Uzma Qaisar

Gene Expression Profiling in Alzheimer's Disease Brain Microvessels

The enigma that is Alzheimers disease (AD) continues to present daunting challenges for effective therapeutic intervention. The lack of disease-modifying therapies may, in part, be attributable to the narrow research focus employed to understand this complex disease. Most studies into disease pathogenesis are based on a priori assumptions about the role of AD lesion-associated proteins such as amyloid-β and tau. However, the complex disease processes at work may not be amenable to single-target therapeutic approaches. Genome-wide expression studies provide an unbiased approach for investigating the pathogenesis of complex diseases like AD. A growing literature suggests a role for cerebrovascular contributions to the pathogenesis of AD. The objective of the current study is to examine human brain microvessels isolated from AD patients and controls by microarray analysis. Differentially expressed genes with more than 2-fold change are used for further data analysis. Gene ontology analysis and pathway analysis algorithms within GeneSpringGX are employed to understand the regulatory networks of differentially expressed genes. Twelve matched pairs of AD and control brain microvessel samples are hybridized to Agilent Human 4 × 44 K arrays in replication. We document that more than 2,000 genes are differentially altered in AD microvessels and that a large number of these genes map to pathways associated with immune and inflammatory response, signal transduction, and nervous system development and function categories. These data may help elucidate heretofore unknown molecular alterations in the AD cerebromicrovasculature.

Serum influences the expression of Pseudomonas aeruginosa quorum-sensing genes and QS-controlled virulence genes during early and late stages of growth

In response to diverse environmental stimuli at different infection sites, Pseudomonas aeruginosa, a serious nosocomial pathogen, coordinates the production of different virulence factors through a complicated network of the hierarchical quorum‐sensing (QS) systems including the las, rhl, and the 2‐alkyl‐4‐quinolone‐related QS systems. We recently showed that at early stages of growth serum alters the expression of numerous P. aeruginosa genes. In this study, we utilized transcriptional analysis and enzyme assays to examine the effect of serum on the QS and QS‐controlled virulence factors during early and late phases of growth of the P. aeruginosa strain PAO1. At early phase, serum repressed the transcription of lasI, rhlI, and pqsA but not lasR or rhlR. However, at late phase, serum enhanced the expression of all QS genes. Serum produced a similar effect on the synthesis of the autoinducers 3OC12‐HSL, C4‐HSL, and HHQ/PQS. Additionally, serum repressed the expression of several QS‐controlled genes in the early phase, but enhanced them in the late phase. Furthermore, serum influenced the expression of different QS‐positive (vqsR, gacA, and vfr) as well as QS‐negative (rpoN, qscR, mvaT, and rsmA) regulatory genes at either early or late phases of growth. However, with the exception of PAOΔvfr, we detected comparable levels of lasI/lasR expression in PAO1 and PAO1 mutants defective in these regulatory genes. At late stationary phase, serum failed to enhance lasI/lasR expression in PAOΔvfr. These results suggest that depending on the phase of growth, serum differentially influenced the expression of P. aeruginosa QS and QS‐controlled virulence genes. In late phase, serum enhanced the expression of las genes through vfr.

The pvc Operon Regulates the Expression of the Pseudomonas aeruginosa Fimbrial Chaperone/Usher Pathway (Cup) Genes

The Pseudomonas aeruginosa fimbrial structures encoded by the cup gene clusters (cupB and cupC) contribute to its attachment to abiotic surfaces and biofilm formation. The P. aeruginosa pvcABCD gene cluster encodes enzymes that synthesize a novel isonitrile functionalized cumarin, paerucumarin. Paerucumarin has already been characterized chemically, but this is the first report elucidating its role in bacterial biology. We examined the relationship between the pvc operon and the cup gene clusters in the P. aeruginosa strain MPAO1. Mutations within the pvc genes compromised biofilm development and significantly reduced the expression of cupB1-6 and cupC1-3, as well as different genes of the cupB/cupC two-component regulatory systems, roc1/roc2. Adjacent to pvc is the transcriptional regulator ptxR. A ptxR mutation in MPAO1 significantly reduced the expression of the pvc genes, the cupB/cupC genes, and the roc1/roc2 genes. Overexpression of the intact chromosomally-encoded pvc operon by a ptxR plasmid significantly enhanced cupB2, cupC2, rocS1, and rocS2 expression and biofilm development. Exogenously added paerucumarin significantly increased the expression of cupB2, cupC2, rocS1 and rocS2 in the pvcA mutant. Our results suggest that pvc influences P. aeruginosa biofilm development through the cup gene clusters in a pathway that involves paerucumarin, PtxR, and different cup regulators.

Mucin inhibits Pseudomonas aeruginosa biofilm formation by significantly enhancing twitching motility

In Pseudomonas aeruginosa, type IV pili (TFP)-dependent twitching motility is required for development of surface-attached biofilm (SABF), yet excessive twitching motility is detrimental once SABF is established. In this study, we show that mucin significantly enhanced twitching motility and decreased SABF formation in strain PAO1 and other P. aeruginosa strains in a concentration-dependent manner. Mucin also disrupted partially established SABF. Our analyses revealed that mucin increased the amount of surface pilin and enhanced transcription of the pilin structural gene pilA. Mucin failed to enhance twitching motility in P. aeruginosa mutants defective in genes within the pilin biogenesis operons pilGHI/pilJK-chpA-E. Furthermore, mucin did not enhance twitching motility nor reduce biofilm development by chelating iron. We also examined the role of the virulence factor regulator Vfr in the effect of mucin. In the presence or absence of mucin, PAOΔvfr produced a significantly reduced SABF. However, mucin partially complemented the twitching motility defect of PAOΔvfr. These results suggest that mucin interferes with SABF formation at specific concentrations by enhancing TFP synthesis and twitching motility, that this effect, which is iron-independent, requires functional Vfr, and only part of the Vfr-dependent effect of mucin on SABF development occurs through twitching motility.

The Pseudomonas aeruginosa extracellular secondary metabolite, Paerucumarin, chelates iron and is not localized to extracellular membrane vesicles

Proteins encoded by the Pseudomonas aeruginosa pvcA-D operon synthesize a novel isonitrile functionalized cumarin termed paerucumarin. The pvcA-D operon enhances the expression of the P. aeruginosa fimbrial chaperone/usher pathway (cup) genes and this effect is mediated through paerucumarin. Whether pvcA-D and/or paerucumarin affect the expression of other P. aeruginosa genes is not known. In this study, we examined the effect of a mutation in pvcA-D operon the global transcriptome of the P. aeruginosa strain PAO1-UW. The mutation reduced the expression of several ironcontrolled genes including pvdS, which is essential for the expression of the pyoverdine genes. Additional transcriptional studies showed that the pvcA-D operon is not regulated by iron. Exogenously added paerucumarin enhanced pyoverdine production and pvdS expression in PAO1-UW. Iron-chelation experiments revealed that purified paerucumarin chelates iron. However, exogenously added paerucumarin significantly reduced the growth of a P. aeruginosa mutant defective in pyoverdine and pyochelin production. In contrast to other secondary metabolite, Pseudomonas quinolone signal (PQS), paerucumarin is not localized to the P. aeruginosa membrane vesicles. These results suggest that paerucumarin enhances the expression of iron-controlled genes by chelating iron within the P. aeruginosa extracellular environment. Although paerucumarin chelates iron, it does not function as a siderophore. Unlike PQS, paerucumarin is not associated with the P. aeruginosa cell envelope.

Studies on involvement of Wrinkled1 transcription factor in the development of extra-long staple in cotton

In the present investigation, fiber transcriptome of Gossypium hirsutum producing short-fiber (JKC703, JKC737, JKC783) and long-fiber (JKC725, JKC777, Guazuncho 2) was compared with the transcriptome of extra-long fiber producing Gossypium barbadense (VH84602). By meta-analysis, we identified 1431 differentially expressed genes among the genotypes of different fibre lengths. Of these, 574 genes were up-regulated while 844 were down regulated in G. barbadense as compared to G. hirsutum. Expression of 5 genes related to fiber development was tested in local germplasm of G. barbadense (Bar14/5) and used G. hirsutum (MNH 886) using real-time RT-PCR. G. arboreum (FDH786) was as a sample with unknown expression. Expression pattern of an ethylene responsive transcription factor wrinkeled-1 (wri1), a vacuolar processing enzyme (vpe) and eceriferum3 (cer3) gene showed close association with fiber lengths. Moreover, cotton wri1 expressed in apical-meristem and bolls and showed significantly higher expression in fiber of G. barbadense as compared to G. hirsutum and G. arboreum during development. Level of Transcription of wri1 was enhanced in fiber during ball development while it remained unchanged in fiber-less seeds.

Techniques of Chromosomal Studies

Chromosomal analysis has been an area of utmost significance for various cytogenetic and medical studies. The techniques to study normal and abnormal chromosomes which initiated as simple observations under the microscope with and without different types of dyes have now developed into more elaborate and invasive techniques. In the early years of cytogenetics, scientists had a difficult time distinguishing individual chromosomes, but over the years, they continued to refine the conditions for preserving and staining chromosomes to the reproducible standard that is now expected in clinical cytogenetics. In today’s procedures, metaphase chromosomes are treated with stains that generate distinctive banding patterns, and chromosome pairs are then arranged into a standardized format known as a karyotype. Over the past few decades, versatile methods based on fluorescence in situ hybridization (FISH) have transformed cytogenetics into a molecular science and provided cytogeneticists with powerful new tools. FISH procedures are now routinely employed in clinical cytogenetics. Although chromosomes may appear to be static structures when viewed under a microscope, cytogeneticists know that chromosomes are actually dynamic assemblies made up of a DNA-protein complex called chromatin. This chapter takes stock of all the prevalent techniques, highlighting the principals involved in each method. Karyotyping, genetic mapping, fluorescence in situ hybridization (FISH), multiplex FISH, spectral karyotyping, flow cytometry, and microarray have been described. New trends in cytogenetics to understand the molecular mechanism have been discussed under new generation sequencing.