Human Synaptic Plasticity Gene Expression Profile and Dendritic Spine Density Changes in HIV-Infected Human CNS Cells: Role in HIV-Associated Neurocognitive Disorders (HAND)

HIV-associated neurocognitive disorders (HAND) is characterized by development of cognitive, behavioral and motor abnormalities, and occur in approximately 50% of HIV infected individuals. Our current understanding of HAND emanates mainly from HIV-1 subtype B (clade B), which is prevalent in USA and Western countries. However very little information is available on neuropathogenesis of HIV-1 subtype C (clade C) that exists in Sub-Saharan Africa and Asia. Therefore, studies to identify specific neuropathogenic mechanisms associated with HAND are worth pursuing to dissect the mechanisms underlying this modulation and to prevent HAND particularly in clade B infection. In this study, we have investigated 84 key human synaptic plasticity genes differential expression profile in clade B and clade C infected primary human astrocytes by using RT2 Profile PCR Array human Synaptic Plasticity kit. Among these, 31 and 21 synaptic genes were significantly (≥3 fold) down-regulated and 5 genes were significantly (≥3 fold) up-regulated in clade B and clade C infected cells, respectively compared to the uninfected control astrocytes. In flow-cytometry analysis, down-regulation of postsynaptic density and dendrite spine morphology regulatory proteins (ARC, NMDAR1 and GRM1) was confirmed in both clade B and C infected primary human astrocytes and SK-N-MC neuroblastoma cells. Further, spine density and dendrite morphology changes by confocal microscopic analysis indicates significantly decreased spine density, loss of spines and decreased dendrite diameter, total dendrite and spine area in clade B infected SK-N-MC neuroblastoma cells compared to uninfected and clade C infected cells. We have also observed that, in clade B infected astrocytes, induction of apoptosis was significantly higher than in the clade C infected astrocytes. In conclusion, this study suggests that down-regulation of synaptic plasticity genes, decreased dendritic spine density and induction of apoptosis in astrocytes may contribute to the severe neuropathogenesis in clade B infection.

Pseudomonas aeruginosa AMPR transcriptional regulatory network

In Enterobacteriaceae, the transcriptional regulator AmpR, a member of the LysR family, regulates the expression of a chromosomal β-lactamase AmpC. The regulatory repertoire of AmpR is broader in Pseudomonas aeruginosa, an opportunistic pathogen responsible for numerous acute and chronic infections including cystic fibrosis. Previous studies showed that in addition to regulating ampC, P. aeruginosa AmpR regulates the sigma factor AlgT/U and production of some quorum sensing (QS)-regulated virulence factors. In order to better understand the ampR regulon, the transcriptional profiles generated using DNA microarrays and RNA-Seq of the prototypic P. aeruginosa PAO1 strain with its isogenic ampR deletion mutant, PAOΔampR were analyzed. Transcriptome analysis demonstrates that the AmpR regulon is much more extensive than previously thought influencing the differential expression of over 500 genes. In addition to regulating resistance to β-lactam antibiotics via AmpC, AmpR also regulates non-β-lactam antibiotic resistance by modulating the MexEF-OprN efflux pump. Virulence mechanisms including biofilm formation, QS-regulated acute virulence, and diverse physiological processes such as oxidative stress response, heat-shock response and iron uptake are AmpR-regulated. Real-time PCR and phenotypic assays confirmed the transcriptome data. Further, Caenorhabditis elegans model demonstrates that a functional AmpR is required for full pathogenicity of P. aeruginosa. AmpR, a member of the core genome, also regulates genes in the regions of genome plasticity that are acquired by horizontal gene transfer. The extensive AmpR regulon included other transcriptional regulators and sigma factors, accounting for the extensive AmpR regulon. Gene expression studies demonstrate AmpR-dependent expression of the QS master regulator LasR that controls expression of many virulence factors. Using a chromosomally tagged AmpR, ChIP-Seq studies show direct AmpR binding to the lasR promoter. The data demonstrates that AmpR functions as a global regulator in P. aeruginosa and is a positive regulator of acute virulence while negatively regulating chronic infection phenotypes. In summary, my dissertation sheds light on the complex regulatory circuit in P. aeruginosa to provide a better understanding of the bacterial response to antibiotics and how the organism coordinately regulates a myriad of virulence factors.

Pseudomonas Aeruginosa AmpR Transcriptional Regulatory Network

In Enterobacteriaceae, the transcriptional regulator AmpR, a member of the LysR family, regulates the expression of a chromosomal β-lactamase AmpC. The regulatory repertoire of AmpR is broader in Pseudomonas aeruginosa, an opportunistic pathogen responsible for numerous acute and chronic infections including cystic fibrosis. Previous studies showed that in addition to regulating ampC, P. aeruginosa AmpR regulates the sigma factor AlgT/U and production of some quorum sensing (QS)-regulated virulence factors. In order to better understand the ampR regulon, the transcriptional profiles generated using DNA microarrays and RNA-Seq of the prototypic P. aeruginosa PAO1 strain with its isogenic ampR deletion mutant, PAO∆ampR were analyzed. Transcriptome analysis demonstrates that the AmpR regulon is much more extensive than previously thought influencing the differential expression of over 500 genes. In addition to regulating resistance to β-lactam antibiotics via AmpC, AmpR also regulates non-β-lactam antibiotic resistance by modulating the MexEF-OprN efflux pump. Virulence mechanisms including biofilm formation, QS-regulated acute virulence, and diverse physiological processes such as oxidative stress response, heat-shock response and iron uptake are AmpR-regulated. Real-time PCR and phenotypic assays confirmed the transcriptome data. Further, Caenorhabditis elegans model demonstrates that a functional AmpR is required for full pathogenicity of P. aeruginosa. AmpR, a member of the core genome, also regulates genes in the regions of genome plasticity that are acquired by horizontal gene transfer. The extensive AmpR regulon included other transcriptional regulators and sigma factors, accounting for the extensive AmpR regulon. Gene expression studies demonstrate AmpR-dependent expression of the QS master regulator LasR that controls expression of many virulence factors. Using a chromosomally tagged AmpR, ChIP-Seq studies show direct AmpR binding to the lasR promoter. The data demonstrates that AmpR functions as a global regulator in P. aeruginosa and is a positive regulator of acute virulence while negatively regulating chronic infection phenotypes. In summary, my dissertation sheds light on the complex regulatory circuit in P. aeruginosa to provide a better understanding of the bacterial response to antibiotics and how the organism coordinately regulates a myriad of virulence factors.

Insulin-like growth factor-I gene expression as a growth indicator in Nile tilapia Oreochromis niloticus L.

Somatic growth in fishes is regulated by a variety of hormones. A central step in this hormonal network is the growth hormone-insulin-like growth factor-I (IGF-I) axis. Studies conducted evaluated the relationship of hepatic IGF-I (hIGF-1) mRNA with growth as affected by feeding regimes (satiation or restricted level; daily or alternate-day feeding), temperatures (high, ambient, low) and by social stress. To develop a cellular means for the quantification of hIGF-I mRNA levels in O. niloticus, hIGF-I cDNA was isolated and cloned. The partial sequence of IGF-I cDNA encodes for signal peptide, mature protein and a portion of the E-domain. A sensitive TaqMan quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay was developed based on the mature IGF-I. Using the developed qRT-PCR assay a significant positive correlation was observed between hIGF-I mRNA levels and growth rate of fish reared under different feeding regimes (r = 0.64) and temperature conditions (r = 0.64). On the dynamics of hIGF-I gene expression in response to elevated temperature, hIGF-I mRNA levels were significantly elevated after at least 2 days of exposure to warm temperature. This validates the concept that hIGF-I gene expressions are sufficiently sensitive to be used as a rapid growth rate indicator for O. niloticus. The hIGF-I levels have a significant positive correlation with specific growth rate (length; r = 0.92), and with condition factor (r = 0.55). On the effect of social stress, differential alterations in growth rates between the dominant and subordinates were observed which was attributed more to behavioral changes as transduced by physiological regulators. The fish's relative position in the social hierarchy was consistently reflected in the levels of hIGF-I mRNA and the eye color pattern. Subordination depressed hIGF-I levels while dominance stimulated it. These findings have shown that hGF-I level remained positively correlated to growth rate as affected by feeding regime, temperature and social stress. This suggests that hIGF-I plays a key role in controlling growth in O. niloticus and indicates that IGF-I mRNA quantification could prove useful for the rapid assessment of growth rate in this species of fish.

Analysis of the Expression Pattern of Wnt Receptor Frizzled 3

Wnt signaling plays a vital role in many developmental processes. Wnt signaling has been implicated in neural crest induction and cell differentiation among other functions. In mice Wnts comprise a family of nineteen glycoproteins that bind to Frizzled (Fzd) receptors and LRP5/6 co-receptors. This activates beta-catenin, which translocates into the nucleus and acts as a transcription factor, resulting in differential gene expression. Specifically, Fzd 3 enhances Wnt 1 signaling. Wnt 1 and Fzd 3 are involved in neural crest induction and in neural crest-derived melanocyte development. We analyzed the expression pattern ofFzd 3 and the LRP 5/6 by in situ hybridization inmouse embryos. Our data suggests a role for these genes in neural crest induction and in melanocyte differentiation in the murine system. Results show Fzd 3 expression in the anterior part of the neural tube and in the hindbrain, while LRP 5 is expressed in the anterior part of the neural tube, in the hindbrain, and in the eye. We conclude that Fzd 3 and LRP 5 are expressed in the neural crest. In addition, Fzd 3 might act as the receptor while LRP 5 might act as the co-receptor for Wntl signaling in the murine system.