Genetically Determined Measures of Striatal D2 Signaling Predict Prefrontal Activity during Working Memory Performance

Background

Variation of the gene coding for D2 receptors (DRD2) has been associated with risk for schizophrenia and with working memory deficits. A functional intronic SNP (rs1076560) predicts relative expression of the two D2 receptors isoforms, D2S (mainly pre-synaptic) and D2L (mainly post-synaptic). However, the effect of functional genetic variation of DRD2 on striatal dopamine D2 signaling and on its correlation with prefrontal activity during working memory in humans is not known.

Methods

Thirty-seven healthy subjects were genotyped for rs1076560 (G>T) and underwent SPECT with [¹²³I]IBZM (which binds primarily to post-synaptic D2 receptors) and with [¹²³I]FP-CIT (which binds to pre-synaptic dopamine transporters, whose activity and density is also regulated by pre-synaptic D2 receptors), as well as BOLD fMRI during N-Back working memory.

Results

Subjects carrying the T allele (previously associated with reduced D2S expression) had striatal reductions of [¹²³I]IBZM and of [¹²³I]FP-CIT binding. DRD2 genotype also differentially predicted the correlation between striatal dopamine D2 signaling (as identified with factor analysis of the two radiotracers) and activity of the prefrontal cortex during working memory as measured with BOLD fMRI, which was positive in GG subjects and negative in GT.

Conclusions

Our results demonstrate that this functional SNP within DRD2 predicts striatal binding of the two radiotracers to dopamine transporters and D2 receptors as well as the correlation between striatal D2 signaling with prefrontal cortex activity during performance of a working memory task. These data are consistent with the possibility that the balance of excitatory/inhibitory modulation of striatal neurons may also affect striatal outputs in relationship with prefrontal activity during working memory performance within the cortico-striatal-thalamic-cortical pathway.     

CHRNA7 Polymorphisms and Response to Cholinesterase Inhibitors in Alzheimer's Disease

Background

CHRNA7 encodes the α7 nicotinic acetylcholine receptor subunit, which is important to Alzheimer''s disease (AD) pathogenesis and cholinergic neurotransmission. Previously, CHRNA7 polymorphisms have not been related to cholinesterase inhibitors (ChEI) response.

Methods

Mild to moderate AD patients received ChEIs were recruited from the neurology clinics of three teaching hospitals from 2007 to 2010 (n = 204). Nine haplotype-tagging single nucleotide polymorphisms of CHRNA7 were genotyped. Cognitive responders were those showing improvement in the Mini-Mental State Examination score ≧2 between baseline and 6 months after ChEI treatment.

Results

AD women carrying rs8024987 variants [GG+GC vs. CC: adjusted odds ratio (AOR) = 3.62, 95% confidence interval (CI) = 1.47–8.89] and GG haplotype in block1 (AOR = 3.34, 95% CI = 1.38–8.06) had significantly better response to ChEIs (false discovery rate <0.05). These variant carriers using galantamine were 11 times more likely to be responders than female non-carriers using donepezil or rivastigmine.

Conclusion

For the first time, this study found a significant association between CHRNA7 polymorphisms and better ChEI response. If confirmed by further studies, CHRNA7 polymorphisms may aid in predicting ChEI response and refining treatment choice.     

Factors Affecting the Biology and Pathogenicity of Heterodera schachtii on Sugarbeet

A direct relationship exists between soil temperature and Heterodera schachtii development. The average developmental period of two nematode populations from Lewiston, Utah, and Rupert, Idaho, from J2 to J3, J4, adult, and the next generation J2 at soil temperatures of 18-28 C were 100, 140,225, and 399 degree-days (base 8 C), respectively. There was a positive relationship (P < 0.05) between nematode Pi, nematode generations, and sugarbeet yields. The greatest sugarbeet growth inhibition (87%) occurred when sugarbeets were exposed to a Pi of 12 eggs/cm³ soil for five generations (1,995 degree-days), compared with a 47% inhibition when plants were exposed to the same Pi for two generations. There was a negative correlation (P < 0.05) between the Pi, Pf, and sugarbeet yield for each population threshold. The smaller the Pi, the greater the sugarbeet yields and the greater the Pf. Root yields were 80 and 29 t /ha and Pf were 8.4 and 3.6 eggs/cm³ soil when sugarbeet seeds were planted at Pi of 0.4 and 7.9 eggs/cm³. respectively, at a soil temperature of 8 C. The number of years rotation with a nonhost crop required to reduce the nematode population density below a damage threshold level of 2 eggs/cm³ depends on the Pi. A Pi of 33.8 eggs/cm³ soil required a 5-year crop rotation, whereas a Pi of 8.4 eggs/cm³ soil required a 2-year crop rotation.     

Time Course of Functional Connectivity in Primate Dorsolateral Prefrontal and Posterior Parietal Cortex during Working Memory

The dorsolateral prefrontal and posterior parietal cortex play critical roles in mediating attention, working memory, and executive function. Despite proposed dynamic modulation of connectivity strength within each area according to task demands, scant empirical data exist about the time course of the strength of effective connectivity, particularly in tasks requiring information to be sustained in working memory. We investigated this question by performing time-resolved cross-correlation analysis for pairs of neurons recorded simultaneously at distances of 0.2–1.5 mm apart of each other while monkeys were engaged in working memory tasks. The strength of effective connectivity determined in this manner was higher throughout the trial in the posterior parietal cortex than the dorsolateral prefrontal cortex. Significantly higher levels of parietal effective connectivity were observed specifically during the delay period of the task. These differences could not be accounted for by differences in firing rate, or electrode distance in the samples recorded in the posterior parietal and prefrontal cortex. Differences were present when we restricted our analysis to only neurons with significant delay period activity and overlapping receptive fields. Our results indicate that dynamic changes in connectivity strength are present but area-specific intrinsic organization is the predominant factor that determines the strength of connections between neurons in each of the two areas.     

Biology of Meloidogyne platani Hirschmann Parasitic on Sycamore, Platanus occidentalis

The development of Meloidogyne platani on sycamore was followed for 40 days (22-28 C). Juveniles penetrated the feeder roots behind the root cap and invaded the vascular cylinder within 3 days after inoculation. All subsequent development of the nematodes and host effects occurred only within the stele. The second juvenile molt and sex differentiation occurred by the 17th day. Young females were observed by the 26th day. Eggs were observed inside the roots by the 35th day and were exposed to the surface of galls by the 40th day. In pathogenicity studies, a significant negative correlation was shown to exist between fresh shoot and root weights and inoculum density. Besides sycamore, white ash was the only hardwood species tested to become infected. Of the herbacious plants tested, tobacco was heavily galled, tomato and watermelon moderately galled, and pepper only slightly galled. Egg production was moderate on tobacco, slight on tomato and watermelon, and absent on pepper.     

BAIAP2 Is Related to Emotional Modulation of Human Memory Strength

Memory performance is the result of many distinct mental processes, such as memory encoding, forgetting, and modulation of memory strength by emotional arousal. These processes, which are subserved by partly distinct molecular profiles, are not always amenable to direct observation. Therefore, computational models can be used to make inferences about specific mental processes and to study their genetic underpinnings. Here we combined a computational model-based analysis of memory-related processes with high density genetic information derived from a genome-wide study in healthy young adults. After identifying the best-fitting model for a verbal memory task and estimating the best-fitting individual cognitive parameters, we found a common variant in the gene encoding the brain-specific angiogenesis inhibitor 1-associated protein 2 (BAIAP2) that was related to the model parameter reflecting modulation of verbal memory strength by negative valence. We also observed an association between the same genetic variant and a similar emotional modulation phenotype in a different population performing a picture memory task. Furthermore, using functional neuroimaging we found robust genotype-dependent differences in activity of the parahippocampal cortex that were specifically related to successful memory encoding of negative versus neutral information. Finally, we analyzed cortical gene expression data of 193 deceased subjects and detected significant BAIAP2 genotype-dependent differences in BAIAP2 mRNA levels. Our findings suggest that model-based dissociation of specific cognitive parameters can improve the understanding of genetic underpinnings of human learning and memory.     

Experimental diabetes exacerbates autophagic flux impairment during myocardial I/R injury through calpain‐mediated cleavage of Atg5/LAMP2

To explore the role of autophagic flux in the increased susceptibility of the experimental diabetic heart to ischaemia‐reperfusion (I/R) injury, we established STZ‐induced diabetic mice and performed I/R. In vitro, neonatal mouse cardiomyocytes were subjected to high glucose and hypoxia/reoxygenation challenge to mimic diabetic I/R injury. We found that experimental diabetes aggravated I/R‐induced injury than compared with nondiabetic mice. Autophagic flux was impaired in I/R hearts, and the impairment was exacerbated in diabetic mice subjected to I/R with defective autophagosome formation and clearance. Calpains, calcium‐dependent thiol proteases, were upregulated and highly activated after I/R of diabetes, while calpain inhibition attenuated cardiac function and cell death and partially restored autophagic flux. The expression levels of Atg5 and LAMP2, two crucial autophagy‐related proteins, were significantly degraded in diabetic I/R hearts, alterations that were associated with calpain activation and could be reversed by calpain inhibition. Co‐overexpression of Atg5 and LAMP2 reduced myocardial injury and normalized autophagic flux. In conclusion, experimental diabetes exacerbates autophagic flux impairment of cardiomyocytes under I/R stress, resulting in worse I/R‐induced injury. Calpain activation and cleavage of Atg5 and LAMP2 at least partially account for the deterioration of autophagic flux impairment.     

Multiband Semimetallic Electronic Structure of Superconducting Ta2PdSe5

We report the electronic structure and related properties of the superconductor Ta₂PdSe₅ as determined from density functional calculations. The Fermi surface has two disconnected sheets, both derived from bands of primarily chalcogenide p states. These are a corrugated hole cylinder and a heavier complex shaped electron sheet. The sheets contain 0.048 holes and a compensating number of electrons per formula unit, making the material a semimetallic superconductor. The results support the presence of two band superconductivity, although a discrepancy in the specific heat is noted. This discrepancy is discussed as a possible consequence of Pd deficiency in samples.     

Novel Alternative Splice Variants of Mouse Cdk5rap2

Autosomal recessive primary microcephaly (MCPH) is a rare neurodevelopmental disorder characterized by a pronounced reduction of brain volume and intellectual disability. A current model for the microcephaly phenotype invokes a stem cell proliferation and differentiation defect, which has moved the disease into the spotlight of stem cell biology and neurodevelopmental science. Homozygous mutations of the Cyclin-dependent kinase-5 regulatory subunit-associated protein 2 gene CDK5RAP2 are one genetic cause of MCPH. To further characterize the pathomechanism underlying MCPH, we generated a conditional Cdk5rap2 LoxP/hCMV Cre mutant mouse. Further analysis, initiated on account of a lack of a microcephaly phenotype in these mutant mice, revealed the presence of previously unknown splice variants of the Cdk5rap2 gene that are at least in part accountable for the lack of microcephaly in the mice.     

In vivo and in vitro Studies of Adaptor-clathrin Interaction

A major endocytic pathway initiates with the formation of clathrin-coated vesicles (CCVs) that transport cargo from the cell surface to endosomes^1-6. CCVs are distinguished by a polyhedral lattice of clathrin that coats the vesicle membrane and serves as a mechanical scaffold. Clathrin coats are assembled during vesicle formation from individual clathrin triskelia , the soluble form of clathrin composed of three heavy and three light chain subunits^7,8. Because the triskelion does not have the ability to bind to the membrane directly, clathrin-binding adaptors are critical to link the forming clathrin lattice to the membrane through association with lipids and/or membrane proteins⁹. Adaptors also package transmembrane protein cargo, such as receptors, and can interact with each other and with other components of the CCV formation machinery⁹.Over twenty clathrin adaptors have been described, several are involved in clathrin mediated endocytosis and others localize to the trans Golgi network or endosomes⁹. With the exception of HIP1R (yeast Sla2p), all known clathrin adaptors bind to the N-terminal -propeller domain of the clathrin heavy chain⁹. Clathrin adaptors are modular proteins consisting of folded domains connected by unstructured flexible linkers. Within these linker regions, short binding motifs mediate interactions with the clathrin N-terminal domain or other components of the vesicle formation machinery⁹. Two distinct clathrin-binding motifs have been defined: the clathrin-box and the W-box⁹. The consensus clathrin-box sequence was originally defined as L[L/I][D/E/N][L/F][D/E]¹⁰ but variants have been subsequently discovered¹¹. The W-box conforms to the sequence PWxxW (where x is any residue).Sla1p (Synthetic Lethal with Actin binding protein-1) was originally identified as an actin associated protein and is necessary for normal actin cytoskeleton structure and dynamics at endocytic sites in yeast cells¹². Sla1p also binds the NPFxD endocytic sorting signal and is critical for endocytosis of cargo bearing the NPFxD signal^13,14. More recently, Sla1p was demonstrated to bind clathrin through a motif similar to the clathrin box, LLDLQ, termed a variant clathrin-box (vCB), and to function as an endocytic clathrin adaptor¹⁵. In addition, Sla1p has become a widely used marker for the endocytic coat in live cell fluorescence microscopy studies¹⁶. Here we use Sla1p as a model to describe approaches for adaptor-clathrin interaction studies. We focus on live cell fluorescence microscopy, GST-pull down, and co-immunoprecipitation methods.Download video file.^{(108M, mov)}     

Identification and Characterization of Prokaryotic Dipeptidyl-peptidase 5 from Porphyromonas gingivalis

Porphyromonas gingivalis, a Gram-negative asaccharolytic anaerobe, is a major causative organism of chronic periodontitis. Because the bacterium utilizes amino acids as energy and carbon sources and incorporates them mainly as dipeptides, a wide variety of dipeptide production processes mediated by dipeptidyl-peptidases (DPPs) should be beneficial for the organism. In the present study, we identified the fourth P. gingivalis enzyme, DPP5. In a dpp4-7-11-disrupted P. gingivalis ATCC 33277, a DPP7-like activity still remained. PGN_0756 possessed an activity indistinguishable from that of the mutant, and was identified as a bacterial orthologue of fungal DPP5, because of its substrate specificity and 28.5% amino acid sequence identity with an Aspergillus fumigatus entity. P. gingivalis DPP5 was composed of 684 amino acids with a molecular mass of 77,453, and existed as a dimer while migrating at 66 kDa on SDS-PAGE. It preferred Ala and hydrophobic residues, had no activity toward Pro at the P1 position, and no preference for hydrophobic P2 residues, showed an optimal pH of 6.7 in the presence of NaCl, demonstrated K_m and k_cat/K_m values for Lys-Ala-MCA of 688 μm and 11.02 μm⁻¹ s⁻¹, respectively, and was localized in the periplasm. DPP5 elaborately complemented DPP7 in liberation of dipeptides with hydrophobic P1 residues. Examinations of DPP- and gingipain gene-disrupted mutants indicated that DPP4, DPP5, DPP7, and DPP11 together with Arg- and Lys-gingipains cooperatively liberate most dipeptides from nutrient oligopeptides. This is the first study to report that DPP5 is expressed not only in eukaryotes, but also widely distributed in bacteria and archaea.     

In-situ Effects of Eutrophication and Overfishing on Physiology and Bacterial Diversity of the Red Sea Coral Acropora hemprichii

Coral reefs of the Central Red Sea display a high degree of endemism, and are increasingly threatened by anthropogenic effects due to intense local coastal development measures. Overfishing and eutrophication are among the most significant local pressures on these reefs, but there is no information available about their potential effects on the associated microbial community. Therefore, we compared holobiont physiology and 16S-based bacterial communities of tissue and mucus of the hard coral Acropora hemprichii after 1 and 16 weeks of in-situ inorganic nutrient enrichment (via fertilizer diffusion) and/or herbivore exclusion (via caging) in an offshore reef of the Central Red Sea. Simulated eutrophication and/or overfishing treatments did not affect coral physiology with respect to coral respiration rates, chlorophyll a content, zooxanthellae abundance, or δ ¹⁵N isotopic signatures. The bacterial community of A. hemprichii was rich and uneven, and diversity increased over time in all treatments. While distinct bacterial species were identified as a consequence of eutrophication, overfishing, or both, two bacterial species that could be classified to the genus Endozoicomonas were consistently abundant and constituted two thirds of bacteria in the coral. Several nitrogen-fixing and denitrifying bacteria were found in the coral specimens that were exposed to experimentally increased nutrients. However, no particular bacterial species was consistently associated with the coral under a given treatment and the single effects of manipulated eutrophication and overfishing could not predict the combined effect. Our data underlines the importance of conducting field studies in a holobiont framework, taking both, physiological and molecular measures into account.     

Drosophila Smad2 Opposes Mad Signaling during Wing Vein Development

In the vertebrates, the BMP/Smad1 and TGF-β/Smad2 signaling pathways execute antagonistic functions in different contexts of development. The differentiation of specific structures results from the balance between these two pathways. For example, the gastrula organizer/node of the vertebrates requires a region of low Smad1 and high Smad2 signaling. In Drosophila, Mad regulates tissue determination and growth in the wing, but the function of dSmad2 in wing patterning is largely unknown. In this study, we used an RNAi loss-of-function approach to investigate dSmad2 signaling during wing development. RNAi-mediated knockdown of dSmad2 caused formation of extra vein tissue, with phenotypes similar to those seen in Dpp/Mad gain-of-function. Clonal analyses revealed that the normal function of dSmad2 is to inhibit the response of wing intervein cells to the extracellular Dpp morphogen gradient that specifies vein formation, as measured by expression of the activated phospho-Mad protein. The effect of dSmad2 depletion in promoting vein differentiation was dependent on Medea, the co-factor shared by Mad and dSmad2. Furthermore, double RNAi experiments showed that Mad is epistatic to dSmad2. In other words, depletion of Smad2 had no effect in Mad-deficient wings. Our results demonstrate a novel role for dSmad2 in opposing Mad-mediated vein formation in the wing. We propose that the main function of dActivin/dSmad2 in Drosophila wing development is to antagonize Dpp/Mad signaling. Possible molecular mechanisms for the opposition between dSmad2 and Mad signaling are discussed.     

In Vitro Interaction of the Housekeeping SecA1 with the Accessory SecA2 Protein of Mycobacterium tuberculosis

The majority of proteins that are secreted across the bacterial cytoplasmic membrane leave the cell via the Sec pathway, which in its minimal form consists of the dimeric ATP-driven motor protein SecA that associates with the protein-conducting membrane pore SecYEG. Some Gram-positive bacteria contain two homologues of SecA, termed SecA1 and SecA2. SecA1 is the essential housekeeping protein, whereas SecA2 is not essential but is involved in the translocation of a subset of proteins, including various virulence factors. Some SecA2 containing bacteria also harbor a homologous SecY2 protein that may form a separate translocase. Interestingly, mycobacteria contain only one SecY protein and thus both SecA1 and SecA2 are required to interact with SecYEG, either individually or together as a heterodimer. In order to address whether SecA1 and SecA2 cooperate during secretion of SecA2 dependent proteins, we examined the oligomeric state of SecA1 and SecA2 of Mycobacterium tuberculosis and their interactions with SecA2 and the cognate SecA1, respectively. We conclude that both SecA1 and SecA2 individually form homodimers in solution but when both proteins are present simultaneously, they form dissociable heterodimers.