Transcription factors interact with RNA to regulate genes.

Transcription factors (TFs) orchestrate the gene expression programs that define each cell's identity. The canonical TF accomplishes this with two domains, one that binds specific DNA sequences and the other that binds protein coactivators or corepressors. We find that at least half of TFs also bind RNA, doing so through a previously unrecognized domain with sequence and functional features analogous to the arginine-rich motif of the HIV transcriptional activator Tat. RNA binding contributes to TF function by promoting the dynamic association between DNA, RNA, and TF on chromatin. TF-RNA interactions are a conserved feature important for vertebrate development and disrupted in disease. We propose that the ability to bind DNA, RNA, and protein is a general property of many TFs and is fundamental to their gene regulatory function.

Distinct chemical environments in biomolecular condensates.

Diverse mechanisms have been described for selective enrichment of biomolecules in membrane-bound organelles, but less is known about mechanisms by which molecules are selectively incorporated into biomolecular assemblies such as condensates that lack surrounding membranes. The chemical environments within condensates may differ from those outside these bodies, and if these differed among various types of condensate, then the different solvation environments would provide a mechanism for selective distribution among these intracellular bodies. Here we use small molecule probes to show that different condensates have distinct chemical solvating properties and that selective partitioning of probes in condensates can be predicted with deep learning approaches. Our results demonstrate that different condensates harbor distinct chemical environments that influence the distribution of molecules, show that clues to condensate chemical grammar can be ascertained by machine learning and suggest approaches to facilitate development of small molecule therapeutics with optimal subcellular distribution and therapeutic benefit.

Enhanced osteochondral repair by leukocyte-depleted platelet-rich plasma in combination with adipose-derived mesenchymal stromal cells encapsulated in a three-dimensional photocrosslinked injectable hydrogel in a rabbit model.

INTRODUCTION: Intra-articular injection of adipose-derived mesenchymal stromal cells (ASCs) and/or platelet-rich plasma (PRP) have been reported to independently and synergistically improve healing of osteochondral lesions in animal models. However, their independent and combined effects when localized to an osteochondral lesion by encapsulation within a photocrosslinkable methacrylated gelatin hydrogel (GelMA) have not been explored. Herein we investigated a unique combination of allogeneic ASCs and PRP embedded in GelMA as a single-stage treatment for osteochondral regeneration in a rabbit model. METHODS: Thirty mature rabbits were divided into six experimental groups: (1) Sham; (2) Defect; (3) GelMA; (4) GelMA + ASCs; (5) GelMA + PRP; and (6) GelMA + ASCs + PRP.At 12 weeks following surgical repair, osteochondral regeneration was assessed on the basis of gross appearance, biomechanical properties, histological and immunohistochemical characteristics, and subchondral bone volume. RESULTS: In terms of mechanical property reflecting the ability of neotissue to bear stress, PRP only group were significantly lower than the Sham group (p = 0.0098). On the other hand, ASCs only and ASCs combined with PRP groups did not exhibit significantly difference, which suggesting that incorporation of ASCs assists in restoring the ability of the neotissue to bear stresses similarly to native tissue (p = 0.346, p = 0.40, respectively). Safranin O in ASCs combined with PRP group was significantly higher than the Defect and GelMA only groups (p = 0.0009, p = 0.0017, respectively). Additionally, ASCs only and ASCs combined with PRP groups presented especially strong staining for collagen type II. Surprisingly, PRP only and PRP + ASCs groups tended to exhibit higher collagen type I and collagen type X staining compared to ASCs only group, suggesting a potential PRP-mediated hypertrophic effect. CONCLUSION: Regeneration of a focal osteochondral defect in a rabbit model was improved by a single-stage treatment of a photocrosslinked hydrogel containing allogenic ASCs and autologous PRP, with the combination of ASCs and PRP producing superior benefit than either alone. No experimental construct fully restored all properties of the native, healthy osteochondral unit, which may require longer follow-up or further modification of PRP and/or ASCs characteristics.

A Mesoscale 3D Culture System for Native and Engineered Biphasic Tissues: Application to the Osteochondral Unit.

Interface tissues are functionally graded tissues characterized by a complex layered structure, which therefore present a great challenge to be reproduced and cultured in vitro. Here, we describe the design and operation of a 3D printed dual-chamber bioreactor as a culturing system for biphasic native or engineered osteochondral tissues. The bioreactor is designed to potentially accommodate a variety of interface tissues and enables the precise study of tissue crosstalk by creating two separate microenvironments while maintaining the tissue compartments in direct contact.

Dissecting the common and compartment-specific features of COVID-19 severity in the lung and periphery with single-cell resolution.

Severe COVID-19 is accompanied by rampant immune dysregulation in the lung and periphery, with immune cells of both compartments contributing to systemic distress. The extent to which immune cells of the lung and blood enter similar or distinct pathological states during severe disease remains unknown. Here, we leveraged 96 publicly available single-cell RNA sequencing datasets to elucidate common and compartment-specific features of severe to critical COVID-19 at the levels of transcript expression, biological pathways, and ligand-receptor signaling networks. Comparing severe patients to milder and healthy donors, we identified distinct differential gene expression signatures between compartments and a core set of co-directionally regulated surface markers. A majority of severity-enriched pathways were shared, whereas TNF and interferon responses were polarized. Severity-specific ligand-receptor networks appeared to be differentially active in both compartments. Overall, our results describe a nuanced response during severe COVID-19 where compartment plays a role in dictating the pathological state of immune cells.

Synthesis and characterization of CaSr-Metal Organic Frameworks for biodegradable orthopedic applications.

Metal-organic frameworks (MOFs) formed from metals and organic ligands, are crystalline materials that are degradable in aqueous medium, and capable of releasing Ca and Sr ions. In this manuscript, the ability of MOFs to degrade and release osteogenic Ca and Sr ions was investigated. MOFs were generated by choosing osteoinductive Ca and Sr metals, and an organic ligand 1,3,5 tricarboxylicbenzene (H3BTC) as a linker. These MOFs were able to induce in vitro biomineralization from pre-osteoblastic MC3T3 cells and human mesenchymal stem cells (hMSCs). Moreover, these MOFs (when loaded with dimethyloxalylglycine (DMOG)) induced vascular endothelial production from hMSCs. qRT-PCR analysis performed on hMSCs (isolated from femoral heads of patients undergoing joint arthroplasty) treated with MOFs crystals suggested that the CaSr-MOFs by themselves can upregulate osteogenic genes in hMSCs, which is the first time to our knowledge that this has been observed from MOFs.

Point-of-Care Procedure for Enhancement of Meniscal Healing in a Goat Model Utilizing Infrapatellar Fat Pad-Derived Stromal Vascular Fraction Cells Seeded in Photocrosslinkable Hydrogel.

BACKGROUND: Large radial tears of the meniscus involving the avascular region can compromise meniscal function and result in poor healing and subsequent osteochondral degeneration. Augmentation of surgical repairs with adipose-derived stromal vascular fraction (SVF), which contains mesenchymal stromal cells, may improve meniscal healing and preserve function (ie, chondroprotection). PURPOSES: (1) To develop a goat model of a radial meniscal tear with resulting osteoarthritis and (2) to explore the efficacy of a 1-step procedure utilizing infrapatellar fat pad-derived SVF cells seeded in a photocrosslinkable hydrogel to enhance meniscal healing and mitigate osteochondral degeneration. STUDY DESIGN: Controlled laboratory study. METHODS: A full-thickness radial tear spanning 90% of the medial meniscal width was made at the junction of the anterior and middle bodies of the goat stifle joint. Tears received 1 of 3 interventions (n = 4 per group): untreated, repair, or repair augmented with photocrosslinkable methacrylated gelatin hydrogel containing 2.0 × 106 SVF cells/mL and 2.0 µg/mL of transforming growth factor ß3. The contralateral (left) joint served as a healthy control. At 6 months, meniscal healing and joint health were evaluated by magnetic resonance imaging (MRI) and assessed by histological and macroscopic scoring. The Whole-Organ Magnetic Resonance Imaging Score and the presence of a residual tear, as evaluated with T2 MRI sequences, were determined by a single blinded orthopaedic surgeon. RESULTS: When compared with tears left untreated or repaired with suture alone, augmented repairs demonstrated increased tissue formation in the meniscal tear site, as seen on MRI and macroscopically. Likewise, the neotissue of augmented repairs possessed a histological appearance more similar, although still inferior, to healthy meniscus. Osteochondral degeneration in the medial compartment, as evaluated by the Whole-Organ Magnetic Resonance Imaging Score and Inoue (macroscopic) scale, revealed increased degeneration in the untreated and repair groups, which was mitigated in the augmented repair group. Histological evaluation with a modified Mankin score showed a similar trend. In all measures of osteochondral degeneration, the augmented repair group did not differ significantly from the uninjured control. CONCLUSION: A radial tear spanning 90% of the medial meniscal width in a goat stifle joint showed poor healing potential and resulted in osteochondral degeneration by 6 months, even if suture repair was performed. Augmentation of the repair with a photocrosslinkable hydrogel containing transforming growth factor ß3 and SVF cells, isolated intraoperatively by rapid enzymatic digestion, improved meniscal healing and mitigated osteoarthritic changes. CLINICAL RELEVANCE: Repair augmentation with an SVF cell-seeded hydrogel may support successful repair of meniscal tears previously considered irreparable.

Enhancing chondrogenesis and mechanical strength retention in physiologically relevant hydrogels with incorporation of hyaluronic acid and direct loading of TGF-ß.

Cell-loaded hydrogels are frequently applied in cartilage tissue engineering for their biocompatibility, ease of application, and ability to conform to various defect sites. As a bioactive adjunct to the biomaterial, transforming growth factor beta (TGF-ß) has been shown to be essential for cell differentiation into a chondrocyte phenotype and maintenance thereof, but the low amounts of endogenous TGF-ß in the in vivo joint microenvironment necessitate a mechanism for controlled delivery and release of this growth factor. In this study, TGF-ß3 was directly loaded with human bone marrow-derived mesenchymal stem cells (MSCs) into poly-d,l-lactic acid/polyethylene glycol/poly-d,l-lactic acid (PDLLA-PEG) hydrogel, or PDLLA-PEG with the addition of hyaluronic acid (PDLLA/HA), and cultured in vitro. We hypothesize that the inclusion of HA within PDLLA-PEG would result in a controlled release of the loaded TGF-ß3 and lead to a robust cartilage formation without the use of TGF-ß3 in the culture medium. ELISA analysis showed that TGF-ß3 release was effectively slowed by HA incorporation, and retention of TGF-ß3 in the PDLLA/HA scaffold was detected by immunohistochemistry for up to 3 weeks. By means of both in vitro culture and in vivo implantation, we found that sulfated glycosaminoglycan production was higher in PDLLA/HA groups with homogenous distribution throughout the scaffold than PDLLA groups. Finally, with an optimal loading of TGF-ß3 at 10 µg/mL, as determined by RT-PCR and glycosaminoglycan production, an almost twofold increase in Young's modulus of the construct was seen over a 4-week period compared to TGF-ß3 delivery in the culture medium. Taken together, our results indicate that the direct loading of TGF-ß3 and stem cells in PDLLA/HA has the potential to be a one-step point-of-care treatment for cartilage injury. STATEMENT OF SIGNIFICANCE: Stem cell-seeded hydrogels are commonly used in cell-based cartilage tissue engineering, but they generally fail to possess physiologically relevant mechanical properties suitable for loading. Moreover, degradation of the hydrogel in vivo with time further decreases mechanical suitability of the hydrogel due in part to the lack of TGF-ß3 signaling. In this study, we demonstrated that incorporation of hyaluronic acid (HA) into a physiologically stiff PDLLA-PEG hydrogel allowed for slow release of one-time preloaded TGF-ß3, and when loaded with adult mesenchymal stem cells and cultured in vitro, it resulted in higher chondrogenic gene expression and constructs of significantly higher mechanical strength than constructs cultured in conventional TGF-ß3-supplemented medium. Similar effects were also observed in constructs implanted in vivo. Our results indicate that direct loading of TGF-ß3 combined with HA in the physiologically stiff PDLLA-PEG hydrogel has the potential to be used for one-step point-of-care treatment of cartilage injury.