The Hsp90 molecular chaperone governs client proteins by targeting intrinsically disordered regions.

Molecular chaperones govern proteome health to support cell homeostasis. An essential eukaryotic component of the chaperone system is Hsp90. Using a chemical-biology approach, we characterized the features driving the Hsp90 physical interactome. We found that Hsp90 associated with ∼20% of the yeast proteome using its three domains to preferentially target intrinsically disordered regions (IDRs) of client proteins. Hsp90 selectively utilized an IDR to regulate client activity as well as maintained IDR-protein health by preventing the transition to stress granules or P-bodies at physiological temperatures. We also discovered that Hsp90 controls the fidelity of ribosome initiation that triggers a heat shock response when disrupted. Our study provides insights into how this abundant molecular chaperone supports a dynamic and healthy native protein landscape.

Inhibiting U1 telescripting: A means to an end for transcription.

In this issue of Molecular Cell, Cugusi et al. (2022) discover that inhibition of U1 telescripting is a novel mechanism that promotes a switch between gene programs in response to heat stress.

Multifaceted Effects of Antigen Valency on B Cell Response Composition and Differentiation In Vivo.

How antigen valency affects B cells in vivo during immune responses is not well understood. Here, using HIV immunogens with defined valencies ranging from 1 to 60, we investigated the role of antigen valency during different phases of B cell responses in vivo. Highly multimerized immunogens preferentially rapidly activated cognate B cells, with little affinity discrimination. This led to strong early induction of the transcription factors IRF4 (interferon regulatory factor 4) and Bcl6, driving both early extrafollicular plasma cell and germinal center responses, in a CD4+ T-cell-dependent manner, involving B cells with a broad range of affinities. Low-valency antigens induced smaller effector B cell responses, with preferential recruitment of high-affinity B cells. Thus, antigen valency has multifaceted effects on B cell responses and can dictate affinity thresholds and competitive landscapes for B cells in vivo, with implications for vaccine design.

The Transcription Factor YY-1 Is an Essential Regulator of T Follicular Helper Cell Differentiation.

T follicular helper (TFH) cells are a specialized subset of CD4 T cells that deliver critical help signals to B cells for the production of high-affinity Abs. Understanding the genetic program regulating TFH differentiation is critical if one wants to manipulate TFH cells during vaccination. A large number of transcription factor (TFs) involved in the regulation of TFH differentiation have been characterized. However, there are likely additional unknown TFs required for this process. To identify new TFs, we screened a large short hairpin RNA library targeting 353 TFs in mice using an in vivo RNA interference screen. Yin Yang 1 (YY-1) was identified as a novel positive regulator of TFH differentiation. Ablation of YY-1 severely impaired TFH differentiation following acute viral infection and protein immunization. We found that the zinc fingers of YY-1 are critical to support TFH differentiation. Thus, we discovered a novel TF involved in the regulation of TFH cells.

Hsp90 and p23 Molecular Chaperones Control Chromatin Architecture by Maintaining the Functional Pool of the RSC Chromatin Remodeler.

Molecular chaperones govern protein homeostasis, being allied to the beginning (folding) and ending (degradation) of the protein life cycle. Yet, the Hsp90 system primarily associates with native factors, including fully assembled complexes. The significance of these connections is poorly understood. To delineate why Hsp90 and its cochaperone p23 interact with a mature structure, we focused on the RSC chromatin remodeler. Both Hsp90 and p23 triggered the release of RSC from DNA or a nucleosome. Although Hsp90 only freed bound RSC, p23 enhanced nucleosome remodeling prior to discharging the complex. In vivo, RSC mobility and remodeling function were chaperone dependent. Our results suggest Hsp90 and p23 contribute to proteostasis by chaperoning mature factors through energetically unfavorable events, thereby maintaining the cellular pool of active native proteins. In the case of RSC, p23 and Hsp90 promote a dynamic action, allowing a limited number of remodelers to effectively maintain chromatin in a pliable state.

The conceptus and its parts: o ntogenetic recapitulation in early human development.

Our understanding of the processes of human development that occur during and just after implantation is still incomplete. The anatomical studies by Erich Blechschmidt (1904-1992) at the University of Göttingen demonstrate the uniqueness and beauty of the early stages of individual human development or ontogeny. The interpretations of human embryology by Blechschmidt offer a simple unifying hypothesis: metabolic and biomechanical events are repeated, and this can be described as an ontogenetic recapitulation. This commentary provides a rationale for using some older terms and introducing new ones in the description of early human development. The product of conception is a conceptus; the outer part of the conceptus is the ectoblast and everything inside is the endoblast; the endocyst arises in the endoblast when the future amniotic fluid is forming. The human embryo arises from the innermost part of the endocyst. Terms such as morula, gastrula, and cyema, which are imported from zoology and ignore the role of the zona pellucida and constrained fluid compartments in the conceptus, can be avoided.

Investigating the Effect of Axial Compression and Distraction on Cervical Facet Mechanics During Supraphysiologic Anterior Shear.

Bilateral cervical facet dislocation (BFD) with facet fracture (Fx) often causes tetraplegia but is rarely recreated experimentally, possibly due to a lack of muscle replication. Intervertebral axial compression (due to muscle activation) or distraction (due to inertial loading), when combined with excessive anterior translation, may influence interfacet contact or separation and the subsequent production of BFD with or without Fx. This paper presents a methodology to produce C6/C7 BFD+Fx using anterior shear motion superimposed with 300 N compression or 2.5 mm distraction. The effect of these superimposed axial conditions on six-axis loads, and C6 inferior facet deflections and surface strains, was assessed. Twelve motion segments (70 ± 13 yr) achieved 2.19 mm of supraphysiologic anterior shear without embedding failure (supraphysiologic shear analysis point; SSP), and BFD+Fx was produced in all five specimens that reached 20 mm of shear. Linear mixed-effects models (α = 0.05) assessed the effect of axial condition. At the SSP, the compressed specimens experienced higher axial forces, facet shear strains, and sagittal facet deflections, compared to the distracted group. Facet fractures had similar radiographic appearance to those that are observed clinically, suggesting that intervertebral anterior shear motion contributes to BFD+Fx.

Lumbar spine loads are reduced for activities of daily living when using a braced arm-to-thigh technique.

PURPOSE: To evaluate the effect of the braced arm-to-thigh technique (BATT) (versus self-selected techniques) on three-dimensional trunk kinematics and spinal loads for three common activities of daily living (ADLs) simulated in the laboratory: weeding (gardening), reaching for an object in a low cupboard, and car egress using the two-legs out technique. METHODS: Ten young healthy males performed each task using a self-selected technique, and then using the BATT. The pulling action of weeding was simulated using a magnet placed on a steel plate. Cupboard and car egress tasks were simulated using custom apparatus representing the dimensions of a kitchen cabinet and a medium-sized Australian car, respectively. Three-dimensional trunk kinematics and L4/L5 spinal loads were estimated using the Lifting Full-Body OpenSim model and compared between techniques. Paired t-tests were used to compare peak values between methods (self-selected vs BATT). RESULTS: The BATT significantly reduced peak extension moments (13-51%), and both compression (27-45%) and shear forces (31-62%) at L4/L5, compared to self-selected techniques for all three tasks (p < 0.05). Lateral bending angles increased with the BATT for weeding and cupboard tasks, but these changes were expected as the BATT inherently introduces asymmetric trunk motion. CONCLUSION: The BATT substantially reduced L4/L5 extension moments, and L4/L5 compression and shear forces, compared to self-selected methods, for three ADLs, in a small cohort of ten young healthy males without prior history of back pain. These study findings can be used to inform safe procedures for these three ADLs, as the results are considered representative of a mature population.

Local Bone Grafting Is Sufficient for Instrumented Adolescent Idiopathic Scoliosis Surgery: A Preliminary Study.

BACKGROUND: Several options for grafting exist; iliac crest bone grafting, allografts, and bone substitutes. Local bone graft (LBG) offers high-quality bone graft and no commercial cost. The aim of this study was to assess the clinical and radiologic results of adolescent idiopathic scoliosis (AIS) surgery with posterior instrumentation and fusion (PIF) in patients using only LBG and to measure the quantities harvested. METHODS: A total of 218 AIS patients who underwent pedicle screw PIF surgery using only LBG with a minimum 1-year follow-up were reviewed. Bone was harvested during surgery from the excised facet joints, spinous processes (not from the end instrumented vertebrae) and decortication of laminae and transverse processes in the operative field. The harvested bone graft weight of 127 patients was recorded prospectively and then computed to graft weight per kilogram body weight (GWPK) and graft weight per motion segment (GWPMS). RESULTS: The median follow-up time was 24.7 months (12.1 to 133 mo) with 128 of the 218 patients having over 2 years follow-up. A total of 280 curves were fused. One hundred fifty-six of the patients had single curve instrumentation and 62 had double curve surgery. The median preoperative primary Cobb angle was 57.0 (31 to 100) degrees and postoperatively was 20.0 (0 to 66) degrees, indicating a median correction of 65.3% (17.5% to 100%). The median graft weight was 30 g (14 to 62 g), GWPK was 0.54 g/kg (0.24 to 1.29 g/kg) and GWPMS was 3.3 g/motion segments (2.3 to 10.0 g/kg). Twelve of 218 patients (5.5%) required subsequent surgery. Only 2 patients developed pseudarthrosis (0.91%), noting that modern segmental instrumentation warrants longer follow-up for increased confidence of complete fusion. CONCLUSIONS: LBG achieved successful fusion in over 99% of patients undergoing PIF for AIS. The described terms GWPK and GWPMS can be insightful for future studies. LBG offers a safe and low-cost solution for bone grafting in AIS surgery. LEVEL OF EVIDENCE: Level IV.

Sclerostin Depletion Induces Inflammation in the Bone Marrow of Mice.

Romosozumab, a humanized monoclonal antibody specific for sclerostin (SOST), has been approved for treatment of postmenopausal women with osteoporosis at a high risk for fracture. Previous work in sclerostin global knockout (Sost-/-) mice indicated alterations in immune cell development in the bone marrow (BM), which could be a possible side effect in romosozumab-treated patients. Here, we examined the effects of short-term sclerostin depletion in the BM on hematopoiesis in young mice receiving sclerostin antibody (Scl-Ab) treatment for 6 weeks, and the effects of long-term Sost deficiency on wild-type (WT) long-term hematopoietic stem cells transplanted into older cohorts of Sost-/- mice. Our analyses revealed an increased frequency of granulocytes in the BM of Scl-Ab-treated mice and WT→Sost-/- chimeras, indicating myeloid-biased differentiation in Sost-deficient BM microenvironments. This myeloid bias extended to extramedullary hematopoiesis in the spleen and was correlated with an increase in inflammatory cytokines TNFα, IL-1α, and MCP-1 in Sost-/- BM serum. Additionally, we observed alterations in erythrocyte differentiation in the BM and spleen of Sost-/- mice. Taken together, our current study indicates novel roles for Sost in the regulation of myelopoiesis and control of inflammation in the BM.

Conserved pathway activation following xenogeneic, heterotypic fusion.

Fusion between cells of different organisms (i.e., xenogeneic hybrids) can occur, and for humans this may occur in the course of tissue transplantation, animal handling, and food production. Previous work shows that conferred advantages are rare in xenogeneic hybrids, whereas risks of cellular dysregulation are high. Here, we explore the transcriptome of individual xenogeneic hybrids of human mesenchymal stem cells and murine cardiomyocytes soon after fusion and ask whether the process is stochastic or involves conserved pathway activation. Toward this end, single-cell RNA sequencing was used to analyze the transcriptomes of hybrid cells with respect to the human and mouse genomes. Consistent with previous work, hybrids possessed a unique transcriptome distinct from either fusion partner but were dominated by the cardiomyocyte transcriptome. New in this work is the documentation that a few genes that were latent in both fusion partners were consistently expressed in hybrids. Specifically, human growth hormone 1, murine ribosomal protein S27, and murine ATP synthase H+ transporting, mitochondrial Fo complex subunit C2 were expressed in nearly all hybrids. The consistent activation of latent genes between hybrids suggests conserved signaling mechanisms that either cause or are the consequence of fusion of these 2 cell types and might serve as a target for limiting unwanted xenogeneic fusion in the future.-Yuan, C., Freeman, B. T., McArdle, T. J., Jung, J. P., Ogle, B. M. Conserved pathway activation following xenogeneic, heterotypic fusion.

The p23 molecular chaperone and GCN5 acetylase jointly modulate protein-DNA dynamics and open chromatin status.

Cellular processes function through multistep pathways that are reliant on the controlled association and disassociation of sequential protein complexes. While dynamic action is critical to propagate and terminate work, the mechanisms used to disassemble biological structures are not fully understood. Here we show that the p23 molecular chaperone initiates disassembly of protein-DNA complexes and that the GCN5 acetyltransferase prolongs the dissociated state through lysine acetylation. By modulating the DNA-bound state, we found that the conserved and essential joint activities of p23 and GCN5 impacted transcription factor activation potential and response time to an environmental cue. Notably, p23 and GCN5 were required to maintain open chromatin regions along the genome, indicating that dynamic protein behavior is a critical feature of various DNA-associated events. Our data support a model in which p23 and GCN5 regulate diverse multistep pathways by controlling the longevity of protein-DNA complexes.

Myocardial Tissue Engineering With Cells Derived From Human-Induced Pluripotent Stem Cells and a Native-Like, High-Resolution, 3-Dimensionally Printed Scaffold.

RATIONALE: Conventional 3-dimensional (3D) printing techniques cannot produce structures of the size at which individual cells interact. OBJECTIVE: Here, we used multiphoton-excited 3D printing to generate a native-like extracellular matrix scaffold with submicron resolution and then seeded the scaffold with cardiomyocytes, smooth muscle cells, and endothelial cells that had been differentiated from human-induced pluripotent stem cells to generate a human-induced pluripotent stem cell-derived cardiac muscle patch (hCMP), which was subsequently evaluated in a murine model of myocardial infarction. METHODS AND RESULTS: The scaffold was seeded with ≈50 000 human-induced pluripotent stem cell-derived cardiomyocytes, smooth muscle cells, and endothelial cells (in a 2:1:1 ratio) to generate the hCMP, which began generating calcium transients and beating synchronously within 1 day of seeding; the speeds of contraction and relaxation and the peak amplitudes of the calcium transients increased significantly over the next 7 days. When tested in mice with surgically induced myocardial infarction, measurements of cardiac function, infarct size, apoptosis, both vascular and arteriole density, and cell proliferation at week 4 after treatment were significantly better in animals treated with the hCMPs than in animals treated with cell-free scaffolds, and the rate of cell engraftment in hCMP-treated animals was 24.5% at week 1 and 11.2% at week 4. CONCLUSIONS: Thus, the novel multiphoton-excited 3D printing technique produces extracellular matrix-based scaffolds with exceptional resolution and fidelity, and hCMPs fabricated with these scaffolds may significantly improve recovery from ischemic myocardial injury.

Global functional map of the p23 molecular chaperone reveals an extensive cellular network.

In parallel with evolutionary developments, the Hsp90 molecular chaperone system shifted from a simple prokaryotic factor into an expansive network that includes a variety of cochaperones. We have taken high-throughput genomic and proteomic approaches to better understand the abundant yeast p23 cochaperone Sba1. Our work revealed an unexpected p23 network that displayed considerable independence from known Hsp90 clients. Additionally, our data uncovered a broad nuclear role for p23, contrasting with the historical dogma of restricted cytosolic activities for molecular chaperones. Validation studies demonstrated that yeast p23 was required for proper Golgi function and ribosome biogenesis, and was necessary for efficient DNA repair from a wide range of mutagens. Notably, mammalian p23 had conserved roles in these pathways as well as being necessary for proper cell mobility. Taken together, our work demonstrates that the p23 chaperone serves a broad physiological network and functions both in conjunction with and sovereign to Hsp90.

Traumatic Aorto-Cisterna Chlyi Fistula with Treatment of Aortic Pseudoaneurysm with CT-Guided Thrombin Injection.

BACKGROUND: Only 3 cases of aorto-cisterna chyli fistula have been described in the literature but none with a resulting pseudoaneurysm (PSA). METHODS: A 68-year-old man presented following a motor vehicle collision. Imaging revealed a retroperitoneal hematoma with enhancement of the cisterna chyli, representing an aortic to cisterna chyli fistula. Three days later, computed tomography angiography showed resolution of the fistula, but revealed a PSA. The patient underwent arteriography that confirmed the PSA, and then a computed tomography-guided thrombin injection was performed. Follow-up imaging showed resolution of the PSA. RESULTS: Only 3 cases of aorto-cisterna chyli fistula have been described. We hypothesize that this fistula was caused from his L2 vertebral body fracture, which avulsed the lumbar artery and injured the cisterna chyli. The cisterna chyli provided an outflow tract for the aortic injury. We believe this type of fistula follows a benign clinical course. Aorto-cisterna chyli fistula is rare, and reports point to spontaneous resolution. Our case is unique in that the patient progressed from a fistula to a PSA. Options for treatment of this PSA include covered stent graft, open repair, coil embolization, or thrombin injection. CONCLUSIONS: This case report describes an extremely rare diagnosis and the natural history of this aorto-cisterna chyli fistula. Furthermore, the resulting aortic PSA was successfully treated with computed tomography-guided thrombin injection, which in the appropriate setting, should be considered an acceptable option.

Ligation of Common Iliac Artery Mycotic Aneurysm in a Neonate and Review of the Literature.

BACKGROUND: Mycotic aortoiliac aneurysms in neonates are rare. Surgical treatment has traditionally been the standard of care, but recent case reports have suggested that endovascular management of mycotic iliac aneurysms may also be safe and effective. In this case, we describe successful management of a mycotic aortoiliac aneurysm in a neonate with exploratory laparotomy and ligation of the left common iliac artery. METHODS: A full-term infant boy of uncomplicated delivery was transferred to our institution on day 2 of life after a barium enema concerning for small left colon syndrome. An umbilical artery catheter had been placed for monitoring but was removed before transfer. During his hospital course, he developed left leg edema and fever. He was found to have a mycotic aneurysm of the left common and internal iliac arteries, causing common iliac venous compression. A repeat ultrasound revealed the aneurysm measured a maximum of 12 mm in diameter and 26 mm in length. RESULTS: Treatment was delayed until the patient was clinically stable. He was monitored with serial ultrasounds, which showed no significant increase in aneurysmal size. A review of the literature supported the perception the aneurysm posed an impending risk to the patient. On day 16 of life, the neonate underwent ligation and excision of the left common iliac artery aneurysm. CONCLUSION: Our experience found ligation of the common iliac artery to be safe and effective, establishing that surgical reconstruction is not required.

A WD-repeat protein stabilizes ORC binding to chromatin.

Origin recognition complex (ORC) plays critical roles in the initiation of DNA replication and cell-cycle progression. In metazoans, ORC associates with origin DNA during G1 and with heterochromatin in postreplicated cells. However, what regulates the binding of ORC to chromatin is not understood. We have identified a highly conserved, leucine-rich repeats and WD40 repeat domain-containing protein 1 (LRWD1) or ORC-associated (ORCA) in human cells that interacts with ORC and modulates chromatin association of ORC. ORCA colocalizes with ORC and shows similar cell-cycle dynamics. We demonstrate that ORCA efficiently recruits ORC to chromatin. Depletion of ORCA in human primary cells and embryonic stem cells results in loss of ORC association to chromatin, concomitant reduction of MCM binding, and a subsequent accumulation in G1 phase. Our results suggest ORCA-mediated association of ORC to chromatin is critical to initiate preRC assembly in G1 and chromatin organization in post-G1 cells.

Testosterone replacement attenuates intimal hyperplasia development in an androgen deficient model of vascular injury.

BACKGROUND: Androgen deficiency (AD) is associated with increased risk of vascular disease. Dysfunctional remodeling of the vessel wall and atypical proliferative potential of vascular smooth muscle cells (VSMCs) are fundamental processes in the development of intimal hyperplasia (IH). We have demonstrated an inverse relationship between dihydrotestosterone (DHT) levels, matrix metalloproteinase activity, and VSMC migration and proliferation in vitro. Here, we investigated the role of AD and testosterone (TST) replacement in IH development in an animal model of vascular injury to elucidate mechanisms modulated by AD that could be playing a role in the development of vascular pathogenesis. METHODS: Aged orchiectomized male rats underwent TST supplementation via controlled release pellet (0.5-35 mg). Young adult and middle-age adult intact (MI) and orchiectomized placebo (Plac) groups served as controls. All groups underwent balloon angioplasty of the left common carotid at a 14-d post-TST. Carotid tissue was collected at a 14-d post-balloon angioplasty and subjected to morphologic and immunohistochemical analyses. Human male VSMCs were treated with DHT (0-3000 nM) for 24 h then subjected to quantitative PCR for gene expression analyses and costained for F-actin and G-actin for visualization of cytoskeletal organization. RESULTS: I:M ratio was increased in Plac, subphysiological, low-physiological, and high pharmacologic level TST animals compared with MI controls but was decreased with high-physiological TST supplementation. Injury-induced expression of previously defined matrix metalloproteinase remodeling enzymes was not significantly affected by TST status. Urotensin (UTS) receptor (UTSR) staining was low in injured vessels of all young adult intact, MI, and Plac controls but was significantly upregulated in all groups receiving exogenous TST supplementation, irrespective of dose. In vitro DHT exposure increased the expression of UTSR in VSMCs in a dose-dependent manner. However, this did not correlate with any change in proliferative markers. F:G actin staining revealed that DHT-induced cytoskeletal organization in a dose-dependent manner. CONCLUSIONS: AD increased IH development in response to vascular injury, whereas physiological TST replacement attenuated this effect. AD-induced IH occurs independent of matrix remodeling mechanisms known to be heavily involved in vascular dysfunction, and AD alone does not affect the UTS and/or UTSR mechanism. Exogenous TST and/or DHT increases UTSR pathway signaling in vitro and in vivo. This modulation correlates to a shift in cytoskeletal organization and may exacerbate vasoconstrictive pathogenesis. While physiological TST replacement attenuates AD-modulated IH development, its UTS-mediated effect on vasotone may prove deleterious to overall vascular function.

Is the duration of pre-operative conservative treatment associated with the clinical outcome following surgical decompression for lumbar spinal stenosis? A study based on the Spine Tango Registry.

BACKGROUND: The incidence of lumbar spinal stenosis (LSS) continues to rise, with both conservative and surgical management representing options for its treatment. The timing of surgery for LSS varies from shortly after the onset of symptoms to several months or years after conservative treatment. The aim of this study was to investigate the association between the duration of pre-operative conservative treatment and the ultimate outcome following surgical interventions for LSS. METHODS: The study was based on prospective multicentre registry data (Spine Tango). Cases of LSS with a documented duration of conservative treatment, undergoing spinal decompression with at least one post-operative patient assessment between 3 and 30 months, were included in the study. Cases of LSS with spondylolisthesis, additional spinal pathology or previous spinal surgery were excluded. Interrogation of the Spine Tango Registry listed 3478 patients meeting the prescribed inclusion criteria. This cohort was stratified into four groups: (1) no previous treatment (n = 497; 14.3%), (2) conservative treatment <6 months (n = 965; 27.8%), (3) conservative treatment between 6 and 12 months (n = 758; 21.8%), and (4) conservative treatment >12 months (n = 1258; 36.1%). Group 4 reference group in regression analysis. The inverse probability of treatment weighting (IPTW) was applied using the propensity score to balance the groups for their characteristics. Outcome measures included achievement of the minimum clinically important change (MCIC) score of 2 points for (a) back pain, (b) leg pain and (c) Core Outcome Measures Index (COMI), and (d) surgical complications, (e) general complications and (f) operation time >2 h. RESULTS: Patient group ("duration of conservative therapy") was not associated with achievement of the MCIC for post-operative relief of leg pain (p = 0.22), achievement of MCIC for the COMI score (p = 0.054), surgical complications (p = 0.11) or general complications (p = 0.14). Only MCIC for post-operative relief of back pain (p = 0.021) and operation time were significantly associated with patient group (p = 0.038). However, compared with the reference group of >12 months of conservative treatment there was no significant difference in the likelihood of achieving the MCIC for those with none, <6 or 6-12 months of conservative treatment. CONCLUSIONS: The duration of pre-operative conservative treatment was not associated with the ultimate outcome of decompression surgery. Further research is required to investigate optimal thresholds/indications for surgery and its appropriate timing in individual patients.