Huntington disease (HD) is caused by an expanded HTT CAG repeat that leads in a length-dependent, completely dominant manner to onset of a characteristic movement disorder. HD also displays early mortality, so we tested whether the expanded CAG repeat exerts a dominant influence on age at death and on the duration of clinical disease. We found that, as with clinical onset, HD age at death is determined by expanded CAG-repeat length and has no contribution from the normal CAG allele. Surprisingly, disease duration is independent of the mutation's length. It is also unaffected by a strong genetic modifier of HD motor onset. These findings suggest two parsimonious alternatives. (1) HD pathogenesis is driven by mutant huntingtin, but before or near motor onset, sufficient CAG-driven damage occurs to permit CAG-independent processes and then lead to eventual death. In this scenario, some pathological changes and their clinical correlates could still worsen in a CAG-driven manner after disease onset, but these CAG-related progressive changes do not themselves determine duration. Alternatively, (2) HD pathogenesis is driven by mutant huntingtin acting in a CAG-dependent manner with different time courses in multiple cell types, and the cellular targets that lead to motor onset and death are different and independent. In this scenario, processes driven by HTT CAG length lead directly to death but not via the striatal pathology associated with motor manifestations. Each scenario has important ramifications for the design and testing of potential therapeutics, especially those aimed at preventing or delaying characteristic motor manifestations.

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We have examined regulation of the E2F transcription factor during differentiation of muscle cells. E2F regulates many genes involved in growth control and is also the target of regulation by diverse cellular signals, including the RB family of growth suppressors (e.g., the retinoblastoma protein [RB], p107, and p130). The following aspects of E2F function and regulation during muscle differentiation were investigated: (i) proteinprotein interactions, (ii) protein levels, (iii) phosphorylation of the E2F protein, and (iv) transcriptional activity. A distinct E2F complex was present in differentiated cells but not in undifferentiated cells. The p130 protein was a prominent component of the E2F complex associated with differentiation. In contrast, in undifferentiated cells, the p107 protein was the prominent component in one of three E2F complexes. In addition, use of a differentiation-defective muscle line provided genetic and biochemical evidence that quiescence and differentiation are separable events. Exclusive formation of the E2F-p130 complex did not occur in this differentiation-defective line; however, E2F complexes diagnostic of quiescence were readily apparent. Thus, sole formation of the E2F-p130 complex is a necessary event in terminal differentiation. Other changes in E2F function and regulation upon differentiation include decreased phosphorylation and increased repression by E2F. These observations suggest that the regulation of E2F function during terminal differentiation may proceed through differential interaction within the RB family and/or phosphorylation.In many cells, the trigger event in differentiation is withdrawal from the cell cycle with subsequent distinct morphological transitions. Muscle cells are an excellent system for examining molecular mechanisms of differentiation because they exhibit both permanent cell cycle withdrawal and a distinctive phenotype. Differentiation involves a transition from myoblasts to myotubes. Myoblasts are undifferentiated cells and are characterized by rapidly growing, mononucleated cells. In contrast, differentiated cells, or myotubes, are multinucleated and tubular. Upon differentiation, myotubes also express several muscle-specific markers. Because differentiated muscle cells (myotubes) are permanently withdrawn from the cell cycle, cellular mechanisms for the initiation and maintenance of the differentiated state must also exist (5).Studies with viral oncogenes have indicated a critical role for the retinoblastoma family of growth suppressors (the retinoblastoma protein [RB], p107, and p130) in the differentiation pathway. The expression of E1A and polyomavirus large T antigen may block muscle differentiation (7,49,66). It is well established that these two divergent viral oncogenes can bind the RB family (69). Importantly, when the binding site for RB family members was mutated, both E1A and polyomavirus large T antigen mutants no longer blocked differentiation (7, 49). These studies suggest that RB family members function in the control of differenti...

Archaea have a self-assembling proteinaceous surface (S-) layer as the primary and outermost boundary of their cell envelopes. The S-layer maintains structural rigidity, protects the organism from adverse environmental elements, and yet provides access to all essential nutrients. We have determined the crystal structure of one of the two “homologous” tandem polypeptide repeats that comprise the Methanosarcina acetivorans S-layer protein and propose a high-resolution model for a microbial S-layer. The molecular features of our hexameric S-layer model recapitulate those visualized by medium resolution electron microscopy studies of microbial S-layers and greatly expand our molecular view of S-layer dimensions, porosity, and symmetry. The S-layer model reveals a negatively charged molecular sieve that presents both a charge and size barrier to restrict access to the cell periplasmic-like space. The β-sandwich folds of the S-layer protein are structurally homologous to eukaryotic virus envelope proteins, suggesting that Archaea and viruses have arrived at a common solution for protective envelope structures. These results provide insight into the evolutionary origins of primitive cell envelope structures, of which the S-layer is considered to be among the most primitive: it also provides a platform for the development of self-assembling nanomaterials with diverse functional and structural properties.

Purpose To ascertain deformation of the optic nerve head (ONH) and peripapillary tissues caused by horizontal duction. Design Prospective, experimental study. Methods Optical coherence tomography of the ONH region was performed in 23 eyes of twelve normal volunteers in central gaze and increasing (10, 20, and 30°) adduction and abduction. Main outcome measures were changes from central gaze in the configuration of the ONH and peripapillary tissues in eccentric gazes. Results Adduction but not abduction was associated with significant, progressive relative posterior displacement of the temporal peripapillary retinal pigment epithelium (tRPE) from its position in central gaze reaching 49±10 μm in 30° adduction (standard error of mean, p<0.0001). Absolute (anterior or posterior) optic cup displacement (OCD) averaged 41±7 μm in 30° adduction. Linear regression showed significant effect of adduction on absolute OCD (slope 1.09±0.36 μm/degree, p=0.0037). In 20° and 30° adduction, all eyes exhibited significant progressive temporal ONH tilting reaching 3.1±0.4° in 30° adduction (p<0.0001). Abduction was not associated with significant peripapillary RPE displacement, OCD, or ONH tilt. Both nasal and temporal peripapillary choroid averaged 9 to 19 μm thinner in adduction and abduction than in central gaze (p<0.02). Conclusions Adduction temporally tilts and displaces the prelaminar ONH and peripapillary tissues. Both adduction and abduction compress the peripapillary choroid. These effects support MRI and biomechanical evidence that adduction imposes strain on the ONH and peripapillary tissues. Repetitive strain from eye movements over decades might in susceptible individuals lead to optic neuropathies such as normal tension glaucoma.

The SARS-CoV-2 variants replacing the first wave strain pose an increased threat by their potential ability to escape pre-existing humoral protection. An angiotensin converting enzyme 2 (ACE2) decoy that competes with endogenous ACE2 for binding of the SARS-CoV-2 spike receptor binding domain (S RBD) and inhibits infection may offer a therapeutic option with sustained efficacy against variants. Here, we used Molecular Dynamics (MD) simulation to predict ACE2 sequence substitutions that might increase its affinity for S RBD and screened candidate ACE2 decoys in vitro. The lead ACE2(T27Y/H34A)-IgG1FC fusion protein with enhanced S RBD affinity shows greater live SARS-CoV-2 virus neutralization capability than wild type ACE2. MD simulation was used to predict the effects of S RBD variant mutations on decoy affinity that was then confirmed by testing of an ACE2 Triple Decoy that included an additional enzyme activity-deactivating H374N substitution against mutated S RBD. The ACE2 Triple Decoy maintains high affinity for mutated S RBD, displays enhanced affinity for S RBD N501Y or L452R, and has the highest affinity for S RBD with both E484K and N501Y mutations, making it a viable therapeutic option for the prevention or treatment of SARS-CoV-2 infection with a high likelihood of efficacy against variants.

Recently there have been major advances in the electro-mechanical design of upper extremity prosthetics. However, the development of control strategies for such prosthetics has lagged significantly behind. Conventional noninvasive myoelectric control strategies rely on the amplitude of electromyography (EMG) signals from flexor and extensor muscles in the forearm. Surface EMG has limited specificity for deep contiguous muscles because of cross talk and cannot reliably differentiate between individual digit and joint motions. We present a novel ultrasound imaging based control strategy for upper arm prosthetics that can overcome many of the limitations of myoelectric control. Real time ultrasound images of the forearm muscles were obtained using a wearable mechanically scanned single element ultrasound system, and analyzed to create maps of muscle activity based on changes in the ultrasound echogenicity of the muscle during contraction. Individual digit movements were associated with unique maps of activity. These maps were correlated with previously acquired training data to classify individual digit movements. Preliminary results using ten healthy volunteers demonstrated this approach could provide robust classification of individual finger movements with 98% accuracy (precision 96%-100% and recall 97%-100% for individual finger flexions). The change in ultrasound echogenicity was found to be proportional to the digit flexion speed (R(2)=0.9), and thus our proposed strategy provided a proportional signal that can be used for fine control. We anticipate that ultrasound imaging based control strategies could be a significant improvement over conventional myoelectric control of prosthetics.

Objective. To restore central vision in patients with atrophic age-related macular degeneration, we replace the lost photoreceptors with photovoltaic pixels, which convert light into current and stimulate the secondary retinal neurons. Clinical trials demonstrated prosthetic acuity closely matching the sampling limit of the 100 μm pixels, and hence smaller pixels are required for improving visual acuity. However, with smaller flat bipolar pixels, the electric field penetration depth and the photodiode responsivity significantly decrease, making the device inefficient. Smaller pixels may be enabled by (a) increasing the diode responsivity using vertical p–n junctions and (b) directing the electric field in tissue vertically. Here, we demonstrate such novel photodiodes and test the retinal stimulation in a vertical electric field. Approach. Arrays of silicon photodiodes of 55, 40, 30, and 20 μm in width, with vertical p–n junctions, were fabricated. The electric field in the retina was directed vertically using a common return electrode at the edge of the device. Optical and electronic performance of the diodes was characterized in-vitro, and retinal stimulation threshold measured by recording the visually evoked potentials in rats with retinal degeneration. Main results. The photodiodes exhibited sufficiently low dark current (<10 pA) and responsivity at 880 nm wavelength as high as 0.51 A W−1, with 85% internal quantum efficiency, independent of pixel size. Field mapping in saline demonstrated uniformity of the pixel performance in the array. The full-field stimulation threshold was as low as 0.057 ± 0.029 mW mm−2 with 10 ms pulses, independent of pixel size. Significance. Photodiodes with vertical p–n junctions demonstrated excellent charge collection efficiency independent of pixel size, down to 20 μm. Vertically oriented electric field provides a stimulation threshold that is independent of pixel size. These results are the first steps in validation of scaling down the photovoltaic pixels for subretinal stimulation.