The Structure of the Polycystic Kidney Disease Channel PKD2 in Lipid Nanodiscs.

The Polycystic Kidney Disease 2 (Pkd2) gene is mutated in autosomal dominant polycystic kidney disease (ADPKD), one of the most common human monogenic disorders. Here, we present the cryo-EM structure of PKD2 in lipid bilayers at 3.0 Å resolution, which establishes PKD2 as a homotetrameric ion channel and provides insight into potential mechanisms for its activation. The PKD2 voltage-sensor domain retains two of four gating charges commonly found in those of voltage-gated ion channels. The PKD2 ion permeation pathway is constricted at the selectivity filter and near the cytoplasmic end of S6, suggesting that two gates regulate ion conduction. The extracellular domain of PKD2, a hotspot for ADPKD pathogenic mutations, contributes to channel assembly and strategically interacts with the transmembrane core, likely serving as a physical substrate for extracellular stimuli to allosterically gate the channel. Finally, our structure establishes the molecular basis for the majority of pathogenic mutations in Pkd2-related ADPKD.

Visualizing the chaperone-mediated folding trajectory of the G protein ß5 ß-propeller.

The Chaperonin Containing Tailless polypeptide 1 (CCT) complex is an essential protein folding machine with a diverse clientele of substrates, including many proteins with ß-propeller domains. Here, we determine the structures of human CCT in complex with its accessory co-chaperone, phosducin-like protein 1 (PhLP1), in the process of folding Gß5, a component of Regulator of G protein Signaling (RGS) complexes. Cryoelectron microscopy (cryo-EM) and image processing reveal an ensemble of distinct snapshots that represent the folding trajectory of Gß5 from an unfolded molten globule to a fully folded ß-propeller. These structures reveal the mechanism by which CCT directs Gß5 folding through initiating specific intermolecular contacts that facilitate the sequential folding of individual ß sheets until the propeller closes into its native structure. This work directly visualizes chaperone-mediated protein folding and establishes that CCT orchestrates folding by stabilizing intermediates through interactions with surface residues that permit the hydrophobic core to coalesce into its folded state.

A ribosome-bound quality control complex triggers degradation of nascent peptides and signals translation stress.

The conserved transcriptional regulator heat shock factor 1 (Hsf1) is a key sensor of proteotoxic and other stress in the eukaryotic cytosol. We surveyed Hsf1 activity in a genome-wide loss-of-function library in Saccaromyces cerevisiae as well as ~78,000 double mutants and found Hsf1 activity to be modulated by highly diverse stresses. These included disruption of a ribosome-bound complex we named the Ribosome Quality Control Complex (RQC) comprising the Ltn1 E3 ubiquitin ligase, two highly conserved but poorly characterized proteins (Tae2 and Rqc1), and Cdc48 and its cofactors. Electron microscopy and biochemical analyses revealed that the RQC forms a stable complex with 60S ribosomal subunits containing stalled polypeptides and triggers their degradation. A negative feedback loop regulates the RQC, and Hsf1 senses an RQC-mediated translation-stress signal distinctly from other stresses. Our work reveals the range of stresses Hsf1 monitors and elucidates a conserved cotranslational protein quality control mechanism.

Headache response after CT-guided fibrin glue occlusion of CSF-venous fistulas.

OBJECTIVE: To assess headache response and patient perception of improvement after computed tomography (CT)-guided fibrin glue occlusion of cerebrospinal fluid-venous fistulas (CVFs) in a large sample size and with a long clinical follow-up. BACKGROUND: CVFs are an increasingly identified type of spinal leak in patients with spontaneous intracranial hypotension (SIH), and CT-guided fibrin glue occlusion has been introduced as a treatment option in a prior small series. METHODS: Retrospective case series review of medical records from a single institution was performed for all patients with CVFs that were treated with CT-guided fibrin glue occlusion between August 2018 and April 2022 in an outpatient or inpatient setting. Pre- and posttreatment Headache Impact Tests (HIT-6) were administered to patients, and a change in scores was evaluated. In some patients, pretreatment HIT-6 tests were not obtained prior to the fibrin glue procedure, and the patient was asked to fill out the pretreatment test based on personal recall of their symptoms prior to treatment. Patients completed a Patient Global Impression of Change (PGIC) scale after treatment. Pre- and posttreatment brain imaging was compared using Bern SIH scores. RESULTS: Thirty-five patients (19 females, 16 males; mean age 60 years) with CVFs treated with CT-guided fibrin glue occlusion met the inclusion criteria. Mean pretreatment and posttreatment HIT-6 scores were 64.7 ± 10.2 and 43.4 ± 9.9 (p < 0.001), respectively. The posttreatment HIT-6 questionnaires were completed on average 10.3 months after treatment, and 20 patients filled out the pretreatment HIT-6 form after their treatment. The mean PGIC score was 6.1 ± 1.3. Mean pretreatment and posttreatment Bern SIH scores were 5.9 ± 2.5 and 1.5 ± 1.5 (p < 0.001), respectively. CONCLUSIONS: We report a large series of patients who underwent CT-guided fibrin glue occlusion of CVFs. We showed that headache scores decreased after treatment, and the majority of patients had high PGIC scores. Posttreatment brain MRIs also showed improved Bern SIH scores.

Spontaneous Intracranial Hypotension in the Critical Patient.

Spontaneous intracranial hypotension typically manifests with orthostatic headaches and is caused by spinal dural tears, ruptured meningeal diverticula, or CSF-venous fistulas. While most patients are diagnosed and treated in the outpatient setting, some patients will occasionally present in the emergent ICU setting due to subdural hematomas, coma, or downward brain herniation. In this review paper, we will discuss the diagnostic and treatment steps that intensivists can undertake to coordinate a team approach to successfully manage these patients. A brief general overview of spontaneous intracranial hypotension will also be discussed.

CT-guided Fibrin Glue Occlusion of Cerebrospinal Fluid-Venous Fistulas.

Background Cerebrospinal fluid-venous fistulas (CVFs) are one of the less common etiologic causes of spontaneous intracranial hypotension. CVFs are most commonly treated with open surgical ligation and have reportedly not responded well to percutaneous treatments. Purpose To study treatment outcomes of CT-guided fibrin glue occlusion for CVFs. Materials and Methods Retrospective review of medical records from two institutions was performed for all patients with CVFs who underwent CT-guided percutaneous fibrin glue occlusion from March to October 2020. CVFs were assessed for resolution or persistence at posttreatment decubitus CT myelography (CTM). Pre- and posttreatment brain MRI scans were reviewed for principal signs of spontaneous intracranial hypotension. Clinical symptoms were documented before and immediately after therapy, and the current symptoms to date after fibrin glue occlusion were documented. Results CT-guided fibrin glue occlusion was performed in 13 patients (mean age, 62 years ± 14 [standard deviation]; eight women) with CVFs. Ten of 10 patients who underwent final posttreatment decubitus CTM examinations showed CVF resolution. All 13 patients showed improvement on posttreatment brain MRI scans. All 13 patients are currently asymptomatic, although three patients were asymptomatic before fibrin glue occlusion. Conclusion CT-guided fibrin glue occlusion is an effective treatment for patients with cerebrospinal fluid-venous fistulas (CVFs). Direct fibrin glue administration within the CVF may be one of the key factors for success. Further studies are needed to determine the long-term efficacy of this treatment. © RSNA, 2021.

Risk factors for neonatal brachial plexus palsy attributed to anatomy, physiology, and evolution.

The inherent variable anatomy of the neonate and the uniquely-shaped maternal birth canal that is associated with the evolution of human bipedalism constitute risk factors for neonatal brachial plexus palsy (NBPP). For example, those neonates with a prefixed brachial plexus (BP) are at greater risk of trauma due to lateral neck traction during delivery than those with a normal or postfixed BP. Compared to adults, neonates also have extremely large and heavy heads (high head: body ratio) set upon necks with muscles and ligaments that are weak and poorly developed. Accordingly, insufficient cranial stability can place large torques on the cervical spinal nerves. In addition, the pelvic changes necessary for habitual bipedal posture resulted in a uniquely-shaped, obstruction-filled, sinusoidal birth canal, requiring the human fetus to complete a complicated series of rotations to successfully traverse it. Furthermore, although there are many risk factors that are known to contribute to NBPP, the specific anatomy and physiology of the neonate, except for macrosomia, is not considered to be one of them. In fact, currently, the amount of lateral traction applied to the neck during delivery is the overwhelming legal factor that is used to evaluate whether a birth attendant is liable in cases of permanent NBPP. Here, we suggest that the specific anatomy and physiology of the neonate and mother, which are clearly not within the control of the birth attendant, should also be considered when assessing liability in cases of NBPP.

The 2017 Nobel Prize in Chemistry: cryo-EM comes of age.

The 2017 Nobel Prize in Chemistry was awarded to Jacques Dubochet, Joachim Frank, and Richard Henderson for "developing cryo-electron microscopy (cryo-EM) for the high-resolution structure determination of biomolecules in solution." This feature article summarizes some of the major achievements leading to the development of cryo-EM and recent technological breakthroughs that have transformed the method into a mainstream tool for structure determination.

Neonatal Magnetic Resonance Imaging Without Sedation Correlates With Injury Severity in Brachial Plexus Birth Palsy.

PURPOSE: Which infants with brachial plexus birth palsy (BPBP) should undergo microsurgical plexus reconstruction remains controversial. The current gold standard for the decision for plexus reconstruction is serial clinical examinations, but this approach obviates the possibility of early surgical treatment. We hypothesize that a new technique using 3-dimensional volumetric proton density magnetic resonance imaging (MRI) without sedation can evaluate the severity of BPBP injury earlier than serial clinical examinations. METHODS: Infants were prospectively enrolled prior to 12 weeks of age and imaged using 3 Tesla MRI without sedation. Clinical scores were collected at all visits. The imaging findings were graded based on the number of injured levels and the severity of each injury, and a radiological score was calculated. All infants were followed at least until the decision for surgery was made based on clinical examination. RESULTS: Nine infants completed the MRI scan and clinical follow-up. The average Toronto score at presentation was 4.4 out of 10 (range, 0-8.2); the average Active Movement Scale score was 50 out of 105 (range, 0-86). Four infants required surgery: 2 because of a flail limb and Horner syndrome and 2 owing to failure to recover antigravity elbow flexion by age 6 months. Radiological scores ranged from 0 to 18 out of a maximum score of 25. The average radiological score for those infants who required surgery was 12 (range, 6.5-18), whereas the average score for infants who did not require surgery was 3.5 (range, 0-8). CONCLUSIONS: Three-dimensional proton density MRI can evaluate spinal nerve roots in infants without the need for radiation, contrast agents, or sedation. These data suggest that MRI can help determine the severity of injury earlier than clinical examination in infants with BPBP, although further study of a larger sample of infants with varying severity of disease is necessary. TYPE OF STUDY/LEVEL OF EVIDENCE: Diagnostic II.

Blunt Intraoral Trauma Resulting in Internal Carotid Artery Dissection and Infarction in a Child.

A 16-month-old child fell forward onto her toothbrush sustaining minor oropharyngeal injury. The following day, she became acutely lethargic with localizing neurologic signs of a cerebrovascular infarct. CTA and MR imaging demonstrated occlusion of the right internal carotid artery with a large right middle cerebral artery territory infarction. She was treated with decompressive craniectomy and anticoagulation but remained weak on the left side. Pediatric oropharyngeal injuries can rarely be complicated by internal carotid artery injury with dissection, thrombosis, or embolization to the cerebral circulation. For the best outcome, carotid dissection treatment requires prompt diagnosis at the initial onset of neurologic symptoms. However, further research is needed to determine the best management and advanced imaging work-up for neurologically intact children.

Clinical scoring system may improve yield of head CT of non-trauma emergency department patients.

The positive rate of head CT in non-trauma patients presenting to the emergency department (ED) is low. Currently, indications for imaging are based on the individual experience of the treating physician, which contributes to overutilization and variability in imaging utilization. The goals of this study are to ascertain the predictors of positive head CT in non-trauma patients and demonstrate feasibility of a clinical scoring algorithm to improve yield. We retrospectively reviewed 500 consecutive ED non-trauma patients evaluated with non-contrast head CT after presenting with headache, altered mentation, syncope, dizziness, or focal neurologic deficit. Medical records were assessed for clinical risk factors: focal neurologic deficit, altered mental status, nausea/vomiting, known malignancy, coagulopathy, and age. Data was analyzed using logistic regression and receiver operator characteristic (ROC) curves and three derived algorithms. Positive CTs were found in 51 of 500 patients (10.2 %). Only two clinical factors were significant: focal neurologic deficit (adjusted odds ratio (OR) 20.7; 95 % confidence interval (CI) 9.4-45.7) and age >55 (adjusted OR 3.08; CI 1.44-6.56). Area under the ROC curve for all three algorithms was 0.73-0.83. In proposed algorithm C, only patients with focal neurologic deficit (major risk factor) or ≥2 of the five minor risk factors (altered mental status, nausea/vomiting, known malignancy, coagulopathy, and age) would undergo CT imaging. This may reduce utilization by 34 % with only a small decrease in sensitivity (98 %). Our simple scoring algorithm utilizing multiple clinical risk factors could help to predict the non-trauma patients who will benefit from CT imaging, resulting in reduced radiation exposure without sacrificing sensitivity.

CrossFit-related cervical internal carotid artery dissection.

CrossFit is a high-intensity strength and conditioning program that has gained popularity over the past decade. Potential injuries associated with CrossFit training have been suggested in past reports. We report three cases of cervical carotid dissection that are associated with CrossFit workouts. Patient 1 suffered a distal cervical internal carotid artery (ICA) dissection near the skull base and a small infarct in Wernicke's area. He was placed on anticoagulation and on follow-up has near complete recovery. Patient 2 suffered a proximal cervical ICA dissection that led to arterial occlusion and recurrent middle cerebral artery territory infarcts and significant neurological sequelae. Patient 3 had a skull base ICA dissection that led to a partial Horner's syndrome but no cerebral infarct. While direct causality cannot be proven, intense CrossFit workouts may have led to the ICA dissections in these patients.

Thromboelastography (TEG)-based algorithm reduces blood product utilization in patients undergoing VAD implant.

OBJECTIVES: Multiple blood products are often required during and after ventricular assist device (VAD) implants. Generally, transfusion therapy is empirically guided by conventional laboratory tests. In this study, we aimed to compare a thromboelastography (TEG)-based algorithm with a laboratory coagulation test-based algorithm with respect to blood product utilization in patients undergoing VAD implant. METHODS: From June 2010 to May 2012, a total of 39 consecutive patients underwent VAD implantation. Patients undergoing VAD implant were retrospectively divided into two groups according to transfusion strategy. In the control group (n=20), the need for blood transfusion was based on clinician's discretion according to standard coagulation test results. In the TEG group (n=19), a strict protocol based on TEG parameters was followed for the usage of all perioperative blood products. Coagulation factors, TEG parameters, and blood transfusions were documented and compared between these two groups. RESULTS: There were no differences in demographic variables with the exception of a decreased CPB time in the TEG group (p=0.019). Prothrombin time (PT) (p<0.001) and international normalized ratio (INR) (p<0.001) in the postprotamine interval were significantly higher in the TEG group than in the control group. No significant difference was detected in any coagulation variable in the postoperative (ICU) period between the two groups. Platelet counts decreased in a linear fashion from baseline to the postoperative period in the two groups (p<0.001). Patients in the TEG group received significantly less fresh-frozen plasma in both the intraoperative (p=0.005) and postoperative (p=0.014) periods. Patients in the TEG group also received significantly less platelets both in the postoperative (p=0.03) period and in total amount (p=0.033). There was no difference in consumption of packed red blood cell units between the two groups. CONCLUSIONS: Our results show that the strict use of a TEG-guided algorithm significantly reduces the consumption of blood products in patients undergoing VAD implant

Protocol to study CCT-mediated folding of Gß5 by single-particle cryo-EM.

The chaperonin CCT mediates folding of many cytosolic proteins, including G protein ß subunits (Gßs). Here, we present a protocol for isolating Gß5 bound to CCT and its co-chaperone PhLP1 and determining the CCT-mediated folding trajectory of Gß5 using single-particle cryoelectron microscopy (cryo-EM) techniques. We describe steps for purifying CCT-Gß5-PhLP1 from human cells, stabilizing the closed CCT conformation, preparing and imaging cryo-EM specimens, and processing data to recover multiple Gß5 folding intermediates. This protocol permits visualization of protein folding by CCT. For complete details on the use and execution of this protocol, please refer to Sass et al.1.

C. elegans Dicer acts with the RIG-I-like helicase DRH-1 and RDE-4 to cleave dsRNA.

Invertebrates use the endoribonuclease Dicer to cleave viral dsRNA during antiviral defense, while vertebrates use RIG-I-like Receptors (RLRs), which bind viral dsRNA to trigger an interferon response. While some invertebrate Dicers act alone during antiviral defense, C. elegans Dicer acts in a complex with a dsRNA binding protein called RDE-4, and an RLR ortholog called DRH-1. We used biochemical and structural techniques to provide mechanistic insight into how these proteins function together. We found RDE-4 is important for ATP-independent and ATP-dependent cleavage reactions, while helicase domains of both DCR-1 and DRH-1 contribute to ATP-dependent cleavage. DRH-1 plays the dominant role in ATP hydrolysis, and like mammalian RLRs, has an N-terminal domain that functions in autoinhibition. A cryo-EM structure indicates DRH-1 interacts with DCR-1's helicase domain, suggesting this interaction relieves autoinhibition. Our study unravels the mechanistic basis of the collaboration between two helicases from typically distinct innate immune defense pathways.

Caenorhabditis elegans Dicer acts with the RIG-I-like helicase DRH-1 and RDE-4 to cleave dsRNA.

Invertebrates use the endoribonuclease Dicer to cleave viral dsRNA during antiviral defense, while vertebrates use RIG-I-like Receptors (RLRs), which bind viral dsRNA to trigger an interferon response. While some invertebrate Dicers act alone during antiviral defense, Caenorhabditis elegans Dicer acts in a complex with a dsRNA binding protein called RDE-4, and an RLR ortholog called DRH-1. We used biochemical and structural techniques to provide mechanistic insight into how these proteins function together. We found RDE-4 is important for ATP-independent and ATP-dependent cleavage reactions, while helicase domains of both DCR-1 and DRH-1 contribute to ATP-dependent cleavage. DRH-1 plays the dominant role in ATP hydrolysis, and like mammalian RLRs, has an N-terminal domain that functions in autoinhibition. A cryo-EM structure indicates DRH-1 interacts with DCR-1's helicase domain, suggesting this interaction relieves autoinhibition. Our study unravels the mechanistic basis of the collaboration between two helicases from typically distinct innate immune defense pathways.

ASPSCR1-TFE3 reprograms transcription by organizing enhancer loops around hexameric VCP/p97.

The t(X,17) chromosomal translocation, generating the ASPSCR1::TFE3 fusion oncoprotein, is the singular genetic driver of alveolar soft part sarcoma (ASPS) and some Xp11-rearranged renal cell carcinomas (RCCs), frustrating efforts to identify therapeutic targets for these rare cancers. Here, proteomic analysis identifies VCP/p97, an AAA+ ATPase with known segregase function, as strongly enriched in co-immunoprecipitated nuclear complexes with ASPSCR1::TFE3. We demonstrate that VCP is a likely obligate co-factor of ASPSCR1::TFE3, one of the only such fusion oncoprotein co-factors identified in cancer biology. Specifically, VCP co-distributes with ASPSCR1::TFE3 across chromatin in association with enhancers genome-wide. VCP presence, its hexameric assembly, and its enzymatic function orchestrate the oncogenic transcriptional signature of ASPSCR1::TFE3, by facilitating assembly of higher-order chromatin conformation structures demonstrated by HiChIP. Finally, ASPSCR1::TFE3 and VCP demonstrate co-dependence for cancer cell proliferation and tumorigenesis in vitro and in ASPS and RCC mouse models, underscoring VCP's potential as a novel therapeutic target.

Sequence and structural characterization of great salt lake bacteriophage CW02, a member of the T7-like supergroup.

Halophage CW02 infects a Salinivibrio costicola-like bacterium, SA50, isolated from the Great Salt Lake. Following isolation, cultivation, and purification, CW02 was characterized by DNA sequencing, mass spectrometry, and electron microscopy. A conserved module of structural genes places CW02 in the T7 supergroup, members of which are found in diverse aquatic environments, including marine and freshwater ecosystems. CW02 has morphological similarities to viruses of the Podoviridae family. The structure of CW02, solved by cryogenic electron microscopy and three-dimensional reconstruction, enabled the fitting of a portion of the bacteriophage HK97 capsid protein into CW02 capsid density, thereby providing additional evidence that capsid proteins of tailed double-stranded DNA phages have a conserved fold. The CW02 capsid consists of bacteriophage lambda gpD-like densities that likely contribute to particle stability. Turret-like densities were found on icosahedral vertices and may represent a unique adaptation similar to what has been seen in other extremophilic viruses that infect archaea, such as Sulfolobus turreted icosahedral virus and halophage SH1.

Factors Predictive of Treatment Success in CT-Guided Fibrin Occlusion of CSF-Venous Fistulas: A Multicenter Retrospective Cross-Sectional Study.

BACKGROUND AND PURPOSE: CSF-to-venous fistulas contribute to spontaneous intracranial hypotension. CT-guided fibrin occlusion has been described as a minimally invasive treatment strategy; however, its reproducibility across different institutions remains unclear. This multi-institution study evaluated the clinical and radiologic outcomes of CT-guided fibrin occlusion, hypothesizing a correlation among cure rates, fibrin injectate spread, and drainage patterns. MATERIALS AND METHODS: A retrospective evaluation was conducted on CT-guided fibrin glue treatment in patients with CSF-to-venous fistulas from 6 US and UK institutions from 2020 to 2023. Patient information, procedural characteristics, and injectate spread and drainage patterns were examined. Clinical improvement assessed through medical records served as the primary outcome. RESULTS: Of 119 patients at a mean follow-up of 5.0 months, fibrin occlusion resulted in complete clinical improvement in 59.7%, partial improvement in 34.5%, and no improvement in 5.9% of patients. Complications were reported in 4% of cases. Significant associations were observed between clinical improvement and concordant injectate spread with the fistula drainage pattern (P = .0089) and pretreatment symptom duration (P < .001). No associations were found between clinical improvement and cyst puncture, intravascular extension, rebound headache, body mass index, age, or number of treatment attempts. CONCLUSIONS: Fibrin occlusion performed across various institutions shows cure when associated with injectate spread matching the CVF drainage pattern and shorter pretreatment symptom duration, emphasizing the importance of accurate injectate placement and early intervention.

Visualization of the Cdc48 AAA+ ATPase protein unfolding pathway.

The Cdc48 AAA+ ATPase is an abundant and essential enzyme that unfolds substrates in multiple protein quality control pathways. The enzyme includes two conserved AAA+ ATPase cassettes, D1 and D2, that assemble as hexameric rings with D1 stacked above D2. Here, we report an ensemble of structures of Cdc48 affinity purified from lysate in complex with the adaptor Shp1 in the act of unfolding substrate. Our analysis reveals a continuum of structural snapshots that spans the entire translocation cycle. These data reveal new elements of Shp1-Cdc48 binding and support a "hand-over-hand" mechanism in which the sequential movement of individual subunits is closely coordinated. D1 hydrolyzes ATP and disengages from substrate prior to D2, while D2 rebinds ATP and re-engages with substrate prior to D1, thereby explaining the dominant role played by D2 in substrate translocation/unfolding.