TDP-43 is a model protein for pathophysiological phase transitions, forming a multitude of intracellular assemblies with different physical properties. Physiological condensation is widely presumed to precede pathological aggregation, but the causal relationships between different modes of assembly in vivo are still unclear. Here we use Distributed Amphifluoric FRET (DAmFRET) and complementary approaches to map the phase space of TDP-43 self-assembly in yeast cells. We discovered that the low-complexity C-terminal domain (CTD) on its own populates dynamically arrested soluble clusters rather than condensates. These clusters uniquely supported amyloid formation, and only when templated by pre-existing amyloids of other proteins. Other features of full-length TDP-43, pathological C-terminal fragments, or fusion partners that self-interacted blocked amyloid nucleation by allowing CTD clusters to further condense. Stress and cotranslational condensation had the same effect. Our findings reveal that TDP-43 amyloid formation occurs only under very specific physical and biological circumstances that could present new opportunities for therapeutic control.

Living proteomes are necessarily far from equilibrium. It is paradoxical, then, that reducing the translation of new proteins -- which should promote equilibration -- instead prolongs life. We investigated the impact of translational flux to nucleation barriers that preserve the solubility of proteins destined to form amyloids or other assemblies. By manipulating translation initiation rates directly or indirectly, across yeast and human cells, and across a variety of supersaturable proteins, we find that accelerating translation initiation broadly accelerates nucleation irrespective of their global concentrations. We showed that this effect was confined to polysomes and was enhanced by N-terminal placement or other features that retained the nascent aggregating domain at polysomes. Finally, we show that intrinsically disordered regions with high tendencies to self-associate are specifically positioned to do so co-translationally, providing evidence that cotranslational nucleation has shaped proteome evolution.

How minute pathogenic signals trigger decisive immune responses is a fundamental question in biology. Classical signaling often relies on ATP-driven enzymatic cascades, but innate immunity frequently employs death fold domain (DFD) self-assembly. The energetic basis of this assembly is unknown. Here, we show that specific DFDs function as energy reservoirs through metastable supersaturation. Characterizing all 109 human DFDs, we identified sequence-encoded nucleation barriers specifically in the central adaptors of inflammatory signalosomes, allowing them to accumulate far above their saturation concentration while remaining soluble and poised for activation. We demonstrate that the inflammasome adaptor ASC is constitutively supersaturated in vivo, retaining energy that powers on-demand cell death. Swapping a non-supersaturable DFD in the apoptosome with a supersaturable one sensitized cells to sublethal stimuli. Mapping all DFD nucleating interactions revealed that supersaturated adaptors are triggered to polymerize specifically by other DFDs in their respective pathways, limiting potentially deleterious crosstalk. Across human cell types, adaptor supersaturation strongly correlates with cell turnover, implicating this thermodynamic principle in the trade-off between immunity and longevity. Profiling homologues from fish and sponge, we find nucleation barriers to be conserved across metazoa. These findings reveal DFD adaptors as biological phase change materials in thermal batteries to power cellular life-or-death decisions on demand.

Killer meiotic drivers are selfish DNA loci that sabotage the gametes that do not inherit them from a driver+/driver- heterozygote. These drivers often employ toxic proteins that target essential cellular functions to cause the destruction of driver- gametes. Identifying the mechanisms of drivers can expand our understanding of infertility and reveal novel insights about the cellular functions targeted by drivers. In this work, we explore the molecular mechanisms underlying thewtffamily of killer meiotic drivers found in fission yeasts. Eachwtfkiller acts using a toxic Wtfpoisonprotein that can be neutralized by a corresponding Wtfantidoteprotein. Thewtfgenes are rapidly evolving and extremely diverse. Here we found that self-assembly of Wtfpoisonproteins is broadly conserved and associated with toxicity across the gene family, despite minimal amino acid conservation. In addition, we found the toxicity of Wtfpoisonassemblies can be modulated by protein tags designed to increase or decrease the extent of the Wtfpoisonassembly, implicating assembly size in toxicity. We also identified a conserved, critical role for the specific co-assembly of the Wtfpoisonand Wtfantidoteproteins in promoting effective neutralization of Wtfpoisontoxicity. Finally, we engineered wtf alleles that encode toxic Wtfpoisonproteins that are not effectively neutralized by their corresponding Wtfantidoteproteins. The possibility of such self-destructive alleles reveals functional constraints onwtfevolution and suggests similar alleles could be cryptic contributors to infertility in fission yeast populations. As rapidly evolving killer meiotic drivers are widespread in eukaryotes, analogous self-killing drive alleles could contribute to sporadic infertility in many lineages.

Most fungi can produce dormant, long-lived cells known as spores (e.g., conidia and ascospores). Spores play a critical role in fungal biology and human health, but much about spores is unknown. Here, we investigate factors affecting spore fitness using ascospores of the fission yeast Schizosaccharomyces pombe as a model. We found that storage conditions affect spore longevity and that spore health declines over time. We identified a delay in dormancy breaking (germination), decreased asymmetry during cell division, and reduced stress tolerance as aging phenotypes. These results support that S. pombe ascospores are affected by both time and experiences during dormancy, highlighting critical features of spore biology and revealing parallels between aging in spores and aging in animal cells. IMPORTANCE Fungi are integral to human health and well-being. They also offer scientists easy experimental systems to study facets of life shared with other eukaryotes, including humans. Most studies of fungal cell biology focus on actively growing cells, but fungi can also produce dormant cells known as spores. Spores promote fungal survival and dispersal and are often the agents of infection in pathogenic fungi. In this work, we characterize the spores produced by an easy-to-study model fungus, Schizosaccharomyces pombe. We find that the longevity and health of S. pombe spores decline over time and in response to heat stress. We characterize several traits associated with stressed and aged spores and identify parallels to aging cells in animals. This study expands the foundation for using S. pombe spores as a model system for fungal spore biology and as a model for the aging of non-dividing cells.

SummaryInnate immunity protects us in youth but turns against us as we age. The reason for this tradeoff is unclear. Seeking a thermodynamic basis, we focused on death fold domains (DFDs), whose ordered polymerization has been stoichiometrically linked to innate immune signal amplification. We hypothesized that soluble ensembles of DFDs function as phase change batteries that store energy via supersaturation and subsequently release it through nucleated polymerization. Using imaging and FRET-based cytometry to characterize the phase behaviors of all 109 human DFDs, we found that the hubs of innate immune signaling networks encode large nucleation barriers that are intrinsically insulated from cross-pathway activation. We showed via optogenetics that supersaturation drives signal amplification and that the inflammasome is constitutively supersaturatedin vivo. Our findings reveal that the soluble “inactive” states of adaptor DFDs function as essential, yet impermanent, kinetic barriers to inflammatory cell death, suggesting a thermodynamic driving force for aging.

The concentrations of individual proteins vary between cells, both developmentally and stochastically. The functional consequences of this variation remain largely unexplored due to limited experimental tools to manipulate the relationship of protein concentration to activity. Here, we introduce a genetically encoded tool based on a tunable amyloid that enables precise control of protein concentration thresholds in cells. By systematically screening dipeptide repeats, we identified poly-threonine alanine (poly-TA) as an ideal candidate due to its unique ability to form amyloid-like assemblies with a negligible nucleation barrier at arbitrarily chosen concentration thresholds. We demonstrate that the saturating concentration (Csat) of poly-TA can be finely tuned by adjusting the length of uninterrupted TA repeats, even while maintaining length and composition, providing a modular system for manipulating protein solubility. This tool offers a powerful approach to investigate the relationship between protein concentration, phase separation, and cellular function, with potential applications in cell-, developmental-, and synthetic biology.

Nucleolar morphology is a well-established indicator of ribosome biogenesis activity that has served as the foundation of many screens investigating ribosome production. Missing from this field of study is a broad-scale investigation of the regulation of ribosomal DNA morphology, despite the essential role of rRNA gene transcription in modulating ribosome output. We hypothesized that the morphology of rDNA arrays reflects ribosome biogenesis activity. We established GapR-GFP, a prokaryotic DNA-binding protein that recognizes transcriptionally-induced overtwisted DNA, as a live visual fluorescent marker for quantitative analysis of rDNA organization in Schizosaccharomyces pombe. We found that the morphology—which we refer to as spatial organization—of the rDNA arrays is dynamic throughout the cell cycle, under glucose starvation, RNA pol I inhibition, and TOR activation. Screening the haploid S. pombe Bioneer deletion collection for spatial organization phenotypes revealed large ribosomal protein (RPL) gene deletions that alter rDNA organization. Further work revealed RPL gene deletion mutants with altered rDNA organization also demonstrate resistance to the TOR inhibitor Torin1. A genetic analysis of signaling pathways essential for this resistance phenotype implicated many factors including a conserved MAPK, Pmk1, previously linked to extracellular stress responses. We propose RPL gene deletion triggers altered rDNA morphology due to compensatory changes in ribosome biogenesis via multiple signaling pathways, and we further suggest compensatory responses may contribute to human diseases such as ribosomopathies. Altogether, GapR-GFP is a powerful tool for live visual reporting on rDNA morphology under myriad conditions.

Background: The second generation of a custom total knee arthroplasty (TKA) implant cast from a 3D-printed mold was introduced into the market in 2012. The purpose of this retrospective study was to investigate short- to mid-term survivorship and complication rates of this novel implant. Methods: This study is a retrospective analysis of 314 TKA procedures (264 patients) performed by a single surgeon using a customized TKA from September 2012 to November 2015. Patient demographics, rate of implant revision, rate of reoperation for any reason, and rate of postoperative complications were recorded. Results: At the time of index surgery, the mean patient age was 64.7 years, and the mean follow-up duration was 3 years. At the final follow-up, implant survivorship free from revision was 98.1%, and survivorship free from reoperation for any reason was 92.4%. Conclusions: Our analysis revealed favorable short- to mid-term survivorship for a customized TKA implant. While the short- to mid-term outcomes for this implant are promising, future studies are required to assess long-term outcomes and durability.

Serial capture affinity purification (SCAP) is a powerful method to isolate a specific protein complex. When combined with cross-linking mass spectrometry and computational approaches, one can build an integrated structural model of the isolated complex. Here, we applied SCAP to dissect a subpopulation of WDR76 in complex with SPIN1, a histone reader that recognizes trimethylated histone H3 lysine4 (H3K4me3). In contrast to a previous SCAP analysis of the SPIN1:SPINDOC complex, histones and the H3K4me3 mark were enriched with the WDR76:SPIN1 complex. Next, interaction network analysis of copurifying proteins and microscopy analysis revealed a potential role of the WDR76:SPIN1 complex in the DNA damage response. Since we detected 149 pairs of cross-links between WDR76, SPIN1, and histones, we then built an integrated structural model of the complex where SPIN1 recognized the H3K4me3 epigenetic mark while interacting with WDR76. Finally, we used the powerful Bayesian Integrative Modeling approach as implemented in the Integrative Modeling Platform to build a model of WDR76 and SPIN1 bound to the nucleosome.

INTRODUCTION:Depression and anxiety are common comorbidities that may exacerbate osteoarthritis (OA)-related pain. We aim to evaluate the effect of pharmacologic treatment of depression/anxiety on hip and knee patient-reported outcome measures (PROMs). METHODS:A multi-institutional PROMs database was queried for patients with depression or anxiety and hip or knee OA who completed a PROMs questionnaire at an initial orthopaedic visit between January 2015 and March 2023. Data on demographics, comorbidities, and duration of pharmacologic treatment of depression/anxiety were obtained. Patients were stratified into three cohorts based on treatment duration. PROMs were compared across cohorts. RESULTS:Two thousand nine hundred sixty patients who completed PROMs at their initial orthopaedic visit had both OA and depression/anxiety. One hundred thirty-four (4.5%) received pharmacologic treatment of depression/anxiety for < 1 year, versus 196 (6.6%) for more than 1 year. In unadjusted analyses, patients with pharmacologic treatment had significantly lower Patient-Reported Outcomes Measurement Information System (PROMIS)-Physical (39.8 [IQR 34.9, 44.9] vs 42.3 [37.4, 47.7], P < 0.001) and PROMIS-Mental (43.5 [36.3, 50.8] vs 48.3 [41.1, 53.3], P < 0.001) scores than those without treatment. After adjusting for demographics and comorbidities, only differences in PROMIS-Mental scores remained statistically significant, with pharmacologic treatment associated with lower scores (β = -2.26, 95% CI, [-3.29, -1.24], P < 0.001). On secondary analysis including duration of pharmacologic treatment, < 1 year of treatment was associated with significantly lower PROMIS-Mental scores than those not treated (β = -4.20, 95% CI [-5.77, -2.62], P < 0.001) while scores of patients with more than 1 year of treatment did not differ significantly from those without treatment. CONCLUSION::Our results indicate that pharmacologic treatment of depression/anxiety is associated with improved psychological health but not with improved physical symptoms related to OA. We observed a nonsignificant trend that patients with depression/anxiety who warrant pharmacologic treatment tend to have worse physical symptoms than those who do not; however, unadjusted analyses suggest this is a complex relationship beyond the isolated effect of pharmacologic treatment.

BackgroundDespite increasing adoption of the direct anterior (DA) approach in total hip arthroplasty (THA), uncertainty persists regarding its outcomes beyond the 1-year mark in comparison to other approaches. We used the reverse fragility index (RFI) to evaluate the robustness of reported findings in the literature.MethodsWe conducted a systematic review of randomized controlled trials (RCTs) comparing implant revision rates between DA and other approaches in THA, defined as all those different from DA. Our primary outcome was the RFI, which gauges the number of events needed for a nonsignificant result to become significant, in the revision rate between DA and other approaches. We also calculated the reverse fragility quotient by dividing the RFI by each study’s sample size. Median values and interquartile ranges (IQRs) were displayed.ResultsA total of 10 RCTs with a total of 971 patients were included. The median RFI was 5 (IQR, 4 to 5), indicating the study’s results would be statistically significant if the outcomes of 5 patients in 1 treatment arm were reversed. The median reverse fragility quotient was 0.049 (IQR, 0.04 to 0.057), indicating that a change of outcome in 4.9% of patients would render the revision rate significant. The median number of patients lost to follow-up was 4 (IQR, 0 to 7). Of the 10 RCTs, 6 had more patients lost to follow-up than their respective RFI values.ConclusionsNotable fragility was evidenced in most studies comparing DA to other approaches for THA. Surgeons should not solely rely on the P value to determine clinical significance and instead use multiple metrics.Level of evidenceII.

Background: The Successful Aging after Elective Surgery (SAGES) II Study was designed to examine the relationship between delirium and Alzheimer's disease and related dementias (AD/ADRD), by capturing novel fluid biomarkers, neuroimaging markers, and neurophysiological measurements. The goal of this paper is to provide the first complete description of the enrolled cohort, which details the baseline characteristics and data completion. We also describe the study modifications necessitated by the COVID-19 pandemic, and lay the foundation for future work using this cohort.Methods: SAGES II is a prospective observational cohort study of community-dwelling adults age 65 and older undergoing major non-cardiac surgery. Participants were assessed preoperatively, throughout hospitalization, and at 1, 2, 6, 12, and 18 months following discharge to assess cognitive and physical functioning. Since participants were enrolled throughout the COVID-19 pandemic, procedural modifications were designed to reduce missing data and allow for high data quality.Results: About 420 participants were enrolled with a mean (standard deviation) age of 73.4 (5.6) years, including 14% minority participants. Eighty-eight percent of participants had either total knee or hip replacements; the most common surgery was total knee replacement with 210 participants (50%). Despite the challenges posed by the COVID-19 pandemic, which required the use of novel procedures such as video assessments, there were minimal missing interviews during hospitalization and up to 1-month follow-up; nearly 90% of enrolled participants completed interviews through 6-month follow-up.Conclusion: While there are many longitudinal studies of older adults, this study is unique in measuring health outcomes following surgery, along with risk factors for delirium through the application of novel biomarkers-including fluid (plasma and cerebrospinal fluid), imaging, and electrophysiological markers. This paper is the first to describe the characteristics of this unique cohort and the data collected, enabling future work using this novel and important resource.

Background: Spinopelvic mechanics are critical in total hip arthroplasty; however, there is no established consensus for adjusting acetabular component positioning based on spinopelvic parameters. This study aimed to (1) validate a recently developed Patient-Specific acetabular safe-zone calculator that factors in spinopelvic parameters and (2) compare differences with hip-spine classification targets. Methods: A total of 3750 patients underwent primary total hip arthroplasty across 3 academic referral centers, with 33 (0.88%) requiring revision for instability. Spinopelvic parameters were measured before initial total hip arthroplasty, and acetabular component position was measured following the index and revision procedures. Most operations employed either computer navigation or robotic assistance (94%). Surgical approaches included both anterior and posterior techniques. Utilizing our recently developed patient-specific safe-zone calculator, theoretical intraoperative positions were calculated and compared to true component positions before and after revision. Results: Among 33 patients who underwent revision, none dislocated at an average follow-up of 5.1 years. In the external validation cohort, the average absolute differences between the patient-specific safe-zone and the median hip-spine classification recommendation were 3.8° ± 2.1° inclination and 5.0° ± 3.2° version. For the pooled cohort, the absolute differences between the patient-specific safe-zone targets and the prerevision component positions were 7.9° ± 5.1° inclination and 11.4° ± 6.9° version. After revision, the mean absolute differences decreased to 3.6° ± 3.1° inclination and 5.8° ± 3.5° version (P < .001). Conclusions: A patient-specific approach improved acetabular component positioning accuracy within 6° of version and 4° of inclination of stable, revised hips. Patient-specific safe zones provide quantitative targets for nuanced spinopelvic preoperative planning that may mitigate risk of instability and may indicate use of assisted technologies.

BACKGROUND:Vitamin D deficiency is associated with poorer functional outcomes and increased complication rates after total knee arthroplasty (TKA). Yet, there is no longer term study evaluating vitamin D levels and supplementation after TKA. Our study aimed to compare quantitative vitamin D levels and supplementation regimens after TKA stratified by patient sex and race. METHODS:A retrospective cohort study of primary TKA patients at a single hospital from 2015 to 2022 was conducted. We analyzed vitamin D preoperatively and postoperatively up to 2 years. Vitamin D deficiency was defined as <30 ng/mL. A subgroup analysis was conducted in patients with vitamin D <21 ng/mL. Supplementation categories included none, low (<1,001 IU), medium (1,001 to 5,000 IU), and high (>5,000 IU). RESULTS:A total of 400 (66.0% female) patients who underwent 430 primary TKA procedures were included, and 65.3% received supplementation. Patients who were vitamin D sufficient preoperatively demonstrated higher vitamin D levels and ability to maintain sufficiency postoperatively using low-dose supplementation compared with no supplementation ( P = 0.004). Those who were vitamin D deficient preoperatively demonstrated higher vitamin D levels postoperatively using medium to high doses ( P = 0.02). For patients who became deficient postoperatively, supplementation was associated with achieving repletion at an average of 10.2 months ( P < 0.001). Black patients demonstrated 2.8 times higher odds of having a vitamin D level less than 30 ng/mL ( P = 0.03). CONCLUSION:Our study demonstrated that low-dose vitamin D supplementation (<1,001 IU) was beneficial for vitamin D-sufficient TKA patients to achieve higher levels and maintain vitamin D sufficiency. Vitamin D-deficient TKA patients benefitted from medium-to-high dose supplementation (1,001 to 5,000+), but only 33.7% achieved vitamin D repletion. This work highlights the need to continue vitamin D surveillance postoperatively and the need to continue vitamin D repletion.

BACKGROUND:Clinical guidelines for performing total joint arthroplasty (TJA) have not been uniformly adopted in practice because research has suggested that they may foster inequities in surgical access, potentially disadvantaging minority sociodemographic groups. The aim of this study was to assess whether undergoing TJA without meeting clinical guidelines affects complication risk and leads to disparities in postoperative outcomes. METHODS:This retrospective cohort study evaluated the records of 11,611 adult patients who underwent primary TJA from January 1, 2010, to December 31, 2020, at an academic hospital network. Based on self-reported race and ethnicity, 89.5% of patients were White, 3.5% were Black, 2.9% were Hispanic, 1.3% were Asian, and 2.8% were classified as other. Patients met institutional guidelines for undergoing TJA if they had a hemoglobin A1c of <8.0% and a body mass index of <40 kg/m 2 and were not currently smoking. A logistic regression model was utilized to identify factors associated with complications, and a mixed-effects model was utilized to identify factors associated with not meeting guidelines for undergoing TJA. RESULTS:During the study period, 11% (1,274) of the 11,611 adults who underwent primary TJA did not meet clinical guidelines. Compared with the group who met guidelines, the group who did not had higher proportions of Black patients (3.2% versus 6.0%; p < 0.001) and Hispanic patients (2.7% versus 4.6%; p < 0.001). An increased risk of not meeting guidelines at the time of surgery was demonstrated among Black patients (odds ratio [OR], 1.60 [95% confidence interval (CI), 1.22 to 2.10]; p = 0.001) and patients insured by Medicaid (OR, 1.75 [95% CI, 1.26 to 2.44]; p = 0.001) or Medicare (OR, 1.22 [95% CI, 1.06 to 1.41]; p = 0.007). Patients who did not meet guidelines had a higher risk of reoperation than those who met guidelines (7.7% [98] versus 5.9% [615]; p = 0.017), including a higher risk of infection-related reoperation (3.1% [40] versus 1.4% [147]; p < 0.001). CONCLUSIONS:We found that patients who underwent TJA despite not meeting institutional preoperative criteria had a higher risk of postoperative complications. These patients were more likely to be from racial and ethnic minority groups, to have a lower socioeconomic status, and to have Medicare or Medicaid insurance. These findings underscore the need for surgery-related shared decision-making that is informed by evidence-based guidelines in order to reduce complication burden. LEVEL OF EVIDENCE:Prognostic Level III . See Instructions for Authors for a complete description of levels of evidence.

The development of cell-type-specific gene therapy vectors for treating neurological diseases holds great promise, but has relied on animal models with limited translational utility. We have adapted an ex vivo organotypic model to evaluate adeno-associated virus (AAV) transduction properties in living slices of human brain tissue. Using fluorescent reporter expression and single-nucleus RNA sequencing, we found that common AAV vectors show broad transduction of normal cell types, with protein expression most apparent in astrocytes; this work introduces a pipeline for identifying and optimizing AAV gene therapy vectors in human brain samples.

BACKGROUND:Previous research has underscored the benefits of geriatrician consultation in improving outcomes for older patients undergoing hip fracture repair, highlighting enhanced functional outcomes and reduced morbidity. However, the impact of geriatrician care in outcomes for patients undergoing elective total joint arthroplasty (TJA) has yet to be described. We aimed to determine whether preoperative or postoperative geriatrician involvement was associated with differences in the length of hospital stay and emergency department (ED) visits after TJA. METHODS:This retrospective cohort study screened the medical records of patients ≥65 years of age undergoing primary elective TJA in a network of tertiary hospitals. Geriatrician consultations occurring within a period spanning 90 days before to 90 days after TJA were recorded. Bivariate analysis and multivariable regression models were used to assess the relationship between receiving these consultations and changes in the length of stay and ED visits. RESULTS:A total of 16,076 patients undergoing primary TJA were included. Of these surgical procedures, 9,677 (60.2%) were total knee arthroplasties and 6,087 (37.9%) were total hip arthroplasties; 1,416 (8.8%) of cases had geriatrician visits. Patients had lower odds of requiring postoperative ED visits when they had at least 1 geriatrician appointment within the week preceding an arthroplasty (odds ratio [OR], 0.97 [95% confidence interval (CI), 0.68 to 0.99]; p = 0.005). This effect was most notable for 65-year-old patients (OR, 0.66 [95% CI, 0.45 to 0.98]). CONCLUSION:This study reports promising evidence supporting the benefits of perioperative geriatrician visits on TJA outcomes. Preoperative visits were shown to be associated with decreased odds of ED visits after TJA in patients for up to 90 days postoperatively. Thus, geriatrician involvement in elective TJAs has the potential to improve outcomes and reduce morbidity and costs for patients and reduce costs for surgeons and institutions. LEVEL OF EVIDENCE:Therapeutic Level III . See Instructions for Authors for a complete description of levels of evidence.