Biomarkers in the Diagnosis and Prediction of Medication Response in Depression and the Role of Nutraceuticals.

Depression continues to be a significant and growing public health concern. In clinical practice, it involves a clinical diagnosis. There is currently no defined or agreed upon biomarker/s for depression that can be readily tested. A biomarker is defined as a biological indicator of normal physiological processes, pathogenic processes, or pharmacological responses to a therapeutic intervention that can be objectively measured and evaluated. Thus, as there is no such marker for depression, there is no objective measure of depression in clinical practice. The discovery of such a biomarker/s would greatly assist clinical practice and potentially lead to an earlier diagnosis of depression and therefore treatment. A biomarker for depression may also assist in determining response to medication. This is of particular importance as not all patients prescribed with medication will respond, which is referred to as medication resistance. The advent of pharmacogenomics in recent years holds promise to target treatment in depression, particularly in cases of medication resistance. The role of pharmacogenomics in routine depression management within clinical practice remains to be fully established. Equally so, the use of pharmaceutical grade nutrients known as nutraceuticals in the treatment of depression in the clinical practice setting is largely unknown, albeit frequently self-prescribed by patients. Whether nutraceuticals have a role in not only depression treatment but also in potentially modifying the biomarkers of depression has yet to be proven. The aim of this review is to highlight the potential biomarkers for the diagnosis, prediction, and medication response of depression.

The trans-SNARE complex VAMP4/Stx6/Stx7/Vti1b is a key regulator of Golgi to late endosome MT1-MMP transport in macrophages.

The activity of the matrix metalloproteinase (MMP) MT1-MMP is strictly regulated by expression and cellular location. In macrophages LPS activation leads to the up-regulation of MT1-MMP and this need to be at the cell surface for them to degrade the dense extracellular matrix (ECM) components to create a path to migrate into injured and infected tissues. Fixed and live imaging shows newly made MT1-MMP is packaged into vesicles that traffic to and fuse with LBPA+ LAMP1+ late endosomes en route to the surface. The R-SNARE VAMP4, found on Golgi-derived vesicles that traffic to late endosomes, forms a trans-SNARE complex with the Q-SNARE complex Stx6/Stx7/Vti1b. The Stx6/Stx7/Vti1b complex has been shown to be up-regulated in lipopolysaccharide (LPS)-activated cells to increase trafficking of key cytokines through the classical pathway and now we show here it is up-regulation also plays a role in the late endosomal pathway of MT1-MMP trafficking. Depletion of any of the SNAREs in this complex reduces surface MT1-MMP and gelatin degradation. Conversely, overexpression of the Stx6/Stx7/Vti1b components increases surface MT1-MMP levels. This suggests that Stx6/Stx7/Vti1b is a key Q-SNARE complex in macrophages during an immune response and in partnership with VAMP4 it regulates transport of newly made MT1-MMP.

Pyrroles as a Potential Biomarker for Oxidative Stress Disorders.

Redox imbalance or oxidative stress that results from both environmental and genetic factors is observed in patients with schizophrenia. Therefore, identifying markers of oxidative stress in the early stages of psychosis and using antioxidant treatments as an adjuvant to antipsychotics has important implications. The reaction of p-N,N-dimethylaminobenzaldehyde (DMAB) with pyrrole moieties has been well studied for well over a century for use as a marker of oxidative stress dysregulation. Throughout this time, pyrroles have been investigated with varying veracity in urine extracts to identify elevated levels in patients diagnosed with schizophrenia. Since the 1960's, various claims have been made with respect to what causes the colour change when DMAB is added to urine extracts. Whilst the substances from this reaction have not been fully elucidated, an objective look at most studies indicates that urobilinogen is likely to be one them. Urobilinogen has also been identified as a major interferent in our results. Both pyrroles and urobilinogen condense the DMAB reaction system (form condensation products) and are quite different. The urobilinogen detected in urine forms when gut microflora chemically reduces the bilirubin content of bile acids. In comparison, evidence suggests that the pyrrole fraction originates from the fragmentation of regulatory haem by reactive oxygen species (ROS) such as hydrogen peroxide and super and nitrous oxides. Clinical studies in our laboratories have established that pyrroles as a urine biomarker have specificity in detecting schizophrenia; however, caution must be applied as the readings are subject to interference by other DMAB active compounds that are present, such as urobilinogen. This review highlights the initial chemistry in isolating pyrroles and provides recommendations for standardised laboratory testing to ensure pyrroles are correctly measured and distinguished from other by-products.

Invasion by activated macrophages requires delivery of nascent membrane-type-1 matrix metalloproteinase through late endosomes/lysosomes to the cell surface.

Macrophage migration into injured or infected tissue is a key aspect in the pathophysiology of many diseases where inflammation is a driving factor. Membrane-type-1 matrix metalloproteinase (MT1-MMP) cleaves extracellular matrix components to facilitate invasion. Here we show that, unlike the constitutive MT1-MMP surface recycling seen in cancer cells, unactivated macrophages express low levels of MT1-MMP. Upon lipopolysaccharide (LPS) activation, MT1-MMP synthesis dramatically increases 10-fold at the surface by 15 hours. MT1-MMP is trafficked from the Golgi complex to the surface via late endosomes/lysosomes in a pathway regulated by the late endosome/lysosome R-SNAREs VAMP7 and VAMP8. These form two separate complexes with the surface Q-SNARE complex Stx4/SNAP23 to regulate MT1-MMP delivery to the plasma membrane. Loss of either one of these SNAREs leads to a reduction in surface MT1-MMP, gelatinase activity and reduced invasion. Thus, inhibiting MT1-MMP transport through this pathway could reduce macrophage migration and the resulting inflammation.

The impact of anticancer drugs on the ocular surface.

Cancer is a global health problem and is one of the leading causes of death worldwide. Pleasingly, the rate of survival has improved and continues in an upward trend mainly due to better diagnosis and treatment modalities. In particular, the development of anticancer drugs including cytotoxic chemotherapy, hormonal agents and targeted therapies have provided the most effective treatment options in combatting cancerous cells. However, the antineoplastic mechanisms of these drugs can also lead to undesirable systemic and ocular side effects resulting from cytotoxicity, inflammation and neurotoxicity. While survival rates are projected to increase with time, the number of patients presenting with these side effects that can substantially impact quality of life will also rise. The current paper reviews the ocular surface and adnexal side effects of anticancer drugs, the appropriate management and possible interactions between drugs for ocular surface pathology treatment and the anticancer drugs.

The effects of both noradrenaline and CGP12177, mediated through human beta1 -adrenoceptors, are reduced by PDE3 in human atrium but PDE4 in CHO cells.

(-)-Noradrenaline and (-)-CGP12177 activate beta(1)-adrenoceptors through a high (H)- and low-affinity (L) site, respectively. The positive inotropic effects of (-)-noradrenaline are blunted by phosphodiesterase4 (PDE4) but not PDE3, while both PDE isoenzymes, acting in concert, prevent the effects of (-)-CGP12177 through beta(1)-adrenoceptors in rat ventricle. We sought to unravel the role of PDE3 and PDE4 on signals through the H and L sites in human myocardium. The kinetics of matching positive inotropic effects of (-)-noradrenaline (20 nM) and (-)-CGP12177 (100 nM) were investigated on human atrial trabeculae in the absence and presence of the PDE3 inhibitor cilostamide (300 nM), PDE4 inhibitor rolipram (1 microM) or both. The influence of cilostamide and rolipram on agonist-evoked cyclic adenosine monophosphate (cAMP) increases were also compared in Chinese hamster ovary (CHO) cells expressing recombinant human beta1 -adrenoceptors. (-)-Noradrenaline and (-)-CGP12177 caused matching inotropic responses that faded during a 60-min time course. Cilostamide, but not rolipram, increased the positive inotropic effects and abolished the time dependent fade of both agonists. In CHO cells, rolipram, but not cilostamide, enhanced the cAMP signals caused by both (-)-noradrenaline and (-)-CGP12177. PDE3, but not PDE4, blunts the positive inotropic effects of both (-)-noradrenaline and (-)-CGP12177 through H and L sites, respectively, of human atrial beta1 -adrenoceptors. However, in CHO cells, PDE4 blunts the cAMP signals of both (-)-noradrenaline and (-)-CGP12177. Neither CHO cells nor the rat ventricle are appropriate models for the beta1 -adrenoceptor-evoked signalling to PDE3 observed in human atrium.

(-)-Adrenaline elicits positive inotropic, lusitropic, and biochemical effects through beta2 -adrenoceptors in human atrial myocardium from nonfailing and failing hearts, consistent with Gs coupling but not with Gi coupling.

Activation of either coexisting beta1- or beta2 -adrenoceptors with noradrenaline or adrenaline, respectively, causes maximum increases of contractility of human atrial myocardium. Previous biochemical work with the beta2 -selective agonist zinterol is consistent with activation of the cascade beta2 -adrenoceptors-->Gsalpha-protein-->adenylyl cyclase-->cAMP-->protein kinase (PKA)-->phosphorylation of phospholamban, troponin I, and C-protein-->hastened relaxation of human atria from nonfailing hearts. However, in feline and rodent myocardium, catecholamines and zinterol usually do not hasten relaxation through activation of beta2 -adrenoceptors, presumably because of coupling of the receptors to Gi protein. It is unknown whether the endogenously occurring beta2 -adrenoceptor agonist adrenaline acts through the above cascade in human atrium and whether its mode of action could be changed in heart failure. We assessed the effects of (-)-adrenaline, mediated through beta2 -adrenoceptors (in the presence of CGP 20712A 300 nM to block beta1 -adrenoceptors), on contractility and relaxation of right atrial trabecula obtained from nonfailing and failing human hearts. Cyclic AMP levels were measured as well as phosphorylation of phospholamban, troponin I, and protein C with Western blots and the back-phosphorylation procedure. For comparison, beta1 -adrenoceptor-mediated effects of (-)-noradrenaline were investigated in the presence of ICI 118,551 (50 nM to block beta2 -adrenoceptors). The positive inotropic effects of both (-)-noradrenaline and (-)-adrenaline were accompanied by reductions in time to peak force and time to reach 50% relaxation. (-)-Adrenaline caused similar positive inotropic and lusitropic effects in atrial trabeculae from failing hearts. However, the inotropic potency, but not the lusitropic potency, of (-)-noradrenaline was reduced fourfold in atrial trabeculae from heart failure patients. Both (-)-adrenaline and (-)-noradrenaline enhanced cyclic AMP levels and produced phosphorylation of phospholamban, troponin I, and C-protein to a similar extent in atrial trabeculae from nonfailing hearts. The hastening of relaxation caused by (-)-adrenaline together with the PKA-catalyzed phosphorylation of the three proteins involved in relaxation, indicate coupling of beta2 -adrenoceptors to Gs protein. The phosphorylation of phospholamban at serine16 and threonine17 evoked by (-)-adrenaline through beta2 -adrenoceptors and by (-)-noradrenaline through beta1 -adrenoceptors was not different in atria from nonfailing and failing hearts. Activation of beta2 -adrenoceptors caused an increase in phosphorylase a activity in atrium from failing hearts further emphasizing the presence of the beta2 -adrenoceptor-Gsalpha-protein pathway in human heart. The positive inotropic and lusitropic potencies of (-)-adrenaline were conserved across Arg16Gly- and Gln27Glu-beta2 -adrenoceptor polymorphisms in the right atrium from patients undergoing coronary artery bypass surgery, chronically treated with beta1 -selective blockers. The persistent relaxant and biochemical effects of (-)-adrenaline through beta2 -adrenoceptors and of (-)-noradrenaline through beta1 -adrenoceptors in heart failure are inconsistent with an important role of coupling of beta2 -adrenoceptors with Gialpha-protein in human atrial myocardium.

Peripheral compartment innate immune response to Haemophilus influenzae and Streptococcus pneumoniae in chronic obstructive pulmonary disease patients.

Alterations in innate immunity that predispose to chronic obstructive pulmonary disease (COPD) exacerbations are poorly understood. We examined innate immunity gene expression in peripheral blood polymorphonuclear leukocytes (PMN) and monocytes stimulated by Haemophilus influenzae and Streptococcus pneumoniae. Thirty COPD patients (15 rapid and 15 non-rapid lung function decliners) and 15 smokers without COPD were studied. Protein expression of IL-8, IL-6, TNF-α and IFN-γ (especially monocytes) increased with bacterial challenge. In monocytes stimulated with S. pneumoniae, TNF-α protein expression was higher in COPD (non-rapid decliners) than in smokers. In co-cultures of monocytes and PMN, mRNA expression of TGF-ß1 and MYD88 was up-regulated, and CD14, TLR2 and IFN-γ down-regulated with H. influenzae challenge. TNF-α mRNA expression was increased with H. influenzae challenge in COPD. Cytokine responses were similar between rapid and non-rapid decliners. TNF-α expression was up-regulated in non-rapid decliners in response to H. influenzae (monocytes) and S. pneumoniae (co-culture of monocytes and PMN). Exposure to bacterial pathogens causes characteristic innate immune responses in peripheral blood monocytes and PMN in COPD. Bacterial exposure significantly alters the expression of TNF-α in COPD patients, although not consistently. There did not appear to be major differences in innate immune responses between rapid and non-rapid decliners.

Arg389Gly-beta1-adrenergic receptors determine improvement in left ventricular systolic function in nonischemic cardiomyopathy patients with heart failure after chronic treatment with carvedilol.

OBJECTIVE: Administration of the beta-adrenergic receptor blocker carvedilol to patients with chronic heart failure leads to clinically significant benefits, including improvement in left ventricular systolic function in some, but not all, patients. We sought to determine the basis of the variable effect obtained with carvedilol in patients with heart failure. Carvedilol blocks both beta1-adrenergic and beta2-adrenergic receptors, and both receptors exist as polymorphisms. We aimed to determine whether these polymorphisms contribute to variability in response to carvedilol in patients with chronic heart failure. METHODS: We retrospectively and prospectively investigated 135 patients with nonischemic cardiomyopathy and chronic stable heart failure (New York Heart Association class II, III) treated with carvedilol. Baseline echocardiography was obtained before introduction of carvedilol and repeated after stabilization of a maximally tolerated dose of carvedilol (50-100 mg/day) for at least 1 year. Polymerase chain reaction and restriction fragment length polymorphism analysis were used to genotype beta1-adrenergic and beta2-adrenergic receptor polymorphisms. RESULTS: When grouped according to receptor polymorphisms patients were well matched for severity of heart failure, comorbidity and treatment. No significant difference was observed in baseline left ventricular ejection fraction (LVEF) between groups (P>0.05). After 1.5 years of treatment with carvedilol patients with Arg389Arg-beta1-adrenergic receptors had a significantly greater improvement in LVEF compared with Gly389 carriers (Arg389Arg 18.8%; Arg389Gly 9.4%; Gly389Gly 6.0%; P<0.001) whereas there were no differences attributable to other beta1-adrenergic and beta2-adrenergic receptor polymorphisms (P>0.05). CONCLUSION: In patients with nonischemic dilated cardiomyopathy, carvedilol leads to a significantly greater improvement in LVEF in patients with the Arg389Arg-beta1 adrenergic receptor phenotype.

Epigenetics of lung cancer.

Epigenetics is the study of heritable changes in gene expression that occur without changes in DNA sequence. It has a role in determining when and where a gene is expressed during development. Perhaps the most well known epigenetic mechanism is DNA methylation whereby cytosines at position 5 in CpG dinucleotides are methylated. Histone modification is another form of epigenetic control, which is quite complex and diverse. Histones and DNA make up the nucleosome which is the structural unit of chromatin which are involved in packaging DNA. Apart from the crucial role epigenetics plays in embryonic development, transcription, chromatin structure, X chromosome inactivation and genomic imprinting, its role in an increasing number of human diseases is more and more recognized. These diseases include cancer, and lung cancer in particular has been increasingly studied for the potential biological role of epigenetic changes with the promise of better and novel diagnostic and therapeutic tools.

Identification of a novel gene, fimV, involved in twitching motility in Pseudomonas aeruginosa.

Transposon mutagenesis was used to identify a new locus required for twitching motility in Pseudomonas aeruginosa. Four Tn5-B21 mutants which lacked twitching motility and a fifth which exhibited impaired motility were found to map to the same KPN:I restriction fragment at approximately 40 min on the P. aeruginosa genome. Cloning and sequencing studies showed that all five transposon insertions occurred within the same 2.8 kb ORF, which was termed fimV. The product of this gene has a putative peptidoglycan-binding domain, predicted transmembrane domains, a highly acidic C terminus and anomalous electrophoretic migration, indicating unusual primary or secondary structure. The P. aeruginosa genome also possesses a paralogue of fimV. Homologues of fimV were also found in the sequenced genomes of the other type-IV-fimbriated bacteria Neisseria gonorrhoeae, Neisseria meningitidis, Legionella pneumophila and Vibrio cholerae, but not in those of other bacteria which lack type IV fimbriae. A fimV homologue was also found in the genome sequence of Shewanella putrefaciens, along with many other homologues of type IV fimbrial genes, indicating that this bacterium is also likely to produce type IV fimbriae. Wild-type twitching motility was restored to fimV mutants by complementation in a dosage-dependent manner. Overexpression of fimV resulted in an unusual phenotype where the cells were massively elongated and migrated in large convoys at the periphery of the colony. It is suggested that FimV may be involved in remodelling of the peptidoglycan layer to enable assembly of the type IV fimbrial structure and machinery.

Quorum sensing is not required for twitching motility in Pseudomonas aeruginosa.

It has been reported that mutations in the quorum-sensing genes lasI and rhlI in Pseudomonas aeruginosa result in, among many other things, loss of twitching motility (A. Glessner, R. S. Smith, B. H. Iglewski, and J. B. Robinson, J. Bacteriol. 181:1623-1629, 1999). We constructed knockouts of lasI and rhlI and the corresponding regulatory genes lasR and rhlR and found no effect on twitching motility. However, twitching-defective variants accumulated during culturing of lasI and rhlI mutants. Further analysis showed that the stable twitching-defective variants of lasI and rhlI mutants had arisen as a consequence of secondary mutations in vfr and algR, respectively, both of which encode key regulators affecting a variety of phenotypes, including twitching motility. In addition, when grown in shaking broth culture, lasI and rhlI mutants, but not the wild-type parent, also accumulated unstable variants that lacked both twitching motility and swimming motility and appeared to be identical in phenotype to the S1 and S2 variants that were recently reported to occur at high frequencies in P. aeruginosa strains grown as a biofilm or in static broth culture (E. Deziel, Y. Comeau, and R. Villemur, J. Bacteriol. 183:1195-1204, 2001). These results indicate that mutations in one regulatory system may create distortions that select during subsequent culturing for compensatory mutations in other regulatory genes within the cellular network. This problem may have compromised some past studies of regulatory hierarchies controlled by quorum sensing and of bacterial regulatory systems in general.

Lateral flagella and swarming motility in Aeromonas species.

Swarming motility, a flagellum-dependent behavior that allows bacteria to move over solid surfaces, has been implicated in biofilm formation and bacterial virulence. In this study, light and electron microscopic analyses and genetic and functional investigations have shown that at least 50% of Aeromonas isolates from the species most commonly associated with diarrheal illness produce lateral flagella which mediate swarming motility. Aeromonas lateral flagella were optimally produced when bacteria were grown on solid medium for approximately 8 h. Transmission and thin-section electron microscopy confirmed that these flagella do not possess a sheath structure. Southern analysis of Aeromonas reference strains and strains of mesophilic species (n = 84, varied sources and geographic regions) with a probe designed to detect lateral flagellin genes (lafA1 and lafA2) showed there was no marked species association of laf distribution. Approximately 50% of these strains hybridized strongly with the probe, in good agreement with the expression studies. We established a reproducible swarming assay (0.5% Eiken agar in Difco broth, 30 degrees C) for Aeromonas spp. The laf-positive strains exhibited vigorous swarming motility, whereas laf-negative strains grew but showed no movement from the inoculation site. Light and scanning electron microscopic investigations revealed that lateral flagella formed bacterium-bacterium linkages on the agar surface. Strains of an Aeromonas caviae isolate in which lateral flagellum expression was abrogated by specific mutations in flagellar genes did not swarm, proving conclusively that lateral flagella are required for the surface movement. Whether lateral flagella and swarming motility contribute to Aeromonas intestinal colonization and virulence remains to be determined.

Phosphorylation of the Pseudomonas aeruginosa response regulator AlgR is essential for type IV fimbria-mediated twitching motility.

The response regulator AlgR is required for both alginate biosynthesis and type IV fimbria-mediated twitching motility in Pseudomonas aeruginosa. In this study, the roles of AlgR signal transduction and phosphorylation in twitching motility and biofilm formation were examined. The predicted phosphorylation site of AlgR (aspartate 54) and a second aspartate (aspartate 85) in the receiver domain of AlgR were mutated to asparagine, and mutant algR alleles were introduced into the chromosome of P. aeruginosa strains PAK and PAO1. Assays of these mutants demonstrated that aspartate 54 but not aspartate 85 of AlgR is required for twitching motility and biofilm initiation. However, strains expressing AlgR D85N were found to be hyperfimbriate, indicating that both aspartate 54 and aspartate 85 are involved in fimbrial biogenesis and function. algD mutants were observed to have wild-type twitching motility, indicating that AlgR control of twitching motility is not mediated via its role in the control of alginate biosynthesis. In vitro phosphorylation assays showed that AlgR D54N is not phosphorylated by the enteric histidine kinase CheA. These findings indicate that phosphorylation of AlgR most likely occurs at aspartate 54 and that aspartate 54 and aspartate 85 of AlgR are required for the control of the molecular events governing fimbrial biogenesis, twitching motility, and biofilm formation in P. aeruginosa.

Characterization of a complex chemosensory signal transduction system which controls twitching motility in Pseudomonas aeruginosa.

Virulence of the opportunistic pathogen Pseudomonas aeruginosa involves the coordinate expression of a wide range of virulence factors including type IV pili which are required for colonization of host tissues and are associated with a form of surface translocation termed twitching motility. Twitching motility in P. aeruginosa is controlled by a complex signal transduction pathway which shares many modules in common with chemosensory systems controlling flagella rotation in bacteria and which is composed, in part, of the previously described proteins PilG, PilH, PilI, PilJ and PilK. Here we describe another three components of this pathway: ChpA, ChpB and ChpC, as well as two downstream genes, ChpD and ChpE, which may also be involved. The central component of the pathway, ChpA, possesses nine potential sites of phosphorylation: six histidine-containing phosphotransfer (HPt) domains, two novel serine- and threonine-containing phosphotransfer (SPt, TPt) domains and a CheY-like receiver domain at its C-terminus, and as such represents one of the most complex signalling proteins yet described in nature. We show that the Chp chemosensory system controls twitching motility and type IV pili biogenesis through control of pili assembly and/or retraction as well as expression of the pilin subunit gene pilA. The Chp system is also required for full virulence in a mouse model of acute pneumonia.