Impact of Inbreeding and Ancestral Inbreeding on Longevity Traits in German Brown Cows.

A recent study on the population structure of the German Brown population found increasing levels of classical and ancestral inbreeding coefficients. Thus, the aim of this study was to determine the effects of inbreeding depression and purging on longevity traits using classical and ancestral inbreeding coefficients according to Kalinowski (2002) (Fa_Kal, FNew), Ballou (1997) (Fa_Bal), and Baumung (2015) (Ahc). For this purpose, uncensored data of 480,440 cows born between 1990 and 2001 were available. We analyzed 17 longevity traits, including herd life, length of productive life, number of calvings, lifetime and effective lifetime production for milk, fat, and protein yield, the survival to the 2nd, 4th, 6th, 8th, and 10th lactation number, and the culling frequencies due to infertility, or udder and foot and leg problems. Inbreeding depression was significant and negative for all traits but for culling due to udder and to foot and leg problems. When expressed in percentages of genetic standard deviations, inbreeding depression per 1% increase in inbreeding was -3.61 to -10.98%, -2.42 to -2.99%, -2.21 to -4.58%, and 5.13% for lifetime production traits, lifetime traits, survival rates, and culling due to infertility, respectively. Heterosis and recombination effects due to US Brown Swiss genes were positive and counteracted inbreeding depression. The effects of FNew were not significantly different from zero, while Fa_Kal had negative effects on lifetime and lifetime production traits. Similarly, the interaction of F with Fa_Bal was significantly negative. Thus, purging effects could not be shown for longevity traits in German Brown. A possible explanation may be seen in the breed history of the German Brown, that through the introgression of US Brown Swiss bulls ancestral inbreeding increased and longevity decreased. Our results show, that reducing a further increase in inbreeding in mating plans is advisable to prevent a further decline in longevity due to inbreeding depression, as purging effects were very unlikely in this population.

Combined proteomics and CRISPR‒Cas9 screens in PDX identify ADAM10 as essential for leukemia in vivo.

BACKGROUND: Acute leukemias represent deadly malignancies that require better treatment. As a challenge, treatment is counteracted by a microenvironment protecting dormant leukemia stem cells. METHODS: To identify responsible surface proteins, we performed deep proteome profiling on minute numbers of dormant patient-derived xenograft (PDX) leukemia stem cells isolated from mice. Candidates were functionally screened by establishing a comprehensive CRISPR‒Cas9 pipeline in PDX models in vivo. RESULTS: A disintegrin and metalloproteinase domain-containing protein 10 (ADAM10) was identified as an essential vulnerability required for the survival and growth of different types of acute leukemias in vivo, and reconstitution assays in PDX models confirmed the relevance of its sheddase activity. Of translational importance, molecular or pharmacological targeting of ADAM10 reduced PDX leukemia burden, cell homing to the murine bone marrow and stem cell frequency, and increased leukemia response to conventional chemotherapy in vivo. CONCLUSIONS: These findings identify ADAM10 as an attractive therapeutic target for the future treatment of acute leukemias.

A Dynamic rRNA Ribomethylome Drives Stemness in Acute Myeloid Leukemia.

The development and regulation of malignant self-renewal remain unresolved issues. Here, we provide biochemical, genetic, and functional evidence that dynamics in ribosomal RNA (rRNA) 2'-O-methylation regulate leukemia stem cell (LSC) activity in vivo. A comprehensive analysis of the rRNA 2'-O-methylation landscape of 94 patients with acute myeloid leukemia (AML) revealed dynamic 2'-O-methylation specifically at exterior sites of ribosomes. The rRNA 2'-O-methylation pattern is closely associated with AML development stage and LSC gene expression signature. Forced expression of the 2'-O-methyltransferase fibrillarin (FBL) induced an AML stem cell phenotype and enabled engraftment of non-LSC leukemia cells in NSG mice. Enhanced 2'-O-methylation redirected the ribosome translation program toward amino acid transporter mRNAs enriched in optimal codons and subsequently increased intracellular amino acid levels. Methylation at the single site 18S-guanosine 1447 was instrumental for LSC activity. Collectively, our work demonstrates that dynamic 2'-O-methylation at specific sites on rRNAs shifts translational preferences and controls AML LSC self-renewal. SIGNIFICANCE: We establish the complete rRNA 2'-O-methylation landscape in human AML. Plasticity of rRNA 2'-O-methylation shifts protein translation toward an LSC phenotype. This dynamic process constitutes a novel concept of how cancers reprogram cell fate and function. This article is highlighted in the In This Issue feature, p. 247.

In vivo PDX CRISPR/Cas9 screens reveal mutual therapeutic targets to overcome heterogeneous acquired chemo-resistance.

Resistance towards cancer treatment represents a major clinical obstacle, preventing cure of cancer patients. To gain mechanistic insights, we developed a model for acquired resistance to chemotherapy by treating mice carrying patient derived xenografts (PDX) of acute lymphoblastic leukemia with widely-used cytotoxic drugs for 18 consecutive weeks. In two distinct PDX samples, tumors initially responded to treatment, until stable disease and eventually tumor re-growth evolved under therapy, at highly similar kinetics between replicate mice. Notably, replicate tumors developed different mutations in TP53 and individual sets of chromosomal alterations, suggesting independent parallel clonal evolution rather than selection, driven by a combination of stochastic and deterministic processes. Transcriptome and proteome showed shared dysregulations between replicate tumors providing putative targets to overcome resistance. In vivo CRISPR/Cas9 dropout screens in PDX revealed broad dependency on BCL2, BRIP1 and COPS2. Accordingly, venetoclax re-sensitized derivative tumors towards chemotherapy, despite genomic heterogeneity, demonstrating direct translatability of the approach. Hence, despite the presence of multiple resistance-associated genomic alterations, effective rescue treatment for polychemotherapy-resistant tumors can be identified using functional testing in preclinical models.

Risk factors associated with the infection of sheep with Dichelobacter nodosus.

Ovine footrot is a highly contagious foot disease caused by the gram-negative bacterium Dichelobacter nodosus (D. nodosus). In a recent report, we showed a prevalence of 42.9% D. nodosus positive swabs across Germany. In this follow-up study, we used real-time PCR results for D. nodosus and footrot scores of 9297 sheep from 208 flocks and collated these data with survey data on herd and animal characteristics and herd management. The aims of the present study were to investigate herd and animal factors associated with D. nodosus infection and footrot scores in individual sheep. Multivariable analyses with generalized mixed models showed that month of recording, breed, herdbook membership, use of antibiotics, and footbaths in the past 3-10 years, signs of footrot in the past 12 months and flock environment of the sheep, modelled as a random farm effect within region, were significant risk factors. Among the 21 different breeds, Romney had the lowest risk of D. nodosus infection, while Swifter had the highest risk and German Merino and German White Heath were the next breeds at highest risk of D. nodosus infection. The variance between farms in the prevalence of D. nodosus was large and accounted for 84% of the total variance in the mixed model analysis. We conclude that specific and as yet unknown effects influencing D. nodosus infections in flocks, as well as breed and weather, are the most important effects on D. nodosus infection in sheep, pointing towards the need to establish adequate infection control at farm level.

Serological Diversity of Dichelobacter nodosus in German Sheep Flocks.

Footrot is one of the major causes of lameness in sheep and leads to decreased animal welfare and high economic losses. The causative agent is the Gram-negative anaerobic bacterium Dichelobacter nodosus. The prevalence of D. nodosus in 207 sheep flocks across Germany was 42.9%. Based on the sequence variation in the type IV fimbrial gene fimA, D. nodosus can be subdivided into ten serogroups (A-I and M). There are commercially available vaccines covering nine serogroups, but the efficacy is low compared to bivalent vaccines. The aim of this study was to investigate the diversity of serogroups in Germany at the flock and animal levels. In total, we detected at least one serogroup in 819 samples out of 969 D. nodosus-positive samples from 83 flocks using serogroup-specific singleplex PCR for the serogroups A-I. Serogroup A was most prevalent at the animal level, followed by serogroups B, H and C. At the flock level, serogroups A and B had the highest prevalence, each with 64%, but only 40% of flocks had both. The average number of serogroups per animal was 1.42 (range one to five) and, per flock, 3.10 (range one to six). The serogrouping showed within-flock specific clusters but were widely distributed, with 50 different combinations across the flocks. The factors associated with the number of serogroups per animal and single serogroups were the load of D. nodosus, footrot score, sheep breed and flock. Our results indicate that efficient vaccination programs would benefit from tailor-made flock-specific vaccines and regular monitoring of circulating serotypes in the flock to be able to adjust vaccine formulations for nationwide progressive control of footrot in Germany.

Modeling and Bioinformatics Identify Responders to G-CSF in Patients With Amyotrophic Lateral Sclerosis.

Objective: Developing an integrative approach to early treatment response classification using survival modeling and bioinformatics with various biomarkers for early assessment of filgrastim (granulocyte colony stimulating factor) treatment effects in amyotrophic lateral sclerosis (ALS) patients. Filgrastim, a hematopoietic growth factor with excellent safety, routinely applied in oncology and stem cell mobilization, had shown preliminary efficacy in ALS. Methods: We conducted individualized long-term filgrastim treatment in 36 ALS patients. The PRO-ACT database, with outcome data from 23 international clinical ALS trials, served as historical control and mathematical reference for survival modeling. Imaging data as well as cytokine and cellular data from stem cell analysis were processed as biomarkers in a non-linear principal component analysis (NLPCA) to identify individual response. Results: Cox proportional hazard and matched-pair analyses revealed a significant survival benefit for filgrastim-treated patients over PRO-ACT comparators. We generated a model for survival estimation based on patients in the PRO-ACT database and then applied the model to filgrastim-treated patients. Model-identified filgrastim responders displayed less functional decline and impressively longer survival than non-responders. Multimodal biomarkers were then analyzed by PCA in the context of model-defined treatment response, allowing identification of subsequent treatment response as early as within 3 months of therapy. Strong treatment response with a median survival of 3.8 years after start of therapy was associated with younger age, increased hematopoietic stem cell mobilization, less aggressive inflammatory cytokine plasma profiles, and preserved pattern of fractional anisotropy as determined by magnetic resonance diffusion tensor imaging (DTI-MRI). Conclusion: Long-term filgrastim is safe, is well-tolerated, and has significant positive effects on disease progression and survival in a small cohort of ALS patients. Developing and applying a model-based biomarker response classification allows use of multimodal biomarker patterns in full potential. This can identify strong individual treatment responders (here: filgrastim) at a very early stage of therapy and may pave the way to an effective individualized treatment option.

Prevalence of Dichelobacter nodosus and Ovine Footrot in German Sheep Flocks.

The bacterium Dichelobacter nodosus (D. nodosus) is the causative agent of ovine footrot. The aim of this field study was to determine the prevalence of D. nodosus in German sheep flocks. The sheep owners participated voluntarily in the study. More than 9000 sheep from 207 flocks were screened for footrot scores using a Footrot Scoring System from 0 to 5 and sampling each sheep using one interdigital swab for all four feet of the sheep. The detection and discrimination between benign and virulent strains was done employing a real-time PCR. Our results showed a mean prevalence of 42.93% of D. nodosus in German sheep on an animal level. Underrunning of hoof horn on at least one foot (Scores 3-5) was detected in 567 sheep (6.13%). Sheep with four clinically healthy feet were found through visual inspection in 47.85% of all animals included in this study. In total, 1117 swabs from sheep with four clinically healthy feet tested positive for D. nodosus. In 90.35% of the positive swabs, virulent D. nodosus were detected. Benign D. nodosus were detected in 4.74% of the D. nodosus-positive swabs while 4.91% tested positive for both, benign and virulent D. nodosus. In 59 flocks D. nodosus were not detected and in 115 flocks only virulent D. nodosus were found while seven flocks tested positive for benign strains.

Genetic Diversity and the Impact of the Breed Proportions of US Brown Swiss in German Brown Cattle.

Increase of inbreeding and loss of genetic diversity have large impact on farm animal genetic resources. Therefore, the aims of the present study were to analyse measures of genetic diversity as well as recent and ancestral inbreeding using pedigree data of the German Brown population, and to identify causes for loss of genetic diversity. The reference population included 922,333 German Brown animals born from 1990 to 2014. Pedigree depth and completeness reached an average number of complete equivalent generations of 6.24. Estimated effective population size for the German Brown reference population was about 112 with a declining trend from 141 to 95 for the birth years. Individual inbreeding coefficients increased from 0.013 to 0.036. Effective number of founders, ancestors and founder genomes of 63.6, 36.23 and 20.34 indicated unequal contributions to the reference population. Thirteen ancestors explained 50% of the genetic diversity. Higher breed proportions of US Brown Swiss were associated with higher levels of individual inbreeding. Ancestral inbreeding coefficients, which are indicative for exposure of ancestors to identical-by-descent alleles, increased with birth years but recent individual inbreeding was higher than ancestral inbreeding. Given the increase of inbreeding and decline of effective population size, measures to decrease rate of inbreeding and increase effective population size through employment of a larger number of sires are advisable.

Crossed-beam slowing to enhance narrow-line ytterbium magneto-optic traps.

We demonstrate a method to enhance the atom loading rate of a ytterbium (Yb) magneto-optic trap (MOT) operating on the 556 nm 1S0 → 3P1 intercombination transition (narrow linewidth Γg = 2π × 182 kHz). Following traditional Zeeman slowing of an atomic beam near the 399 nm 1S0 → 1P1 transition (broad linewidth Γp = 2π × 29 MHz), two laser beams in a crossed-beam geometry, frequency tuned near the same transition, provide additional slowing immediately prior to the MOT. Using this technique, we observe an improvement by a factor of 6 in the atom loading rate of a narrow-line Yb MOT. The relative simplicity and generality of this approach make it readily adoptable to other experiments involving narrow-line MOTs. We also present a numerical simulation of this two-stage slowing process, which shows good agreement with the observed dependence on experimental parameters, and use it to assess potential improvements to the method.

Inducible transgene expression in PDX models in vivo identifies KLF4 as a therapeutic target for B-ALL.

BACKGROUND: Clinically relevant methods are not available that prioritize and validate potential therapeutic targets for individual tumors, from the vast amount of tumor descriptive expression data. METHODS: We established inducible transgene expression in clinically relevant patient-derived xenograft (PDX) models in vivo to fill this gap. RESULTS: With this technique at hand, we analyzed the role of the transcription factor Krüppel-like factor 4 (KLF4) in B-cell acute lymphoblastic leukemia (B-ALL) PDX models at different disease stages. In competitive preclinical in vivo trials, we found that re-expression of wild type KLF4 reduced the leukemia load in PDX models of B-ALL, with the strongest effects being observed after conventional chemotherapy in minimal residual disease (MRD). A nonfunctional KLF4 mutant had no effect on this model. The re-expression of KLF4 sensitized tumor cells in the PDX model towards systemic chemotherapy in vivo. It is of major translational relevance that azacitidine upregulated KLF4 levels in the PDX model and a KLF4 knockout reduced azacitidine-induced cell death, suggesting that azacitidine can regulate KLF4 re-expression. These results support the application of azacitidine in patients with B-ALL as a therapeutic option to regulate KLF4. CONCLUSION: Genetic engineering of PDX models allows the examination of the function of dysregulated genes like KLF4 in a highly clinically relevant translational context, and it also enables the selection of therapeutic targets in individual tumors and links their functions to clinically available drugs, which will facilitate personalized treatment in the future.

Critical phenomena in the temperature-pressure-crowding phase diagram of a protein.

In the cell, proteins fold and perform complex functions through global structural rearrangements. Function requires a protein to be at the brink of stability to be susceptible to small environmental fluctuations, yet stable enough to maintain structural integrity. These apparently conflicting behaviors are exhibited by systems near a critical point, where distinct phases merge-a concept beyond previous studies indicating proteins have a well-defined folded/unfolded phase boundary in the pressure-temperature plane. Here, by modeling the protein phosphoglycerate kinase (PGK) on the temperature (T), pressure (P), and crowding volume-fraction (ϕ) phase diagram, we demonstrate a critical transition where phases merge, and PGK exhibits large structural fluctuations. Above the critical point, the difference between the intermediate and unfolded phases disappears. When ϕ increases, the critical point moves to lower T c. We verify the calculations with experiments mapping the T-P-ϕ space, which likewise reveal a critical point at 305 K and 170 MPa that moves to lower T c as ϕ increases. Crowding places PGK near a critical line in its natural parameter space, where large conformational changes can occur without costly free energy barriers. Specific structures are proposed for each phase based on simulation.

Value of fluid-attenuated inversion recovery MRI data analyzed by the lesion segmentation toolbox in amyotrophic lateral sclerosis.

BACKGROUND: MRI fluid-attenuated inversion recovery (FLAIR) studies reported hyperintensity in the corticospinal tract and corpus callosum of patients with amyotrophic lateral sclerosis (ALS). PURPOSE: To evaluate the lesion segmentation toolbox (LST) for the objective quantification of FLAIR lesions in ALS patients. STUDY TYPE: Retrospective. POPULATION: Twenty-eight ALS patients (eight females, mean age: 50 range: 24-73, mean ALSFRS-R sum score: 36) were compared with 31 age-matched healthy controls (12 females, mean age: 45, range: 25-67). ALS patients were treated with riluzole and additional G-CSF (granulocyte-colony stimulating factor) on a named patient basis. FIELD STRENGTH/SEQUENCE: 1.5 T, FLAIR, T1 -weighted MRI. ASSESSMENT: The lesion prediction algorithm (LPA) of the LST enabled the extraction of individual binary lesion maps, total lesion volume (TLV), and number (TLN). Location and overlap of FLAIR lesions across patients were investigated by registration to FLAIR average space and an atlas. ALS-specific functional rating scale revised (ALSFRS-R), disease progression, and survival since diagnosis served as clinical correlates. STATISTICAL TESTS: Univariate analysis of variance (ANOVA), repeated-measures ANOVA, t-test, Bravais-Pearson correlation, Chi-square test of independence, Kaplan-Meier analysis, Cox-regression analysis. RESULTS: Both ALS patients and healthy controls exhibited FLAIR alterations. TLN significantly depended on age (F(1,54) = 24.659, P < 0.001) and sex (F(1,54) = 5.720, P = 0.020). ALS patients showed higher TLN than healthy controls depending on sex (F(1, 54) = 5.076, P = 0.028). FLAIR lesions were small and most pronounced in male ALS patients. FLAIR alterations were predominantly detected in the superior and posterior corona radiata, anterior capsula interna, and posterior thalamic radiation. Patients with pyramidal tract (PT) lesions exhibited significantly inferior survival than patients without PT lesions (P = 0.013). Covariate age exhibited strong prognostic value for survival (P = 0.015). DATA CONCLUSION: LST enables the objective quantification of FLAIR alterations and is a potential prognostic biomarker for ALS. LEVEL OF EVIDENCE: 3 Technical Efficacy: Stage 2 J. Magn. Reson. Imaging 2019;50:552-559.

Combinatory Biomarker Use of Cortical Thickness, MUNIX, and ALSFRS-R at Baseline and in Longitudinal Courses of Individual Patients With Amyotrophic Lateral Sclerosis.

Objective: Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative process affecting upper and lower motor neurons as well as non-motor systems. In this study, precentral and postcentral cortical thinning detected by structural magnetic resonance imaging (MRI) were combined with clinical (ALS-specific functional rating scale revised, ALSFRS-R) and neurophysiological (motor unit number index, MUNIX) biomarkers in both cross-sectional and longitudinal analyses. Methods: The unicenter sample included 20 limb-onset classical ALS patients compared to 30 age-related healthy controls. ALS patients were treated with standard Riluzole and additional long-term G-CSF (Filgrastim) on a named patient basis after written informed consent. Combinatory biomarker use included cortical thickness of atlas-based dorsal and ventral subdivisions of the precentral and postcentral cortex, ALSFRS-R, and MUNIX for the musculus abductor digiti minimi (ADM) bilaterally. Individual cross-sectional analysis investigated individual cortical thinning in ALS patients compared to age-related healthy controls in the context of state of disease at initial MRI scan. Beyond correlation analysis of biomarkers at cross-sectional group level (n = 20), longitudinal monitoring in a subset of slow progressive ALS patients (n = 4) explored within-subject temporal dynamics of repeatedly assessed biomarkers in time courses over at least 18 months. Results: Cross-sectional analysis demonstrated individually variable states of cortical thinning, which was most pronounced in the ventral section of the precentral cortex. Correlations of ALSFRS-R with cortical thickness and MUNIX were detected. Individual longitudinal biomarker monitoring in four slow progressive ALS patients revealed evident differences in individual disease courses and temporal dynamics of the biomarkers. Conclusion: A combinatory use of structural MRI, neurophysiological and clinical biomarkers allows for an appropriate and detailed assessment of clinical state and course of disease of ALS.

White Matter Connectivity of the Visual-Vestibular Cortex Examined by Diffusion-Weighted Imaging.

The parieto-insular vestibular cortex (PIVC) and the posterior insular cortex (PIC) are key regions of the cortical vestibular network, both located in the midposterior section of the lateral sulcus. Little is known about the structural connectivity pattern of these areas. We used probabilistic fiber tracking based on diffusion-weighted magnetic resonance imaging (MRI) and compared the ipsilateral connectivity of PIVC and PIC. Seed areas for the tracking algorithm were identified in each brain by functional MRI activity during caloric and visual motion stimulation, respectively. Cortical track terminations were investigated by a surface-based approach. Both PIVC and PIC shared ipsilateral connections to the insular/lateral sulcus, superior temporal cortex, and inferior frontal gyrus. However, PIVC showed significantly more connections than PIC with the anterior insula and Heschl's gyrus in both hemispheres and with the precuneus, intraparietal sulcus, and posterior callosum of the right hemisphere. In contrast, PIC connectivity was more pronounced with the supramarginal gyrus and superior temporal sulcus. Subcortical tracks were examined by a region-of-interest-based approach, which was validated on cortico-thalamic motor tracts. Both PIVC and PIC were connected with lateral nuclei of the thalamus and the basal ganglia (primarily putamen). PIVC tracks but not PIC tracks showed a right-hemispheric lateralization in cortical and subcortical connectivity. Overall, these results suggest that human PIVC and PIC share cortical and even subcortical connections. Nevertheless, they also differ in their primary connectivity pattern: PIVC is linked with posterior parietal and inferior frontal cortex, whereas PIC is linked with superior temporal and inferior parietal cortex.

Visual-vestibular processing in the human Sylvian fissure.

Unlike other sensory systems, the cortical organization of the human vestibular system is not well established. A central role is assumed for the region of the posterior Sylvian fissure, close to the posterior insula. At this site, activation during vestibular stimulation has been observed in previous imaging studies and labeled as the parieto-insular vestibular cortex area (PIVC). However, vestibular responses are found in other parts of the Sylvian fissure as well, including a region that is referred to as the posterior insular cortex (PIC). The anatomical and functional relationship between PIC and PIVC is still poorly understood, because both areas have never been compared in the same participants. Therefore, to better understand the apparently more complex organization of vestibular cortex in the Sylvian fissure, we employed caloric and visual object motion stimuli during functional magnetic resonance imaging and compared location and function of PIVC and PIC in the same participants. Both regions responded to caloric vestibular stimulation, but only the activation pattern in right PIVC reliably represented the direction of the caloric stimulus. Conversely, activity in PIVC was suppressed during stimulation with visual object motion, whereas PIC showed activation. Area PIC is located at a more posterior site in the Sylvian fissure than PIVC. Our results suggest that PIVC and PIC should be considered separate areas in the vestibular Sylvian network, both in terms of location and function.

The effect of educational information about treatments for schizophrenia on stigmatizing perceptions.

BACKGROUND: A promising strategy for reducing stigmatizing perceptions towards people with schizophrenia is education about treatment and recovery. The effects of different kinds of treatment information on stigmatizing perceptions, however, have yet to be compared directly. This study compared three different educational interventions focusing on medication, CBT, and psychodynamic psychotherapy in their potential to reduce stigmatizing perceptions towards people with schizophrenia. METHODS: In an online experiment 178 participants received one of three psychoeducation texts that focused on medication, CBT or psychodynamic therapy. The effects on stereotypical beliefs about psychosis (dangerousness, unpredictability, blame, prognostic pessimism) and emotional responses towards people with schizophrenia (anxiety, anger, sympathy) were tested. RESULTS: Perceptions of dangerousness, unpredictability, and anxiety towards people with schizophrenia were reduced in all conditions. Prognostic pessimism was reduced only after reading the CBT information. LIMITATIONS: No neutral control group was included. The sample was not representative with respect to level of education or gender. CONCLUSIONS: Stigmatizing perceptions may be reduced by receiving information about any type of treatment for psychosis and without producing negative side-effects, although this needs to be replicated in a controlled study. However, information on CBT seems most suitable to reduce stigma, since it was able to reduce prognostic pessimism.

ReAsH as a Quantitative Probe of In-Cell Protein Dynamics.

The tetracysteine (tc) tag/biarsenical dye system (FlAsH or ReAsH) promises to combine the flexibility of fluorescent protein tags with the small size of dye labels, allowing in-cell study of target proteins that are perturbed by large protein tags. Quantitative thermodynamic and kinetic studies in-cell using FlAsH and ReAsH have been hampered by methodological complexities presented by the fluorescence properties of the tag-dye complex probed by either Förster resonance energy transfer (FRET) or direct excitation. We label the model protein phosphoglycerate kinase (PGK) with AcGFP1 and ReAsH for direct comparison with AcGFP1/mCherry-labeled PGK. We find that fast relaxation imaging (FReI), combining millisecond temperature jump kinetics with fluorescence microscopy detection, circumvents many of the difficulties encountered working with the ReAsH system, allowing us to obtain quantitative FRET measurements of protein stability and kinetics both in vitro and in cells. We also demonstrate the to us surprising result that fluorescence from directly excited, unburied ReAsH at the C-terminus of the model protein also reports on folding in vitro and in cells. Comparing the ReAsH-labeled protein to a construct labeled with two fluorescent protein tags allows us to evaluate how a bulkier protein tag affects protein dynamics in cells and in vitro. We find that the average folding rate in the cell is closer to the in vitro rate with the smaller tag, highlighting the effect of tags on quantitative in-cell measurements.

Comparing Fast Pressure Jump and Temperature Jump Protein Folding Experiments and Simulations.

The unimolecular folding reaction of small proteins is now amenable to a very direct mechanistic comparison between experiment and simulation. We present such a comparison of microsecond pressure and temperature jump refolding kinetics of the engineered WW domain FiP35, a model system for ß-sheet folding. Both perturbations produce experimentally a faster and a slower kinetic phase, and the "slow" microsecond phase is activated. The fast phase shows differences between perturbation methods and is closer to the downhill limit by temperature jump, but closer to the transiently populated intermediate limit by pressure jump. These observations make more demands on simulations of the folding process than just a rough comparison of time scales. To complement experiments, we carried out several pressure jump and temperature jump all-atom molecular dynamics trajectories in explicit solvent, where FiP35 folded in five of the six simulations. We analyzed our pressure jump simulations by kinetic modeling and found that the pressure jump experiments and MD simulations are most consistent with a 4-state kinetic mechanism. Together, our experimental and computational data highlight FiP35's position at the boundary where activated intermediates and downhill folding meet, and we show that this model protein is an excellent candidate for further pressure jump molecular dynamics studies to compare experiment and modeling at the folding mechanism level.

Temporal variation of a protein folding energy landscape in the cell.

Chemical reaction rate coefficients and free energies are usually time-independent quantities. Protein folding in vitro is one such reaction with a fixed energy landscape. However, in the milieu of the cell, the energy landscape can be modulated in space and time by fluctuations in the intracellular environment such as cytoskeletal rearrangements, changes in biomolecule concentrations, and large scale cellular reorganization. We studied the time dependence of the folding landscape of a FRET-labeled enzyme, yeast phosphoglycerate kinase (PGK-FRET). Living U2OS cells served as our test tube, and the mammalian cell cycle, a process strictly regulated in time, served as our clock. We found that both the rate of folding and the thermodynamic stability of PGK-FRET are cell cycle-dependent. We also assayed folding rates of PGK-FRET in spatial proximity to and far away from mitotic chromosomes. Our results show that expedited folding in DNA-rich regions cannot account for the faster rate of PGK-FRET folding in mitotic cells.