SKP2 inactivation suppresses prostate tumorigenesis by mediating JARID1B ubiquitination.

Aberrant elevation of JARID1B and histone H3 lysine 4 trimethylation (H3K4me3) is frequently observed in many diseases including prostate cancer (PCa), yet the mechanisms on the regulation of JARID1B and H3K4me3 through epigenetic alterations still remain poorly understood. Here we report that Skp2 modulates JARID1B and H3K4me3 levels in vitro in cultured cells and in vivo in mouse models. We demonstrated that Skp2 inactivation decreased H3K4me3 levels, along with a reduction of cell growth, cell migration and malignant transformation of Pten/Trp53 double null MEFs, and further restrained prostate tumorigenesis of Pten/Trp53 mutant mice. Mechanistically, Skp2 decreased the K63-linked ubiquitination of JARID1B by E3 ubiquitin ligase TRAF6, thus decreasing JARID1B demethylase activity and in turn increasing H3K4me3. In agreement, Skp2 deficiency resulted in an increase of JARID1B ubiquitination and in turn a reduction of H3K4me3, and induced senescence through JARID1B accumulation in nucleoli of PCa cells and prostate tumors of mice. Furthermore, we showed that the elevations of Skp2 and H3K4me3 contributed to castration-resistant prostate cancer (CRPC) in mice, and were positively correlated in human PCa specimens. Taken together, our findings reveal a novel network of SKP2-JARID1B, and targeting SKP2 and JARID1B may be a potential strategy for PCa control.

Misclassification of Plasmodium infections by conventional microscopy and the impact of remedial training on the proficiency of laboratory technicians in species identification.

BACKGROUND: Malaria diagnosis is largely dependent on the demonstration of parasites in stained blood films by conventional microscopy. Accurate identification of the infecting Plasmodium species relies on detailed examination of parasite morphological characteristics, such as size, shape, pigment granules, besides the size and shape of the parasitized red blood cells and presence of cell inclusions. This work explores misclassifications of four Plasmodium species by conventional microscopy relative to the proficiency of microscopists and morphological characteristics of the parasites on Giemsa-stained blood films. CASE DESCRIPTION: Ten-day malaria microscopy remedial courses on parasite detection, species identification and parasite counting were conducted for public health and research laboratory personnel. Proficiency in species identification was assessed at the start (pre) and the end (post) of each course using known blood films of Plasmodium falciparum, Plasmodium malariae, Plasmodium ovale and Plasmodium vivax infections with densities ranging from 1,000 to 30,000 parasites/µL. Outcomes were categorized as false negative, positive without speciation, P. falciparum, P. malariae, P. ovale, P. vivax and mixed infections. DISCUSSION AND EVALUATION: Reported findings are based on 1,878 P. falciparum, 483 P. malariae, 581 P. ovale and 438 P. vivax cumulative results collated from 2008 to 2010 remedial courses. Pre-training false negative and positive misclassifications without speciation were significantly lower on P. falciparum infections compared to non-falciparum infections (p < 0.0001). Post-training misclassifications decreased significantly compared to pre- training misclassifications which in turn led to significant improvements in the identification of the four species. However, P. falciparum infections were highly misclassified as mixed infections, P. ovale misclassified as P. vivax and P. vivax similarly misclassified as P. ovale (p < 0.05). CONCLUSION: These findings suggest that the misclassification of malaria species could be a common occurrence especially where non-falciparum infections are involved due to lack of requisite skills in microscopic diagnosis and variations in morphological characteristics within and between Plasmodium species. Remedial training might improve reliability of conventional light microscopy with respect to differentiation of Plasmodium infections.

Lack of fetuin-A (alpha2-HS-glycoprotein) reduces mammary tumor incidence and prolongs tumor latency via the transforming growth factor-beta signaling pathway in a mouse model of breast cancer.

The present analyses were done to define the role of fetuin-A (Fet) in mammary tumorigenesis using the polyoma middle T antigen (PyMT) transgenic mouse model. We crossed Fet-null mice in the C57BL/6 background with PyMT mice in the same background and after a controlled breeding protocol obtained PyMT/Fet+/+, PyMT/Fet+/-, and PyMT/Fet-/- mice that were placed in control and experimental groups. Whereas the control group (PyMT/Fet+/+) formed mammary tumors 90 days after birth, tumor latency was prolonged in the PyMT/Fet-/- and PyMT/Fet+/- mice. The majority of the PyMT/Fet-/- mice were tumor-free at the end of the study, at approximately 40 weeks. The pathology of the mammary tumors in the Fet-null mice showed extensive fibrosis, necrosis, and squamous metaplasia. The preneoplastic mammary tissues of the PyMT/Fet-/- mice showed intense phopho-Smad2/3 staining relative to control tissues, indicating that transforming growth factor-ß signaling is enhanced in these tissues in the absence of Fet. Likewise, p19ARF and p53 were highly expressed in tumor tissues of PyMT/Fet-/- mice relative to the controls in the absence of Fet. The phosphatidylinositol 3-kinase/Akt signaling pathway that we previously showed to be activated by Fet, on the other hand, was unaffected by the absence of Fet. The data indicate that Fet is a powerful modulator of breast tumorigenesis in this model system and has the potential to modulate breast cancer progression in humans.

The levels of CD16/Fc gamma receptor IIIA on CD14+ CD16+ monocytes are higher in children with severe Plasmodium falciparum anemia than in children with cerebral or uncomplicated malaria.

Fc gamma receptor IIIA (CD16/Fc gamma RIIIA) on monocytes/macrophages may play an important role in the pathogenesis of severe malarial anemia (SMA) by promoting phagocytosis of IgG-coated uninfected red cells and by allowing the production of tumor necrosis factor alpha (TNF-alpha) upon cross-linking by immune complexes (ICs). However, not much is known about the differential expression of this receptor on monocytes of children with severe malaria and uncomplicated malaria. Therefore, we investigated the expression of CD16/Fc gamma RIIIA on monocytes of children with SMA, cerebral malaria (CM), and their age-matched uncomplicated malaria controls by flow cytometry. Since CD14 low (CD14(+)) monocytes are considered more mature and macrophage-like than CD14 high (CD14(++)) monocytes, we also compared the level of expression of CD16/Fc gamma RIIIA according to the CD14 level and studied the relationship between CD16/Fc gamma RIIIA expression and intracellular TNF-alpha production upon stimulation by ICs. CD16/Fc gamma RIIIA expression was the highest overall on CD14(+) CD16(+) monocytes of children with SMA at enrollment. At convalescence, SMA children were the only ones to show a significant decline in the same parameter. In contrast, there were no significant differences among groups in the expression of CD16/Fc gamma RIIIA on CD14(++) CD16(+) monocytes. A greater percentage of CD14(+) CD16(+) monocytes produced TNF-alpha upon stimulation than any other monocyte subset, and the amount of intracellular TNF-alpha correlated positively with CD16/Fc gamma RIIIA expression. Furthermore, there was an inverse correlation between hemoglobin levels and CD16/Fc gamma RIIIA expression in children with SMA and their controls. These data suggest that monocytes of children with SMA respond differently to Plasmodium falciparum infection by overexpressing CD16/Fc gamma RIIIA as they mature, which could enhance erythrophagocytosis and TNF-alpha production.

A novel function for the atypical small G protein Rab-like 5 in the assembly of the trypanosome flagellum.

The atypical small G protein Rab-like 5 has been shown to traffic in sensory cilia of Caenorhabditis elegans, where it participates in signalling processes but not in cilia construction. In this report, we demonstrate that RABL5 colocalises with intraflagellar transport (IFT) proteins at the basal body and in the flagellum matrix of the protist Trypanosoma brucei. RABL5 fused to GFP exhibits anterograde movement in the flagellum of live trypanosomes, suggesting it could be associated with IFT. Accordingly, RABL5 accumulates in the short flagella of the retrograde IFT140(RNAi) mutant and is restricted to the basal body region in the IFT88(RNAi) anterograde mutant, a behaviour that is identical to other IFT proteins. Strikingly, RNAi silencing reveals an essential role for RABL5 in trypanosome flagellum construction. RNAi knock-down produces a phenotype similar to inactivation of retrograde IFT with formation of short flagella that are filled with a high amount of IFT proteins. These data reveal for the first time a functional difference for a conserved flagellar matrix protein between two different ciliated species and raise questions related to cilia diversity.

Increased deposition of C3b on red cells with low CR1 and CD55 in a malaria-endemic region of western Kenya: implications for the development of severe anemia.

BACKGROUND: Severe anemia due to Plasmodium falciparum malaria is a major cause of mortality among young children in western Kenya. The factors that lead to the age-specific incidence of this anemia are unknown. Previous studies have shown an age-related expression of red cell complement regulatory proteins, which protect erythrocytes from autologous complement attack and destruction. Our primary objective was to determine whether in a malaria-endemic area red cells with low levels of complement regulatory proteins are at increased risk for complement (C3b) deposition in vivo. Secondarily, we studied the relationship between red cell complement regulatory protein levels and hemoglobin levels. METHODS: Three hundred and forty-two life-long residents of a malaria-holoendemic region of western Kenya were enrolled in a cross-sectional study and stratified by age. We measured red cell C3b, CR1, CD55, and immune complex binding capacity by flow cytometry. Individuals who were positive for malaria were treated and blood was collected when they were free of parasitemia. Analysis of variance was used to identify independent variables associated with the %C3b-positive red cells and the hemoglobin level. RESULTS: Individuals between the ages of 6 and 36 months had the lowest red cell CR1, highest %C3b-positive red cells, and highest parasite density. Malaria prevalence also reached its peak within this age group. Among children 6 to

Reduced immune complex binding capacity and increased complement susceptibility of red cells from children with severe malaria-associated anemia.

Plasmodium falciparum malaria causes 1-2 million deaths per year. Most deaths occur as a result of complications such as severe anemia and cerebral malaria (CM) (coma). Red cells of children with severe malaria-associated anemia (SMA) have acquired deficiencies in the complement regulatory proteins complement receptor 1 (CR1, CD35) and decay accelerating factor (DAF, CD55). We investigated whether these deficiencies affect the ability of erythrocytes to bind immune complexes (ICs) and regulate complement activation. We recruited 75 children with SMA (Hb < or = 6 g/dL) from the holoendemic malaria region of the Lake Victoria basin, western Kenya, and 74 age- and gender-matched uncomplicated malaria controls. In addition, we recruited 32 children with CM and 52 age- and gender-matched controls. Deficiencies in red cell CR1 and CD55 in children with SMA were accompanied by a marked decline in IC binding capacity and increased C3b deposition in vivo and ex vivo. Importantly, these changes were specific because they were not seen in red cells of children with CM or their controls. These data suggest that the declines in red cell CR1 and CD55 seen in children with SMA are of physiologic significance and may predispose erythrocytes to complement-mediated damage and phagocytosis in vivo.

Establishing a malaria diagnostics centre of excellence in Kisumu, Kenya.

BACKGROUND: Malaria microscopy, while the gold standard for malaria diagnosis, has limitations. Efficacy estimates in drug and vaccine malaria trials are very sensitive to small errors in microscopy endpoints. This fact led to the establishment of a Malaria Diagnostics Centre of Excellence in Kisumu, Kenya. The primary objective was to ensure valid clinical trial and diagnostic test evaluations. Key secondary objectives were technology transfer to host countries, establishment of partnerships, and training of clinical microscopists. CASE DESCRIPTION: A twelve-day "long" and a four-day "short" training course consisting of supervised laboratory practicals, lectures, group discussions, demonstrations, and take home assignments were developed. Well characterized slides were developed and training materials iteratively improved. Objective pre- and post-course evaluations consisted of 30 slides (19 negative, 11 positive) with a density range of 50-660 parasites/mul, a written examination (65 questions), a photographic image examination (30 images of artifacts and species specific characteristics), and a parasite counting examination. DISCUSSION AND EVALUATION: To date, 209 microscopists have participated from 11 countries. Seventy-seven experienced microscopists participated in the "long" courses, including 47 research microscopists. Sensitivity improved by a mean of 14% (CI 9-19%) from 77% baseline (CI 73-81 %), while specificity improved by a mean of 17% (CI 11-23%) from 76% (CI 70-82%) baseline. Twenty-three microscopists who had been selected for a four-day refresher course showed continued improvement with a mean final sensitivity of 95% (CI 91-98%) and specificity of 97% (CI 95-100%). Only 9% of those taking the pre-test in the "long" course achieved a 90% sensitivity and 95% specificity, which increased to 61% of those completing the "short" course. All measures of performance improved substantially across each of the five organization types and in each course offered. CONCLUSION: The data clearly illustrated that false positive and negative malaria smears are a serious problem, even with research microscopists. Training dramatically improved performance. Quality microscopy can be provided by the Centre of Excellence concept. This concept can be extended to other diagnostics of public health importance, and comprehensive disease control strategies.

The two cytoplasmic dynein-2 isoforms in Leishmania mexicana perform separate functions.

Eukaryotic organisms with cilia or flagella typically express two non-axonemal or "cytoplasmic" dyneins, dynein-1 and dynein-2. Interestingly, we find that Leishmania mexicana is unusual and contains two distinct cytoplasmic dynein-2 heavy chain genes (designated LmxDHC2.1 and LmxDHC2.2) along with a single dynein-1 heavy chain (LmxDHC1). Disruption of LmxDHC2.2 resulted in immotile parasites that had a rounded cell body. Although they assume amastigote morphology, immunoblot analysis of these cells demonstrates protein expression consistent with the promastigote stage. Ultrastructural analysis revealed non-emergent flagella that lacked the paraflagellar rod and an axoneme with deficiencies in several components. We confirmed the absence of paraflagellar rod proteins PFR1 and PFR2. These results show that LmxDHC2.2 is required for flagellar assembly and also participates in the maintenance of promastigote cell shape. In contrast to the results with LmxDHC2.2, we were unable to generate homologous disruptions of LmxDHC2.1. This result suggests that, unlike LmxDHC2.2, LmxDHC2.1 is an essential gene in Leishmania. Together, these findings demonstrate that the two dynein-2 heavy chain isoforms in Leishmania perform distinct functions. The observation that the genomes of Leishmania major, Leishmania infantum and Trypanosoma brucei also contain two dynein-2 isoforms suggests that this unusual aspect of cytoplasmic dynein is a conserved feature of the kinetoplastids.