Target Specific Changes Research Articles

Optimizing transcranial focused ultrasound for modulating cognitive, affective, and motor and symptoms in neurodegenerative disease

Abstract Pulsed transcranial focused ultrasound (tFUS) offers many advantages of other non-invasive neuromodulation technologies. However, to date, there are few studies in humans demonstrating changes in MRI and behavioral outcomes. Further, the optimization of these metrics for specific brain targets and the neuropsychological constructs they mediate is limited. Our group has completed a double-blind, cross over study in healthy humans demonstrating brain target-specific changes in perfusion, functional connectivity and behavior. Using this study as a foundation, our group further conducted a parameter optimization study to determine appropriate tFUS parameters for engagement of medial temporal lobe memory structures with associated memory effects. Thereafter, we began a study using these parameters in participants with diagnoses of amnestic mild cognitive impairment. Further, our group is conducting a double-blind, sham-controlled study on tFUS in participants with diagnoses of Parkinson’s disease, assessing MRI, behavioral, and motor effects. In this symposium talk, we will review these findings and the opportunities and limitations they suggest for the field of tFUS and the future of tFUS as a potential clinical neuromodulation tool. Research Category and Technology and Methods Translational Research: 6. Pulsed Ultrasound (pUS) Keywords: Ultrasound, Memory, Anxiety, MRI

Adrenomedullary responses to non-invasive cortical stimulation in humans

Abstract Introduction: The discovery of cortical adrenomedullary projections in cebus monkeys suggests that similar pathways exist in humans. Notably, the major corticoadrenomedullary projection regions have human equivalents that can be stimulated non-invasively with repetitive transcranial magnetic stimulation (rTMS). If rTMS can be used to selectively modulate adrenomedullary activity, there may be significant therapeutic benefits for stress-, affective-, or neuroimmune-related disorders. Purpose: To assess resting and stress-induced adrenomedullary neuroendocrine responses to continuous (cTBS) and intermittent (iTBS) theta burst TMS in humans. Methods: 39 healthy young adults (14W) were randomly assigned to three counterbalanced sessions of iTBS (n=19) or cTBS (n=20). Stimulation was applied to motor cortical areas that contain the majority of adrenomedullary projections in non-human primates (primary motor cortex trunk region (M1TRUNK) and presupplementary motor area (preSMA)). The M1 leg region (M1LEG) was used as a spatial control. Stimulation locations were determined based on TMS-evoked electromyographic responses in the rectus abdominus (M1TRUNK) and vastus lateralis (M1LEG) or using a combination of fMRI activity and metanalytical structural coordinates (preSMA). All TMS procedures were performed with a 96mm curved double coil and stereotactic navigation. TBS was applied as 600 biphasic TMS pulses at 80% AMT. Corticoadrenomedullary activity and corticospinal excitability (CSE) were assayed before and after TBS, immediately after maximal exercise, and 60min and 90min after TBS based on plasma epinephrine concentrations and median MEP AUC, respectively. Initial analysis of adrenomedullary responses to rTMS were made with a 2 (protocol) x 3 (visit) x 5 (time-point) mixed methods ANOVA. Results and Conclusions: As expected, exercise resulted in a large epinephrine increase (p<0.0001, 8-fold) overall. Ongoing analysis indicates that target-specific changes in adrenomedullary activity are likely, and similar to CSE, may often be paradoxical. Our preliminary results also suggest that adrenomedullary responses to subthreshold rTMS are principally stress inducible. Research Category and Technology and Methods Translational Research: 10. Transcranial Magnetic Stimulation (TMS) Keywords: Adrenal Medulla, Cortex, Sympathetic Nervous System, Non-Invasive Brain Stimulation

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COVER ILLUSTRATION Neural changes accompanying dosage intensive treatments targeting voice or articulation in patients with Parkinson's dysphonia. Voice treatment (yellow) targeted larynx and respiration while articulation treatment (blue) targeted mouth and lips. Behaviorally, voice treatment but not articulation treatment resulted in improvement in voice quality. However, treatment target specific changes in the speech motor network were observed during a reading task immediately following each treatment. Brain areas altered by voice treatment are shown in yellow and brain areas showing changes following articulation treatment are shown in blue. Left hemisphere activations are clear and right hemisphere activations are checkered. Voice treatment increased activity in left premotor and bilateral auditory cortices while articulation treatment increased activity in bilateral premotor and left insular cortices. The intensive dosage contributed to persistent changes at 7-month follow up. These results indicate that dysphonia and the speech motor network can be differentially modified by treatment targets, while intensive dosage contribute to long lasting effects of speech treatments.

An interdisciplinary examination of attentional focus strategies used during running gait retraining.

The aim was to investigate the biomechanical, physiological, and perceptual responses to different motor learning strategies derived to elicit a flatter foot contact. Twenty-eight rearfoot-striking recreational runners (age 24.9±2.8years; body mass 78.8±13.6kg; height 1.79±0.09m) were matched by age, mass, and height and assigned to one verbal cue group: internal focus of attention (IF), external focus of attention (EF), and a clinically derived condition (CLIN) incorporating an IF followed by an EF statement. Participants completed two treadmill runs at 10kmh-1 for 6minutes each: normal running (control) followed by the experimental condition (IF, EF, or CLIN). Lower limb kinematics, oxygen consumption ( ), and central and peripheral ratings of perceived exertion (RPE) were recorded for each run. Compared to the control condition, foot angle was reduced in the IF (difference=5.86°, d=2.58) and CLIN (difference=3.00°, d=1.31) conditions, but unchanged in the EF condition (difference=0.33°, d=0.14), while greater knee flexion at initial contact in the EF and CLIN conditions was observed (difference=-5.19°, d=1.97; difference=-3.66°, d=1.39, respectively). A higher was observed in the CLIN condition (difference=-4.56mLkg-1 min-1 , d=2.29), but unchanged in the IF (difference=-1.87mLkg-1 min-1 , d=0.94) and EF conditions (difference=-0.37mLkg-1 min-1 , d=0.19). All experimental conditions increased central and peripheral RPE (difference=-1.08, d=0.54 and difference=-2.39, d=1.33, respectively). Providing gait retraining instructions using an internally directed focus of attention was the most effective way to target specific changes in running kinematics, with no detrimental effect on physiological responses. Yet, perceptual effort responses increased regardless of the type of cue provided.

Diaminothiazoles evade multidrug resistance in cancer cells and xenograft tumour models and develop transient specific resistance: understanding the basis of broad-spectrum versus specific resistance.

Acquired drug resistance poses a challenge in cancer therapy. Drug efflux is the most common mechanism of resistance displayed by hydrophobic drugs beyond a certain size. However, target specific changes and imbalance between the pro- and anti-apoptotic proteins are also found quite often in many tumours. A number of small antimitotic agents show high potential for multidrug resistant tumours, mainly because they are able to evade the efflux pumps. However, these compounds are also likely to suffer from resistance upon prolonged treatment. Thus, it is important to find out agents that are sensitive to resistant tumours and to know the resistance mechanisms against small molecules so that proper combinations can be planned. In this report, we have studied the efficiency of diaminothiazoles, a novel class of tubulin targeting potential anticancer compounds of small size, in multidrug resistant cancer. Studies in model cell lines raised against taxol and the lead diaminothiazole, DAT1 [4-amino-5-benzoyl-2-(4-methoxy phenyl amino) thiazole], and the xenograft tumours derived from them, show that diaminothiazoles are highly promising against multidrug resistant cancers. They were able to overcome the expression of efflux protein MDR1 and certain tubulin isotypes, could sensitize improper apoptotic machinery and ablated checkpoint proteins Bub1 and Mad2. Further, we have found that the resistance against microtubule binding compounds with higher size is broad-spectrum and emerges due to multiple factors including overexpression of transmembrane pumps. However, resistance against small molecules is transient, specific and is contributed by target specific changes and variations in apoptotic factors.

Fear Extinction Causes Target-Specific Remodeling of Perisomatic Inhibitory Synapses

A more complete understanding of how fear extinction alters neuronal activity and connectivity within fear circuits may aid in the development of strategies to treat human fear disorders. Using a c-fos-based transgenic mouse, we found that contextual fear extinction silenced basal amygdala (BA) excitatory neurons that had been previously activated during fear conditioning. We hypothesized that the silencing of BA fear neurons was caused by an action of extinction on BA inhibitory synapses. In support of this hypothesis, we found extinction-induced target-specific remodeling of BA perisomatic inhibitory synapses originating from parvalbumin and cholecystokinin-positive interneurons. Interestingly, the predicted changes in the balance of perisomatic inhibition matched the silent and active states of the target BA fear neurons. These observations suggest that target-specific changes in perisomatic inhibitory synapses represent a mechanism through which experience can sculpt the activation patterns within a neural circuit.

A single methyltransferase YefA (RlmCD) catalyses both m5U747 and m5U1939 modifications in Bacillus subtilis 23S rRNA

Methyltransferases that use S-adenosylmethionine (AdoMet) as a cofactor to catalyse 5-methyl uridine (m5U) formation in tRNAs and rRNAs are widespread in Bacteria and Eukaryota, and are also found in certain Archaea. These enzymes belong to the COG2265 cluster, and the Gram-negative bacterium Escherichia coli possesses three paralogues. These comprise the methyltransferases TrmA that targets U54 in tRNAs, RlmC that modifies U747 in 23S rRNA and RlmD that is specific for U1939 in 23S rRNA. The tRNAs and rRNAs of the Gram-positive bacterium Bacillus subtilis have the same three m5U modifications. However, as previously shown, the m5U54 modification in B. subtilis tRNAs is catalysed in a fundamentally different manner by the folate-dependent enzyme TrmFO, which is unrelated to the E. coli TrmA. Here, we show that methylation of U747 and U1939 in B. subtilis rRNA is catalysed by a single enzyme, YefA that is a COG2265 member. A recombinant version of YefA functions in an E. coli m5U-null mutant adding the same two rRNA methylations. The findings suggest that during evolution, COG2265 enzymes have undergone a series of changes in target specificity and that YefA is closer to an archetypical m5U methyltransferase. To reflect its dual specificity, YefA is renamed RlmCD.

Specificity shifts in the rRNA and tRNA nucleotide targets of archaeal and bacterial m5U methyltransferases.

Methyltransferase enzymes that use S-adenosylmethionine as a cofactor to catalyze 5-methyl uridine (m(5)U) formation in tRNAs and rRNAs are widespread in Bacteria and Eukaryota, but are restricted to the Thermococcales and Nanoarchaeota groups amongst the Archaea. The RNA m(5)U methyltransferases appear to have arisen in Bacteria and were then dispersed by horizontal transfer of an rlmD-type gene to the Archaea and Eukaryota. The bacterium Escherichia coli has three gene paralogs and these encode the methyltransferases TrmA that targets m(5)U54 in tRNAs, RlmC (formerly RumB) that modifies m(5)U747 in 23S rRNA, and RlmD (formerly RumA) the archetypical enzyme that is specific for m(5)U1939 in 23S rRNA. The thermococcale archaeon Pyrococcus abyssi possesses two m(5)U methyltransferase paralogs, PAB0719 and PAB0760, with sequences most closely related to the bacterial RlmD. Surprisingly, however, neither of the two P. abyssi enzymes displays RlmD-like activity in vitro. PAB0719 acts in a TrmA-like manner to catalyze m(5)U54 methylation in P. abyssi tRNAs, and here we show that PAB0760 possesses RlmC-like activity and specifically methylates the nucleotide equivalent to U747 in P. abyssi 23S rRNA. The findings indicate that PAB0719 and PAB0760 originated as RlmD-type m(5)U methyltransferases and underwent changes in target specificity after their acquisition by a Thermococcales ancestor from a bacterial source.

Construction of intragenic synthetic riboswitches for detection of a small molecule

Bacterial sensors, based on ligand-mediated genetic control systems, are promising for on-site chemical detection because sensing targets and generating signals do not require costly instrumentation. Here, we have constructed intragenic synthetic riboswitches without relying on high-throughput screening and demonstrated that the riboswitches can be harnessed to develop bacterial sensors displaying readily visible reporter signals in response to theophylline. In vivo imaging using the riboswitch showed target-specific changes in the expression of a green fluorescence protein reporter, which was visible even to the naked eye.

Examination of the structural and functional versatility of glmS ribozymes by using in vitro selection.

Self-cleaving ribozymes associated with the glmS genes of many Gram-positive bacteria are activated by binding to glucosamine-6-phosphate (GlcN6P). Representatives of the glmS ribozyme class function as metabolite-sensing riboswitches whose self-cleavage activities down-regulate the expression of GlmS enzymes that synthesizes GlcN6P. As with other riboswitches, natural glmS ribozyme isolates are highly specific for their target metabolite. Other small molecules closely related to GlcN6P, such as glucose-6-phosphate, cannot activate self-cleavage. We applied in vitro selection methods in an attempt to identify variants of a Bacillus cereus glmS ribozyme that expand the range of compounds that induce self-cleavage. In addition, we sought to increase the number of variant ribozymes of this class to further examine the proposed secondary structure model. Although numerous variant ribozymes were obtained that efficiently self-cleave, none exhibited changes in target specificity. These findings are consistent with the hypothesis that GlcN6P is used by the ribozyme as a coenzyme for RNA cleavage, rather than an allosteric effector.

Evolution of aptamers with a new specificity and new secondary structures from an ATP aptamer.

Small changes in target specificity can sometimes be achieved, without changing aptamer structure, through mutation of a few bases. Larger changes in target geometry or chemistry may require more radical changes in an aptamer. In the latter case, it is unknown whether structural and functional solutions can still be found in the region of sequence space close to the original aptamer. To investigate these questions, we designed an in vitro selection experiment aimed at evolving specificity of an ATP aptamer. The ATP aptamer makes contacts with both the nucleobase and the sugar. We used an affinity matrix in which GTP was immobilized through the sugar, thus requiring extensive changes in or loss of sugar contact, as well as changes in recognition of the nucleobase. After just five rounds of selection, the pool was dominated by new aptamers falling into three major classes, each with secondary structures distinct from that of the ATP aptamer. The average sequence identity between the original aptamer and new aptamers is 76%. Most of the mutations appear to play roles either in disrupting the original secondary structure or in forming the new secondary structure or the new recognition loops. Our results show that there are novel structures that recognize a significantly different ligand in the region of sequence space close to the ATP aptamer. These examples of the emergence of novel functions and structures from an RNA molecule with a defined specificity and fold provide a new perspective on the evolutionary flexibility and adaptability of RNA.

Human natural killer cells: a convenient purification procedure and the influence of cryopreservation on cytotoxic activity

The recognition of natural killer cells as a lymphoid subpopulation with a distinct set of surface markers has led to the development of a variety of antibody-based purification methods. In this paper we describe a rapid, three-step negative selection protocol for the purification of human natural killer (NK) cells from the mononuclear cell fraction, which is obtained by the centrifugation of peripheral blood on Ficoll-Paque. Subsequently, monocytes and B lymphocytes are removed by adherence to nylon wool and T lymphocytes by panning with anti-CD3. With this procedure, CD3 −, CD16/56 + NK cells are purified about five-fold, from 12 ± 3% in the starting population to a final purity of 61 ± 11%. A further increase to ≧ 70% is obtained, if an extra Ficoll centrifugation step is included. The recovery of NK cells (50%) is significantly higher than is usually achieved by previously described procedures. Furthermore, we show that activation of cytotoxicity, with concomitant changes in target specificity, occurs when frozen/thawed NK effector cells are kept in culture in order to regain their pre-freezing cytotoxicity levels.