Roles of myosin 1e and the actin cytoskeleton in kidney functions and familial kidney disease.

Mammalian kidneys are responsible for removing metabolic waste and maintaining fluid and electrolyte homeostasis via selective filtration. One of the proteins closely linked to selective renal filtration is myosin 1e (Myo1e), an actin-dependent molecular motor found in the specialized kidney epithelial cells involved in the assembly and maintenance of the renal filter. Point mutations in the gene encoding Myo1e, MYO1E, have been linked to familial kidney disease, and Myo1e knockout in mice leads to the disruption of selective filtration. In this review, we discuss the role of the actin cytoskeleton in renal filtration, the known and hypothesized functions of Myo1e, and the possible explanations for the impact of MYO1E mutations on renal function.

Steroid-Resistant Nephrotic Syndrome-Associated MYO1E Mutations Have Differential Effects on Myosin 1e Localization, Dynamics, and Activity.

BACKGROUND: Myo1e is a nonmuscle motor protein enriched in podocytes. Mutations in MYO1E are associated with steroid-resistant nephrotic syndrome (SRNS). Most of the MYO1E variants identified by genomic sequencing have not been functionally characterized. Here, we set out to analyze two mutations in the Myo1e motor domain, T119I and D388H, which were selected on the basis of protein sequence conservation. METHODS: EGFP-tagged human Myo1e constructs were delivered into the Myo1e-KO mouse podocyte-derived cells via adenoviral infection to analyze Myo1e protein stability, Myo1e localization, and clathrin-dependent endocytosis, which is known to involve Myo1e activity. Furthermore, truncated Myo1e constructs were expressed using the baculovirus expression system and used to measure Myo1e ATPase and motor activity in vitro. RESULTS: Both mutants were expressed as full-length proteins in the Myo1e-KO cells. However, unlike wild-type (WT) Myo1e, the T119I variant was not enriched at the cell junctions or clathrin-coated vesicles (CCVs). In contrast, D388H variant localization was similar to that of WT. The rate of dissociation of the D388H variant from cell-cell junctions and CCVs was decreased, suggesting this mutation affects Myo1e interactions with binding partners. ATPase activity and ability to translocate actin filaments were drastically reduced for the D388H mutant, supporting findings from cell-based experiments. CONCLUSIONS: T119I and D388H mutations are deleterious to Myo1e functions. The experimental approaches used in this study can be applied to future characterization of novel MYO1E variants associated with SRNS.

Involvement of L-selectin expression in Burkholderia pseudomallei-infected monocytes invading the brain during murine melioidosis.

The development of neurologic melioidosis was linked to the elicitation of Burkholderia pseudomallei-infected L-selectinhiCD11b+ BALB/c cells in our previous study. However, whether monocytic L-selectin (CD62L, encoded by the sell gene) is a key factor remains uncertain. In the present study, after establishing multi-organ foci via hematogenous routes, we demonstrated that B. pseudomallei GFP steadily persisted in blood, splenic, hepatic and bone marrow (BM) Ly6C monocytes; however, the circulating CD16/32+CD45hiGFP+ brain-infiltrating leukocytes (BILs) derived from the blood Ly6C monocytes were expanded in BALB/c but not in C57BL/6 bacteremic melioidosis. Consistent with these results, 60% of BALB/c mice but only 10% of C57BL/6 mice exhibited neurologic melioidosis. In a time-dependent manner, B. pseudomallei invaded C57BL/6 BM-derived phagocytes and monocytic progenitors by 2 d. The number of Ly6C+CD62L+GFP+ inflamed cells that had expanded in the BM and that were ready for emigration peaked on d 21 post-infection. Hematogenous B. pseudomallei-loaded sell+/+Ly6C monocytes exacerbated the bacterial loads and the proportion of Ly6C+GFP+ BILs in the recipient brains compared to sell-/- infected Ly6C cells when adoptively transferred. Moreover, a neutralizing anti-CD62L antibody significantly depleted the bacterial colonization of the brain following adoptive transfer of B. pseudomallei-loaded C57BL/6 or BALB/c Ly6C cells. Our data thus suggest that Ly6C+CD62L+ infected monocytes served as a Trojan horse across the cerebral endothelium to induce brain infection. Therefore, CD62L should be considered as not only a temporally elicited antigen but also a disease-relevant leukocyte marker during the development of neurologic melioidosis.

Genomic Sequence of Burkholderia multivorans NKI379, a Soil Bacterium That Inhibits the Growth of Burkholderia pseudomallei.

Burkholderia multivorans NKI379 is a soil bacterium that exhibits an antagonistic effect against the growth of Burkholderia pseudomallei, the causative agent of the infectious disease melioidosis. We report the draft genomic sequence of B. multivorans NKI379, which has a G+C content of 67% and 5,203 candidate protein-encoding genes.

Comparison of Whole-Genome Sequences from Two Colony Morphovars of Burkholderia pseudomallei.

The entire genomes of two isogenic morphovars (vgh16W and vgh16R) of Burkholderia pseudomallei were sequenced. A comparison of the sequences from both strains indicates that they show 99.99% identity, are composed of 22 tandem repeated sequences with <100 bp of indels, and have 199 single-base variants.

Airborne Transmission of Melioidosis to Humans from Environmental Aerosols Contaminated with B. pseudomallei.

Melioidosis results from an infection with the soil-borne pathogen Burkholderia pseudomallei, and cases of melioidosis usually cluster after rains or a typhoon. In an endemic area of Taiwan, B. pseudomallei is primarily geographically distributed in cropped fields in the northwest of this area, whereas melioidosis cases are distributed in a densely populated district in the southeast. We hypothesized that contaminated cropped fields generated aerosols contaminated with B. pseudomallei, which were carried by a northwesterly wind to the densely populated southeastern district. We collected soil and aerosol samples from a 72 km2 area of land, including the melioidosis-clustered area and its surroundings. Aerosols that contained B. pseudomallei-specific TTSS (type III secretion system) ORF2 DNA were well distributed in the endemic area but were rare in the surrounding areas during the rainy season. The concentration of this specific DNA in aerosols was positively correlated with the incidence of melioidosis and the appearance of a northwesterly wind. Moreover, the isolation rate in the superficial layers of the contaminated cropped field in the northwest was correlated with PCR positivity for aerosols collected from the southeast over a 2-year period. According to pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) analyses, PFGE Type Ia (ST58) was the predominant pattern linking the molecular association among soil, aerosol and human isolates. Thus, the airborne transmission of melioidosis moves from the contaminated soil to aerosols and/or to humans in this endemic area.

Improvement in T helper 1-related immune responses in BALB/c mice immunized with an HIV-1 gag plasmid combined with a chimeric plasmid encoding interleukin-18 and flagellin.

Both flagellin (fliC) and IL-18 (INF-γ-inducing factor) have been developed as adjuvants for improving immunogenicity in DNA-vaccinated hosts. An HIV-1 gag plasmid encodes a protein harboring broad epitopes for cytotoxic T-lymphocytes. In this study, the immunogenicity of BALB/c mice immunized with an HIV-1 gag plasmid (pVAX/gag) combined with a chimeric plasmid encoding IL-18 fused to flagellin (pcDNA3/IL-18_fliC) or a single plasmid encoding IL-18 (pcDNA3/IL-18) and/or flagellin (pcDNA3/fliC) was assessed. Through in vitro transcription and translation, it was demonstrated that both mRNA and protein were appropriately expressed by each construct. The IL-18 and flagellin fusion protein, which could be detected in supernatants from transfected cells, was effective in inducing IFN-γ by lymphocytes. Following i.m. immunization, expressions of flagellin or IL-18 were detected in muscle cells by immunohistochemistry analysis from 72 hr. At 12 weeks post-immunization, both gag-specific IgG in sera and spleen cell proliferation were high in all murine groups. However, the IgG2a/IgG1 ratio, Th1 cytokine (IL-2 and IFN-γ) production and proportion of gag-specific CD3(+) CD8(+) IFN-γ-secreting cells were significantly higher in the murine group co-immunized with pVAX/gag plasmid and pcDNA3/IL-18_fliC than in the mice immunized with pVAX/gag plasmid combined with either pcDNA3/fliC or pcDNA3/IL-18 plasmid or both. These findings suggest that a chimeric plasmid encoding IL-18 fused to flagellin can be used as an adjuvant-like plasmid to improve the Th1 immune response, particularly for induction of CD3(+) CD8(+) IFN-γ-secreting cells in gag plasmid-vaccinated mice.

Whole-Genome Sequence of an Epidemic Strain of Burkholderia pseudomallei vgh07 in Taiwan.

Here, we report the complete genome sequence of B. pseudomallei vgh07. This is an epidemic strain that was isolated from a melioidosis patient with arthro-osteomyelitis in Taiwan.

[Control strategies of nitrogen removal process in a pilot test of the southern WWTP based on the nitrogen balance].

By building the mass balance of nitrogen in A2/O process, the nitrogen model which raised some strategies on how to control sludge return ratio and mixed liquid return ratio to make the effluent nitrogen achieve the national standard A under different influent total nitrogen (TN) , was set up. And the presumed parameters were verified by the pilot test of the Wuhan's Longwangzui WWTP. The result showed that when the temperature and the TN were over 15 degrees C and below 30 mg x L(-1) respectively, the mixed liquid return ratio was 0. When the temperature was between 10 degrees C and 15 degrees C and TN was over 30 mg x L(-1), higher MLSS and DO elevated N removal. When the temperature was far below 10 degrees C, the mixed liquid return ratio was also at a higher level. Based on the Wuhan's Longwangzui WWTP influent water quality, measures of adjusting the return ratio were well adapted to obtain acceptable nitrogen effluent.

Induction of mouse melioidosis with meningitis by CD11b+ phagocytic cells harboring intracellular B. pseudomallei as a Trojan horse.

BACKGROUND: Approximately 3-5% of patients with melioidosis manifest CNS symptoms; however, the clinical data regarding neurological melioidosis are limited. METHODS AND FINDINGS: We established a mouse model of melioidosis with meningitis characterized by neutrophil infiltration into the meninges histologically and B. pseudomallei in the cerebrospinal fluid (CSF) by bacteriological culturing methods. As the disease progresses, the bacteria successively colonize the spleen, liver, bone marrow (BM) and brain and invade splenic and BM cells by days 2 and 6 post-infection, respectively. The predominant cell types intracellularly infected with B. pseudomallei were splenic and BM CD11b(+) populations. The CD11b(+)Ly6C(high) inflamed monocytes, CD11b(+)Ly6C(low) resident monocytes, CD11b(+)Ly6G(+) neutrophils, CD11b(+)F4/80(+) macrophages and CD11b(+)CD19(+) B cells were expanded in the spleen and BM during the progression of melioidosis. After adoptive transfer of CD11b populations harboring B. pseudomallei, the infected CD11b(+) cells induced bacterial colonization in the brain, whereas CD11b(-) cells only partially induced colonization; extracellular (free) B. pseudomallei were unable to colonize the brain. CD62L (selectin) was absent on splenic CD11b(+) cells on day 4 but was expressed on day 10 post-infection. Adoptive transfer of CD11b(+) cells expressing CD62L (harvested on day 10 post-infection) resulted in meningitis in the recipients, but transfer of CD11b(+) CD62L-negative cells did not. CONCLUSIONS/SIGNIFICANCE: We suggest that B. pseudomallei-infected CD11b(+) selectin-expressing cells act as a Trojan horse and are able to transmigrate across endothelial cells, resulting in melioidosis with meningitis.

Use of 3-hydroxy fatty acid concentrations in a murine air pouch infection model as a surrogate marker for LPS activity: a feasibility study using environmental Burkholderia cenocepacia isolates.

Using a murine hypodermic air pouch infection model designed to mimic the release of bacterial products at physiological levels, 3-hydroxy fatty acid (3-OH FA) and endotoxin unit levels from Burkholderia cenocepacia isolates were assessed. The B. cenocepacia environmental isolates (n=35) survived in the hypodermic air pouch but did not invade across the peritoneal epithelial layer during a 72-h infection. For all 35 strains, when the molar ratio of C(14:0) 3-OH FA to C(16:0) 3-OH FA in the air pouch fluid wash samples was between 1.4 and 2.5, the concentrations of C(14:0) 3-OH FA were correlated with the endotoxin unit levels. However, both surrogate markers exhibited different correlations to the inflammatory response. The linear regression coefficient was 0.4234 for C(14:0) 3-OH FA concentrations vs. NO productions, 0.223 for endotoxin unit levels vs. NO productions, 0.5008 for C(14:0) 3-OH FA concentrations vs. TNF-alpha productions and 0.2869 for endotoxin unit levels vs. TNF-alpha productions. Therefore, C(14:0) 3-OH FA concentrations, rather than endotoxin unit levels, acted as an immunostimulatory indicator for LPS in the B. cenocepacia isolates.

The scale of ethnic experience: development and psychometric properties.

The Scale of Ethnic Experience (SEE) is a new self-report instrument designed to measure multiple ethnicity-related cognitive constructs across ethnic groups. We present the development and psychometric properties here. We generated and refined an item pool using expert consultants and culturally diverse focus groups. We derived a final 32-item version of the SEE based on separate factor analyses of data from college students in 4 ethnic groups: African Americans, Caucasian Americans, Filipino Americans, and Mexican Americans. Four factors were consistent across the ethnic groups: Ethnic Identity, Perceived Discrimination, Mainstream Comfort, and Social Affiliation. We found evidence of test-retest reliability, internal consistency, and criterion and construct validity for all groups. Finally, we cross-validated the factor structure of the SEE in a culturally diverse sample. Results support the reliability and validity of the SEE as a multidimensional measure of ethnicity-related cognitive constructs that can be used across American ethnic groups.