High-Throughput Phenotypic Characterization of Pseudomonas aeruginosa Membrane Transport Genes

The deluge of data generated by genome sequencing has led to an increasing reliance on bioinformatic predictions, since the traditional experimental approach of characterizing gene function one at a time cannot possibly keep pace with the sequence-based discovery of novel genes. We have utilized Biolog phenotype MicroArrays to identify phenotypes of gene knockout mutants in the opportunistic pathogen and versatile soil bacterium Pseudomonas aeruginosa in a relatively high-throughput fashion. Seventy-eight P. aeruginosa mutants defective in predicted sugar and amino acid membrane transporter genes were screened and clear phenotypes were identified for 27 of these. In all cases, these phenotypes were confirmed by independent growth assays on minimal media. Using qRT-PCR, we demonstrate that the expression levels of 11 of these transporter genes were induced from 4- to 90-fold by their substrates identified via phenotype analysis. Overall, the experimental data showed the bioinformatic predictions to be largely correct in 22 out of 27 cases, and led to the identification of novel transporter genes and a potentially new histamine catabolic pathway. Thus, rapid phenotype identification assays are an invaluable tool for confirming and extending bioinformatic predictions

Functional Divergence of Heme-Thiolate Proteins: A Classification Based on Spectroscopic Attributes

1. INTRODUCTION Heme proteins are among the most versatile players in the biological milieu; their functions range widely and include electron transfer, catalysis, and small-molecule sensing and transport.1 This broad functional diversity may be attributed to the protein environment that constitutes the heme cofactor’s binding site, particularly those amino acid residues that serve as axial ligands to the heme iron atom. For example, cytochromes, globins, heme oxygenases, and the majority of peroxidases employ the imidazole moiety of a histidine (His) residue as an axial ligand. Certain cytochromes are also coordinated by the thioether of methionine (Met) or, rarely, the polypeptide Nterminal amino group.2 Catalases utilize a tyrosine (Tyr) phenolate.3 An ever-growing class of heme proteins employs a cysteine (Cys) thiolate as an axial ligand to heme b (ironprotoporphyrin IX, Figure 1). Differences in solvent exposure, hydrophobicity, iron atom oxidation and spin state, and the identity of the iron atom axial ligand(s) give rise to a wide variety of spectroscopic characteristics. These spectroscopic signatures provide useful fingerprinting handles with which to classify Cys(thiolate)-ligated hemoproteins. Two distinct classes of heme thiolate proteins emerge, which cluster according to their biological functions. We call these two classes the type-1 and type-2 heme thiolates. Herein, we demonstrate how type-1 and type-2 heme thiolate proteins differ in coordination numbers, spin states, and propensities to undergo ligand switching when reduced. Type- 1 heme thiolates have a vacant or labile axial coordination site trans to the thiolate ligand, and they retain the thiolate ligand when reduced. The high-spin, five-coordinate Fe(II) heme is functionally essential in small-molecule activation. Type-2 heme thiolates are always low-spin with two axial ligands; however, the thiolate ligand is replaced upon heme reduction. A second ligand replacement at the low-spin, six-coordinate Fe(II) heme enables function in small-molecule sensing. The differences in spin and coordination states between type-1 and type-2 heme thiolates give rise to their distinctive spectroscopic properties, as described in sections 4−8, and correlate with their functions in small molecule activation or sensing, as described in sections 9−11. Because the spectroscopic signatures arise from variations in electronic structure at the heme cofactor, it follows that each type of Cys(thiolate)-ligated hemoprotein displays distinct reactivity. This difference in reactivity is then manifested in the two classes of thiolate-ligated hemoproteins: type-1 that retains its Cys- (thiolate) ligand throughout changes in the heme iron oxidation state and type-2 with a propensity to lose its Cys(thiolate) upon reduction of the heme iron (Figure 2). It is the differences in coordination behavior that give rise to the distinct spectroscopic signatures that allow their classification. Importantly, these two types of reactivity allow heme-thiolate proteins to play disparate roles in nature: as catalysts (type-1) or small-molecule sensors/ transporters (type-2). The purpose of this Review is to identify, separate, and classify these two types of heme-thiolate proteins on the basis of their distinct spectroscopic and functional properties. Because the classification system is based on spectroscopic attributes, and because the function is dependent on the distinctive reactivity arising from the heme electronic structure, spectroscopic and reactivity differences are presented before functional differences. This Review draws on nearly six decades of spectroscopic and functional data from well-studied and well-reviewed hemethiolate monooxygenases, but its new classification system is inspired by the emergence of new families of thiolate-ligated hemoproteins that function in numerous signaling pathways. Amazingly, these divergent and independent functions are possible despite the fact that each protein uses the exact same cofactor (heme b) and a common thiolate ligand in the firstcoordination sphere of the heme iron. Such diversity is made possible by utilizing the protein scaffold to modulate how the heme cofactor changes spin and coordination states upon substrate binding, gas binding, and/or changes in the redox state of the heme iron (Figure 2). This Review attempts to explain how nature utilizes the versatility of heme-thiolate proteins to play multiple indispensible roles in life processes

Network topology and functional connectivity disturbances precede the onset of Huntington’s disease

Cognitive, motor and psychiatric changes in prodromal Huntington’s disease have nurtured the emergent need for early interventions. Preventive clinical trials for Huntington’s disease, however, are limited by a shortage of suitable measures that could serve as surrogate outcomes. Measures of intrinsic functional connectivity from resting-state functional magnetic resonance imaging are of keen interest. Yet recent studies suggest circumscribed abnormalities in resting-state functional magnetic resonance imaging connectivity in prodromal Huntington’s disease, despite the spectrum of behavioural changes preceding a manifest diagnosis. The present study used two complementary analytical approaches to examine whole-brain resting-state functional magnetic resonance imaging connectivity in prodromal Huntington’s disease. Network topology was studied using graph theory and simple functional connectivity amongst brain regions was explored using the network-based statistic. Participants consisted of gene-negative controls (n = 16) and prodromal Huntington’s disease individuals (n = 48) with various stages of disease progression to examine the influence of disease burden on intrinsic connectivity. Graph theory analyses showed that global network interconnectivity approximated a random network topology as proximity to diagnosis neared and this was associated with decreased connectivity amongst highly-connected rich-club network hubs, which integrate processing from diverse brain regions. However, functional segregation within the global network (average clustering) was preserved. Functional segregation was also largely maintained at the local level, except for the notable decrease in the diversity of anterior insula intermodular-interconnections (participation coefficient), irrespective of disease burden. In contrast, network-based statistic analyses revealed patterns of weakened frontostriatal connections and strengthened frontal-posterior connections that evolved as disease burden increased. These disturbances were often related to long-range connections involving peripheral nodes and interhemispheric connections. A strong association was found between weaker connectivity and decreased rich-club organization, indicating that whole-brain simple connectivity partially expressed disturbances in the communication of highly-connected hubs. However, network topology and network-based statistic connectivity metrics did not correlate with key markers of executive dysfunction (Stroop Test, Trail Making Test) in prodromal Huntington’s disease, which instead were related to whole-brain connectivity disturbances in nodes (right inferior parietal, right thalamus, left anterior cingulate) that exhibited multiple aberrant connections and that mediate executive control. Altogether, our results show for the first time a largely disease burden-dependent functional reorganization of whole-brain networks in prodromal Huntington’s disease. Both analytic approaches provided a unique window into brain reorganization that was not related to brain atrophy or motor symptoms. Longitudinal studies currently in progress will chart the course of functional changes to determine the most sensitive markers of disease progression

Functional Connectivity of Primary Motor Cortex Is Dependent on Genetic Burden in Prodromal Huntington Disease

Subtle changes in motor function have been observed in individuals with prodromal Huntington disease (prHD), but the underlying neural mechanisms are not well understood nor is the cumulative effect of the disease (disease burden) on functional connectivity. The present study examined the resting-state functional magnetic resonance imaging (rs-fMRI) connectivity of the primary motor cortex (M1) in 16 gene-negative (NEG) controls and 48 gene-positive prHD participants with various levels of disease burden. The results showed that the strength of the left M1 connectivity with the ipsilateral M1 and somatosensory areas decreased as disease burden increased and correlated with motor symptoms. Weakened M1 connectivity within the motor areas was also associated with abnormalities in long-range connections that evolved with disease burden. In this study, M1 connectivity was decreased with visual centers (bilateral cuneus), but increased with a hub of the default mode network (DMN; posterior cingulate cortex). Changes in connectivity measures were associated with worse performance on measures of cognitive–motor functioning. Short- and long-range functional connectivity disturbances were also associated with volume loss in the basal ganglia, suggesting that weakened M1 connectivity is partly a manifestation of striatal atrophy. Altogether, the results indicate that the prodromal phase of HD is associated with abnormal interhemispheric interactions among motor areas and disturbances in the connectivity of M1 with visual centers and the DMN. These changes may, respectively, contribute to increased motor symptoms, visuomotor integration problems, and deficits in the executive control of movement as individuals approach a manifest diagnosis

Functional connectivity of primary motor cortex is dependent on genetic burden in prodromal Huntington disease.

Subtle changes in motor function have been observed in individuals with prodromal Huntington disease (prHD), but the underlying neural mechanisms are not well understood nor is the cumulative effect of the disease (disease burden) on functional connectivity. The present study examined the resting-state functional magnetic resonance imaging (rs-fMRI) connectivity of the primary motor cortex (M1) in 16 gene-negative (NEG) controls and 48 gene-positive prHD participants with various levels of disease burden. The results showed that the strength of the left M1 connectivity with the ipsilateral M1 and somatosensory areas decreased as disease burden increased and correlated with motor symptoms. Weakened M1 connectivity within the motor areas was also associated with abnormalities in long-range connections that evolved with disease burden. In this study, M1 connectivity was decreased with visual centers (bilateral cuneus), but increased with a hub of the default mode network (DMN; posterior cingulate cortex). Changes in connectivity measures were associated with worse performance on measures of cognitive-motor functioning. Short- and long-range functional connectivity disturbances were also associated with volume loss in the basal ganglia, suggesting that weakened M1 connectivity is partly a manifestation of striatal atrophy. Altogether, the results indicate that the prodromal phase of HD is associated with abnormal interhemispheric interactions among motor areas and disturbances in the connectivity of M1 with visual centers and the DMN. These changes may, respectively, contribute to increased motor symptoms, visuomotor integration problems, and deficits in the executive control of movement as individuals approach a manifest diagnosis

Cross-sectional and longitudinal multimodal structural imaging in prodromal Huntington's disease.

ObjectivesDiffusivity in white-matter tracts is abnormal throughout the brain in cross-sectional studies of prodromal Huntington's disease. To date, longitudinal changes have not been observed. The present study investigated cross-sectional and longitudinal changes in white-matter diffusivity in relationship to the phase of prodromal Huntington's progression, and compared them with changes in brain volumes and clinical variables that track disease progression.MethodsDiffusion MRI profiles were studied for 2 years in 37 gene-negative controls and 64 prodromal Huntington's disease participants in varied phases of disease progression. To estimate the relative importance of diffusivity metrics in the prodromal phase, group effects were rank ordered relative to those obtained from analyses of brain volumes, motor, cognitive, and sensory variables.ResultsFirst, at baseline diffusivity was abnormal throughout all tracts, especially as individuals approached a manifest Huntington's disease diagnosis. Baseline diffusivity metrics in 6 tracts and basal ganglia volumes best distinguished among the groups. Second, group differences in longitudinal change in diffusivity were localized to the superior fronto-occipital fasciculus, most prominently in individuals closer to a diagnosis. Group differences were also observed in longitudinal changes of most brain volumes, but not clinical variables. Last, increases in motor symptoms across time were associated with greater changes in the superior fronto-occipital fasciculus diffusivity and corpus callosum, cerebrospinal fluid, and lateral ventricle volumes.ConclusionsThese novel findings provide new insights into changes within 2 years in different facets of brain structure and their clinical relevance to changes in symptomatology that is decisive for a manifest Huntington's diagnosis. © 2016 International Parkinson and Movement Disorder Society

Disruption of response inhibition circuits in prodromal Huntington disease

Cognitive changes in the prodromal phase of Huntington disease (prHD) are found in multiple domains, yet their neural bases are not well understood. One component process that supports cognition is inhibitory control. In the present fMRI study, we examined brain circuits involved in response inhibition in 65 prHD participants and 36 gene-negative (NEG) controls using the stop signal task (SST). PrHD participants were subdivided into three groups (LOW, MEDIUM, HIGH) based on their CAG-Age Product (CAP) score, an index of genetic exposure and a proxy for expected time to diagnosis. Poorer response inhibition (stop signal duration) correlated with CAP scores. When response inhibition was successful, activation of the classic frontal inhibitory-network was normal in prHD, yet stepwise reductions in activation with proximity to diagnosis were found in the posterior ventral attention network (inferior parietal and temporal cortices). Failures in response inhibition in prHD were related to changes in inhibition centers (supplementary motor area (SMA)/anterior cingulate and inferior frontal cortex/insula) and ventral attention networks, where activation decreased with proximity to diagnosis. The LOW group showed evidence of early compensatory activation (hyperactivation) of right-hemisphere inhibition and attention reorienting centers, despite an absence of cortical atrophy or deficits on tests of executive functioning. Moreover, greater activation for failed than successful inhibitions in an ipsilateral motor-control network was found in the control group, whereas such differences were markedly attenuated in all prHD groups. The results were not related to changes in cortical volume and thickness, which did not differ among the groups. However, greater hypoactivation of classic right-hemisphere inhibition centers [inferior frontal gyrus (IFG)/insula, SMA/anterior cingulate cortex (ACC)] during inhibition failures correlated with greater globus pallidus atrophy. These results are the first to demonstrate that response inhibition in prHD is associated with altered functioning in brain networks that govern inhibition, attention, and motor control

Cross‐sectional and longitudinal multimodal structural imaging in prodromal Huntington's disease

ObjectivesDiffusivity in white-matter tracts is abnormal throughout the brain in cross-sectional studies of prodromal Huntington's disease. To date, longitudinal changes have not been observed. The present study investigated cross-sectional and longitudinal changes in white-matter diffusivity in relationship to the phase of prodromal Huntington's progression, and compared them with changes in brain volumes and clinical variables that track disease progression.MethodsDiffusion MRI profiles were studied for 2 years in 37 gene-negative controls and 64 prodromal Huntington's disease participants in varied phases of disease progression. To estimate the relative importance of diffusivity metrics in the prodromal phase, group effects were rank ordered relative to those obtained from analyses of brain volumes, motor, cognitive, and sensory variables.ResultsFirst, at baseline diffusivity was abnormal throughout all tracts, especially as individuals approached a manifest Huntington's disease diagnosis. Baseline diffusivity metrics in 6 tracts and basal ganglia volumes best distinguished among the groups. Second, group differences in longitudinal change in diffusivity were localized to the superior fronto-occipital fasciculus, most prominently in individuals closer to a diagnosis. Group differences were also observed in longitudinal changes of most brain volumes, but not clinical variables. Last, increases in motor symptoms across time were associated with greater changes in the superior fronto-occipital fasciculus diffusivity and corpus callosum, cerebrospinal fluid, and lateral ventricle volumes.ConclusionsThese novel findings provide new insights into changes within 2 years in different facets of brain structure and their clinical relevance to changes in symptomatology that is decisive for a manifest Huntington's diagnosis. © 2016 International Parkinson and Movement Disorder Society