Baseline unfolded protein response signaling adjusts the timing of the mammalian cell cycle.

The endoplasmic reticulum (ER) is a single-copy organelle that cannot be generated de novo, suggesting coordination between the mechanisms overseeing ER integrity and those controlling the cell cycle to maintain organelle inheritance. The Unfolded Protein Response (UPR) is a conserved signaling network that regulates ER homeostasis. Here, we show that pharmacological and genetic inhibition of the UPR sensors IRE1, ATF6, and PERK in unstressed cells delays the cell cycle, with PERK inhibition showing the most penetrant effect, which was associated with a slowdown of the G1-to-S/G2 transition. Treatment with the small molecule ISRIB to bypass the effects of PERK-dependent phosphorylation of the translation initiation factor eIF2α had no such effect, suggesting that cell cycle timing depends on PERK's kinase activity but is independent of eIF2α phosphorylation. Using complementary light and electron microscopy and flow cytometry-based analyses, we also demonstrate that the ER enlarges before mitosis. Together, our results suggest coordination between UPR signaling and the cell cycle to maintain ER physiology during cell division.

Role of trafficking protein particle complex 2 in medaka development.

The skeletal dysplasia spondyloepiphyseal dysplasia tarda (SEDT) is caused by mutations in the TRAPPC2 gene, which encodes Sedlin, a component of the trafficking protein particle (TRAPP) complex that we have shown previously to be required for the export of type II collagen (Col2) from the endoplasmic reticulum. No vertebrate model for SEDT has been generated thus far. To address this gap, we generated a Sedlin knockout animal by mutating the orthologous TRAPPC2 gene (olSedl) of Oryzias latipes (medaka) fish. OlSedl deficiency leads to embryonic defects, short size, diminished skeletal ossification and altered Col2 production and secretion, resembling human defects observed in SEDT patients. Moreover, SEDT knock-out animals display photoreceptor degeneration and gut morphogenesis defects, suggesting a key role for Sedlin in the development of these organs. Thus, by studying Sedlin function in vivo, we provide evidence for a mechanistic link between TRAPPC2-mediated membrane trafficking, Col2 export, and developmental disorders.

Signaling plasticity in the integrated stress response.

The Integrated Stress Response (ISR) is an essential homeostatic signaling network that controls the cell's biosynthetic capacity. Four ISR sensor kinases detect multiple stressors and relay this information to downstream effectors by phosphorylating a common node: the alpha subunit of the eukaryotic initiation factor eIF2. As a result, general protein synthesis is repressed while select transcripts are preferentially translated, thus remodeling the proteome and transcriptome. Mounting evidence supports a view of the ISR as a dynamic signaling network with multiple modulators and feedback regulatory features that vary across cell and tissue types. Here, we discuss updated views on ISR sensor kinase mechanisms, how the subcellular localization of ISR components impacts signaling, and highlight ISR signaling differences across cells and tissues. Finally, we consider crosstalk between the ISR and other signaling pathways as a determinant of cell health.

Optogenetic control of the integrated stress response reveals proportional encoding and the stress memory landscape.

The integrated stress response (ISR) is a conserved signaling network that detects aberrations and computes cellular responses. Dissecting these computations has been difficult because physical and chemical inducers of stress activate multiple parallel pathways. To overcome this challenge, we engineered a photo-switchable control over the ISR sensor kinase PKR (opto-PKR), enabling virtual, on-target activation. Using light to control opto-PKR dynamics, we traced information flow through the transcriptome and for key downstream ISR effectors. Our analyses revealed a biphasic, proportional transcriptional response with two dynamic modes, transient and gradual, that correspond to adaptive and terminal outcomes. We then constructed an ordinary differential equation (ODE) model of the ISR, which demonstrated the dependence of future stress responses on past stress. Finally, we tested our model using high-throughput light-delivery to map the stress memory landscape. Our results demonstrate that cells encode information in stress levels, durations, and the timing between encounters. A record of this paper's transparent peer review process is included in the supplemental information.

Signaling by the integrated stress response kinase PKR is fine-tuned by dynamic clustering.

The double-stranded RNA sensor kinase PKR is one of four integrated stress response (ISR) sensor kinases that phosphorylate the α subunit of eukaryotic initiation factor 2 (eIF2α) in response to stress. The current model of PKR activation considers the formation of back-to-back PKR dimers as a prerequisite for signal propagation. Here we show that PKR signaling involves the assembly of dynamic PKR clusters. PKR clustering is driven by ligand binding to PKR's sensor domain and by front-to-front interfaces between PKR's kinase domains. PKR clusters are discrete, heterogeneous, autonomous coalescences that share some protein components with processing bodies. Strikingly, eIF2α is not recruited to PKR clusters, and PKR cluster disruption enhances eIF2α phosphorylation. Together, these results support a model in which PKR clustering may limit encounters between PKR and eIF2α to buffer downstream signaling and prevent the ISR from misfiring.

Correction of oxidative stress enhances enzyme replacement therapy in Pompe disease.

Pompe disease is a metabolic myopathy due to acid alpha-glucosidase deficiency. In addition to glycogen storage, secondary dysregulation of cellular functions, such as autophagy and oxidative stress, contributes to the disease pathophysiology. We have tested whether oxidative stress impacts on enzyme replacement therapy with recombinant human alpha-glucosidase (rhGAA), currently the standard of care for Pompe disease patients, and whether correction of oxidative stress may be beneficial for rhGAA therapy. We found elevated oxidative stress levels in tissues from the Pompe disease murine model and in patients' cells. In cells, stress levels inversely correlated with the ability of rhGAA to correct the enzymatic deficiency. Antioxidants (N-acetylcysteine, idebenone, resveratrol, edaravone) improved alpha-glucosidase activity in rhGAA-treated cells, enhanced enzyme processing, and improved mannose-6-phosphate receptor localization. When co-administered with rhGAA, antioxidants improved alpha-glucosidase activity in tissues from the Pompe disease mouse model. These results indicate that oxidative stress impacts on the efficacy of enzyme replacement therapy in Pompe disease and that manipulation of secondary abnormalities may represent a strategy to improve the efficacy of therapies for this disorder.

Hybrid Robotics for Endoscopic Transnasal Skull Base Surgery: Single-Centre Case Series.

BACKGROUND: Only preclinical studies and case reports have described robotic surgery for endoscopic transnasal skull base surgery. OBJECTIVE: To evaluate the role of a novel robotic endoscope holder, developed for transsphenoidal surgery. METHODS: Patients were prospectively enrolled for 3 mo at the Neurosurgery Unit of Brescia. Endoscope Robot® was used to assist during the sphenoidal phase of the approach, tumor removal, and skull base reconstruction. A Likert scale questionnaire was given to all surgeons after each procedure. Patients who underwent robotic-assisted surgery were matched with nonrobotic ones for pathology and type of procedure. All surgical videos were evaluated during bimanual phases. RESULTS: Twenty-one patients underwent robot-assisted, endoscopic transsphenoidal surgery for different pathologies (16 pituitary adenomas, 3 chordomas, 1 craniopharyngioma, 1 pituitary exploration for Cushing disease) for a total of 23 procedures (1 patient underwent 2 endoscopic revisions of a skull base reconstruction). Subjective advantages reported by surgeons included smoothness of movement, image steadiness, and improvement of maneuvers in narrow spaces and with angled endoscopes; as the main limitation, Endoscope Robot® appeared to be relatively heavy during the initial endoscope positioning. A comparative analysis with a historical matched cohort documented similar clinical outcomes, while endoscope lens cleaning and position readjustments were significantly less frequent in robotic procedures. CONCLUSION: Although confirmation in larger studies is needed, Endoscope Robot® was a safe and effective tool, especially advantageous in lengthy interventions through deep and narrow corridors.

Comparison of Severe Acute Respiratory Syndrome Coronavirus 2 Screening Using Reverse Transcriptase-Quantitative Polymerase Chain Reaction or CRISPR-Based Assays in Asymptomatic College Students.

Importance: The reopening of colleges and universities in the US during the coronavirus disease 2019 (COVID-19) pandemic is a significant public health challenge. The development of accessible and practical approaches for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection in the college population is paramount for deploying recurrent surveillance testing as an essential strategy for virus detection, containment, and mitigation. Objective: To determine the prevalence of SARS-CoV-2 in asymptomatic participants in a university community by using CREST (Cas13-based, rugged, equitable, scalable testing), a CRISPR-based test developed for accessible and large-scale viral screening. Design, Setting, and Participants: For this cohort study, a total of 1808 asymptomatic participants were screened for SARS-CoV-2 using a CRISPR-based assay and a point-of-reference reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) test. Viral prevalence in self-collected oropharyngeal swab samples collected from May 28 to June 11, 2020, and from June 23 to July 2, 2020, was evaluated. Exposures: Testing for SARS-CoV-2. Main Outcomes and Measures: SARS-CoV-2 status, viral load, and demographic information of the study participants were collected. Results: Among the 1808 participants (mean [SD] age, 27.3 [11.0] years; 955 [52.8%] female), 732 underwent testing from May to early June (mean [SD] age, 28.4 [11.7] years; 392 [53.6%] female). All test results in this cohort were negative. In contrast, 1076 participants underwent testing from late June to early July (mean [SD] age, 26.6 [10.5] years; 563 [52.3%] female), with 9 positive results by RT-qPCR. Eight of these positive samples were detected by the CRISPR-based assay and confirmed by Clinical Laboratory Improvement Amendments-certified diagnostic testing. The mean (SD) age of the positive cases was 21.7 (3.3) years; all 8 individuals self-identified as students. These metrics showed that a CRISPR-based assay was effective at capturing positive SARS-CoV-2 cases in this student population. Notably, the viral loads detected in these asymptomatic cases resemble those seen in clinical samples, highlighting the potential of covert viral transmission. The shift in viral prevalence coincided with the relaxation of stay-at-home measures. Conclusions and Relevance: These findings reveal a shift in SARS-CoV-2 prevalence in a young and asymptomatic population and uncover the leading edge of a local outbreak that coincided with rising case counts in the surrounding county and the state of California. The concordance between CRISPR-based and RT-qPCR testing suggests that CRISPR-based assays are reliable and offer alternative options for surveillance testing and detection of SARS-CoV-2 outbreaks, as is required to resume operations in higher-education institutions in the US and abroad.

Hybrid Robotics for Endoscopic Skull Base Surgery: Preclinical Evaluation and Surgeon First Impression.

BACKGROUND: A robotic endoscope holder should theoretically provide various advantages in transnasal endoscopic skull base surgery, but only recently has a robotic system become commercially available. The objective of this study was to provide a preclinical evaluation of potential advantages and surgeon first impression of this robotic hybrid solution. METHODS: Thirty skull base surgeons, attending the Joint European Diploma of Endoscopic Skull Base Surgery 2018-2019 in Paris, France, were enrolled. A questionnaire, mainly concerning personal surgical experience and habits, was administered. The test phase consisted of 2 different dry-lab tasks, performed with and without EndoscopeRobot, according to randomization and on 2 different days. A modified NASA Task Load Index test was subsequently administered via e-mail to all participants. Completion times and modified Global Evaluative Assessment of Robotic Skills in Endoscopy scores of the videotaped tasks were recorded. RESULTS: Nineteen otorhinolaryngologic surgeons and 11 neurosurgeons, with different surgical habits and endoscopic experience, were enrolled. No one appeared unfavorable a priori to robotic endoscopic surgery. Although the robot did not provide an advantage in the simple grasping task 1, a trend toward better completion times and efficacy was evident in the bimanual task 2, when performed with the robot and bimanually. According to the modified NASA Task Load Index test, surgeons felt more successful with the robot in task 2, finding it less stressful and mentally demanding. CONCLUSIONS: Endoscopic skull base surgeons seem to view a hybrid robotic solution positively. EndoscopeRobot seems to provide a benefit to the single surgeon with experience in bimanual endoscopic surgery. Further preclinical and clinical evaluation of this technology is necessary.

Hijacking intracellular membranes to feed autophagosomal growth.

Autophagy is widely considered as a housekeeping mechanism that enables cells to survive stress conditions and, in particular, nutrient deprivation. Autophagy begins with the formation of the phagophore that expands and closes around cytosolic material and damaged organelles destined for degradation. The execution of this complex machinery is guaranteed by the coordinated action of more than 40 ATG (autophagy-related) proteins that control the entire process at different stages from the biogenesis of the autophagosome to cargo sequestration and fusion with lysosomes. Autophagosome biogenesis occurs at multiple intracellular sites, such as the endoplasmic reticulum (ER) and the plasma membrane. Soon after the formation of the phagophore, the nascent autophagosome progressively grows in size and ultimately closes by recruiting intracellular membranes. In this review, we focus on the contribution of three membrane sources - the ER, the ER-Golgi intermediate compartment, and the Golgi complex - to autophagosome biogenesis and expansion. We also highlight the interplay between the secretory pathway and autophagy in cells when nutrients are scarce.

The TRAPP complex mediates secretion arrest induced by stress granule assembly.

The TRAnsport Protein Particle (TRAPP) complex controls multiple membrane trafficking steps and is strategically positioned to mediate cell adaptation to diverse environmental conditions, including acute stress. We have identified the TRAPP complex as a component of a branch of the integrated stress response that impinges on the early secretory pathway. The TRAPP complex associates with and drives the recruitment of the COPII coat to stress granules (SGs) leading to vesiculation of the Golgi complex and arrest of ER export. The relocation of the TRAPP complex and COPII to SGs only occurs in cycling cells and is CDK1/2-dependent, being driven by the interaction of TRAPP with hnRNPK, a CDK substrate that associates with SGs when phosphorylated. In addition, CDK1/2 inhibition impairs TRAPP complex/COPII relocation to SGs while stabilizing them at ER exit sites. Importantly, the TRAPP complex controls the maturation of SGs. SGs that assemble in TRAPP-depleted cells are smaller and are no longer able to recruit RACK1 and Raptor, two TRAPP-interactive signaling proteins, sensitizing cells to stress-induced apoptosis.

Three-Dimensional High-Definition Ventriculoscope: Single-Center Case Series.

OBJECTIVE: Three-dimensional (3D), high-definition (HD) endoscopy has been recently introduced in neurosurgery, and its value has been discussed extensively in endonasal skull base surgery. Because there has been no reported clinical series on the use of a recent 3D-HD ventriculoscope, the aim of this study was to describe our initial experience with this novel device. METHODS: Patients consecutively operated on from June 2016 to June 2018 with a 3D-HD ventriculoscope were prospectively collected. The system is a 6-mm, 0-degree optic with a 105-degree field of view, with a central working channel of 2.2-mm diameter and 2 side channels of 1.3-mm diameter. Patients' demographic data, preoperative symptoms, and neurologic status; neuroradiologic data; type of surgery; operative time; intraoperative and postoperative complications, and follow-up data were prospectively recorded and retrospectively reviewed. RESULTS: Twenty-four patients (age range: 3-84 years) underwent 25 procedures including endoscopic third ventriculocisternostomy, biopsy, and cyst fenestration. The technical goal of surgery was obtained in all patients. There were no intraoperative complications, expect for 1 intraoperative epileptic seizure. Postoperative complications included asymptomatic subdural collections in 2 patients, infection, and delayed endoscopic third ventriculocisternostomy closure in 1 patient each. Relative limits of the system are its size and the availability of only a 0-degree optic. Image quality appeared satisfactory in all procedures. The lack of a dedicated introducer was resolved, exploiting a vascular "peel-away" system. CONCLUSIONS: 3D-HD technology seems to provide potential advantages in ventricular surgery. This initial experience is promising but must be confirmed by larger series.

The Golgi complex in disease and therapy.

The Golgi complex occupies a strategic position in the endomembrane system and acts not only as a key trafficking and sorting station and a vital biosynthetic center for glycoproteins and lipids, but also as an active signaling hub. As such, the Golgi complex participates in the establishment and maintenance of cell compartmentalization and in general, cell processes such as cell growth and apoptosis. The different functions of the Golgi complex are executed by composite molecular machineries that have been exhaustively dissected over the last three decades. These machineries can become dysfunctional as a result of mutations in the respective encoding genes or may be hijacked by infectious agents or misregulated in the course of multifactorial diseases such as neurodegeneration and cancer. Small molecules targeting components of these machineries have been instrumental in dissecting their functions in in vitro studies and some of them have been developed or are currently under development for clinical use.

Brain tumors in eloquent areas: A European multicenter survey of intraoperative mapping techniques, intraoperative seizures occurrence, and antiepileptic drug prophylaxis.

Intraoperative mapping and monitoring techniques for eloquent area tumors are routinely used world wide. Very few data are available regarding mapping and monitoring methods and preferences, intraoperative seizures occurrence and perioperative antiepileptic drug management. A questionnaire was sent to 20 European centers with experience in intraoperative mapping or neurophysiological monitoring for the treatment of eloquent area tumors. Fifteen centers returned the completed questionnaires. Data was available on 2098 patients. 863 patients (41.1%) were operated on through awake surgery and intraoperative mapping, while 1235 patients (58.8%) received asleep surgery and intraoperative electrophysiological monitoring or mapping. There was great heterogeneity between centers with some totally AW oriented (up to 100%) and other almost totally ASL oriented (up to 92%) (31% SD). For awake surgery, 79.9% centers preferred an asleep-awake-asleep anesthesia protocol. Only 53.3% of the centers used ECoG or transcutaneous EEG. The incidence of intraoperative seizures varied significantly between centers, ranging from 2.5% to 54% (p < 0.001). It there appears to be a statistically significant link between the mastery of mapping technique and the risk of intraoperative seizures. Moreover, history of preoperative seizures can significantly increase the risk of intraoperative seizures (p < 0.001). Intraoperative seizures occurrence was similar in patients with or without perioperative drugs (12% vs. 12%, p = 0.2). This is the first European survey to assess intraoperative functional mapping and monitoring protocols and the management of peri- and intraoperative seizures. This data can help identify specific aspects that need to be investigated in prospective and controlled studies.