Helminth egg derivatives as proregenerative immunotherapies.

The immune system is increasingly recognized as an important regulator of tissue repair. We developed a regenerative immunotherapy from the helminth Schistosoma mansoni soluble egg antigen (SEA) to stimulate production of interleukin (IL)-4 and other type 2-associated cytokines without negative infection-related sequelae. The regenerative SEA (rSEA) applied to a murine muscle injury induced accumulation of IL-4-expressing T helper cells, eosinophils, and regulatory T cells and decreased expression of IL-17A in gamma delta (γδ) T cells, resulting in improved repair and decreased fibrosis. Encapsulation and controlled release of rSEA in a hydrogel further enhanced type 2 immunity and larger volumes of tissue repair. The broad regenerative capacity of rSEA was validated in articular joint and corneal injury models. These results introduce a regenerative immunotherapy approach using natural helminth derivatives.

A Thermodynamic Atlas of Proteomes Reveals Energetic Innovation across the Tree of Life.

Protein stability is a fundamental molecular property enabling organisms to adapt to their biological niches. How this is facilitated and whether there are kingdom specific or more general universal strategies are unknown. A principal obstacle to addressing this issue is that the vast majority of proteins lack annotation, specifically thermodynamic annotation, beyond the amino acid and chromosome information derived from genome sequencing. To address this gap and facilitate future investigation into large-scale patterns of protein stability and dynamics within and between organisms, we applied a unique ensemble-based thermodynamic characterization of protein folds to a substantial portion of extant sequenced genomes. Using this approach, we compiled a database resource focused on the position-specific variation in protein stability. Interrogation of the database reveals: 1) domains of life exhibit distinguishing thermodynamic features, with eukaryotes particularly different from both archaea and bacteria; 2) the optimal growth temperature of an organism is proportional to the average apolar enthalpy of its proteome; 3) intrinsic disorder content is also proportional to the apolar enthalpy (but unexpectedly not the predicted stability at 25 °C); and 4) secondary structure and global stability information of individual proteins is extractable. We hypothesize that wider access to residue-specific thermodynamic information of proteomes will result in deeper understanding of mechanisms driving functional adaptation and protein evolution. Our database is free for download at https://afc-science.github.io/thermo-env-atlas/ (last accessed January 18, 2022).

Phosphorylation Increases Persistence Length and End-to-End Distance of a Segment of Tau Protein.

Intrinsically disordered regions of proteins, which lack unique tertiary structure under physiological conditions, are enriched in phosphorylation sites and in significant local bias toward the polyproline II conformation. The overrepresented coincidence of this posttranslational regulatory signal and local conformational bias within unstructured regions raises a question: can phosphorylation serve to manipulate the conformational preferences of a disordered protein? In this study, we use time-resolved fluorescence resonance energy transfer and a, to our knowledge, novel data analysis method to directly measure the end-to-end distance distribution of a phosphorylatable peptide derived from the human microtubule associated protein tau. Our results show that phosphorylation at threonine or serine extends the end-to-end distance and increases the effective persistence length of the tested model peptides. Unexpectedly, the extension is independent of salt concentration, suggestive of a nonelectrostatic origin. The phosphorylation extension and stiffening effect provides a peptide-scale physical interpretation for the posttranslational regulation of the highly abundant protein-protein interactions found in disordered proteins, as well as a potential insight into the regulatory mechanism of the tau protein's microtubule binding activity.

Senolytic treatment reduces oxidative protein stress in an aging male murine model of post-traumatic osteoarthritis.

Senolytic drugs are designed to selectively clear senescent cells (SnCs) that accumulate with injury or aging. In a mouse model of osteoarthritis (OA), senolysis yields a pro-regenerative response, but the therapeutic benefit is reduced in aged mice. Increased oxidative stress is a hallmark of advanced age. Therefore, here we investigate whether senolytic treatment differentially affects joint oxidative load in young and aged animals. We find that senolysis by a p53/MDM2 interaction inhibitor, UBX0101, reduces protein oxidative modification in the aged arthritic knee joint. Mass spectrometry coupled with protein interaction network analysis and biophysical stability prediction of extracted joint proteins revealed divergent responses to senolysis between young and aged animals, broadly suggesting that knee regeneration and cellular stress programs are contrarily poised to respond as a function of age. These opposing responses include differing signatures of protein-by-protein oxidative modification and abundance change, disparate quantitative trends in modified protein network centrality, and contrasting patterns of oxidation-induced folding free energy perturbation between young and old. We develop a composite sensitivity score to identify specific key proteins in the proteomes of aged osteoarthritic joints, thereby nominating prospective therapeutic targets to complement senolytics.

Senescent cells in tissue engineering.

Tissue engineers have long worked to develop cells, biomaterial matrices, and signaling molecules designed to restore or promote the repair of lost or damaged tissue. Senescent cells (SnCs), that is, cells that have entered permanent cell-cycle arrest, exert powerful cell non-autonomous effects on their local environments. As such, SnCs influence cell fates and pathologies in adult tissue, including in settings where tissue engineers have directed their efforts. Here, we compare transient SnCs in tissue repair, contrasted with chronic SnCs in osteoarthritic pathology and the foreign-body response. Then, we discuss recent advances in strategies to control the presence and downstream effects of SnCs in tissues, such as immunomodulatory biomaterials, human trials of senolytic molecules, and senescent-cell-directed CAR-T therapy.

Phylogenetic convergence of phase separation and mitotic function in the disordered protein BuGZ.

Intrinsically disordered proteins (IDPs) effect biological function despite their sequence-encoded lack of preference for stable three-dimensional structure. Among their many functions, IDPs form membraneless cellular compartments through liquid-liquid phase separation (LLPS), also termed biomolecular condensation. The extent to which LLPS has been evolutionarily selected remains largely unknown, as the complexities of IDP evolution hamper progress. Unlike structured proteins, rapid sequence divergence typical of IDPs confounds inference of their biophysical or biological functions from comparative sequence analyses. Here, we leverage mitosis as a universal eukaryotic feature to interrogate condensate evolutionary history. We observe that evolution has conserved the ability for six homologs of the mitotic IDP BuGZ to undergo LLPS and to serve the same mitotic function, despite low sequence conservation. We also observe that cellular context may tune LLPS. The phylogenetic correlation of LLPS and mitotic function in one protein raises the possibility of an ancient evolutionary interplay between LLPS and biological function, dating back at least 1.6 billion years to the last common ancestor of plants and animals.

Mechanical stimulation enhances development of scaffold-free, 3D-printed, engineered heart tissue grafts.

Current efforts to engineer a clinically relevant tissue graft from human-induced pluripotent stem cells (hiPSCs) have relied on the addition or utilization of external scaffolding material. However, any imbalance in the interactions between embedded cells and their surroundings may hinder the success of the resulting tissue graft. Therefore, the goal of our study was to create scaffold-free, 3D-printed cardiac tissue grafts from hiPSC-derived cardiomyocytes (CMs), and to evaluate whether or not mechanical stimulation would result in improved graft maturation. To explore this, we used a 3D bioprinter to produce scaffold-free cardiac tissue grafts from hiPSC-derived CM cell spheroids. Static mechanical stretching of these grafts significantly increased sarcomere length compared to unstimulated free-floating tissues, as determined by immunofluorescent image analysis. Stretched tissue was found to have decreased elastic modulus, increased maximal contractile force, and increased alignment of formed extracellular matrix, as expected in a functionally maturing tissue graft. Additionally, stretched tissues had upregulated expression of cardiac-specific gene transcripts, consistent with increased cardiac-like cellular identity. Finally, analysis of extracellular matrix organization in stretched grafts suggests improved remodeling by embedded cardiac fibroblasts. Taken together, our results suggest that mechanical stretching stimulates hiPSC-derived CMs in a 3D-printed, scaffold-free tissue graft to develop mature cardiac material structuring and cellular fates. Our work highlights the critical role of mechanical conditioning as an important engineering strategy toward developing clinically applicable, scaffold-free human cardiac tissue grafts.

Type 2 immunity induced by bladder extracellular matrix enhances corneal wound healing.

The avascular nature of cornea tissue limits its regenerative potential, which may lead to incomplete healing and formation of scars when damaged. Here, we applied micro- and ultrafine porcine urinary bladder matrix (UBM) particulate to promote type 2 immune responses in cornea wounds. Results demonstrated that UBM particulate substantially reduced corneal haze formation as compared to the saline-treated group. Flow cytometry and gene expression analysis showed that UBM particulate suppressed the differentiation of corneal stromal cells into α-smooth muscle actin-positive (αSMA+) myofibroblasts. UBM treatments up-regulated interleukin-4 (IL-4) produced primarily by eosinophils in the wounded corneas and CD4+ T cells in draining lymph nodes, suggesting a cross-talk between local and peripheral immunity. Gata1-/- mice lacking eosinophils did not respond to UBM treatment and had impaired wound healing. In summary, stimulating type 2 immune responses in the wounded cornea can promote proregenerative environments that lead to improved wound healing for vision restoration.

Adherence to Emergency Public Health Measures for Bioevents: Review of US Studies.

The frequency of bioevents is increasing worldwide. In the United States, as elsewhere, control of contagion may require the cooperation of community members with emergency public health measures. The US general public is largely unfamiliar with these measures, and our understanding of factors that influence behaviors in this context is limited. The few previous reviews of research on this topic focused on non-US samples. For this review, we examined published research on the psychosocial influences of adherence in US sample populations. Of 153 articles identified, only 9 met the inclusion criteria. Adherence behaviors were categorized into 2 groups: self-protective behaviors (personal hygiene, social distancing, face mask use, seeking out health care advice, and vaccination) and protecting others (isolation, temperature screening, and quarantine). A lack of uniformity across studies regarding definitions and measures was noted. Only 5 of the 9 articles reported tests of association between adherence with emergency measures and psychosocial factors; perceived risk and perceived seriousness were found to be significantly associated with adherence or adherence intentions. Although it is well documented that psychosocial factors are important predictors of protective health behaviors in general, this has not been rigorously studied in the context of bioevents. (Disaster Med Public Health Preparedness. 2018;12:528-535).

What's in an Average? An Ensemble View of Phosphorylation Effects.

The number of examples of functional post-translational modulation of disordered proteins rapidly grows. In a recent issue of Structure, Jie et al. (2017) show that phosphorylation of dynein intermediate chain alters partner binding but retains its dynamic, disorder character. An ensemble model offers an organizing framework to relate function, conformation, and phosphorylation in disordered and ordered proteins alike.

Using biomimetic polymers in place of noncollagenous proteins to achieve functional remineralization of dentin tissues.

In calcified tissues such as bones and teeth, mineralization is regulated by an extracellular matrix, which includes non-collagenous proteins (NCP). This natural process has been adapted or mimicked to restore tissues following physical damage or demineralization by using polyanionic acids in place of NCPs, but the remineralized tissues fail to fully recover their mechanical properties. Here we show that pre-treatment with certain amphiphilic peptoids, a class of peptide-like polymers consisting of N-substituted glycines that have defined monomer sequences, enhances ordering and mineralization of collagen and induces functional remineralization of dentin lesions in vitro. In the vicinity of dentin tubules, the newly formed apatite nano-crystals are co-aligned with the c-axis parallel to the tubular periphery and recovery of tissue ultrastructure is accompanied by development of high mechanical strength. The observed effects are highly sequence-dependent with alternating polar and non-polar groups leading to positive outcomes while diblock sequences have no effect. The observations suggest aromatic groups interact with the collagen while the hydrophilic side chains bind the mineralizing constituents and highlight the potential of synthetic sequence-defined biomimetic polymers to serve as NCP mimics in tissue remineralization.

Effect of Diffusion on Resonance Energy Transfer Rate Distributions: Implications for Distance Measurements.

Intrinsically disordered protein regions and many other biopolymers lack the three-dimensional structure that could be determined by X-ray crystallography or NMR, which encourages the application of alternative experimental methods. Time-resolved resonance energy transfer data are often used to measure distances between two fluorophores attached to a flexible biopolymer. This is complicated by the rotational and translational diffusion of the fluorophores and by nonmonoexponential donor decay in the absence of the acceptor. Equation I(DA)(t) = I(D)(t)·F(t) is derived here, which is applicable regardless of whether I(D)(t) is monoexponential. I(D)(t) and I(DA)(t) are the δ-excitation donor emission decays in the absence and in the presence of the acceptor; F(t) contains information about energy transfer, donor-acceptor distance distribution, and diffusion dynamics. It is shown that in the absence of rotational and translational diffusion, F(t) is a continuous distribution of exponentials, whereas in the presence of rotational and translational diffusion, F(t) is a sum of discrete exponentials. For each case it is shown how F(t) is related to the distance distribution. Experimental data obtained with a flexible tetradecapeptide in aqueous solution clearly demonstrate that F(t) is a sum of discrete exponential terms. A partial differential equation describing resonance energy transfer in the presence of both rotational and translational diffusion of the donor and acceptor tethered to the ends of a semiflexible chain is solved in this work using a combination of analytical and numerical methods; the solution is used to fit time-resolved emission of the donor, which makes it possible to determine the model parameters: contour length, persistence length, and the end-to-end translational diffusion coefficient.

In vitro evaluation of adhesive characteristics of 4-META/MMA-TBB resin with organic filler.

OBJECTIVES: A commercial restorative material, BondfillSB (BF), is a modification of 4-META/MMA-TBB resin cement. BF uses a self-etching primer and added pre-polymerized organic fillers. We compared BF with another self-etching system, EasyBond (EB), in shear bond strength, bonded interface characteristics to human dentin and contraction gap when used in bulk-filling. METHODS: Shear bond strength of BF and EB + Z100 (Z), bonded by different experience-level operators, was evaluated. Bonded interfaces were characterized by SEM, AFM, and AFM based nano-indentation. Contraction gaps (CG) at 0h and 24h after polymerization were evaluated for BF or EB bulk filled class I cavities. To meet the clinical recommendation, BF's powder was replaced by experimental radioopaque powder (BFO) for the CG study. EB was used with Z (EBZ) or with a resin marketed for bulk-fill base (SureFil-SDR-flow (EBSF)). RESULTS: Shear bond strengths (Mean ± Standard Deviation (S.D.)) of BF (37.4 ± 2.6 MPa; n=36) were higher and less variable than EBZ (18.2 ± 7.6 MPa; n=36) (p<0.0001, One-way ANOVA). Weibull characteristic strength (η) differed significantly between materials (p<0.0001) but not between operators (p=0.90). EBZ often had non-uniform interfaces and a wider band of reduced elastic modulus (E) of greater than 20 µm across the interface. BF had uniform interfaces and a smaller width of affected dentin under the interface (∼1 µm). There was a difference in dentin-E between EBZ and BF up to 9 µm from the interface (mixed-effects model; P=0.03). A stratified linear regression model used for CG. EBSF and BFO showed significantly smaller CG than EBZ at time 0. None of three combinations showed any significant change between 0h-CG and 24h-CG. SIGNIFICANCE: BF possessed bonding characteristics required to serve as a restorative.