Comparison of the HIV-1 VPU interaction with CD4 and CD74

A thesis submitted to the Faculty of Health Sciences, University of the Witwatersrand, In fulfilment of the requirements for the degree of Doctor of Philosophy Johannesburg, February 2016Viral protein U (Vpu) is a versatile accessory protein of human immunodeficiency virus type 1 (HIV-1) and some simian immunodeficiency virus isolates. Amongst other functions, Vpu targets human CD4 for ubiquitination and proteasomal degradation. This process requires the presence of phospho-serine residues within the Vpu cytoplasmic domain in order to allow for the recruitment of beta-transducin repeat-containing protein (β-TrCP) and ultimately the E3 ligase complex. Similarly, it has been shown that the Vpu cytoplasmic domain also interacts with the human CD74 cytoplasmic region. CD74, the invariant chain of major histocompatibility complex class II (MHCII), is involved in the presentation of foreign antigens to CD4+ T cells. The Vpu/CD74 interaction may prevent the maturation of the MCHII complex resulting in the downmodulation of cell surface levels of MHCII. There are several structural similarities between the human CD4 and CD74 host proteins. Consequently, we hypothesised that HIV-1 Vpu targets human CD74 for proteasomal degradation through the binding of β-TrCP similar to that of Vpu-mediated CD4 proteasomal degradation. In view of this, this study aimed to delineate the binding interaction between HIV-1 Vpu and CD74 and to determine whether Vpu also targets CD74 for proteasomal degradation. An in silico homology study determined that the α-helices found within the cytoplasmic domains of CD4 and CD74 were similar with an RMSD of 1.437 Å. Recombinant Vpu::GFP fusion proteins were expressed in HEK 293T cells and purified in sufficient quantities and purity for use in subsequent in vitro assays. Using ELISAs, it was found that CD74 effectively prevented the binding of an anti-Vpu antibody, but not an anti-GFP antibody, to the Vpu::GFP fusion protein with an IC50 of 888.67 ± 130.08 nM. Further to this, using CD74 overlapping peptides of increasing lengths in combination with a Vpu overlapping peptide set, it was determined that the sequences “RIRERAEDSGNESEG” and “EDQKP” in the Vpu and CD74 cytoplasmic regions, respectively, contain the binding sites for this interaction. While the CD4 and CD74 cytoplasmic peptides yielded different responses after binding Vpu peptides, these two host proteins were shown to compete for binding to the Vpu protein. This provides evidence that steric hindrance has an effect on binding. Finally, total CD74 levels including cell membrane and intracellular fractions were significantly downregulated by Vpu at 24 and 48 hours (p = 0.0270 and 0.0062, respectively) as indicated by immunoprecipitation and flow cytometry in Vpu-transfected U937 cells. Notably, Vpu phosphorylation mutants (S52A and S52/56A) were unable to reduce CD74 levels within U937 cells, indicating that CD74 is not targeted for proteasomal degradation. In conclusion, this study has defined the specific Vpu/CD74 binding site and provides novel evidence that CD74 is downregulated in the presence of Vpu. Moreover, CD74 is not targeted for proteasomal degradation through the binding of β-TrCP and the recruitment of the E3 ligase complex. These findings have future relevance for HIV-1 treatment and management as CD74 downregulation is yet another mechanism by which HIV-1 impairs the host immune response.MT201

Light Emission in Silicon from Carbon Nanotubes

The use of optics in microelectronic circuits to overcome the limitation of metallic interconnects is more and more considered as a viable solution. Among future silicon compatible materials, carbon nanotubes are promising candidates thanks to their ability to emit, modulate and detect light in the wavelength range of silicon transparency. We report the first integration of carbon nanotubes with silicon waveguides, successfully coupling their emission and absorption properties. A complete study of this coupling between carbon nanotubes and silicon waveguides was carried out, which led to the demonstration of the temperature-independent emission from carbon nanotubes in silicon at a wavelength of 1.3 {\mu}m. This represents the first milestone in the development of photonics based on carbon nanotubes on silicon

From Smalltalk to Silicon: Towards a methodology to turn Smalltalk code into FPGA

International audienceDue to their ability to combine high performances along with flexibility, FPGAs (Field Programmable Gate Array) are used in robotic applications nowadays, especially in case of realtime applications. The FPGA circuits are often designed and configured using the Hardware Description Languages (HDLs) like VHDL or Verilog. However, although these languages provide abstractions up to the functionality level, they lack many features of todays modern languages that make them unsuited for high-level models and systems. In this paper, we present an overview of a methodology that uses a Dynamic Reflective Language, such as Smalltalk, for high level hardware/software co-design on FPGAs

Los antidepresivos en pediatría: ¿el mayor fracaso de la asistencia sanitaria?

Se dice que los servicios de salud mental infantil son el mayor fracaso de la sanidad británica; en particular, en lo que respecta al tratamiento de la depresión y las conductas suicidas en niños y adolescentes. El uso de los antidepresivos en niños y adolescentes ilustra la mayor brecha existente en los servicios sanitarios entre práctica médica y evidencias científicas, entre estudios de diseño abierto que reivindican beneficios y un gran número de ensayos clínicos aleatorizados (ECA) que señalan lo contrario, y entre los estudios ECA tal como se publican y publicitan y lo que en realidad demuestran sus datos. Sin embargo, los antidepresivos se usan de forma habitual y, probablemente, son los fármacos más prescritos en la adolescencia. Se examina el contexto del uso de antidepresivos en la infancia, el análisis de las pruebas, los daños y riesgos, el aumento de conductas suicidas y otras cuestiones de práctica clínica asociadas. El lugar para la clínica de los ISRS puede estar asociado a su posible efecto “serénico”, distinto al efecto ansiolítico de las benzodiacepinas y antipsicóticos. Considerar este “principio terapéutico” como resultado primario permitiría una mejor comprensión de los datos de los ensayos clínicos y una práctica clínica más equilibrada en su balance riesgo/beneficio. Su aplicación obligaría a un trabajo colaborativo con los pacientes y a un importante grado de autonomía respecto a lo que recogen las guías clínicas. Se analizan los problemas generales de los servicios sanitarios, su sostenibilidad, la promoción de la salud y el uso de medicamentos, ilustrado por el diagnóstico y tratamiento de la depresión infanto-juvenil y otras afecciones, como la osteoporosis, el asma o la hipertensión arterial. Finalmente, se aboga por una campaña de acceso libre a los datos de los ensayos clínicos

Variability and short-term determinants of walking capacity in patients with intermittent claudication

OBJECTIVE: Global positioning system (GPS) recordings can provide valid information on walking capacity in patients with peripheral arterial disease (PAD) and intermittent claudication (IC) during community-based outdoor walking. This study used GPS to determine the variability of the free-living walking distance between two stops (WDBS), induced by lower-limb pain, which may exist within a single stroll in PAD patients with IC and the potential associated parameters obtained from GPS analysis.METHODS: This cross-sectional study of 57 PAD patients with IC was conducted in a university hospital. The intervention was a 1-hour free-living walking in a flat public park with GPS recording at 0.5 Hz. GPS-computed parameters for each patient were WDBS, previous stop duration (PSD), cumulated time from the beginning of the stroll, and average walking speed for each walking bout. The coefficient of variation of each parameter was calculated for patients with the number of walking bouts (N(WB)) >or=5 during their stroll. A multivariate analysis was performed to correlate WDBS with the other parameters. RESULTS: Mean (SD) maximal individual WDBS was 1905 (1189) vs 550 (621) meters for patients with N(WB) <5 vs N(WB) >or= 5, respectively (P < .001). In the 36 patients with N(WB) >or= 5, the coefficient of variation for individual WDBS was 43%. Only PSD and cumulated time were statistically associated with WDBS in 16 and 5 patients, respectively. CONCLUSIONS: A wide short-term variability of WDBS exists and likely contributes to the difficulties experienced by patients with IC to estimate their maximal walking distance at leisurely pace. Incomplete recovery from a preceding walk, as estimated through PSD, seems to dominantly account for the WDBS in patients with IC

The Inter- and Intra-Unit Variability of a Low-Cost GPS Data Logger/Receiver to Study Human Outdoor Walking in View of Health and Clinical Studies

PURPOSE: The present study evaluates the intra- and inter-unit variability of the GlobalSat(R) DG100 GPS data logger/receiver (DG100) when estimating outdoor walking distances and speeds. METHODS: Two experiments were performed using healthy subjects walking on a 400 m outdoor synthetic track. The two experiments consisted of two different outdoor prescribed walking protocols with distances ranging from 50 to 400 m. Experiment 1 examined the intra-unit variability of the DG100 (test-retest reproducibility) when estimating walking distances. Experiment 2 examined the inter-unit variability of four DG100 devices (unit to unit variability) when estimating walking distances and speeds. RESULTS: The coefficient of variation [95% confidence interval], for the reliability of estimating walking distances, was 2.8 [2.5-3.2] %. The inter-unit variability among the four DG100 units tested ranged from 2.8 [2.5-3.2] % to 3.9 [3.5-4.4] % when estimating distances and from 2.7 [2.4-3.0] % to 3.8 [3.4-4.2] % when estimating speeds. CONCLUSION: The present study indicates that the DG100, an economical and convenient GPS data logger/receiver, can be reliably used to study human outdoor walking activities in unobstructed conditions. This device let facilitate the use of GPS in studies of health and disease