Acanthamoeba castellanii induces host cell death via a phosphatidylinositol 3-kinase-dependent mechanism

Granulomatous amoebic encephalitis due to Acanthamoeba castellanii is a serious human infection with fatal consequences, but it is not clear how the circulating amoebae interact with the blood-brain barrier and transmigrate into the central nervous system. We studied the effects of an Acanthamoeba encephalitis isolate belonging to the T1 genotype on human brain microvascular endothelial cells, which constitute the blood-brain barrier. Using an apoptosis-specific enzyme-linked immunosorbent assay, we showed that Acanthamoeba induces programmed cell death in brain microvascular endothelial cells. Next, we observed that Acanthamoeba specifically activates phosphatidylinositol 3-kinase. Acanthamoeba-mediated brain endothelial cell death was abolished using LY294002, a phosphatidylinositol 3-kinase inhibitor. These results were further confirmed using brain microvascular endothelial cells expressing dominant negative forms of phosphatidylinositol 3-kinase. This is the first demonstration that Acanthamoeba-mediated brain microvascular endothelial cell death is dependent on phosphatidylinositol 3-kinase

Post-mortem culture of Balamuthia mandrillaris from the brain and cerebrospinal fluid of a case of granulomatous amoebic meningoencephalitis, using human brain microvascular endothelial cells

The first isolation in the UK of Balamuthia mandrillaris amoebae from a fatal case of granulomatous amoebic meningoencephalitis is reported. Using primary cultures of human brain microvascular endothelial cells (HBMECs), amoebae were isolated from the brain and cerebrospinal fluid (CSF). The cultures showed a cytopathic effect at 20–28 days, but morphologically identifiable B. mandrillaris amoebae were seen in cleared plaques in subcultures at 45 days. The identification of the organism was later confirmed using PCR on Chelex-treated extracts. Serum taken while the patient was still alive reacted strongly with slide antigen prepared from cultures of the post-mortem isolate, and also with those from a baboon B. mandrillaris strain at 1 : 10 000 in indirect immunofluorescence, but with Acanthamoeba castellanii (Neff) at 1 : 160, supporting B. mandrillaris to be the causative agent. If the presence of amoebae in the post-mortem CSF reflects the condition in life, PCR studies on CSF and on biopsies of cutaneous lesions may also be a valuable tool. The role of HBMECs in understanding the interactions of B. mandrillaris with the blood–brain barrier is discussed

Acanthamoeba genotype T4 from the UK and Iran and isolation of the T2 genotype from clinical isolates

The majority of the keratitis-causing Acanthamoeba isolates are genotype T4. In an attempt to determine whether predominance of T4 isolates in Acanthamoeba keratitis is due to greater virulence or greater prevalence of this genotype, Acanthamoeba genotypes were determined for 13 keratitis isolates and 12 environmental isolates from Iran. Among 13 clinical isolates, eight (61.5 %) belonged to T4, two (15.3 %) belonged to T3 and three (23 %) belonged to the T2 genotype. In contrast, the majority of 12 environmental isolates tested in the present study belonged to T2 (7/12, 58.3 %), followed by 4/12 T4 isolates (33.3 %). In addition, the genotypes of six new Acanthamoeba isolates from UK keratitis cases were determined. Of these, five (83.3 %) belonged to T4 and one was T3 (16.6 %), supporting the expected high frequency of T4 in Acanthamoeba keratitis. In total, the genotypes of 24 Acanthamoeba keratitis isolates from the UK and Iran were determined. Of these, 17 belonged to T4 (70.8 %), three belonged to T2 (12.5 %), three belonged to T3 (12.5 %) and one belonged to T11 (4.1 %), confirming that T4 is the predominant genotype (S2 = 4.167; P = 0.0412) in Acanthamoeba keratitis

Acanthamoeba induces cell-cycle arrest in host cells

Acanthamoeba can cause fatal granulomatous amoebic encephalitis (GAE) and eye keratitis. However, the pathogenesis and pathophysiology of these emerging diseases remain unclear. In this study, the effects of Acanthamoeba on the host cell cycle using human brain microvascular endothelial cells (HBMEC) and human corneal epithelial cells (HCEC) were determined. Two isolates of Acanthamoeba belonging to the T1 genotype (GAE isolate) and T4 genotype (keratitis isolate) were used, which showed severe cytotoxicity on HBMEC and HCEC, respectively. No tissue specificity was observed in their ability to exhibit binding to the host cells. To determine the effects of Acanthamoeba on the host cell cycle, a cell-cycle-specific gene array was used. This screened for 96 genes specific for host cell-cycle regulation. It was observed that Acanthamoeba inhibited expression of genes encoding cyclins F and G1 and cyclin-dependent kinase 6, which are proteins important for cell-cycle progression. Moreover, upregulation was observed of the expression of genes such as GADD45A and p130 Rb, associated with cell-cycle arrest, indicating cell-cycle inhibition. Next, the effect of Acanthamoeba on retinoblastoma protein (pRb) phosphorylation was determined. pRb is a potent inhibitor of G1-to-S cell-cycle progression; however, its function is inhibited upon phosphorylation, allowing progression into S phase. Western blotting revealed that Acanthamoeba abolished pRb phosphorylation leading to cell-cycle arrest at the G1-to-S transition. Taken together, these studies demonstrated for the first time that Acanthamoeba inhibits the host cell cycle at the transcriptional level, as well as by modulating pRb phosphorylation using host cell-signalling mechanisms. A complete understanding of Acanthamoeba–host cell interactions may help in developing novel strategies to treat Acanthamoeba infections

Post-mortem culture of Balamuthia mandrillaris from the brain and cerebrospinal fluid of a case of granulomatous amoebic meningoencephalitis, using human brain microvascular endothelial cells.

The first isolation in the UK of Balamuthia mandrillaris amoebae from a fatal case of granulomatous amoebic meningoencephalitis is reported. Using primary cultures of human brain microvascular endothelial cells (HBMECs), amoebae were isolated from the brain and cerebrospinal fluid (CSF). The cultures showed a cytopathic effect at 20-28 days, but morphologically identifiable B. mandrillaris amoebae were seen in cleared plaques in subcultures at 45 days. The identification of the organism was later confirmed using PCR on Chelex-treated extracts. Serum taken while the patient was still alive reacted strongly with slide antigen prepared from cultures of the post-mortem isolate, and also with those from a baboon B. mandrillaris strain at 1:10,000 in indirect immunofluorescence, but with Acanthamoeba castellanii (Neff) at 1:160, supporting B. mandrillaris to be the causative agent. If the presence of amoebae in the post-mortem CSF reflects the condition in life, PCR studies on CSF and on biopsies of cutaneous lesions may also be a valuable tool. The role of HBMECs in understanding the interactions of B. mandrillaris with the blood-brain barrier is discussed

Three dimensional surface preserving smoothing

We propose a new anisotropic diffusion process for removing noise from MRI images without distorting the edges. The method is based on a simple principle: any diffusion that increases a gradient at neighbouring pixels should be prohibited. From this principle, we deduce an inequality that allows diffusion along the edges but not across them. We introduce promising results using synthetic data with various types of noise as well as real MRI scans.Three dimensional surface preserving smoothingacceptedVersio

Improved color edge preserving smoothing

An edge preserving smoothing algorithm is presented. To prevent smoothing over edges, the algorithm requires that diffusion in a direction should not result in an increase in neighbouring gradients. Intuitively, we think that smoothing reduces gradients. This is, however, not true in the vicinity of strong edges where smoothing over the edge results in surface deformation. The possibility of reducing the calculation cost is explored by reducing the frequency of calculating the edge strength

PENEGAKAN HUKUM PIDANA PRAKTIK PROSTITUSI ONLINE BAGI MUCIKARI DAN PELACUR DALAM UNDANG-UNDANG NOMOR 19 TAHUN 2016 TENTANG INFORMASI DAN TRANSAKSI ELEKTRONIK

Tujuan penulisan ini adalah untuk mengetahui cara penegakan hukum efektif bagi mucikari dan pelacur dalam pelaksanaan prostitusi online, metode penulisan yang digunakan adalah yuridis normative dengan kesimpulan 1. Penegakan Hukum dalam praktik prostitusi online adalah Pasal 506 KUHP, Pasal 298 KUHP, UndangUndang No. 11 Tahun 2008 tentang Informasi dan Transaksi Elektronik jo Undang-Undang No. 19 Tahun 2016 tentang Perubahan atas UndangUndang No.11 Tahun 2008, Undang-Undang No. 44 Tahun 2008 tentang Pornografi, UndangUndang No.21 Tahun 2007 tentang Pemberantasan Tindak Pidana Perdagangan Orang, dan Undang-Undang UU No. 23 Tahun 2002 tentang Perlindungan Anak jo. UndangUndang No. 35 Tahun 2014 tentang Perubahan atas UU No. 23 Tahun 2002 tentang Perlindungan Anak 2. Faktor penghambat dalam pelaksanaan penegakan hukum terhadap kegiatan prostitusi online adalah peraturan perundangan tidak sertamerta dapat melakukan penangkapan dan penahanan kepada para pihak karena dalam KUHP belum mengatur tentang prostitusi online, serta belum adanya aturan huhum yang efektif yang mengatur tentang penegakan hukum terhadap pelaku prostitusi onlin

Enhancing employability through hospital placements for Biomedical Science students: A Case Study from the University of Essex, UK

Collaboration between Universities and hospitals has provided the National Health Service (NHS) with many excellent Biomedical Scientists through the placement year scheme. Here, we document the number of students joining the placement scheme and the number and type of hospital departments offering student placements over a 10-year period. Prior to 2012, students were able to join fully-funded placements through the Higher Education Funding Council for England (HEFCE). Since then, there has been a fluctuation in numbers completing a placement year at the University of Essex, but the employability of these graduates remains consistently higher than our 3-year graduates. We demonstrate the positive impact of completing a placement year in an NHS hospital laboratory for students, and the contribution to university metrics in good degrees and graduate outcomes as well as the provision of much needed, qualified biomedical science staff to hospitals