Resting-state brain network connectivity is an independent predictor of responsiveness to language therapy in chronic post-stroke aphasia.

Post-stroke aphasia recovery, especially in the chronic phase, is challenging to predict. Functional integrity of the brain and brain network topology have been suggested as biomarkers of language recovery. This study sought to investigate functional connectivity in four predefined brain networks (i.e., language, default mode, dorsal attention, and salience networks), in relation to aphasia severity and response to language therapy. Thirty patients with chronic post-stroke aphasia were recruited and received a treatment targeting word finding. Structural and functional brain scans were acquired at baseline and resting state functional connectivity for each network was calculated. Additionally, graph measures quantifying network properties were calculated for each network. These included global efficiency for all networks and average strength and clustering coefficient for the language network. Linear mixed effects models showed that mean functional connectivity in the default mode, dorsal attention, and salience networks as well as graph measures of all four networks are independent predictors of response to therapy. While greater mean functional connectivity and global efficiency of the dorsal attention and salience networks predicted greater treatment response, greater mean functional connectivity and global efficiency in the default mode network predicted poorer treatment response. Results for the language network were more nuanced with more efficient network configurations (as reflected in graph measures), but not mean functional connectivity, predicting greater treatment response. These findings highlight the prognostic value of resting-state functional connectivity in chronic treatment-induced aphasia recovery.

Air-liquid interface culture promotes maturation and allows environmental exposure of pluripotent stem cell-derived alveolar epithelium.

Type 2 alveolar epithelial cells (AT2s), facultative progenitor cells of the lung alveolus, play a vital role in the biology of the distal lung. In vitro model systems that incorporate human cells, recapitulate the biology of primary AT2s, and interface with the outside environment could serve as useful tools to elucidate functional characteristics of AT2s in homeostasis and disease. We and others recently adapted human induced pluripotent stem cell-derived AT2s (iAT2s) for air-liquid interface (ALI) culture. Here, we comprehensively characterize the effects of ALI culture on iAT2s and benchmark their transcriptional profile relative to both freshly sorted and cultured primary human fetal and adult AT2s. We find that iAT2s cultured at ALI maintain an AT2 phenotype while upregulating expression of transcripts associated with AT2 maturation. We then leverage this platform to assay the effects of exposure to clinically significant, inhaled toxicants including cigarette smoke and electronic cigarette vapor.

Deuteration of nonexchangeable protons on proteins affects their thermal stability, side-chain dynamics, and hydrophobicity.

We have investigated the effect of deuteration of non-exchangeable protons on protein global thermal stability, hydrophobicity, and local flexibility using well-known thermostable model systems such as the villin headpiece subdomain (HP36) and the third immunoglobulin G-binding domain of protein G (GB3). Reversed-phase high-performance liquid chromatography (RP-HPLC) measurements as a function of temperature probe global thermal stability in the presence of acetonitrile, while differential scanning calorimetry determines thermal stability in solution. Both indicate small but measurable changes in the order of several degrees. RP-HPLC also permitted quantification of the effect of deuteration of just three core phenylalanine side chains of HP36. NMR dynamics investigation has focused on methyl axes motions using cross-correlated relaxation measurements. The analysis of order parameters provided a complex picture indicating that deuteration generally increases motional amplitudes of sub-nanosecond motion in GB3 but decreases those in HP36. Combined with earlier dynamics measurements at Cα -Cß sites and backbone sites of GB3, which probed slower time scales, the results point to the need to probe multiple atoms in the protein and variety of time scales to the discern the full complexity of the effects of deuteration on dynamics.

Optimized purification of a fusion protein by reversed-phase high performance liquid chromatography informed by the linear solvent strength model.

Fusion protein systems are commonly used for expression of small proteins and peptides. An important criterion for a fusion protein system to be useful is the ability to separate the protein of interest from the tag. Additionally, because no protease cleaves fusion proteins with 100% efficiency, the ability to separate the desired peptide from any remaining uncleaved protein is also necessary. This is likely to be the more difficult task as at least a portion of the sequence of the fusion protein is identical to that of the protein of interest. When a high level of purity is required, gradient elution reversed-phase HPLC is frequently used as a final purification step. Shallow gradients are often advantageous for maximizing both the purity and yield of the final product; however, the relationship between relative retention times at shallow gradients and those at steeper gradients typically used for analytical HPLC are not always straightforward. In this work, we report reversed-phase HPLC results for the fusion protein system consisting of the N-terminal domain of ribosomal protein L9 (NTL9) and the 36-residue villin headpiece subdomain (HP36) linked by a recognition sequence for the protease factor Xa. This system represents an excellent example of the difficulties in purification that may arise from this unexpected elution behavior at shallow gradients. Additionally, we report on the sensitivity of this elution behavior to the concentration of the additive trifluoroacetic acid in the mobile phase and present optimized conditions for separating HP36 from the full fusion protein by reversed-phase HPLC using a shallow gradient. Finally, we suggest that these findings are relevant to the purification of other fusion protein systems, for which similar problems may arise, and support this suggestion using insights from the linear solvent strength model of gradient elution liquid chromatography.

Solvent-Driven Dynamical Crossover in the Phenylalanine Side-Chain from the Hydrophobic Core of Amyloid Fibrils Detected by 2H NMR Relaxation.

Aromatic residues are important markers of dynamical changes in proteins' hydrophobic cores. In this work we investigated the dynamics of the F19 side-chain in the core of amyloid fibrils across a wide temperature range of 300 to 140 K. We utilized solid-state 2H NMR relaxation to demonstrate the presence of a solvent-driven dynamical crossover between different motional regimes, often also referred to as the dynamical transition. In particular, the dynamics are dominated by small-angle fluctuations at low temperatures and by π-flips of the aromatic ring at high temperatures. The crossover temperature is more than 43 degrees lower for the hydrated state of the fibrils compared to the dry state, indicating that interactions with water facilitate π-flips. Further, crossover temperatures are shown to be very sensitive to polymorphic states of the fibrils, such as the 2-fold and 3-fold symmetric morphologies of the wild-type protein as well as D23N mutant protofibrils. We speculate that these differences can be attributed, at least partially, to enhanced interactions with water in the 3-fold polymorph, which has been shown to have a water-accessible cavity. Combined with previous studies of methyl group dynamics, the results highlight the presence of multiple dynamics modes in the core of the fibrils, which was originally believed to be quite rigid.

Fast Motions of Key Methyl Groups in Amyloid-ß Fibrils.

Amyloid-ß (Aß) peptide is the major component of plaques found in Alzheimer's disease patients. Using solid-state 2H NMR relaxation performed on selectively deuterated methyl groups, we probed the dynamics in the threefold symmetric and twofold symmetric polymorphs of native Aß as well as the protofibrils of the D23N mutant. Specifically, we investigated the methyl groups of two leucine residues that belong to the hydrophobic core (L17 and L34) as well as M35 residues belonging to the hydrophobic interface between the cross-ß subunits, which has been previously found to be water-accessible. Relaxation measurements performed over 310-140 K and two magnetic field strengths provide insights into conformational variability within and between polymorphs. Core packing variations within a single polymorph are similar to what is observed for globular proteins for the core residues, whereas M35 exhibits a larger degree of variability. M35 site is also shown to undergo a solvent-dependent dynamical transition in which slower amplitude motions of methyl axes are activated at high temperature. The motions, modeled as a diffusion of methyl axis, have activation energy by a factor of 2.7 larger in the twofold compared with the threefold polymorph, whereas D23N protofibrils display a value similar to the threefold polymorph. This suggests enhanced flexibility of the hydrophobic interface in the threefold polymorph. This difference is only observed in the hydrated state and is absent in the dry fibrils, highlighting the role of solvent at the cavity. In contrast, the dynamic behavior of the core is hydration-independent.

Flexibility and Solvation of Amyloid-ß Hydrophobic Core.

Amyloid fibril deposits found in Alzheimer disease patients are composed of amyloid-ß (Aß) protein forming a number of hydrophobic interfaces that are believed to be mostly rigid. We have investigated the µs-ms time-scale dynamics of the intra-strand hydrophobic core and interfaces of the fibrils composed of Aß1-40 protein. Using solid-state (2)H NMR line shape experiments performed on selectively deuterated methyl groups, we probed the 3-fold symmetric and 2-fold symmetric polymorphs of native Aß as well as the protofibrils of D23N Iowa mutant, associated with an early onset of Alzheimer disease. The dynamics of the hydrophobic regions probed at Leu-17, Leu-34, Val-36, and Met-35 side chains were found to be very pronounced at all sites and in all polymorphs of Aß, with methyl axis motions persisting down to 230-200 K for most of the sites. The dominant mode of motions is the rotameric side chain jumps, with the Met-35 displaying the most complex multi-modal behavior. There are distinct differences in the dynamics among the three protein variants, with the Val-36 site displaying the most variability. Solvation of the fibrils does not affect methyl group motions within the hydrophobic core of individual cross-ß subunits but has a clear effect on the motions at the hydrophobic interface between the cross-ß subunits, which is defined by Met-35 contacts. In particular, hydration activates transitions between additional rotameric states that are not sampled in the dry protein. Thus, these results support the existence of water-accessible cavity recently predicted by molecular dynamics simulations and suggested by cryo-EM studies.