A novel method to titrate Herpes simplex virus-1 (HSV-1) using laser-based scanning of near-infrared fluorophores conjugated antibodies

Among several strategies used for Herpes simplex virus (HSV) detection in biological specimens, standard plaque assay (SPA) remains the most reliable method to evaluate virus infectivity and quantify viral replication. However, it is a manual procedure, thereby affected by operator subjectivity, and it may be particularly laborious for multiple sample analysis. Here we describe an innovative method to perform the titration of HSV type 1 (HSV-1) in different samples, using the “In-Cell WesternTM” Assay (ICW) from LI-COR, a quantitative immunofluorescence assay that exploits laser-based scanning of near infrared (NIR). In particular, we employed NIR-immunodetection of viral proteins to monitor foci of HSV-1 infection in cell monolayers, and exploited an automated detection of their fluorescence intensity to evaluate virus titre. This innovative method produced similar and superimposable values compared to SPA, but it is faster and can be performed in 96 well plate, thus allowing to easily and quickly analyze and quantify many samples in parallel. These features make our method particularly suitable for the screening and characterization of antiviral compounds, as we demonstrated by testing acyclovir (ACV), the main anti-HSV-1 drug. Moreover, we developed a new data analysis system that allowed to overcome potential bias due to unspecific florescence signals, thus improving data reproducibility. Overall, our method may represents a useful tool for both clinical and research purposes

Population genetics of identity by descent

Recent improvements in high-throughput genotyping and sequencing technologies have afforded the collection of massive, genome-wide datasets of DNA information from hundreds of thousands of individuals. These datasets, in turn, provide unprecedented opportunities to reconstruct the history of human populations and detect genotype-phenotype association. Recently developed computational methods can identify long-range chromosomal segments that are identical across samples, and have been transmitted from common ancestors that lived tens to hundreds of generations in the past. These segments reveal genealogical relationships that are typically unknown to the carrying individuals. In this work, we demonstrate that such identical-by-descent (IBD) segments are informative about a number of relevant population genetics features: they enable the inference of details about past population size fluctuations, migration events, and they carry the genomic signature of natural selection. We derive a mathematical model, based on coalescent theory, that allows for a quantitative description of IBD sharing across purportedly unrelated individuals, and develop inference procedures for the reconstruction of recent demographic events, where classical methodologies are statistically underpowered. We analyze IBD sharing in several contemporary human populations, including representative communities of the Jewish Diaspora, Kenyan Maasai samples, and individuals from several Dutch provinces, in all cases retrieving evidence of fine-scale demographic events from recent history. Finally, we expand the presented model to describe distributions for those sites in IBD shared segments that harbor mutation events, showing how these may be used for the inference of mutation rates in humans and other species.Comment: Ph.D. thesi

ELECTRICAL POWER GENERATION FROM NON-CONTINUOUS FLOW IN A SELF-CONTAINED BREATHING APPARATUS

In recent times, the number of electronic devices utilized by firefighters has greatly increased. Currently, all of this equipment relies heavily upon batteries as a source of power. The purpose of this research was to investigate alternate methods for powering the electrical functions of an SCBA (self-contained breathing apparatus). Replacing the batteries with a self-sufficient source of constant power is attractive for its characteristics of both reliability and cost efficiency. Maintenance of charged batteries and the logistics problem of an SCBA arriving at the scene unable to operate create an environment which is certainly unreliable and potentially quite dangerous. By relying on a maintenance-free, constant source of power, the jobs of fire departments could be made easier, and the lives of firefighters safer.The energy source for the power generation was to be the pressurized air stored in tanks on the firefighters' backs. Many methods were considered for the ground-up design, but selected was the concept of an inline unit comprised of a small DC dynamo coupled with a pneumatic motor. The generator would be propelled by the air flow resulting from each inhalation breath. Multiple tests were performed to prove the generator's capability in both adequate continuous power generation and in its implementation without affecting the existing performance of the SCBA. An accumulator was added to the system as a corrective method to keep the internal mask pressure at an acceptable level.The final design of the power generation cell includes a non-reversible air motor, coupled with the appropriate DC motor, contained within a sealed cell which acts as an accumulator, protective enclosure, and a means of leak containment for the unit. The unit is self-contained, easily added to an SCBA, and has no negative effects on the current design of the system. Adequate levels of continuous power were reached, ranging from 4 to 7 Watts depending on the rate of breathing

Herpes simplex virus-type1 (HSV-1) impairs DNA repair in cortical neurons

Several findings suggest that Herpes simplex virus-1 (HSV-1) infection plays a role in the neurodegenerative processes that characterize Alzheimer's disease (AD), but the underlying mechanisms have yet to be fully elucidated. Here we show that HSV-1 productive infection in cortical neurons causes the accumulation of DNA lesions that include both single (SSBs) and double strand breaks (DSBs), which are reported to be implicated in the neuronal loss observed in neurodegenerative diseases. We demonstrate that HSV-1 downregulates the expression level of Ku80, one of the main components of non-homologous end joining (NHEJ), a major pathway for the repair of DSBs. We also provide data suggesting that HSV-1 drives Ku80 for proteasomal degradation and impairs NHEJ activity, leading to DSB accumulation. Since HSV-1 usually causes life-long recurrent infections, it is possible to speculate that cumulating damages, including those occurring on DNA, may contribute to virus induced neurotoxicity and neurodegeneration, further suggesting HSV-1 as a risk factor for neurodegenerative conditions

The DNA damage response promotes Polyomavirus JC infection by nucleus to cytoplasm NF-Kappa B activation.

Background: Infection of glial cells by human neurotropic polyomavirus JC (JCV), the causative agent of the CNS demyelinating disease progressive multifocal leukoencephalopathy (PML), rapidly inflicts damage to cellular DNA. This activates DNA damage response (DDR) signaling including induction of expression of DNA repair factor Rad51. We previously reported that Rad51 co-operates with the transcription factor NF-κB p65 to activate JCV early transcription. Thus Rad51 induction by JCV infection may provide positive feedback for viral activation early in JCV infection. DDR is also known to stimulate NF-κB activity, a phenomenon known as nucleus to cytoplasm or “insideout” NF-κB signaling, which is initiated by Ataxia telangiectasia mutated (ATM) protein, a serine/threonine kinase recruited and activated by DNA double-strand breaks. Downstream of ATM, there occurs a series of posttranslational modifications of NF-κB essential modulator (NEMO), the γ regulatory subunit of inhibitor of NF-κB (IκB) kinase (IKK), resulting in NF-κB activation. Methods: We analyzed the effects of downstream pathways in the DDR by phosphospecific Western blots and analysis of the subcellular distribution of NEMO by cell fractionation and immunocytochemistry. The role of DDR in JCV infection was analyzed using a small molecule inhibitor of ATM (KU-55933). NEMO sumoylation was investigated by Western and association of ATM and NEMO by immunoprecipitation/Western blots. Results: We show that JCV infection caused phosphorylation and activation of ATM while KU-55933 inhibited JCV replication. JCV infection caused a redistribution of NEMO from cytoplasm to nucleus. Co-expression of JCV large Tantigen and FLAG-tagged NEMO showed the occurrence of sumoylation of NEMO, while co-expression of ATM and FLAG-NEMO demonstrated physical association between ATM and NEMO. Conclusions: We propose a model where JCV infection induces both overexpression of Rad51 protein and activation of the nucleus to cytoplasm NF-κB signaling pathway, which then act together to enhance JCV gene expression

Effects of vertical vibration on hopper flows of granular material

The discharge of granular material from a hopper subject to vertical sinusoidal oscillations was investigated using experiments and discrete element computer simulations. With the hopper exit closed, side-wall convection cells are observed, oriented such that particles move up along the inclined walls of the hopper and down at the center line. The convection cells are a result of the granular bed dilation during free fall and the subsequent interaction with the hopper walls. The mass discharge rate for a vibrating hopper scaled by the discharge rate without vibration reaches a maximum value at a dimensionless velocity amplitude just greater than 1. Further increases in the velocity decrease the discharge rate. The decrease occurs due to a decrease in the bulk density of the discharging material when vibration is applied