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Background: The subthalamic nucleus (STN) is considered a key structure in motor, behavioral, and emotional control. Although identification of the functional topography of the STN has therapeutic implications in the treatment of the motor features of Parkinson's disease (PD), the details of its functional and somatotopic organization in humans are not well understood. Objective: The aim of this study was to characterize the functional organization of the STN and its correlation with the motor outcomes induced by subthalamotomy. Methods: We used diffusion‐weighted imaging to assess STN connectivity patterns in 23 healthy control subjects and 86 patients with PD, of whom 39 received unilateral subthalamotomy. Analytical tractography was used to reconstruct structural cortico‐subthalamic connectivity. A diffusion‐weighted imaging/functional magnetic resonance imaging–driven somatotopic parcellation of the STN was defined to delineate the representation of the upper and lower limb in the STN. Results: We confirmed a connectional gradient to sensorimotor, supplementary‐motor, associative, and limbic cortical regions, spanning from posterior‐dorsal‐lateral to anterior‐ventral‐medial portions of the STN, with intermediate overlapping zones. Functional magnetic resonance imaging–driven parcellation demonstrated dual segregation of motor cortico‐subthalamic projections in humans. Moreover, the relationship between lesion topography and functional anatomy of the STN explains specific improvement in bradykinesia, rigidity, and tremor induced by subthalamotomy. Conclusions: Our results support an interplay between segregation and integration of cortico‐subthalamic projections, suggesting the coexistence of parallel and convergent information processing. Identifying the functional topography of the STN will facilitate better definition of the optimal location for functional neurosurgical approaches, that is, electrode placement and lesion location, and improve specific cardinal features in PD. © 2021 International Parkinson and Movement Disorder Society [ABSTRACT FROM AUTHOR]

Background: The high acoustic impedance of the skull limits the performance of transcranial magnetic resonance-guided focused ultrasound (tcMRgFUS) therapy. Subject suitability screening is based on skull parameters estimated from computed tomography (CT) scans.Purpose: To assess the feasibility of screening for tcMRgFUS based on zero echo time (ZTE) MRI, and to explore the influence of measurable skull parameters in treatment performance.Study Type: Retrospective.Population: Sixteen patients treated with tcMRgFUS thalamotomy for tremor.Sequence: ZTE on a 3.0T GE scanner.Assessment: Baseline CT and ZTE images were processed to extract skull measures associated with treatment success: skull density ratio (SDR), skull thickness, and angle of incidence. Eight new metrics were proposed. CT and ZTE-based measures were compared. Each subject's energy-temperature curve was processed to extract a global estimate of efficiency and a measure of nonlinearity. These parameters were then correlated with the skull measures.Statistical Tests: Linear regression analysis to compare ZTE vs. CT-based measures, measures vs. efficiency, and measures vs. nonlinearity. Paired t-test to assess nonlinearity.Results: CT and ZTE-based measures were significantly correlated (P < 0.01). In particular, classical metrics were robustly replicated (P < 0.001). The energy-temperature curves showed a nonlinear (logarithmic) relationship (P < 0.01). This nonlinearity was greater for thicker skulls (P < 0.01). Efficiency was correlated with skull thickness (P < 0.001) and SDR (P < 0.05).Data Conclusion: The feasibility of ZTE-based screening has been proven, potentially making it possible to avoid ionizing radiation and the extra imaging session required for CT. The characterization of the influence that skull properties have on tcMRgFUS may serve to develop patient-specific heating models, potentially improving control over the treatment outcome. The relationship of skull thickness with efficiency and nonlinearity empowers the role of this metric in the definition of such models. In addition, the lower association of SDR with the energy-temperature curves emphasizes the need of revisiting this metric.Level Of Evidence: 3 Technical Efficacy: Stage 2 J. Magn. Reson. Imaging 2019;50:1583-1592. [ABSTRACT FROM AUTHOR]

Multiple sclerosis (MS)-related tremor is frequent and can often be refractory to medical treatment, which makes it a potential source of major disability. Functional neurosurgery approaches such as thalamic deep brain stimulation (DBS) or radiofrequency thalamotomy are proven to be effective, but the application of invasive techniques in MS tremor has so far been limited. Magnetic resonance (MR)-guided focused ultrasound thalamotomy, which has already been approved for treating essential and parkinsonian tremor, provides a minimally invasive approach that could be useful in the management of MS tremor. We report for the first time a patient with medically refractory MS-associated tremor successfully treated by focused ultrasound thalamotomy. [ABSTRACT FROM AUTHOR]

Polypeptides exiting the ribosome must fold and assemble in the crowded environment of the cell. Chaperones and other protein homeostasis factors interact with newly translated polypeptides to facilitate their folding and correct localization. Despite the extensive efforts, little is known about the specificity of the chaperones and other factors that bind nascent polypeptides. To address this question we present an approach that systematically identifies cotranslational chaperone substrates through the mRNAs associated with ribosome-nascent chain-chaperone complexes. We here focused on two Saccharomyces cerevisiae chaperones: the Signal Recognition Particle (SRP), which acts cotranslationally to target proteins to the ER, and the Nascent chain Associated Complex (NAC), whose function has been elusive. Our results provide new insights into SRP selectivity and reveal that NAC is a general cotranslational chaperone. We found surprising differential substrate specificity for the three subunits of NAC, which appear to recognize distinct features within nascent chains. Our results also revealed a partial overlap between the sets of nascent polypeptides that interact with NAC and SRP, respectively, and showed that NAC modulates SRP specificity and fidelity in vivo. These findings give us new insight into the dynamic interplay of chaperones acting on nascent chains. The strategy we used should be generally applicable to mapping the specificity, interplay, and dynamics of the cotranslational protein homeostasis network. [ABSTRACT FROM AUTHOR]

Multiple sclerosis (MS)-related tremor is frequent and can often be refractory to medical treatment, which makes it a potential source of major disability. Functional neurosurgery approaches such as thalamic deep brain stimulation (DBS) or radiofrequency thalamotomy are proven to be effective, but the application of invasive techniques in MS tremor has so far been limited. Magnetic resonance (MR)-guided focused ultrasound thalamotomy, which has already been approved for treating essential and parkinsonian tremor, provides a minimally invasive approach that could be useful in the management of MS tremor. We report for the first time a patient with medically refractory MS-associated tremor successfully treated by focused ultrasound thalamotomy.

In this chapter we compile a battery of biophysical and imaging methods suitable to investigate adenovirus structural stability, structure, and assembly. Some are standard methods with a long history of use in virology, such as embedding and sectioning of infected cells, negative staining, or immunoelectron microscopy, as well as extrinsic fluorescence. The newer cryo-electron microscopy technique, which combined with advanced image processing tools has recently yielded an atomic resolution picture of the complete virion, is also described. Finally, we detail the procedure for imaging and interacting with single adenovirus virions using the atomic force microscope in liquid conditions. We provide examples of the kind of data obtained with each technique.

The capsid protein of HIV-1 (p24) (CA) forms the mature capsid of the human immunodeficiency virus. Capsid assembly involves hexamerization of the N-terminal domain and dimerization of the C-terminal domain of CA (CAC), and both domains constitute potential targets for anti-HIV therapy. CAC homodimerization occurs mainly through its second helix, and it is abolished when its sole tryptophan is mutated to alanine. This mutant, CACW40A, resembles a transient monomeric intermediate formed during dimerization. Its tertiary structure is similar to that of the subunits in the dimeric, non-mutated CAC, but the segment corresponding to the second helix samples different conformations. The present study comprises a comprehensive examination of the CACW40A internal dynamics. The results obtained, with movements sampling a wide time regime (from pico- to milliseconds), demonstrate the high flexibility of the whole monomeric protein. The conformational exchange phenomena on the micro-to-millisecond time scale suggest a role for internal motions in the monomer-monomer interactions and, thus, flexibility of the polypeptide chain is likely to contribute to the ability of the protein to adopt different conformational states, depending on the biological environment.

The protein CA forms the mature capsid of human immunodeficiency virus. Hexamerization of the N-terminal domain and dimerization of the C-terminal domain, CAC, occur during capsid assembly, and both domains constitute potential targets for anti-HIV inhibitors. CAC homodimerization occurs mainly through its second helix, and is abolished when its sole tryptophan is mutated to alanine. Previous thermodynamic data obtained with the dimeric and monomeric forms of CAC indicate that the structure of the mutant resembles that of a monomeric intermediate found in the folding and association reactions of CAC. We have solved the three-dimensional structure in aqueous solution of the monomeric mutant. The structure is similar to that of the subunits in the dimeric, nonmutated CAC, except the segment corresponding to the second helix, which is highly dynamic. At the end of this region, the polypeptide chain is bent to bury several hydrophobic residues and, as a consequence, the last two helices are rotated 90 degrees when compared to their position in dimeric CAC. The previously obtained thermodynamic data are consistent with the determined structure of the monomeric mutant. This extraordinary ability of CAC to change its structure may contribute to the different modes of association of CA during HIV assembly, and should be taken into account in the design of new drugs against this virus.

Previous studies on the self-assembly of capsid protein CA of human immunodeficiency virus type 1 (HIV-1) in vitro have provided important insights on the structure and assembly of the mature HIV-1 capsid. However, CA polymerization in vitro was previously observed to occur only at very high ionic strength. Here, we have analyzed the effects on CA assembly in vitro of adding unrelated, inert macromolecules (crowding agents), aimed at mimicking the crowded (very high macromolecular effective concentration) environment within the HIV-1 virion. Crowding agents induced fast and efficient polymerization of CA even at low (close to physiological) ionic strength. The hollow cylinders thus assembled were indistinguishable in shape and dimensions from those formed in dilute protein solutions at high ionic strength. However, two important differences were noted: (i) disassembly by dilution of the capsid-like particles was undetectable at very high ionic strength, but occurred rapidly at low ionic strength in the presence of a crowding agent, and (ii) a variant CA from a presumed infectious HIV-1 with mutations at the CA dimerization interface was unable to assemble at any ionic strength in the absence of a crowding agent; in contrast, this mutation allowed efficient assembly, even at low ionic strength, when a crowding agent was used. The use of a low ionic strength and inert macromolecules to mimic the crowded environment inside the HIV-1 virion may lead to a better in vitro evaluation of the effects of conditions, mutations or/and other molecules, including potential antiviral compounds, on HIV-1 capsid assembly, stability and disassembly.

In previous studies, thermodynamic dissection of the dimerization interface in CA-C, the C-terminal domain of the capsid protein of human immunodeficiency virus type 1, revealed that individual mutation to alanine of Ser178, Glu180, Glu187 or Gln192 led to significant increases in dimerization affinity. Four related aspects derived from this observation have been now addressed, and the results can be summarized as follows: (i) thermodynamic analyses indicate the presence of an intersubunit electrostatic repulsion between both Glu180 residues. (ii) The mutation Glu180 to Ala was detected in nearly all type 2 human immunodeficiency virus variants, and in several simian immunodeficiency viruses analyzed. However, this mutation was strictly co-variant with mutations Ser178Asp in a neighboring residue, and Glu187Gln. Thermodynamic analysis of multiple mutants showed that Ser178Asp compensated, alone or together with Glu187Gln, the increase in affinity caused by the mutation Glu180Ala, and restored a lower dimerization affinity. (iii) The increase in the affinity constant caused by the multiple mutation to Ala of Ser178, Glu180, Glu187 and Gln192 was more than one order of magnitude lower than predicted if additivity were present, despite the fact that the 178/180 pair and the two other residues were located more than 10A apart. (iv) Mutations in CA-C that caused non-additive increases in dimerization affinity also caused a non-additive increase in the capacity of the isolated CA-C domain to inhibit the assembly of capsid-like HIV-1 particles in kinetic assays. In summary, the study of a protein-protein interface involved in the building of a viral capsid has revealed unusual features, including intersubunit electrostatic repulsions, co-variant, compensatory mutations that may evolutionarily preserve a low association constant, and long-range, large magnitude non-additive effects on association.

Pseudomonas sp. strain Y2 is a styrene-degrading bacterium, which initiates the catabolism of this compound via its transformation into phenylacetate by the sequential oxidation of the vinyl side chain. The styrene upper catabolic gene cluster (sty genes) had been localized in a 9.2-kb chromosomal region. This report describes the isolation, sequencing and analysis of an adjacent 20.5-kb chromosomal region that contains the genes of the styrene lower degradative pathway (paa genes), which are involved in the transformation of phenylacetate into aliphatic compounds that can enter the Krebs cycle. Hence, Pseudomonas sp. strain Y2 becomes the first microorganism whose entire styrene catabolic cluster has been completely characterized. Analysis of the paa gene cluster has revealed the presence of 17 open reading frames as well as gene duplications and gene reorganizations that are absent in other phenylacetate catabolic clusters described so far. The functionality of these genes has been proved by means of both complementation experiments on Pseudomonas putida mutants and in vitro enzymatic assays. Moreover, a DNA cassette encoding the whole styrene lower pathway has been constructed and has been used to expand the ability of Pseudomonas strains to degrade phenylacetic acid. For the first time, two functional phenylacetate-CoA ligases have been identified in an aerobic phenylacetic acid degradation pathway. Although the upper and lower styrene catabolic clusters are adjacent in the Pseudomonas sp. strain Y2 chromosome, their particular base composition and codon usage suggest a distinct evolutionary history.

Homo-dimerization of the capsid protein CA of human immunodeficiency virus through its C-terminal domain constitutes an early crucial step in the virion assembly pathway and a potential target for antiviral inhibitors. We have truncated to alanine the 20 amino acid side chains per monomer that participate in intersubunit contacts at the CA dimer interface and analyzed their individual energetic contribution to protein association and stability. About half of the side chains in the contact epitope are critically involved in the energetic epitope as their truncation essentially prevented dimerization. However, dimerization affinity is kept low partly because of the presence of interfacial side chains whose individual truncation improves affinity by 2-20-fold. Many side chains at the interface are energetically important also for the folding of a monomeric intermediate and for its conformational rearrangement during dimerization. The thermodynamic description of this low affinity interface (dissociation constant of approximately 10 microm) was compared with those obtained for the other protein-protein interfaces, nearly all of them of much higher affinity, that have been systematically analyzed by mutation. The results reveal differences that may have been evolutionary selected and that may be exploited for the design of an effective interfacial inhibitor of human immunodeficiency virus assembly.