The bromodomain protein Brd4 is a positive regulatory component of P-TEFb and stimulates RNA polymerase II-dependent transcription.

In eukaryotes, the core promoter serves as a platform for the assembly of transcription preinitiation complex (PIC) that includes TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH, and RNA polymerase II (pol II), which function collectively to specify the transcription start site. PIC formation usually begins with TFIID binding to the TATA box, initiator, and/or downstream promoter element (DPE) found in most core promoters, followed by the entry of other general transcription factors (GTFs) and pol II through either a sequential assembly or a preassembled pol II holoenzyme pathway. Formation of this promoter-bound complex is sufficient for a basal level of transcription. However, for activator-dependent (or regulated) transcription, general cofactors are often required to transmit regulatory signals between gene-specific activators and the general transcription machinery. Three classes of general cofactors, including TBP-associated factors (TAFs), Mediator, and upstream stimulatory activity (USA)-derived positive cofactors (PC1/PARP-1, PC2, PC3/DNA topoisomerase I, and PC4) and negative cofactor 1 (NC1/HMGB1), normally function independently or in combination to fine-tune the promoter activity in a gene-specific or cell-type-specific manner. In addition, other cofactors, such as TAF1, BTAF1, and negative cofactor 2 (NC2), can also modulate TBP or TFIID binding to the core promoter. In general, these cofactors are capable of repressing basal transcription when activators are absent and stimulating transcription in the presence of activators. Here we review the roles of these cofactors and GTFs, as well as TBP-related factors (TRFs), TAF-containing complexes (TFTC, SAGA, SLIK/SALSA, STAGA, and PRC1) and TAF variants, in pol II-mediated transcription, with emphasis on the events occurring after the chromatin has been remodeled but prior to the formation of the first phosphodiester bond.

The general transcription machinery and general cofactors.

Drug development is a lengthy and costly process that proceeds through several stages from target identification to lead discovery and optimization, preclinical validation and clinical trials culminating in approval for clinical use. An important step in this process is high-throughput screening (HTS) of small compound libraries for lead identification. Currently, the majority of cell-based HTS is being carried out on cultured cells propagated in two-dimensions (2D) on plastic surfaces optimized for tissue culture. At the same time, compelling evidence suggests that cells cultured in these non-physiological conditions are not representative of cells residing in the complex microenvironment of a tissue. This discrepancy is thought to be a significant contributor to the high failure rate in drug discovery, where only a low percentage of drugs investigated ever make it through the gamut of testing and approval to the market. Thus, three-dimensional (3D) cell culture technologies that more closely resemble in vivo cell environments are now being pursued with intensity as they are expected to accommodate better precision in drug discovery. Here we will review common approaches to 3D culture, discuss the significance of 3D cultures in drug resistance and drug repositioning and address some of the challenges of applying 3D cell cultures to high-throughput drug discovery.

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Three-Dimensional in Vitro Cell Culture Models in Drug Discovery and Drug Repositioning.

Cell adhesion to the extracellular matrix is fundamental to tissue integrity and human health Integrins are the main cellular adhesion receptors that through multifaceted roles as signalling molecules, mechanotransducers and key components of the cell migration machinery are implicated in nearly every step of cancer progression from primary tumour development to metastasis Altered integrin expression is frequently detected in tumours, where integrins have roles in supporting oncogenic growth factor receptor (GFR) signalling and GFR-dependent cancer cell migration and invasion In addition, integrins determine colonization of metastatic sites and facilitate anchorage-independent survival of circulating tumour cells Investigations describing integrin engagement with a growing number of versatile cell surface molecules, including channels, receptors and secreted proteins, continue to lead to the identification of novel tumour-promoting pathways Integrin-mediated sensing, stiffening and remodelling of the tumour stroma are key steps in cancer progression supporting invasion, acquisition of cancer stem cell characteristics and drug resistance Given the complexity of integrins and their adaptable and sometimes antagonistic roles in cancer cells and the tumour microenvironment, therapeutic targeting of these receptors has been a challenge However, novel approaches to target integrins and antagonism of specific integrin subunits in stringently stratified patient cohorts are emerging as potential ways forward

/pdf/every-step-of-the-way-integrins-in-cancer-progression-and-bjuke49q8c.pdf

Every step of the way: integrins in cancer progression and metastasis.

The human positive transcription elongation factor P-TEFb, consisting of a CDK9/cyclin T1 heterodimer, functions as both a general and an HIV-1 Tat-specific transcription factor. P-TEFb activates transcription by phosphorylating RNA polymerase (Pol) II, leading to the formation of processive elongation complexes. As a Tat cofactor, P-TEFb stimulates HIV-1 transcription by interacting with Tat and the transactivating responsive (TAR) RNA structure located at the 5' end of the nascent viral transcript. Here we identified 7SK, an abundant and evolutionarily conserved small nuclear RNA (snRNA) of unknown function, as a specific P-TEFb-associated factor. 7SK inhibits general and HIV-1 Tat-specific transcriptional activities of P-TEFb in vivo and in vitro by inhibiting the kinase activity of CDK9 and preventing recruitment of P-TEFb to the HIV-1 promoter. 7SK is efficiently dissociated from P-TEFb by treatment of cells with ultraviolet irradiation and actinomycin D. As these two agents have been shown to significantly enhance HIV-1 transcription and phosphorylation of Pol II (refs 6,7,8), our data provide a mechanistic explanation for their stimulatory effects. The 7SK/P-TEFb interaction may serve as a principal control point for the induction of cellular and HIV-1 viral gene expression during stress-related responses. Our studies demonstrate the involvement of an snRNA in controlling the activity of a Cdk-cyclin kinase.

The 7SK small nuclear RNA inhibits the CDK9/cyclin T1 kinase to control transcription

Tissue development is orchestrated by the coordinated activities of both chemical and physical regulators. While much attention has been given to the role that chemical regulators play in driving development, researchers have recently begun to elucidate the important role that the mechanical properties of the extracellular environment play. For instance, the stiffness of the extracellular environment has a role in orienting cell division, maintaining tissue boundaries, directing cell migration, and driving differentiation. In addition, extracellular matrix stiffness is important for maintaining normal tissue homeostasis, and when matrix mechanics become imbalanced, disease progression may ensue. In this article, we will review the important role that matrix stiffness plays in dictating cell behavior during development, tissue homeostasis, and disease progression.

https://www.tandfonline.com/doi/pdf/10.1080/15476278.2015.1019687

Tissue Stiffness Dictates Development, Homeostasis, and Disease Progression

Tat stimulates human immunodeficiency virus type 1 (HIV-1) transcriptional elongation by recruitment of carboxyl-terminal domain (CTD) kinases to the HIV-1 promoter. Using an immobilized DNA template assay, we have analyzed the effect of Tat on kinase activity during the initiation and elongation phases of HIV-1 transcription. Our results demonstrate that cyclin-dependent kinase 7 (CDK7) (TFIIH) and CDK9 (P-TEFb) both associate with the HIV-1 preinitiation complex. Hyperphosphorylation of the RNA polymerase II (RNAP II) CTD in the HIV-1 preinitiation complex, in the absence of Tat, takes place at CTD serine 2 and serine 5. Analysis of preinitiation complexes formed in immunodepleted extracts suggests that CDK9 phosphorylates serine 2, while CDK7 phosphorylates serine 5. Remarkably, in the presence of Tat, the substrate specificity of CDK9 is altered, such that the kinase phosphorylates both serine 2 and serine 5. Tat-induced CTD phosphorylation by CDK9 is strongly inhibited by low concentrations of 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole, an inhibitor of transcription elongation by RNAP II. Analysis of stalled transcription elongation complexes demonstrates that CDK7 is released from the transcription complex between positions +14 and +36, prior to the synthesis of transactivation response (TAR) RNA. In contrast, CDK9 stays associated with the complex through +79. Analysis of CTD phosphorylation indicates a biphasic modification pattern, one in the preinitiation complex and the other between +36 and +79. The second phase of CTD phosphorylation is Tat-dependent and TAR-dependent. These studies suggest that the ability of Tat to increase transcriptional elongation may be due to its ability to modify the substrate specificity of the CDK9 complex.

Tat modifies the activity of CDK9 to phosphorylate serine 5 of the RNA polymerase II carboxyl-terminal domain during human immunodeficiency virus type 1 transcription

During the past three decades, internal fixation has become increasingly popular for fracture management and limb reconstruction. As a result, during their training, orthopaedic surgeons receive less formal instruction in the art of extremity immobilization and cast application and removal. Casting is not without risks and complications (eg, stiffness, pressure sores, compartment syndrome); the risk of morbidity is higher when casts are applied by less experienced practitioners. Certain materials and methods of ideal cast and splint application are recommended to prevent morbidity in the patient who is at high risk for complications with casting and splinting. Those at high risk include the obtunded or comatose multitrauma patient, the patient under anesthesia, the very young patient, the developmentally delayed patient, and the patient with spasticity.

Cast and splint immobilization: complications.

Tendon healing follows a complex series of coordinated events, which ultimately produces a mechanically inferior tissue more scar-like than native tendon. More regenerative healing occurs when anti-inflammatory M2 macrophages play a more dominant role. Mesenchymal stromal/stem cells (MSCs) are able to polarize macrophages to an M2 immunophenotype via paracrine mechanisms. We previously reported that coculture of CD14+ macrophages (MQs) with MSCs resulted in a unique M2-like macrophage. More recently, we generated M2-like macrophages using only extracellular vesicles (EVs) isolated from MSCs creating "EV-educated macrophages" (also called exosome-educated macrophages [EEMs]), thereby foregoing direct use of MSCs. For the current study, we hypothesized that cell therapy with EEMs would improve in vivo tendon healing by modulating tissue inflammation and endogenous macrophage immunophenotypes. We evaluated effects of EEMs using a mouse Achilles tendon rupture model and compared results to normal tendon healing (without any biologic intervention), MSCs, MQs, or EVs. We found that exogenous administration of EEMs directly into the wound promoted a healing response that was significantly more functional and more regenerative. Injured tendons treated with exogenous EEMs exhibited (a) improved mechanical properties, (b) reduced inflammation, and (c) earlier angiogenesis. Treatment with MSC-derived EVs alone were less effective functionally but stimulated a biological response as evidenced by an increased number of endothelial cells and decreased M1/M2 ratio. Because of their regenerative and immunomodulatory effects, EEM treament could provide a novel strategy to promote wound healing in this and various other musculoskeletal injuries or pathologies where inflammation and inadequate healing is problematic. Stem Cells 2019;37:652-662.

https://stemcellsjournals.onlinelibrary.wiley.com/doi/epdf/10.1002/stem.2988

Extracellular Vesicle-Educated Macrophages Promote Early Achilles Tendon Healing.

Background: Current trends in the treatment of idiopathic clubfoot have shifted from extensive surgical release to more conservative techniques. The purpose of the present study was to prospectively compare the results of the Ponseti method with those of surgical releases for the correction of clubfoot deformity.

Methods: We prospectively compared patients who had idiopathic clubfoot deformities that were treated at a single institution either with the Ponseti method or with below-the-knee casting followed by surgical release. The clinical records of the patients with a minimum duration of follow-up of two years were reviewed. All scheduled and completed operative interventions and associated complications were recorded.

Results: Fifty-five patients with eighty-six clubfeet were treated; forty feet were included in the group that was treated with the Ponseti method, and forty-six feet were included in the group that was treated with below-the-knee casts followed by surgery (with three of these feet requiring casting only). There was no difference between the groups in terms of sex, ethnicity, age at the time of first casting, pretreatment Pirani score (average, 5.2 in both groups), or family history. The average number of casts was six in the Ponseti group and thirteen in the surgical group. Of the feet that were treated with below-the-knee casts, forty-three underwent surgery, with forty-two undergoing major surgery (posterior release [eleven] or posteromedial release [thirty-one]). In the Ponseti group, fourteen feet required fifteen operative interventions for recurrences, with only one foot requiring revision surgery. Four of these fifteen were major (necessitating posterior [one] or posteromedial release [three]) while eleven were minor. Thirteen feet in the surgical group required fourteen surgical revisions. Two postoperative complications were seen in each group.

Conclusions: While both cohorts had a relatively high recurrence rate, the Ponseti cohort was managed with significantly less operative intervention and required less revision surgery. The Ponseti method has now been adopted as the primary treatment for clubfoot at our institution.

Level of Evidence: Therapeutic Level II. See Instructions to Authors for a complete description of levels of evidence.

Matthew A. Halanski

Papers

Tissue Stiffness Dictates Development, Homeostasis, and Disease Progression

Tat modifies the activity of CDK9 to phosphorylate serine 5 of the RNA polymerase II carboxyl-terminal domain during human immunodeficiency virus type 1 transcription

Cast and splint immobilization: complications.

Extracellular Vesicle-Educated Macrophages Promote Early Achilles Tendon Healing.

Ponseti method compared with surgical treatment of clubfoot: a prospective comparison.