Novel 2,6-disubstituted benzofuran-3-one analogues improve cerebral ischemia/reperfusion injury via neuroprotective and antioxidative effects.

There are no highly effective and safe medicines for clinical treatment of ischemic stroke, although the natural product 3-n-butylphthalide (NBP) has been approved in China for mild and moderate ischemic stroke. To discover more potent anti-cerebral ischemic agents and overcome the low stability by phthalide derivatives, benzofuran-3-one was selected as a core moiety and two types of nitric oxide (NO)-donating groups were incorporated into the structure. In this work, a series of 2,6-disubstituted benzofuran-3-one derivatives were designed and synthesised as NBP analogues, and tested as neuroprotective and antioxidative agents. Compounds 5 (without an NO donor) and 16 (with an NO donor) displayed more potent neuroprotective effects than the established clinical drugs Edaravone and NBP. More importantly, 5 and 16 also exhibited good antioxidative activity without cytotoxicity in rat primary neuronal and PC12 cells. Most active compounds showed good blood-brain barrier permeability in a parallel artificial membrane permeability assay. Furthermore, compound 5 reduced the ischemic infarct area significantly in rats subjected to ischemia/reperfusion injury, downregulated ionised calcium-binding adaptor molecule 1 and glial fibrillary acidic protein in inflammatory cells, and upregulated nerve growth factor.

Canonical and early lineage-specific stem cell types identified in planarian SirNeoblasts.

BACKGROUND: The pluripotent stem cells in planarians, a model for tissue and cellular regeneration, remain further identification. We recently developed a method to enrich piwi-1+ cells in Schmidtea mediterranea, by staining cells with SiR-DNA and Cell Tracker Green, named SirNeoblasts that permits their propagation and subsequent functional study in vivo. Since traditional enrichment for planarian neoblasts by Hoechst 33342 staining generates X1 cells, blocking the cell cycle and inducing cytotoxicity, this method by SiR-DNA and Cell Tracker Green represents a complementary technological advance for functional investigation of cell fate and regeneration. However, the similarities in heterogeneity of cell subtypes between SirNeoblasts and X1 remain unknown. RESULTS: In this work, we performed single cell RNA sequencing of SirNeoblasts for comparison with differential expression patterns in a publicly available X1 single cell RNA sequencing data. We found first that all of the lineage-specific progenitor cells in X1 were present in comparable proportions in SirNeoblasts. In addition, SirNeoblasts contain an early muscle progenitor that is unreported in X1. Analysis of new markers for putative pluripotent stem cells identified here, with subsequent sub-clustering analysis, revealed earlier lineages of epidermal, muscular, intestinal, and pharyngeal progenitors than have been observed in X1. Using the gcm as a marker, we also identified a cell subpopulation resided in previously identified tgs-1+ neoblasts. Knockdown of gcm impaired the neoblast repopulation, suggesting a function of gcm in neoblasts. CONCLUSIONS: In summary, the use of SirNeoblasts will enable broad experimental advances in regeneration and cell fate specification, given the possibility for propagation and transplantation of recombinant and mutagenized pluripotent stem cells that are not previously afforded to this rapid and versatile model system.

Cyclooctatetraene-conjugated cyanine mitochondrial probes minimize phototoxicity in fluorescence and nanoscopic imaging.

Modern fluorescence-imaging methods promise to unveil organelle dynamics in live cells. Phototoxicity, however, has become a prevailing issue when boosted illumination applies. Mitochondria are representative organelles whose research heavily relies on optical imaging, yet these membranous hubs of bioenergy are exceptionally vulnerable to photodamage. We report that cyclooctatetraene-conjugated cyanine dyes (PK Mito dyes), are ideal mitochondrial probes with remarkably low photodynamic damage for general use in fluorescence cytometry. In contrast, the nitrobenzene conjugate of Cy3 exhibits enhanced photostability but unaffected phototoxicity compared to parental Cy3. PK Mito Red, in conjunction with Hessian-structural illumination microscopy, enables 2000-frame time-lapse imaging with clearly resolvable crista structures, revealing rich mitochondrial dynamics. In a rigorous stem cell sorting and transplantation assay, PK Mito Red maximally retains the stemness of planarian neoblasts, exhibiting excellent multifaceted biocompatibility. Resonating with the ongoing theme of reducing photodamage using optical approaches, this work advocates the evaluation and minimization of phototoxicity when developing imaging probes.

Low prevalence of viable Toxoplasma gondii in fresh, unfrozen American pasture-raised pork and lamb from retail meat stores in the United States.

In a national survey of fresh, unfrozen, American pasture-raised lamb and pork, the prevalence of viable Toxoplasma gondii was determined in 1500 samples selected by random multistage sampling (750 pork, 750 lamb) obtained from 250 retail meat stores from 10 major geographic areas in the USA. Each sample consisted of a minimum of 500g of meat purchased from the retail meat case. To detect viable T. gondii, 50g meat samples of each of 1500 samples were bioassayed in mice. Viable T. gondii was isolated from 2 of 750 lamb samples (unweighted: 0.19%, 0.00-0.46%; weighted: 0.04%, 0.00-0.11%) and 1 of 750 pork samples (unweighted: 0.12%, 0.00-0.37%; weighted: 0.18%, 0.00-0.53%) samples. Overall, the prevalence of viable T. gondii in these retail meats was very low. Nevertheless, consumers, especially pregnant women, should be aware that they can acquire T. gondii infection from ingestion of undercooked meat. Cooking meat to an internal temperature of 66°C kills T. gondii.

Prevalence and genetic characterization of Toxoplasma gondii in free-range chickens from grocery stores and farms in Maryland, Ohio and Massachusetts, USA.

Chickens are considered important in the epidemiology of Toxoplasma gondii. Chicken hearts (n = 1185) obtained from grocery stores were tested for T. gondii infection. Antibodies to T. gondii were assayed in fluid removed from the heart cavity using the modified agglutination test (MAT) at 1:5, 1:25, and 1:100 dilutions. MAT antibodies were detected in 222 hearts at 1:5 dilution and 8 hearts at 1:25 dilution, but none were positive at 1:100 dilution. Seropositive (n = 230, 19.4%) chicken hearts were bioassayed in mice and seronegative (n = 157) chickens were bioassayed in cats. Viable T. gondii was not isolated from any hearts by bioassays in mice. The 2 cats fed 60 and 97 hearts did not excrete T. gondii oocysts. The results indicate a low prevalence of viable T. gondii in chickens from grocery stores. Molecular typing of 23 archived T. gondii strains isolated from free-range chickens from Ohio and Massachusetts using the 10 PCR-RFLP markers including SAG1, SAG2 (5'-3'SAG2 and altSAG2), SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico revealed that seven were ToxoDB PCR-RFLP genotype #1, 11 were genotype #2, one was genotype #3, three were genotype #170, and one was mixed genotype. These results indicate that the clonal genotypes #1 (type II), #2 (type III), and #3 (type II variant) are common in free-range chickens.

Qualitative Assessment for Toxoplasma gondii Exposure Risk Associated with Meat Products in the United States.

Toxoplasma gondii is a global protozoan parasite capable of infecting most warm-blooded animals. Although healthy adult humans generally have no symptoms, severe illness does occur in certain groups, including congenitally infected fetuses and newborns, immunocompromised individuals including transplant patients. Epidemiological studies have demonstrated that consumption of raw or undercooked meat products is one of the major sources of infection with T. gondii. The goal of this study was to develop a framework to qualitatively estimate the exposure risk to T. gondii from various meat products consumed in the United States. Risk estimates of various meats were analyzed by a farm-to-retail qualitative assessment that included evaluation of farm, abattoir, storage and transportation, meat processing, packaging, and retail modules. It was found that exposure risks associated with meats from free-range chickens, nonconfinement-raised pigs, goats, and lamb are higher than those from confinement-raised pigs, cattle, and caged chickens. For fresh meat products, risk at the retail level was similar to that at the farm level unless meats had been frozen or moisture enhanced. Our results showed that meat processing, such as salting, freezing, commercial hot air drying, long fermentation times, hot smoking, and cooking, are able to reduce T. gondii levels in meat products. whereas nitrite and/or nitrate, spice, low pH, and cold storage have no effect on the viability of T. gondii tissue cysts. Raw-fermented sausage, cured raw meat, meat that is not hot-air dried, and fresh processed meat were associated with higher exposure risks compared with cooked meat and frozen meat. This study provides a reference for meat management control programs to determine critical control points and serves as the foundation for future quantitative risk assessments.

Isolation and genetic characterization of viable Toxoplasma gondii from tissues and feces of cats from the central region of China.

Cats are important in the epidemiology of toxoplasmosis because they are the only definitive hosts that excrete environmentally resistant Toxoplasma gondii oocysts. Little is known of feline toxoplasmosis in China and most of the literature is in Chinese. Here we summarized all published reports on feline toxoplasmosis in English and report first identification of oocyst excretion by naturally infected cats in China. Unfrozen tissues of 42 cats and feces of 360 cats from China were bioassayed in mice for isolation of T. gondii. Antibodies to T. gondii were found in 21 of 42 (50%) of cats by the modified agglutination test (cut-off 1:25). Viable T. gondii was isolated from tissues of eight of 21 seropositive but not from 21 seronegative (<1:25) cats. Viable T. gondii was isolated from feces of one cat. DNA derived from cell cultured tachyzoites of all nine T. gondii isolates was characterized by PCR-RFLP at 10 loci (SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico). Four genotypes were found; the genotypes of tissue isolates were ToxoDB #9 in six, ToxoDB #2 in one, and ToxoDB #17 in one. The fecal isolate was ToxoDB #1.To our knowledge, the present study is the first isolation of T. gondii from cat feces from China.

Low prevalence of Neospora caninum and Toxoplasma gondii antibodies in dogs in Jilin, Henan and Anhui Provinces of the People's Republic of China.

BACKGROUND: Neospora caninum and Toxoplasma gondii are important pathogens of worldwide distribution. N. caninum is a major cause of abortion in cattle and dogs are main reservoirs because they excrete the environmentally resistant oocysts. Toxoplasmosis is a worldwide zoonosis and dogs are considered as sentinels for this parasite because of their close contact with people and cats; additionally dog meat is also used for human consumption in China. The aim of the present study was to assess the prevalence of N. caninum and T. gondii infection in dogs from China. A total of 425 countryside dog hearts in Jilin, Henan and Anhui provinces of the People's Republic of China were collected from slaughter houses in two batches; the first batch of 96 in October 2013, and the second batch of 329 in April 2014. Serum samples extracted from 96 dog hearts were tested for antibodies to N. caninum and from 425 dog hearts were tested for T. gondii antibodies in the modified agglutination tests (cut-off 1:25 for both), using respective antigens. RESULTS: Antibodies to N. caninum were 6 of 96 (6.25%) of dogs with titers of 1:25 in 2, 1:50 in 3, and 1:100 in 1. All seropositive dogs were more than 1 year old. Antibodies to T. gondii were found in 35 of 425 (8.24%) dogs with titers of 1:25 in 15, 1:50 in 14; and 1:100 in 6. CONCLUSION: The results of the present study indicated low prevalence of N. caninum and T. gondii antibodies in dogs of China, compared with Europe and America. Identification of the risk factors that underlie these differences may help prevention of neosporosis and toxoplasmosis. This is the first report of N. caninum infection in dogs from China.