AAV-Mediated Clarin-1 Expression in the Mouse Retina: Implications for USH3A Gene Therapy

Usher syndrome type III (USH3A) is an autosomal recessive disorder caused by mutations in clarin-1 (CLRN1) gene, leading to progressive retinal degeneration and sensorineural deafness. Efforts to develop therapies for preventing photoreceptor cell loss are hampered by the lack of a retinal phenotype in the existing USH3 mouse models and by conflicting reports regarding the endogenous retinal localization of clarin-1, a transmembrane protein of unknown function. In this study, we used an AAV-based approach to express CLRN1 in the mouse retina in order to determine the pattern of its subcellular localization in different cell types. We found that all major classes of retinal cells express AAV-delivered CLRN1 driven by the ubiquitous, constitutive small chicken β-actin promoter, which has important implications for the design of future USH3 gene therapy studies. Within photoreceptor cells, AAV-expressed CLRN1 is mainly localized at the inner segment region and outer plexiform layer, similar to the endogenous expression of other usher proteins. Subretinal delivery using a full strength viral titer led to significant loss of retinal function as evidenced by ERG analysis, suggesting that there is a critical limit for CLRN1 expression in photoreceptor cells. Taken together, these results suggest that CLRN1 expression is potentially supported by a variety of retinal cells, and the right combination of AAV vector dose, promoter, and delivery method needs to be selected to develop safe therapies for USH3 disorder.     

Comparative Screening of Digestion Tract Toxic Genes in Proteus mirabilis

Proteus mirabilis is a common urinary tract pathogen, and may induce various inflammation symptoms. Its notorious ability to resist multiple antibiotics and to form urinary tract stones makes its treatment a long and painful process, which is further challenged by the frequent horizontal gene transferring events in P. mirabilis genomes. Three strains of P. mirabilis {"type":"entrez-nucleotide","attrs":{"text":"C02011","term_id":"1434241"}}C02011/{"type":"entrez-nucleotide","attrs":{"text":"C04010","term_id":"1467261"}}C04010/C04013 were isolated from a local outbreak of a food poisoning event in Shenzhen, China. Our hypothesis is that new genes may have been acquired horizontally to exert the digestion tract infection and toxicity. The functional characterization of these three genomes shows that each of them independently acquired dozens of virulent genes horizontally from the other microbial genomes. The representative strain {"type":"entrez-nucleotide","attrs":{"text":"C02011","term_id":"1434241"}}C02011 induces the symptoms of both vomit and diarrhea, and has recently acquired a complete type IV secretion system and digestion tract toxic genes from the other bacteria.     

An extract of Ulmus macrocarpa improves cellular immunity in immuno-suppressed models

An extract of Ulmus macrocarpa Hance, commonly known as the large-fruited elm, has been prescribed as a traditional medicine. In this study, we aimed to investigate the cellular immune effects of U. macrocarpa stem cortex extracts on cyclophosphamide (CY)-treated splenocytes and mice. A methanol extract showed an improved survival rate of splenocytes after 72?h. The extract was successively partitioned with dichloromethane, ethyl acetate, n-butanol, and water; and the fractions so obtained were also examined for their proliferative activity. Among them, the water fraction of U. macrocarpa showed the highest proliferation of splenocytes and was used throughout the present study. We tested the survival rate of splenocytes through dose-dependent treatment of CY and the suppression of the survival effect by CY was recovered by treatment with the water extract of U. macrocarpa. To determine the mechanism involved, we examined the expression of B-cell lymphoma-extra large (Bcl-xL) anti-apoptotic protein. CY decreased Bcl-xL protein levels in resting splenocyte cultures, whereas splenocytes were exposed to water extracts of U. macrocarpa in the presence of CY; however, elevations in Bcl-xL were observed at 96?h. Mice splenocytes treated with water extract of U. macrocarpa for cellular immunity showed an increase in the activity of the mixed lymphocyte reaction (MLR), cytotoxic T lymphocytes (CTLs), and natural killer (NK) cells. In addition, mice receiving a water extract of U. macrocarpa recovered the CTL, NK, and MLR activities suppressed by CY administration. Consequently, U. macrocarpa improves the cell-mediated immune response and provides an insight on cell-based tonic materials.     

Anti-Inflammatory and Antinociceptive Properties of Flavonoids from the Fruits of Black Mulberry (Morus nigra L.)

We analyzed the anti-inflammatory and antinociceptive activities of total flavonoids (TF) found in black mulberry fruits. The TF content was 20.9 mg/g (dry weight). Two anthocyanins, cyanidin-3-O-glucoside (8.3 mg/g) and cyanidin-3-O-rutinoside (2.9 mg/g), were identified in the fruits by UPLC. The TF of black mulberry fruits had significant reducing power and radical (OH^-,O2.−, DPPH and ABTS) scavenging activities that was demonstrated in a dose-response curve. The TF had inhibitory activities on xylene-induced ear edema and carrageenan-induced paw edema in mice. In addition, TF had antinociceptive activities in the two nociceptive phases of formalin test. We used ELISA to detect the pro-inflammatory cytokines IL-1β, TNF-α, IFN-γ, and NO in the serum of mice. These cytokines were significantly inhibited or scavenged by TF (50 and 100 mg/kg). The results demonstrated that TF of black mulberry possess anti-inflammatory and analgesic effects that might correlate to its antioxidant activities and inhibition of pro-inflammatory cytokines.     

Control of Methicillin-Resistant Staphylococcus aureus Pneumonia Utilizing TLR2 Agonist Pam3CSK4

The spread of methicillin-resistant Staphylococcus aureus (MRSA) is a critical health issue that has drawn greater attention to the potential use of immunotherapy. Toll-like receptor 2 (TLR2), a pattern recognition receptor, is an essential component in host innate defense system against S. aureus infection. However, little is known about the innate immune response, specifically TLR2 activation, against MRSA infection. Here, we evaluate the protective effect and the mechanism of MRSA murine pneumonia after pretreatment with Pam3CSK4, a TLR2 agonist. We found that the MRSA-pneumonia mouse model, pretreated with Pam3CSK4, had reduced bacteria and mortality in comparison to control mice. As well, lower protein and mRNA levels of TNF-α, IL-1β and IL-6 were observed in lungs and bronchus of the Pam3CSK4 pretreatment group. Conversely, expression of anti-inflammatory cytokine IL-10, but not TGF-β, increased in Pam3CSK4-pretreated mice. Our additional studies showed that CXCL-2 and CXCL1, which are necessary for neutrophil recruitment, were less evident in the Pam3CSK4-pretreated group compared to control group, whereas the expression of Fcγ receptors (FcγⅠ/Ⅲ) and complement receptors (CR1/3) increased in murine lungs. Furthermore, we found that increased survival and improved bacterial clearance were not a result of higher levels of neutrophil infiltration, but rather a result of enhanced phagocytosis and bactericidal activity of neutrophils in vitro and in vivo as well as increased robust oxidative activity and release of lactoferrin. Our cumulative findings suggest that Pam3CSK4 could be a novel immunotherapeutic candidate against MRSA pneumonia.     

Whole-Genome Sequencing for the Investigation of a Hospital Outbreak of MRSA in China

Staphylococcus aureus is a globally disseminated drug-resistant bacterial species. It remains a leading cause of hospital-acquired infection, primarily among immunocompromised patients. In 2012, the Affiliated People’s Hospital of Jiangsu University experienced a putative outbreak of methicillin-resistant S. aureus (MRSA) that affected 12 patients in the Neurosurgery Department. In this study, whole-genome sequencing (WGS) was used to gain insight into the epidemiology of the outbreak caused by MRSA, and traditional bacterial genotyping approaches were also applied to provide supportive evidence for WGS. We sequenced the DNA from 6 isolates associated with the outbreak. Phylogenetic analysis was constructed by comparing single-nucleotide polymorphisms (SNPs) in the core genome of 6 isolates in the present study and another 3 referenced isolates from GenBank. Of the 6 MRSA sequences in the current study, 5 belonged to the same group, clustering with T0131, while the other one clustered closely with TW20. All of the isolates were identified as ST239-SCCmecIII clones. Whole-genome analysis revealed that four of the outbreak isolates were more tightly clustered into a group and SA13002 together with SA13009 were distinct from the outbreak strains, which were considered non-outbreak strains. Based on the sequencing results, the antibiotic-resistance gene status (present or absent) was almost perfectly concordant with the results of phenotypic susceptibility testing. Various toxin genes were also analyzed successfully. Our analysis demonstrates that using traditional molecular methods and WGS can facilitate the identification of outbreaks and help to control nosocomial transmission.     

Women Are Seen More than Heard in Online Newspapers

Feminist news media researchers have long contended that masculine news values shape journalists’ quotidian decisions about what is newsworthy. As a result, it is argued, topics and issues traditionally regarded as primarily of interest and relevance to women are routinely marginalised in the news, while men’s views and voices are given privileged space. When women do show up in the news, it is often as “eye candy,” thus reinforcing women’s value as sources of visual pleasure rather than residing in the content of their views. To date, evidence to support such claims has tended to be based on small-scale, manual analyses of news content. In this article, we report on findings from our large-scale, data-driven study of gender representation in online English language news media. We analysed both words and images so as to give a broader picture of how gender is represented in online news. The corpus of news content examined consists of 2,353,652 articles collected over a period of six months from more than 950 different news outlets. From this initial dataset, we extracted 2,171,239 references to named persons and 1,376,824 images resolving the gender of names and faces using automated computational methods. We found that males were represented more often than females in both images and text, but in proportions that changed across topics, news outlets and mode. Moreover, the proportion of females was consistently higher in images than in text, for virtually all topics and news outlets; women were more likely to be represented visually than they were mentioned as a news actor or source. Our large-scale, data-driven analysis offers important empirical evidence of macroscopic patterns in news content concerning the way men and women are represented.     

Alagille Syndrome Candidates for Liver Transplantation: Differentiation from End-Stage Biliary Atresia Using Preoperative CT

Purpose

To compare preoperative CT findings before liver transplantation between patients with Alagille syndrome (AGS) and those with end-stage biliary atresia (BA).

Materials and Methods

The institutional review board approved this retrospective study. Eleven children with AGS (median age, 19.0 ± 13.0 months; male to female ratio, 3:8) and 109 children with end-stage BA (median age, 17.9 ± 25.8 months; male to female ratio, 37:72) who underwent abdomen CT as candidates for liver transplant were included. CT images were reviewed focusing on hepatic parenchymal changes, vascular changes, presence of focal lesions, and signs of portal hypertension.

Results

Hepatic parenchymal changes were present in 27% (3/11) of AGS patients and 100% (109/109) of end-stage BA patients (P < .001). The hepatic artery diameter was significantly smaller (1.9 mm versus 3.6 mm, P = 008), whereas portal vein diameter was larger (6.8 mm versus 5.0 mm, P < .001) in patients with AGS compared with patients with end-stage BA. No focal lesion was seen in patients with AGS, whereas 44% (48/109) of patients with end-stage BA had intrahepatic biliary cysts (39%, 43/109) and hepatic tumors (8%, 9/109) (P = .008). Splenomegaly was commonly seen in both groups (P = .082), and ascites (9% [1/11] versus 50% [54/109], P = .010) and gastroesophageal varix (0% [0/11] versus 80% [87/109], P < .001) were less common in patients with AGS than in patients with end-stage BA.

Conclusion

Fibrotic or cirrhotic changes of the liver, presence of focal lesions, and relevant portal hypertension were less common in patients with AGS than in patients with end-stage BA.