Functional partitioning of transcriptional regulators by patterned charge blocks.

Components of transcriptional machinery are selectively partitioned into specific condensates, often mediated by protein disorder, yet we know little about how this specificity is achieved. Here, we show that condensates composed of the intrinsically disordered region (IDR) of MED1 selectively partition RNA polymerase II together with its positive allosteric regulators while excluding negative regulators. This selective compartmentalization is sufficient to activate transcription and is required for gene activation during a cell-state transition. The IDRs of partitioned proteins are necessary and sufficient for selective compartmentalization and require alternating blocks of charged amino acids. Disrupting this charge pattern prevents partitioning, whereas adding the pattern to proteins promotes partitioning with functional consequences for gene activation. IDRs with similar patterned charge blocks show similar partitioning and function. These findings demonstrate that disorder-mediated interactions can selectively compartmentalize specific functionally related proteins from a complex mixture of biomolecules, leading to regulation of a biochemical pathway.

Phosphorylation-mediated regulation of the Bacillus anthracis phosphoglycerate mutase by the Ser/Thr protein kinase PrkC.

Bacillus anthracis Ser/Thr protein kinase PrkC is necessary for phenotypic memory and spore germination, and the loss of PrkC-dependent phosphorylation events affect the spore development. During sporulation, Bacillus sp. can store 3-Phosphoglycerate (3-PGA) that will be required at the onset of germination when ATP will be necessary. The Phosphoglycerate mutase (Pgm) catalyzes the isomerization of 2-PGA and 3-PGA and is important for spore germination as a key metabolic enzyme that maintains 3-PGA pool at later events. Therefore, regulation of Pgm is important for an efficient spore germination process and metabolic switching. While the increased expression of Pgm in B. anthracis decreases spore germination efficiency, it remains unexplored if PrkC could directly influence Pgm activity. Here, we report the phosphorylation and regulation of Pgm by PrkC and its impact on Pgm stability and catalytic activity. Mass spectrometry revealed Pgm phosphorylation on seven threonine residues. In silico mutational analysis highlighted the role of Thr459 residue towards metal and substrate binding. Altogether, we demonstrated that PrkC-mediated Pgm phosphorylation negatively regulates its activity that is essential to maintain Pgm in its apo-like isoform before germination. This study advances the role of Pgm regulation that represents an important switch for B. anthracis resumption of metabolism and spore germination.

Polyphenols in combination with ß-cyclodextrin can inhibit and disaggregate α-synuclein amyloids under cell mimicking conditions: A promising therapeutic alternative.

Parkinson's disease is characterized by the presence of insoluble and neurotoxic aggregates (amyloid fibrils) of an intrinsically disordered protein α-synuclein. In this study we have examined the effects of four naturally occurring polyphenols in combination with ß-cyclodextrin (ß-CD) on the aggregation of α-synuclein in the presence of macromolecular crowding agents. Our results reveal that even at sub-stoichiometric concentrations of the individual components, the polyphenol-ß-CD combination(s) not only inhibited the aggregation of the proteins but was also effective in disaggregating preformed fibrils. Curcumin was found to be the most efficient, followed by baicalein with (-)-epigallocatechin gallate and resveratrol coming in next, the latter two exhibiting very similar effects. Our results suggest that the efficiency of curcumin results from a balanced composition of the phenolic OH groups, benzene rings and flexibility. The latter ensures proper positioning of the functional groups to maximize the underlying interactions with both the monomeric form of α-synuclein and its aggregates. The uniqueness of ß-CD was reinforced by the observation that none of the other cyclodextrin variants [α-CD and HP-ß-CD] used was as effective, in spite of these possessing better water solubility. Moreover, the fact that the combinations remained effective under conditions of macromolecular crowding suggests that these have the potential to be developed into viable drug compositions in the near future. MTT assays on cell viability independently confirmed this hypothesis wherein these combinations (and the polyphenols alone too) appreciably impeded the toxicity of the prefibrillar α-synuclein aggregates on the mouse neuroblastoma cell lines (N2a cells).

Coactivator condensation drives cardiovascular cell lineage specification.

During development, cells make switch-like decisions to activate new gene programs specifying cell lineage. The mechanisms underlying these decisive choices remain unclear. Here, we show that the cardiovascular transcriptional coactivator myocardin (MYOCD) activates cell identity genes by concentration-dependent and switch-like formation of transcriptional condensates. MYOCD forms such condensates and activates cell identity genes at critical concentration thresholds achieved during smooth muscle cell and cardiomyocyte differentiation. The carboxyl-terminal disordered region of MYOCD is necessary and sufficient for condensate formation. Disrupting this region's ability to form condensates disrupts gene activation and smooth muscle cell reprogramming. Rescuing condensate formation by replacing this region with disordered regions from functionally unrelated proteins rescues gene activation and smooth muscle cell reprogramming. Our findings demonstrate that MYOCD condensate formation is required for gene activation during cardiovascular differentiation. We propose that the formation of transcriptional condensates at critical concentrations of cell type-specific regulators provides a molecular switch underlying the activation of key cell identity genes during development.

Prion protein transcription is auto-regulated through dynamic interactions with G-quadruplex motifs in its own promoter.

Cellular prion protein (PrP) misfolds into an aberrant and infectious scrapie form (PrPSc) that lead to fatal transmissible spongiform encephalopathies (TSEs). Association of prions with G-quadruplex (GQ) forming nucleic acid motifs has been reported, but implications of these interactions remain elusive. Herein, we show that the promoter region of the human prion gene (PRNP) contains two putative GQ motifs (Q1 and Q2) that assume stable, hybrid, intra-molecular quadruplex structures and bind with high affinity to PrP. Here, we investigate the ability of PrP to bind to the quadruplexes in its own promoter. We used a battery of techniques including SPR, NMR, CD, MD simulations and cell culture-based reporter assays. Our results show that PrP auto-regulates its expression by binding and resolving the GQs present in its own promoter. Furthermore, we map this resolvase-like activity to the N-terminal region (residues 23-89) of PrP. Our findings highlight a positive transcriptional-translational feedback regulation of the PRNP gene by PrP through dynamic unwinding of GQs in its promoter. Taken together, our results shed light on a yet unknown mechanism of regulation of the PRNP gene. This work provides the necessary framework for a plethora of studies on understanding the regulation of PrP levels and its implications in prion pathogenesis.

Role of Suction Drain after Knee Arthroplasty in the Tranexamic Acid Era: A Randomized Controlled Study.

BACKGROUND: Postoperative suction drains are used after total knee arthroplasty to avoid intra-articular hematoma formation although they can increase blood loss due to a negative suction effect. The use of tranexamic acid to reduce blood loss may nullify this. The aim of this study was to compare outcomes in patients undergoing total knee arthroplasty with or without drains and to analyze whether the drain's diameter also has an impact. METHODS: This is a prospective randomized study of patients undergoing unilateral total knee arthroplasty performed by a single surgeon. The study population was divided into three groups (A, 10G drain; B, 12G drain; and C, no drain). Pain, blood loss, swelling, wound-related complications, functional outcomes and questionnaire-based outcomes were assessed postoperatively. RESULTS: Each group had 35 patients comparable in most demographic and pre- and intraoperative characteristics. During the first 6 hours postoperatively, opioid consumption was significantly higher when the drain was not used (p = 0.036). At 3 months postoperatively, new Knee Society Score (NKSS) was highest with the use of 12G drain (p = 0.018). However, NKSS at 1 year was comparable across the three groups. With the use of tranexamic acid, blood loss and incidence of soakage of dressing were unaffected by the presence or absence of a drain. The calf girth, suprapatellar girth, soakage of dressing and range of motion were comparable in all three groups. There was no incidence of surgical site infection or deep vein thrombosis. CONCLUSIONS: Presence of a suction drain significantly reduces opioid consumption during the first 6 hours after total knee arthroplasty. Use of a drain made no difference to the functional outcome at 1 year postoperatively. With the use of tranexamic acid in total knee arthroplasty, the total blood loss and the requirement of blood transfusion were unaffected by the presence or absence of closed suction drainage or by the bore of the drain used. The clinical parameters such as swelling, range of motion, infection and deep vein thrombosis also remained the same.

A facile microfluidic method for production of liposomes.

BACKGROUND: Ethanol injection is widely used in liposome preparation. However, the parameters determining particle size distribution of the liposomal preparation has not been fully defined. MATERIALS AND METHODS: A syringe pump-driven microfluidic injection device was used to produce liposomes under different conditions. RESULTS: Particle size of the liposomes was decreased with decrease in needle diameter (or increase in hydrodynamic pressure), decrease in lipid concentration in the alcohol solution, decrease in phase transition temperature (T(m)) of the lipid bilayer and the absence of cholesterol (or decrease in, membrane rigidity). CONCLUSION: The device used is simple to adopt and can be used for affordable production of liposomes with tunable particle size.