Competing Protein-RNA Interaction Networks Control Multiphase Intracellular Organization.

Liquid-liquid phase separation (LLPS) mediates formation of membraneless condensates such as those associated with RNA processing, but the rules that dictate their assembly, substructure, and coexistence with other liquid-like compartments remain elusive. Here, we address the biophysical mechanism of this multiphase organization using quantitative reconstitution of cytoplasmic stress granules (SGs) with attached P-bodies in human cells. Protein-interaction networks can be viewed as interconnected complexes (nodes) of RNA-binding domains (RBDs), whose integrated RNA-binding capacity determines whether LLPS occurs upon RNA influx. Surprisingly, both RBD-RNA specificity and disordered segments of key proteins are non-essential, but modulate multiphase condensation. Instead, stoichiometry-dependent competition between protein networks for connecting nodes determines SG and P-body composition and miscibility, while competitive binding of unconnected proteins disengages networks and prevents LLPS. Inspired by patchy colloid theory, we propose a general framework by which competing networks give rise to compositionally specific and tunable condensates, while relative linkage between nodes underlies multiphase organization.

Liquid Nuclear Condensates Mechanically Sense and Restructure the Genome.

Phase transitions involving biomolecular liquids are a fundamental mechanism underlying intracellular organization. In the cell nucleus, liquid-liquid phase separation of intrinsically disordered proteins (IDPs) is implicated in assembly of the nucleolus, as well as transcriptional clusters, and other nuclear bodies. However, it remains unclear whether and how physical forces associated with nucleation, growth, and wetting of liquid condensates can directly restructure chromatin. Here, we use CasDrop, a novel CRISPR-Cas9-based optogenetic technology, to show that various IDPs phase separate into liquid condensates that mechanically exclude chromatin as they grow and preferentially form in low-density, largely euchromatic regions. A minimal physical model explains how this stiffness sensitivity arises from lower mechanical energy associated with deforming softer genomic regions. Targeted genomic loci can nonetheless be mechanically pulled together through surface tension-driven coalescence. Nuclear condensates may thus function as mechano-active chromatin filters, physically pulling in targeted genomic loci while pushing out non-targeted regions of the neighboring genome. VIDEO ABSTRACT.

Nucleation landscape of biomolecular condensates.

All structures within living cells must form at the right time and place. This includes condensates such as the nucleolus, Cajal bodies and stress granules, which form via liquid-liquid phase separation of biomolecules, particularly proteins enriched in intrinsically disordered regions (IDRs)1,2. In non-living systems, the initial stages of nucleated phase separation arise when thermal fluctuations overcome an energy barrier due to surface tension. This phenomenon can be modelled by classical nucleation theory (CNT), which describes how the rate of droplet nucleation depends on the degree of supersaturation, whereas the location at which droplets appear is controlled by interfacial heterogeneities3,4. However, it remains unknown whether this framework applies in living cells, owing to the multicomponent and highly complex nature of the intracellular environment, including the presence of diverse IDRs, whose specificity of biomolecular interactions is unclear5-8. Here we show that despite this complexity, nucleation in living cells occurs through a physical process similar to that in inanimate materials, but the efficacy of nucleation sites can be tuned by their biomolecular features. By quantitatively characterizing the nucleation kinetics of endogenous and biomimetic condensates in living cells, we find that key features of condensate nucleation can be quantitatively understood through a CNT-like theoretical framework. Nucleation rates can be substantially enhanced by compatible biomolecular (IDR) seeds, and the kinetics of cellular processes can impact condensate nucleation rates and specificity of location. This quantitative framework sheds light on the intracellular nucleation landscape, and paves the way for engineering synthetic condensates precisely positioned in space and time.

Composition-dependent thermodynamics of intracellular phase separation.

Intracellular bodies such as nucleoli, Cajal bodies and various signalling assemblies represent membraneless organelles, or condensates, that form via liquid-liquid phase separation (LLPS)1,2. Biomolecular interactions-particularly homotypic interactions mediated by self-associating intrinsically disordered protein regions-are thought to underlie the thermodynamic driving forces for LLPS, forming condensates that can facilitate the assembly and processing of biochemically active complexes, such as ribosomal subunits within the nucleolus. Simplified model systems3-6 have led to the concept that a single fixed saturation concentration is a defining feature of endogenous LLPS7-9, and has been suggested as a mechanism for intracellular concentration buffering2,7,8,10. However, the assumption of a fixed saturation concentration remains largely untested within living cells, in which the richly multicomponent nature of condensates could complicate this simple picture. Here we show that heterotypic multicomponent interactions dominate endogenous LLPS, and give rise to nucleoli and other condensates that do not exhibit a fixed saturation concentration. As the concentration of individual components is varied, their partition coefficients change in a manner that can be used to determine the thermodynamic free energies that underlie LLPS. We find that heterotypic interactions among protein and RNA components stabilize various archetypal intracellular condensates-including the nucleolus, Cajal bodies, stress granules and P-bodies-implying that the composition of condensates is finely tuned by the thermodynamics of the underlying biomolecular interaction network. In the context of RNA-processing condensates such as the nucleolus, this manifests in the selective exclusion of fully assembled ribonucleoprotein complexes, providing a thermodynamic basis for vectorial ribosomal RNA flux out of the nucleolus. This methodology is conceptually straightforward and readily implemented, and can be broadly used to extract thermodynamic parameters from microscopy images. These approaches pave the way for a deeper understanding of the thermodynamics of multicomponent intracellular phase behaviour and its interplay with the nonequilibrium activity that is characteristic of endogenous condensates.

Condensate interfaces can accelerate protein aggregation.

Protein aggregates, formed from the assembly of aberrant, misfolded proteins, are a hallmark of neurodegenerative diseases. Disease-associated aggregates such as mutant Huntingtin polyQ inclusions, are typically enriched in p62/SQSTM1, an oligomeric protein that binds to and sequesters aberrant proteins. p62 has been suggested to sequester proteins through formation of liquid-like biomolecular condensates, but the physical mechanisms by which p62 condensates may regulate pathological protein aggregation remain unclear. Here, we use a light-inducible biomimetic condensate system to show that p62 condensates enhance coarsening of mutant polyQ aggregates through interface-mediated sequestration, which accelerates polyQ accumulation into larger aggregates. However, the resulting large aggregates accumulate polyubiquitinated proteins, which depletes free p62, ultimately suppressing further p62 condensation. This dynamic interplay between interface-mediated coarsening of solid aggregates and downstream consequences on the phase behavior of associated regulatory proteins could contribute to the onset and progression of protein aggregation diseases.

Reamed compared with unreamed nailing of tibial shaft fractures: Does the initial method of nail insertion influence outcome in patients requiring reoperations?

BACKGROUND: Patients with a tibial shaft fracture experiencing their first postoperative complication following treatment with intramedullary nails may be at greater risk of subsequent complications than the whole population. We aimed to determine whether the initial method of nail insertion influences outcome in patients with a tibial shaft fracture requiring multiple reoperations. METHODS: Using the Study to Prospectively Evaluate Reamed Intramedullary Nails in Tibial Shaft Fractures trial data, we categorized patients as those not requiring reoperation, those requiring a single reoperation and those requiring multiple reoperations, and we compared them by nail insertion technique (reamed v. unreamed) and fracture type (open v. closed). We then determined the number of patients whose first reoperation was in response to infection, and we compared other clinical outcomes between the reamed and unreamed groups. RESULTS: Among 1226 patients included in this analysis, 175 (14.27%) experienced a single reoperation and 44 patients (3.59%) underwent multiple reoperations. Nail insertion techniques (reamed v. unreamed) did not play a role in the need to perform multiple reoperations. Seventy-five percent of patients requiring multiple reoperations had open tibial shaft fractures. An equal number of these were reamed and unreamed insertions. The majority of patients had their course complicated by infection and almost 50% of patients whose first reoperation was for infection required more than 2 reoperations for management. The rest required multiple procedures for nonunion or bone loss. CONCLUSION: Our findings corroborate those of other studies, in which open fracture type rather than nail insertion technique was found to be the cause of morbidity following intramedullary nailing of tibial fractures. CLINICAL TRIAL REGISTRATION: www. CLINICALTRIALS: gov, no. NCT00038129.

Preoperative photography improves patient satisfaction following hallux valgus surgery.

BACKGROUND: Patient satisfaction is variable following hallux valgus (HV) surgery. This prospective, blinded, randomized trial endeavored to determine whether showing patients a preoperative photograph would improve satisfaction following HV corrective surgery. METHODS: Adult patients undergoing HV surgery were randomized to a picture group (P) or a no picture group (NP). P-group patients were shown their preoperative photograph for 5 min at each postoperative visit. Outcome measures included the Foot Function Index (FFI), the Foot Ankle Outcome Score (FAOS), and a patient satisfaction questionnaire. RESULTS: Twenty-nine patients were enrolled in the study (15P, 14 NP). At 3, and 6 months postoperatively, patients in the P-group were more likely to be completely satisfied with the appearance of their foot. There were no differences between groups with respect to postoperative HVA, IMA, or FFI, and FAOS scores. CONCLUSION: Patient satisfaction is increased following hallux valgus corrective surgery by reminding patients of the preoperative appearance of their foot through the use of photographs. LEVEL OF EVIDENCE: Level I, randomized control trial.

Early radiographic outcomes following deltoid ligament repair in bimalleolar equivalent ankle fractures.

BACKGROUND: Indications for deltoid ligament repair in bimalleolar equivalent ankle fractures are unclear. This study compared radiographic outcomes in bimalleolar equivalent ankle fractures undergoing open reduction internal fixation (ORIF) +/- deltoid ligament repair. METHODS: A retrospective review of 1024 ankle fractures was performed. Bimalleolar equivalent injuries treated with ORIF +/- deltoid ligament repair were included. Radiographic assessment was performed preoperatively, and at three months postoperatively. RESULTS: One hundred and forty-seven ankle fractures met inclusion criteria with 46 undergoing deltoid ligament repairs. There was a significant decrease in medial clear space (1.93 ± 0.65 mm vs. 2.26 ± 0.64 mm, p = 0.01), and tibiofibular clear space (3.89 ± 1.20 mm vs. 4.87 ± 1.37 mm, p = 0.0001) at 3 months postoperative in the deltoid repair group compared to the no repair group. When syndesmotic fixation was performed, there were no differences between groups. CONCLUSION: Deltoid ligament repair in bimalleolar equivalent ankle fractures resulted in reduced medial clear space, and tibiofibular clear space in the early postoperative period. These differences were small and remained within established normal limits. LEVEL OF CLINICAL EVIDENCE: Level III, retrospective cohort study.

Impact of centre volume, surgeon volume, surgeon experience and geographic location on reoperation after intramedullary nailing of tibial shaft fractures

Background: Tibial shaft fractures are the most common long-bone injury, with a reported annual incidence of more than 75 000 in the United States. This study aimed to determine whether patients with tibial fractures managed with intramedullary nails experience a lower rate of reoperation if treated at higher-volume hospitals, or by higher-volume or more experienced surgeons. Methods: The Study to Prospectively Evaluate Reamed Intramedullary Nails in Patients with Tibial Fractures (SPRINT) was a multicentre randomized clinical trial comparing reamed and nonreamed intramedullary nailing on rates of reoperation to promote fracture union, treat infection or preserve the limb in patients with open and closed fractures of the tibial shaft. Using data from SPRINT, we quantified centre and surgeon volumes into quintiles. We performed analyses adjusted for type of fracture (open v. closed), type of injury (isolated v. multitrauma), gender and age for the primary outcome of reoperation using multivariable logistic regression. Results: There were no significant differences in the odds of reoperation between high- and low-volume centres (p = 0.9). Overall, surgeon volume significantly affected the odds of reoperation (p = 0.03). The odds of reoperation among patients treated by moderate-volume surgeons were 50% less than those among patients treated by verylow-volume surgeons (odds ratio [OR] 0.50, 95% confidence interval [CI] 0.28­0.88), and the odds of reoperation among patients treated by high-volume surgeons were 47% less than those among patients treated by very-low-volume surgeons (OR 0.53, 95% CI 0.30­0.93). Conclusion: There appears to be no significant additional patient benefit in treatment by a higher-volume centre for intramedullary fixation of tibial shaft fractures. Additional research on the effects of surgical and clinical site volume in tibial shaft fracture management is needed to confirm this finding. The odds of reoperation were higher in patients treated by very-low-volume surgeons; this finding may be used to optimize the results of tibial shaft fracture management. Clinical trial registration: ClinicalTrials.gov, NCT00038129

Contexte: La fracture de la diaphyse tibiale est la plus commune des fractures des os longs, avec une incidence annuelle déclarée de plus 75 000 cas aux États-Unis. Cette étude visait à déterminer si les patients traités par enclouage intramédullaire pour une fracture du tibia sont moins souvent réopérés quand l'intervention est effectuée dans des établissements qui traitent de plus forts volumes de cas ou par des chirurgiens opérant un plus fort volume de cas ou plus expérimentés. Méthodes: L'étude SPRINT (Study to Prospectively Evaluate Reamed Intramedullary Nails in Patients with Tibial Fractures) est un essai clinique multicentrique randomisé qui a comparé l'effet de l'enclouage alésé c. non alésé sur le taux des réinterventions visant à promouvoir la consolidation osseuse de la fracture, à traiter une infection ou à préserver le membre chez des patients victimes de fractures fermées ou ouvertes de la diaphyse tibiale. À partir des données de l'étude SPRINT, nous avons classé les établissements et les chirurgiens en quintiles selon les volumes de cas traités. Nous avons effectué des analyses ajustées en fonction du type de fracture (ouverte c. fermée), du type de blessure (isolée c. polytraumatisme), du sexe et de l'âge, pour établir le taux de réintervention (paramètre principal), en utilisant la régression logistique multivariée. Résultats: On n'a noté aucune différence significative quant au risque de réintervention entre les centres qui traitaient des volumes élevés c. bas (p = 0,9). Dans l'ensemble le volume d'opérations des chirurgiens a significativement influé sur le risque de réintervention (p = 0,03). Le risque de réintervention chez les patients traités par des chirurgiens dont le volume d'interventions était moyen était de 50 % de moins que chez les patients traités par des chirurgiens dont le volume était très bas (risque relatif [RR] 0,50, intervalle de confiance [IC] à 95 % 0,28­0,88) et le risque de réintervention chez les patients traités par des chirurgiens dont le volume était très élevé était de 47 % de moins que chez les patients traités par des chirurgiens dont le volume était très bas (RR 0,53, IC à 95 % 0,30­0,93). Conclusion: Il ne semble y avoir aucun bienfait additionnel significatif au fait d'être opéré dans un centre où le volume d'interventions pour enclouage intramédullaire des fractures de la diaphyse tibiale est élevé. Il faudra approfondir la recherche sur les effets du volume chirurgical et de l'expérience clinique des établissements pour confirmer cette observation. Le risque de réintervention a été plus élevé chez les patients traités par des chirurgiens dont le volume d'interventions était très bas; cette observation pourrait être utilisée pour optimiser l'issue du traitement des fractures de la diaphyse tibiale. Enregistrement de l'essai clinique : ClinicalTrials. gov, NCT00038129.

Propagation of prions causing synucleinopathies in cultured cells.

Increasingly, evidence argues that many neurodegenerative diseases, including progressive supranuclear palsy (PSP), are caused by prions, which are alternatively folded proteins undergoing self-propagation. In earlier studies, PSP prions were detected by infecting human embryonic kidney (HEK) cells expressing a tau fragment [TauRD(LM)] fused to yellow fluorescent protein (YFP). Here, we report on an improved bioassay using selective precipitation of tau prions from human PSP brain homogenates before infection of the HEK cells. Tau prions were measured by counting the number of cells with TauRD(LM)-YFP aggregates using confocal fluorescence microscopy. In parallel studies, we fused α-synuclein to YFP to bioassay α-synuclein prions in the brains of patients who died of multiple system atrophy (MSA). Previously, MSA prion detection required ∼120 d for transmission into transgenic mice, whereas our cultured cell assay needed only 4 d. Variation in MSA prion levels in four different brain regions from three patients provided evidence for three different MSA prion strains. Attempts to demonstrate α-synuclein prions in brain homogenates from Parkinson's disease patients were unsuccessful, identifying an important biological difference between the two synucleinopathies. Partial purification of tau and α-synuclein prions facilitated measuring the levels of these protein pathogens in human brains. Our studies should facilitate investigations of the pathogenesis of both tau and α-synuclein prion disorders as well as help decipher the basic biology of those prions that attack the CNS.

Tau Trimers Are the Minimal Propagation Unit Spontaneously Internalized to Seed Intracellular Aggregation.

Tau amyloid assemblies propagate aggregation from the outside to the inside of a cell, which may mediate progression of the tauopathies. The critical size of Tau assemblies, or "seeds," responsible for this activity is currently unknown, but this could be important for the design of effective therapies. We studied recombinant Tau repeat domain (RD) and Tau assemblies purified from Alzheimer disease (AD) brain composed largely of full-length Tau. Large RD fibrils were first sonicated to create a range of assembly sizes. We confirmed our ability to resolve stable assemblies ranging from n = 1 to >100 units of Tau using size exclusion chromatography, fluorescence correlation spectroscopy, cross-linking followed by Western blot, and mass spectrometry. All recombinant Tau assemblies bound heparan sulfate proteoglycans on the cell surface, which are required for Tau uptake and seeding, because they were equivalently sensitive to inhibition by heparin and chlorate. However, cells only internalized RD assemblies of n ≥ 3 units. We next analyzed Tau assemblies from AD or control brains. AD brains contained aggregated species, whereas normal brains had predominantly monomer, and no evidence of large assemblies. HEK293 cells and primary neurons spontaneously internalized Tau of n ≥ 3 units from AD brain in a heparin- and chlorate-sensitive manner. Only n ≥ 3-unit assemblies from AD brain spontaneously seeded intracellular Tau aggregation in HEK293 cells. These results indicate that a clear minimum size (n = 3) of Tau seed exists for spontaneous propagation of Tau aggregation from the outside to the inside of a cell, whereas many larger sizes of soluble aggregates trigger uptake and seeding.

Liberal versus restrictive blood transfusion strategy: 3-year survival and cause of death results from the FOCUS randomised controlled trial.

BACKGROUND: Blood transfusion might affect long-term mortality by changing immune function and thus potentially increasing the risk of subsequent infections and cancer recurrence. Compared with a restrictive transfusion strategy, a more liberal strategy could reduce cardiac complications by lowering myocardial damage, thereby reducing future deaths from cardiovascular disease. We aimed to establish the effect of a liberal transfusion strategy on long-term survival compared with a restrictive transfusion strategy. METHODS: In the randomised controlled FOCUS trial, adult patients aged 50 years and older, with a history of or risk factors for cardiovascular disease, and with postoperative haemoglobin concentrations lower than 100 g/L within 3 days of surgery to repair a hip fracture, were eligible for enrolment. Patients were recruited from 47 participating hospitals in the USA and Canada, and eligible participants were randomly allocated in a 1:1 ratio by a central telephone system to either liberal transfusion in which they received blood transfusion to maintain haemoglobin level at 100 g/L or higher, or restrictive transfusion in which they received blood transfusion when haemoglobin level was lower than 80 g/L or if they had symptoms of anaemia. In this study, we analysed the long-term mortality of patients assigned to the two transfusion strategies, which was a secondary outcome of the FOCUS trial. Long-term mortality was established by linking the study participants to national death registries in the USA and Canada. Treatment assignment was not masked, but investigators who ascertained mortality and cause of death were masked to group assignment. Analyses were by intention to treat. The FOCUS trial is registered with ClinicalTrials.gov, number NCT00071032. FINDINGS: Between July 19, 2004, and Feb 28, 2009, 2016 patients were enrolled and randomly assigned to the two treatment groups: 1007 to the liberal transfusion strategy and 1009 to the restrictive transfusion strategy. The median duration of follow-up was 3·1 years (IQR 2·4-4·1 years), during which 841 (42%) patients died. Long-term mortality did not differ significantly between the liberal transfusion strategy (432 deaths) and the restrictive transfusion strategy (409 deaths) (hazard ratio 1·09 [95% CI 0·95-1·25]; p=0·21). INTERPRETATION: Liberal blood transfusion did not affect mortality compared with a restrictive transfusion strategy in a high-risk group of elderly patients with underlying cardiovascular disease or risk factors. The underlying causes of death did not differ between the trial groups. These findings do not support hypotheses that blood transfusion leads to long-term immunosuppression that is severe enough to affect long-term mortality rate by more than 20-25% or cause of death. FUNDING: National Heart, Lung, and Blood Institute.

Liberal or restrictive transfusion in high-risk patients after hip surgery.

BACKGROUND: The hemoglobin threshold at which postoperative red-cell transfusion is warranted is controversial. We conducted a randomized trial to determine whether a higher threshold for blood transfusion would improve recovery in patients who had undergone surgery for hip fracture. METHODS: We enrolled 2016 patients who were 50 years of age or older, who had either a history of or risk factors for cardiovascular disease, and whose hemoglobin level was below 10 g per deciliter after hip-fracture surgery. We randomly assigned patients to a liberal transfusion strategy (a hemoglobin threshold of 10 g per deciliter) or a restrictive transfusion strategy (symptoms of anemia or at physician discretion for a hemoglobin level of <8 g per deciliter). The primary outcome was death or an inability to walk across a room without human assistance on 60-day follow-up. RESULTS: A median of 2 units of red cells were transfused in the liberal-strategy group and none in the restrictive-strategy group. The rates of the primary outcome were 35.2% in the liberal-strategy group and 34.7% in the restrictive-strategy group (odds ratio in the liberal-strategy group, 1.01; 95% confidence interval [CI], 0.84 to 1.22), for an absolute risk difference of 0.5 percentage points (95% CI, -3.7 to 4.7). The rates of in-hospital acute coronary syndrome or death were 4.3% and 5.2%, respectively (absolute risk difference, -0.9%; 99% CI, -3.3 to 1.6), and rates of death on 60-day follow-up were 7.6% and 6.6%, respectively (absolute risk difference, 1.0%; 99% CI, -1.9 to 4.0). The rates of other complications were similar in the two groups. CONCLUSIONS: A liberal transfusion strategy, as compared with a restrictive strategy, did not reduce rates of death or inability to walk independently on 60-day follow-up or reduce in-hospital morbidity in elderly patients at high cardiovascular risk. (Funded by the National Heart, Lung, and Blood Institute; FOCUS ClinicalTrials.gov number, NCT00071032.).

Factors affecting transfusion requirement after hip fracture: can we reduce the need for blood?

BACKGROUND: Hip fractures are common injuries that result in blood loss and frequently require the transfusion of blood products. We sought to identify risk factors leading to increased blood transfusion in patients presenting with hip fractures, especially those factors that are modifiable. METHODS: We retrospectively reviewed the cases of all patients who had fixation of their hip fractures between October 2005 and February 2010. The need for transfusion was correlated with potential risk factors, including age, sex, preoperative hemoglobin, fracture type, fixation method and more. RESULTS: A total of 835 patients had fixation of their hip fractures during the study period; 631 met the inclusion criteria and 249 of them (39.5%) were transfused. We found an association between need for blood transfusion and female sex (p = 0.018), lower preoperative hemoglobin (p < 0.001), fracture type (p < 0.001) and fixation method (p < 0.001). Compared with femoral neck fractures, there was a 2.37 times greater risk of blood transfusion in patients with intertrochanteric fractures (p < 0.001) and a 4.03 times greater risk in those with subtrochanteric fractures (p < 0.001). Dynamic hip screw (DHS) fixation decreased the risk of transfusion by about half compared with intramedullary nail or hemiarthroplasty. We found no association with age, delay to operation (p = 0.17) or duration of surgery (p = 0.30). CONCLUSION: The only modifiable risk factor identified was fixation method. When considering blood transfusion requirements in isolation, we suggest a potential benefit in using a DHS for intertrochanteric and femoral neck fractures amenable to DHS fixation.

CONTEXTE: La fracture de la hanche est un traumatisme fréquent, qui cause une perte sanguine et nécessite souvent la transfusion de produits sanguins. Nous avons tenté d'identifier les facteurs de risque associés à une hausse du nombre des transfusions sanguines chez des patients ayant subi une fracture de la hanche, en particulier les facteurs modifiables. MÉTHODES: Au cours d'une étude rétrospective, on a revu les cas de tous les patients chez qui on avait pratiqué une ostéosynthèse pour une fracture de la hanche survenue entre octobre 2005 et février 2010. La nécessité d'une transfusion sanguine a été associée à d'éventuels facteurs de risque, dont l'âge, le sexe, le taux d'hémoglobine préopératoire, le type de fracture, la technique d'ostéosynthèse, et d'autres facteurs encore. RÉSULTATS: Au total, 835 patients avaient subi une ostéosynthèse pour fracture de la hanche au cours de la période à l'étude; 631 satisfaisaient les critères d'inclusion à l'étude et parmi eux, 249 (39,5 %) ont reçu une transfusion sanguine. On a observé l'existence d'un lien entre la nécessité d'une transfusion sanguine et le sexe féminin (p = 0,018), une plus faible concentration d'hémoglobine préopératoire (p < 0,001), le type de fracture (p <0,001) et la technique d'ostéosynthèse (p < 0,001). Par rapport aux fractures du col fémoral, le risque de transfusion sanguine était 2,37 fois plus élevé chez les patients présentant une fracture intertrochantérienne (p < 0,001) et 4,03 fois plus élevé chez ceux présentant une fracture sous-trochantérienne (p <0,001). En utilisant une vis dynamique de hanche, le risque de transfusion sanguine a diminué d'environ 50 % par rapport à l'enclouage centromédullaire ou à l'hémiarthroplastie. Aucun lien n'a été observé avec l'âge, le délai de l'intervention chirurgicale (p = 0,17), ni avec sa durée (p = 0,30). CONCLUSION: La technique d'ostéosynthèse est l'unique facteur de risque modifiable ayant été identifié. Mais lorsqu'on évalue la nécessité d'une transfusion sanguine sans tenir compte des facteurs de risque, nos résultats semblent indiquer qu'on aurait avantage à utiliser une vis dynamique de hanche pour consolider les fractures intertrochantériennes et les fractures du col fémoral.

DnaJC7 specifically regulates tau seeding.

Neurodegenerative tauopathies are caused by accumulation of toxic tau protein assemblies. This appears to involve template-based seeding events, whereby tau monomer changes conformation and is recruited to a growing aggregate. Several large families of chaperone proteins, including Hsp70s and J domain proteins (JDPs) cooperate to regulate the folding of intracellular proteins such as tau, but the factors that coordinate this activity are not well known. The JDP DnaJC7 binds tau and reduces its intracellular aggregation. However, it is unknown whether this is specific to DnaJC7 or if other JDPs might be similarly involved. We used proteomics within a cell model to determine that DnaJC7 co-purified with insoluble tau and colocalized with intracellular aggregates. We individually knocked out every possible JDP and tested the effect on intracellular aggregation and seeding. DnaJC7 knockout decreased aggregate clearance and increased intracellular tau seeding. This depended on the ability of the J domain (JD) of DnaJC7 to bind to Hsp70, as JD mutations that block binding to Hsp70 abrogated the protective activity. Disease-associated mutations in the JD and substrate binding site of DnaJC7 also abrogated its protective activity. DnaJC7 thus specifically regulates tau aggregation in cooperation with Hsp70.

Size distributions of intracellular condensates reflect competition between coalescence and nucleation.

Phase separation of biomolecules into condensates has emerged as a mechanism for intracellular organization and affects many intracellular processes, including reaction pathways through the clustering of enzymes and pathway intermediates. Precise and rapid spatiotemporal control of reactions by condensates requires tuning of their sizes. However, the physical processes that govern the distribution of condensate sizes remain unclear. Here we show that both native and synthetic condensates display an exponential size distribution, which is captured by Monte Carlo simulations of fast nucleation followed by coalescence. In contrast, pathological aggregates exhibit a power-law size distribution. These distinct behaviours reflect the relative importance of nucleation and coalescence kinetics. We demonstrate this by utilizing a combination of synthetic and native condensates to probe the underlying physical mechanisms determining condensate size. The appearance of exponential distributions for abrupt nucleation versus power-law distributions under continuous nucleation may reflect a general principle that determines condensate size distributions.

DnaJC7 specifically regulates tau seeding.

Neurodegenerative tauopathies are caused by accumulation of toxic tau protein assemblies. This appears to involve template-based seeding events, whereby tau monomer changes conformation and is recruited to a growing aggregate. Several large families of chaperone proteins, including Hsp70s and J domain proteins (JDPs), cooperate to regulate the folding of intracellular proteins such as tau, but the factors that coordinate this activity are not well known. The JDP DnaJC7 binds tau and reduces its intracellular aggregation. However, it is unknown whether this is specific to DnaJC7 or if other JDPs might be similarly involved. We used proteomics within a cell model to determine that DnaJC7 co-purified with insoluble tau and colocalized with intracellular aggregates. We individually knocked out every possible JDP and tested the effect on intracellular aggregation and seeding. DnaJC7 knockout decreased aggregate clearance and increased intracellular tau seeding. This depended on the ability of the J domain (JD) of DnaJC7 to stimulate Hsp70 ATPase activity, as JD mutations that block this interaction abrogated the protective activity. Disease-associated mutations in the JD and substrate binding site of DnaJC7 also abolished its protective activity. DnaJC7 thus specifically regulates tau aggregation in cooperation with Hsp70.

The AAA+ chaperone VCP disaggregates Tau fibrils and generates aggregate seeds in a cellular system.

Amyloid-like aggregates of the microtubule-associated protein Tau are associated with several neurodegenerative disorders including Alzheimer's disease. The existence of cellular machinery for the removal of such aggregates has remained unclear, as specialized disaggregase chaperones are thought to be absent in mammalian cells. Here we show in cell culture and in neurons that the hexameric ATPase valosin-containing protein (VCP) is recruited to ubiquitylated Tau fibrils, resulting in their efficient disaggregation. Aggregate clearance depends on the functional cooperation of VCP with heat shock 70 kDa protein (Hsp70) and the ubiquitin-proteasome machinery. While inhibition of VCP activity stabilizes large Tau aggregates, disaggregation by VCP generates seeding-active Tau species as byproduct. These findings identify VCP as a core component of the machinery for the removal of neurodegenerative disease aggregates and suggest that its activity can be associated with enhanced aggregate spreading in tauopathies.