phylogenetic analyses showed that the four Parascaris species clustered in a clade, indicating that P. equorum and P. univalens are very closely related. These mt genome datasets provide genetic evidence that P. equorum and P. univalens may represent the same species, which will be of use in further studies of the taxonomy, systematics and population genetics of ascarids and other nematodes.

Comparative analyses of the complete mitochondrial genomes of Cyathostomum pateratum and Cyathostomum catinatum provide new molecular data for the evolution of Cyathostominae nematodes.

The parasite Cyathostomum pateratum, which occurs in the large intestine of equines, is a common species of the subfamily Cyathostominae. Cyathostominae nematodes are a complex nematode group for which only limited genetic information has been reported. To re-examine the phylogenetic relationships among Cyathostominae nematodes, we sequenced the complete mitochondrial (mt) genome of Cy. pateratum and compared it with the mt genome of the congeneric species Cyathostomum catinatum. The complete mtDNA sequence of Cy. pateratum was 13,822 bp in length, 16 bp shorter than that of Cy. catinatum. The mtDNA sequences of both species contained 12 protein-coding genes, two rRNA genes and 22 tRNA genes, and all 36 genes were transcribed in the same direction and in the same strand. Pairwise comparisons of the 12 predicted amino acid sequences between Cy. catinatum and Cy. pateratum revealed differences of 0.4-3.1%; the least conserved sequence was that of cytochrome c oxidase subunit 3 (cox3). Phylogenetic analysis of the concatenated amino acid sequences using Bayesian inference and maximum likelihood methods showed that Cy. catinatum and Cy. pateratum clustered together with very high nodal support, and Cylicostephanus goldi was closer to the Cyathostomum nematodes than to other Cyathostominae nematodes. The mtDNA sequence of Cy. pateratum is reported here for the first time. The study will shed some light on the genetic evolution among parasitic nematodes in Cyathostomum.

Systemic lupus erythematosus-associated diffuse alveolar haemorrhage: a single-centre experience in Han Chinese patients.

OBJECTIVE: Despite aggressive therapeutic regimens, diffuse alveolar haemorrhage (DAH) is still associated with a high mortality rate in systemic lupus erythematosus (SLE). This study was carried out in patients with SLE-associated DAH with a focus on their therapeutic modality. METHOD: A retrospective review was performed in 839 Han Chinese lupus patients hospitalized for their DAH manifestation from May 2006 to December 2016. RESULTS: There were 24 episodes in 17 cases (2.0% incidence), 15 females and two males aged 19-67 years (mean ± sd 38.2 ± 15.1 years). High disease activity [Systemic Lupus Erythematosus Disease Activity Index 2000 (SLEDAI-2K) 12-31, 19.8 ± 5.6] was found at the onset of DAH. All patients were treated with high-dose corticosteroid, followed by pulse methylprednisolone (70.6%), plasmapheresis (41.2%), pulse cyclophosphamide (35.3%), and rituximab (23.5%). Six patients (35.3%), including three with extracorporeal membrane oxygenation, died owing to acute respiratory failure. All patients receiving rituximab treatment survived with a follow-up period of 12-58 months (40.8 ± 21.1 months), and no further relapse was noted in three cases with a history of recurrent DAH episodes. In addition, there was a significant decrease in their lupus activity (SLEDAI-2K 21.5 ± 6.0 to 6.3 ± 1.7, p = 0.0286). CONCLUSION: In this single-centre series with SLE-associated DAH in Han Chinese patients, a beneficial effect of rituximab therapy was observed.

Characterization of the complete nuclear ribosomal DNA sequences of Eurytrema pancreaticum.

Eurytrema pancreaticum is one of the most common trematodes of cattle and sheep, and also infects humans occasionally, causing great economic losses and medical costs. In this study, the sequences of the complete nuclear ribosomal DNA (rDNA) repeat units of five E. pancreaticum individuals were determined for the first time. They were 8306-8310 bp in length, including the small subunit (18S) rDNA, internal transcribed spacer 1 (ITS1), 5.8S rDNA, internal transcribed spacer 2 (ITS2), large subunit (28S) rDNA and intergenic spacer (IGS). There were no length variations in any of the investigated 18S (1996 bp), ITS1 (1103 bp), 5.8S (160 bp), ITS2 (231 bp) or 28S (3669 bp) rDNA sequences, whereas the IGS rDNA sequences of E. pancreaticum had a 4-bp length variation, ranging from 1147 to 1151 bp. The intraspecific variations within E. pancreaticum were 0-0.2% for 18S rDNA, 0-0.5% for ITS1, 0% for 5.8S rDNA and ITS2, 0-0.2% for 28S rDNA and 2.9-20.2% for IGS. There were nine types of repeat sequences in ITS1, two types in 28S rDNA, but none in IGS. A phylogenetic analysis based on the 18S rDNA sequences classified E. pancreaticum in the family Dicrocoeliidae of Plagiorchiata, closely related to the suborder Opisthorchiata. These results provide useful information for the further study of Dicrocoeliidae trematodes.

[Risk factors for lower extremity amputation in patients with diabetic foot].

Objective: To explore the risk factors for lower extremity amputation in patients with diabetic foot. Methods: The clinical data of 1 771 patients with diabetic foot at the Air Force General Hospital of PLA from November 2001 to April 2015 were retrospectively analyzed. The patients were divided into the non-amputation and amputation groups. Within the amputation group, subjects were further divided into the minor and major amputation subgroups. Binary logistic regression analyses were used to assess the association between risk factors and lower extremity amputation. Results: Among 1 771 patients with diabetic foot, 323 of them (18.24%) were in the amputation group (major amputation: 41; minor amputation: 282) and 1 448 (81.76%) in the non-amputation group. Compared with non-amputation patients, those in the amputation group had a longer hospital stay and higher estimated glomerular filtration rate(eGFR)levels. Fasting plasma glucose (FPG), glycosylated hemoglobin (HbA1c), C-reaction protein (CRP), ESR, ferritin, fibrinogen and WBC levels of the amputation group were higher, while hemoglobin albumin, transferrin, TC, TG, HDL-C and LDL-C were lower than those of the non-amputation group (all P<0.05). The proportion of hypertension(52.48% vs 59.98%), peripheral vascular disease (PAD)(68.11% vs 25.04%), and coronary heart disease(21.33% vs 28.71%)were different between the amputation and non-amputation groups (all P<0.05). Multivariable logistic regression analyses showed that Wagner's grade, PAD and CRP were the independent risk factors associated with lower extremity amputation in hospitalized patients with diabetic foot. Conclusion: Wagner's grade, ischemia of lower limbs and infection are closely associated with amputation of diabetic foot patients.

[Intervention of edaravone against renal injury induced by acute paraquat poisoning in rats].

Objective: To investigate the dynamic change of paraquant-induced kidney injury in rats and the protective effect of edaravone. Methods: Eighty SD rats were randomly divided into 4 groups: the normal control group, paraquat poisoning group, edaravone treatment group and edaravone control group. The normal control group of 8 rats were given 1 ml of 0.9% sodium chloride through the abdominal cavity, and the same amount of fluid into the abdominal cavity after 30 minutes. The paraquat poisoning group of 24 rats were given 1 ml of paraquat solution (20 mg/kg) through the abdominal cavity to build poisoning models, and the same amount of 0.9% sodium chloride was injected into the abdominal cavity after 30 minutes. The edaravone treatment group of 24 rats were given edaravone (5 mg/kg) through the abdominal cavity after 30 minutes when the poisoning models were set up. The edaravone control group of 24 rats were given 1 ml of 0.9% sodium chloride through the abdominal cavity, and edaravone (5 mg/kg) was injected into the abdominal cavity after 30 minutes. In addition to the normal control group, the other groups processed 1 times a day to mantain 7 d. On 1, 3, 7, 21 d several rats in each group were excuted and the kidney tissue and serum samples were collected, then each pathological changes of the kidney were observed with light microscopy. Serum creatinine, KIM-1, NGAL were measured by ELISA, the expression of HSP70 protein in kidney were observed with immunohistochemical staining. Results: The pathological examination reveald that the damage of kidney tissue in the paraquat group was the most serious on 3 d, and the damage was consistently alleviated in edaravone treatment group at the same time, renal fibrosisn was unseen in each group until 21 d. Compared with normal control group, there was no statistically significant in edaravone control group (P>0.05) . The KIM-1 in blood and kidney in paraquat poisoning group were markedly increased in 1 d (P<0.05) . The NGAL in blood and creatinine were markedly increased in d7 (P<0.05) . The NGAL in kidney increased over time, but had no statistically difference with the control group (P>0.05) .Compared with paraquat poisoning group, the serum creatinine, KIM-1 in blood and kidney, the KIM-1 in kidney had decreased significantly in edaravone treatment group (P<0.05) . The NGAL in kidney has no statistically significant compared with the poisoning group (P>0.05) . HSP70 expression of kidney tissue in edaravone treatment group had significantly increased in d3 compared with the paraquat poisoning group (P<0.05) . Conclusion: Edaravone can prompt a significant rise of HSP70 in kidney tissue, reduce KIM-1 and NGAL levels, and play a protective role in kidney injury of acute paraquat poisoning.

Active gratings tuned by thermoplasmonics-induced phase transition in vanadium dioxide thin films.

We propose and investigate an active grating of gold metallic structure on vanadium dioxide (VO2) thin film illuminated by an intense light. Nonuniform phase transition in VO2 film is expected due to the thermoplasmonics effect where the plasmonic-induced light absorption features an enhanced local heat generation at nanometer-scale. The spatial profiles of the electric field, the heat generation, and the temperature distribution, as well as the temperature-dependent dielectric parameters in VO2 film, are solved numerically in a self-consistent manner. Our results show that the evolution of the metallic and semiconducting phases of VO2 changes the effective dielectric environment of the grating and modifies its optical response in a controlled way. The interplay of the thermoplasmonics effect and the phase transition processes can thus provide another degree of freedom in designing optical modulators or switches which are remotely tunable via incident light.

Nucleotide variation in the Toxoplasma gondii micronemal protein 8 gene.

Toxoplasma gondii is a successful opportunistic protozoan distributed worldwide, which can infect all vertebrates, leading to serious infection, blindness, and abortion. Micronemal (MIC) proteins are critically important for T. gondii infection, as they participate in various stages of the Toxoplasma life cycle, including invasion and attachment to host cells. MIC8 secretion relies on the concentration of intracellular calcium, and can mediate the invasion of T. gondii by interacting with soluble MIC3. To investigate genetic diversity of the MIC8 gene, 16 T. gondii strains from different hosts and geographical locations, and two reference isolates (ToxoDB: TGME49_245490 and TGVEG_245490) were examined in this study. The results showed that all the examined MIC8 genes are 2055 bp, with an A+T content ranging from 50.2 to 50.6%. Conversely, lower levels of variation were detected within their nucleotide and amino acid sequences. Phylogenetic analyses indicated that three classical genotypes of T. gondii and the ToxoDB#9 genotype did not group exclusively via Bayesian inference, maximum parsimony, neighbor joining, and/or maximum likelihood assays based on the nucleotide and amino acid sequences of the MIC8 gene. In summary, the T. gondii MIC8 gene is not a suitable marker for population genetic studies of this parasite.

Orthogonal design in the optimization of a start codon targeted (SCoT) PCR system in Roegneria kamoji Ohwi.

Roegneria kamoji Ohwi is an excellent forage grass due to its high feeding value and high resistance to some biotic and abiotic stresses. However, the start codon targeted (SCoT) polymorphism has not been conducted on R. kamoji. In this study, an orthogonal L16 (45) design was employed to investigate the effects of five factors (Mg2+, dNTPs, Taq DNA polymerase, primer, and template DNA) on the polymerase chain reaction (PCR) to determine the optimal SCoT-PCR system for R. kamoji. The results showed that the most suitable conditions for SCoT-PCR in R. kamoji included 1.5 mM Mg2+, 0.15 mM dNTPs, 1.0 U Taq DNA polymerase, 0.4 pM primer, and 40 ng template DNA. SCoT primers 39 and 41 were used to verify the stability of the optimal reaction system, and amplification bands obtained from diverse samples were found to be clear, rich, and stable in polymorphisms, indicating that this reaction system can be used for SCoT-PCR analysis of R. kamoji. We have developed a simple and rapid way to study the mutual effects of factors and to obtain positive results through the use of an orthogonal design L16 (45) to optimize the SCoT-PCR system. This method may provide basic information for molecular marker-assisted breeding and analyses of genetic diversity in R. kamoji.

Sequence variability in internal transcribed spacers of nuclear ribosomal DNA among isolates of the oxyurid nematodes Syphacia obvelata and Aspiculuris tetraptera from mice reared in laboratories in China.

This study examined sequence variability in internal transcribed spacers (ITS) of nuclear ribosomal DNA among Syphacia obvelata and Aspiculuris tetraptera isolates from laboratory mice from different geographical locations in China. ITS1, 5.8S and ITS2 rDNA were amplified separately from adult S. obvelata and A. tetraptera individuals by polymerase chain reaction (PCR), and the amplicons were subjected to sequencing from both directions. The lengths of the sequences of ITS1, 5.8S and ITS2 rDNA from both nematodes were 314 bp and 456 bp, 157 bp, and 273 bp and 419 bp, respectively. The intraspecific sequence variations in S. obvelata ITS1 were 0-0.3%. For A. tetraptera they were 0-0.7% in ITS1 and 0-1.0% in ITS2. However, the interspecific sequence differences among members of the infraorder Oxyuridomorpha were significantly higher, being 54.0-65.5% for ITS1 and 55.3-64.1% for ITS2. Phylogenetic analysis based on the combined partial sequences of ITS1 and ITS2 using three inference methods - Bayesian inference, maximum likelihood and maximum parsimony - revealed that all the S. obvelata and A. tetraptera samples formed independent monophyletic groups. Syphacia obvelata was closer to Syphacia muris than to A. tetraptera, consistent with morphological classification. These results demonstrate that ITS1 and ITS2 rDNA sequences are useful markers for population genetic studies of oxyurid nematodes.

Amelioration of experimental arthritis by intra-articular injection of an epidermal growth factor receptor tyrosine kinase inhibitor.

OBJECTIVES: Selectively targeting signalling pathways represents a promising pharmacological approach in rheumatoid arthritis (RA). Abundant levels of epidermal growth factor receptor (EGFR) are expressed in the synovial lining layers, and the anti-arthritis effect of erlotinib and lapatinib, small-molecule EGFR tyrosine kinase inhibitors (TKIs), has been demonstrated through the systemic administration on experimental arthritis models. Nevertheless, their therapeutic responses by the intra-articular (i.a.) route remain to be explored in rheumatoid joint. METHODS: The administration of an EGFR TKI (a gefitinib analogue) was explored in two in vivo models of collagen-induced arthritis (CIA) and in vitro experiments by using synovial fibroblasts (SF) from RA patients and CIA rats. RESULTS: There was a significant reduction of arthritis scores in CIA mice receiving the daily intraperitoneal injection. After the onset of arthritis in CIA rats, ankle joints receiving a single i.a. injection had significant lower articular indexes with reduced synovial inflammation, pannus formation and erosion on cartilage and bone as well as total histological scores by histopathological analyses. In CIASF or RASF, upon in vitro human EGF stimulation, there was a dose-dependent increase in cell proliferation and Akt activation with suppressed responses under the EGFR TKI treatment. CONCLUSIONS: These findings demonstrate the effect of i.a. injection of an EGFR TKI on amelioration of rheumatoid joint through the suppression of synovial inflammation, pannus formation and erosion on cartilage and bone in experimental arthritis, implicating targeting the i.a. EGFR signalling transduction as a pharmacological strategy.

LPS induces pro-inflammatory response in mastitis mice and mammary epithelial cells: Possible involvement of NF-κB signaling and OPN.

BACKGROUND: Lipopolysaccharide (LPS) has pro-inflammatory properties. This study was conducted to determine whether the LPS induced pro-inflammatory response in a model of mastitis and in mouse mammary epithelial cells (MEC). METHODS: To investigate the effects of LPS in vivo, 50 µL of a solution of LPS (20 ng/µL) were infused into the mammary glands of mice. To study the effects of LPS in vitro, MEC were exposed to LPS (20 µg/mL) for 24h. Activation of nuclear factor kB (NF-κB) and myeloperoxidase (MPO) were studied. Production of pro-inflammatory cytokines (interleukin-6 [IL-6], tumor necrosis factor-alpha [TNF-alpha], interleukin-1 beta [IL-1 beta]) and expression of osteopontin (OPN) were also evaluated. RESULTS: After LPS administration, route of NF-κB signaling is activated and the activity of MPO is increased. Furthermore, LPS increases the expression of OPN and production of TNF-alpha, IL-6 and IL-1 beta. CONCLUSIONS: Present results demonstrate that LPS induces a pro-inflammatory response in a murine model of mastitis and suggest the involvement of the NF-κB pathway and OPN.

Sequence variation in ROP8 gene among Toxoplasma gondii isolates from different hosts and geographical localities.

The protozoan parasite Toxoplasma gondii has a worldwide distribution; it can cause serious diseases in humans and almost all other warm-blooded animals. Different genotypes of T. gondii result in different lesions in the same host. T. gondii rhoptry protein 8 (TgROP8) is a major factor of T. gondii acute virulence. We examined sequence variation in the TgROP8 gene among T. gondii isolates from different hosts and geographical localities. The TgROP8 gene was amplified from individual isolates and sequenced. A phylogenetic tree was constructed using Bayesian inference, maximum parsimony, and maximum likelihood based on the sequences obtained plus TgME49 from the ToxoDB database. The TgROP8 gene was 1728 bp in length for all the examined T. gondii strains, and their A+T contents were 45.37-45.95%. Sequence analysis detected 140 (0.06-5.56%) variable nucleotide positions resulting in 96 (0-10.78%) amino acid substitutions. Sequence variations in the TgROP8 gene resulted in polymorphic restriction sites for endonucleases BstBI, BsaI, and XhoI, which allowed the differentiation of the three classical genotype strains (types I, II, and III) by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). However, phylogenetic analyses indicated that the TgROP8 gene is not a suitable genetic marker for population studies of T. gondii.

Huntingtin gene CAG repeat numbers in Chinese patients with Huntington's disease and controls.

BACKGROUND AND PURPOSE: Huntington's disease is due to a CAG triplet repeat elongation in the huntingtin gene. Boundaries in CAG numbers have been found between healthy people with and without risk to pass the disorder to the next generation, and between people without, with a mild, or with a fully penetrant phenotype. These data have been generated in western populations and it is not clear whether they are also valid amongst Chinese. METHODS: In order to establish normative data in the huntingtin gene for Chinese people, 966 chromosomes from normal controls were tested. Further, the range of CAG repeats was examined in a cohort from six centres and a total of 368 patients with the disease were included. RESULTS: The CAG triplet repeat range in normal controls was between 9 and 35 (mean 18.9, SD 2.57). Triplets in the range between 26 and 35 were found in 2.5%. In the patient cohort, triplet repeats in the shorter allele were between 8 and 37 (mean 17.7, SD 1.6). In the longer allele, a range between 36 and 120 was found. There was a negative correlation (-0.65, r = 0.42) between age at onset and the number of triplet repeats in the larger allele. The mean age at onset was 38 years, with a range between 2 and 70 years. In 23 patients (6%) a childhood or juvenile onset was noted. CONCLUSION: These data show comparable ranges of huntingtin gene CAG triplet repeats in normal people and in patients with Huntington's disease as in western populations.