Structural Principles in Robo Activation and Auto-inhibition.

Proper brain function requires high-precision neuronal expansion and wiring, processes controlled by the transmembrane Roundabout (Robo) receptor family and their Slit ligands. Despite their great importance, the molecular mechanism by which Robos' switch from "off" to "on" states remains unclear. Here, we report a 3.6 Å crystal structure of the intact human Robo2 ectodomain (domains D1-8). We demonstrate that Robo cis dimerization via D4 is conserved through hRobo1, 2, and 3 and the C. elegans homolog SAX-3 and is essential for SAX-3 function in vivo. The structure reveals two levels of auto-inhibition that prevent premature activation: (1) cis blocking of the D4 dimerization interface and (2) trans interactions between opposing Robo receptors that fasten the D4-blocked conformation. Complementary experiments in mouse primary neurons and C. elegans support the auto-inhibition model. These results suggest that Slit stimulation primarily drives the release of Robo auto-inhibition required for dimerization and activation.

Ubiquitin-independent proteasomal degradation driven by C-degron pathways.

Although most eukaryotic proteins are targeted for proteasomal degradation by ubiquitination, a subset have been demonstrated to undergo ubiquitin-independent proteasomal degradation (UbInPD). However, little is known about the molecular mechanisms driving UbInPD and the degrons involved. Utilizing the GPS-peptidome approach, a systematic method for degron discovery, we found thousands of sequences that promote UbInPD; thus, UbInPD is more prevalent than currently appreciated. Furthermore, mutagenesis experiments revealed specific C-terminal degrons required for UbInPD. Stability profiling of a genome-wide collection of human open reading frames identified 69 full-length proteins subject to UbInPD. These included REC8 and CDCA4, proteins which control proliferation and survival, as well as mislocalized secretory proteins, suggesting that UbInPD performs both regulatory and protein quality control functions. In the context of full-length proteins, C termini also play a role in promoting UbInPD. Finally, we found that Ubiquilin family proteins mediate the proteasomal targeting of a subset of UbInPD substrates.

Geographical Inequalities in Access to Bellwether Procedures in Brazil.

BACKGROUND: Brazil is a middle-income country that aims to provide universal health coverage, but its surgical system's efficiency has rarely been analyzed. In an effort to strengthen surgical national systems, the Lancet Commission on Global Surgery proposed bellwether procedures as quality indicators of surgical workforces. This study aims to evaluate regional inequalities in access to bellwether procedures and their associated mortality across the five Brazilian geographical regions. METHODS: Using DATASUS, Brazil's national healthcare database, data were collected on the total amount of performed bellwether procedures-cesarean section, laparotomy, and open fracture management-and their associated mortality, by geographical region. We evaluated the years 2018-2020, both in emergent and elective conditions. Statistical analysis was performed by one-way ANOVA test and Tukey's multiple comparisons test. RESULTS: During this period, DATASUS registered 2,687,179 cesarean sections, 1,036,841 laparotomies, and 648,961 open fracture treatments. The access and associated mortality related to these procedures were homogeneous between the regions in elective care. There were significant geographical inequalities in access and associated mortality in emergency care (p < 0.05, 95% CI) for all bellwether procedures. The Southeast, the most economically developed region of the country, was the region with the lowest amount of bellwether procedures per 100,000 inhabitants. CONCLUSION: Brazil's public surgical system is competent at promoting elective surgical care, but more effort is needed to fortify emergency care services. Public policies should encourage equity in the geographic allocation of the surgical workforce.

A duplex structure of SARM1 octamers stabilized by a new inhibitor.

In recent years, there has been growing interest in SARM1 as a potential breakthrough drug target for treating various pathologies of axon degeneration. SARM1-mediated axon degeneration relies on its TIR domain NADase activity, but recent structural data suggest that the non-catalytic ARM domain could also serve as a pharmacological site as it has an allosteric inhibitory function. Here, we screened for synthetic small molecules that inhibit SARM1, and tested a selected set of these compounds in a DRG axon degeneration assay. Using cryo-EM, we found that one of the newly discovered inhibitors, a calmidazolium designated TK106, not only stabilizes the previously reported inhibited conformation of the octamer, but also a meta-stable structure: a duplex of octamers (16 protomers), which we have now determined to 4.0 Å resolution. In the duplex, each ARM domain protomer is engaged in lateral interactions with neighboring protomers, and is further stabilized by contralateral contacts with the opposing octamer ring. Mutagenesis of the duplex contact sites leads to a moderate increase in SARM1 activation in cultured cells. Based on our data we propose that the duplex assembly constitutes an additional auto-inhibition mechanism that tightly prevents pre-mature activation and axon degeneration.

Comment on "Bi-layering at ionic liquid surfaces: a sum - frequency generation vibrational spectroscopy - and molecular dynamics simulation-based study" by T. Iwahashi, T. Ishiyama, Y. Sakai, A. Morita, D. Kim and Y. Ouchi, Phys. Chem. Chem. Phys., 2020, 22, 12565.

This Comment raises several questions concerning the surface structure concluded in the paper referenced in the title. Specifically, that paper ignores previous experiments and simulations which demonstrate for the same ionic liquids depth-decaying, multilayered surface-normal density profiles rather than the claimed molecular mono- or bi-layers. We demonstrate that the claimed structure does not reproduce the measured X-ray reflectivity, which probes directly the surface-normal density profile. The measured reflectivities are found, however, to be well-reproduced by a multilayered density model. These results, and previous experimental and simulation results, cast severe doubt on the validity of the surface structure claimed in the paper referenced in the title.

Surface structure evolution in a homologous series of ionic liquids.

Interfaces of room temperature ionic liquids (RTILs) are important for both applications and basic science and are therefore intensely studied. However, the evolution of their interface structure with the cation's alkyl chain length [Formula: see text] from Coulomb to van der Waals interaction domination has not yet been studied for even a single broad homologous RTIL series. We present here such a study of the liquid-air interface for [Formula: see text], using angstrom-resolution X-ray methods. For [Formula: see text], a typical "simple liquid" monotonic surface-normal electron density profile [Formula: see text] is obtained, like those of water and organic solvents. For [Formula: see text], increasingly more pronounced nanoscale self-segregation of the molecules' charged moieties and apolar chains yields surface layering with alternating regions of headgroups and chains. The layering decays into the bulk over a few, to a few tens, of nanometers. The layering periods and decay lengths, their linear [Formula: see text] dependence, and slopes are discussed within two models, one with partial-chain interdigitation and the other with liquid-like chains. No surface-parallel long-range order is found within the surface layer. For [Formula: see text], a different surface phase is observed above melting. Our results also impact general liquid-phase issues like supramolecular self-aggregation and bulk-surface structure relations.

Structural History of Human SRGAP2 Proteins.

In the development of the human brain, human-specific genes are considered to play key roles, conferring its unique advantages and vulnerabilities. At the time of Homo lineage divergence from Australopithecus, SRGAP2C gradually emerged through a process of serial duplications and mutagenesis from ancestral SRGAP2A (3.4-2.4 Ma). Remarkably, ectopic expression of SRGAP2C endows cultured mouse brain cells, with human-like characteristics, specifically, increased dendritic spine length and density. To understand the molecular mechanisms underlying this change in neuronal morphology, we determined the structure of SRGAP2A and studied the interplay between SRGAP2A and SRGAP2C. We found that: 1) SRGAP2A homo-dimerizes through a large interface that includes an F-BAR domain, a newly identified F-BAR extension (Fx), and RhoGAP-SH3 domains. 2) SRGAP2A has an unusual inverse geometry, enabling associations with lamellipodia and dendritic spine heads in vivo, and scaffolding of membrane protrusions in cell culture. 3) As a result of the initial partial duplication event (∼3.4 Ma), SRGAP2C carries a defective Fx-domain that severely compromises its solubility and membrane-scaffolding ability. Consistently, SRGAP2A:SRAGP2C hetero-dimers form, but are insoluble, inhibiting SRGAP2A activity. 4) Inactivation of SRGAP2A is sensitive to the level of hetero-dimerization with SRGAP2C. 5) The primal form of SRGAP2C (P-SRGAP2C, existing between ∼3.4 and 2.4 Ma) is less effective in hetero-dimerizing with SRGAP2A than the modern SRGAP2C, which carries several substitutions (from ∼2.4 Ma). Thus, the genetic mutagenesis phase contributed to modulation of SRGAP2A's inhibition of neuronal expansion, by introducing and improving the formation of inactive SRGAP2A:SRGAP2C hetero-dimers, indicating a stepwise involvement of SRGAP2C in human evolutionary history.

Self-segregated nanostructure in room temperature ionic liquids.

The nanosegregated bulk structure, and its evolution with the cation's alkyl length n, are studied by X-ray scattering for an unprecedentedly broad homologous series of a model room-temperature ionic liquid, [CnMIM][NTf2] (n = 4-22). A tri-periodic local structure is found, with the lateral periodicities, dII and dIII independent of n, and a longitudinal one, dI, linearly increasing with n. The results are consistent with a local structure comprising alternating layers of polar headgroups and apolar, interdigitated, partly overlapping, cations' alkyl tails, of an average macroscopic mass density close to that of liquid alkanes. A slope decrease in the linear dI(n) suggests a change from a lower to a higher rate of increase with n of chain overlap for n ≥ 12. The order decay lengths of the layering, and of the lateral chain packing, increase with n, as expected from the increasing van der Waals interaction's domination of the structure. The headgroups' lateral packing decay length decreases with n, due to increasing frustration between the longer lateral periodicity preferred by the headgroups, and the shorter lateral periodicity preferred by the chains. A comparison of the bulk and surface structures highlights the surface's ordering effect, which, however, does not induce here a surface phase different from the bulk, as it does in liquid crystals and liquid alkanes.

Correction: Self-segregated nanostructure in room temperature ionic liquids.

Correction for 'Self-segregated nanostructure in room temperature ionic liquids' by Diego Pontoni et al., Soft Matter, 2017, DOI: 10.1039/c7sm01464c.

Development of a New Device for Postoperative Self-Irrigation of the Maxillary and Frontal Sinus.

OBJECTIVES: Nasal saline irrigation is the corner stone of postoperative care after functional endoscopic sinus surgery (FESS) for chronic rhinosinusitis (CRS). However, intrasinus penetration of the saline solution can be challenging and may require difficult head position, particularly for the frontal sinus. Our aim was to evaluate a novel device for direct intrasinus self-irrigation, usable at home for both maxillary and frontal sinus. METHODS: Thirty devices were implemented in 23 patients: in the maxillary sinus for 18 patients and in the frontal sinus for 5 patients. The device was removed after 7 days on average (5-10 days), and nasal saline irrigation was carried on with a squeeze bottle for 6 weeks. Retrospective evaluation of the device included: device-related complication, patient satisfaction, and ostial or middle turbinate synechiae at 3 months. RESULTS: No device-related complication (obstruction, displacement, infection, bleeding) occurred. Twenty-one (91.3%) patients were satisfied with the device. Two patients required the help of a nurse for irrigation. No ostial of middle turbinate synechiae was visualized at 3 months. This new endonasal device enables direct intrasinus self-irrigation after FESS for CRS. CONCLUSION: This preliminary study showed that this device is safe and easy to use. However, further investigations are required to assess its potential role to reduce the risk of synechiae and revision surgery.

Role of Neural Plasticity of Motor Cortex in Gliomas Evaluated by Brain Imaging and Mapping Techniques in Pre- and Postoperative Period: A Systematic Review.

BACKGROUND: Resection of infiltrative neuroepithelial primary brain tumors, such as low-grade gliomas (LGGs) remains a neurosurgical challenge. Usual lack of clinical deficit despite LGGs growing in eloquent brain areas may be explained by reshaping and reorganization of functional networks. The development of modern diagnostic imaging techniques could disclose better understanding of the rearrangement of the brain cortex; however, mechanisms underlying such compensation and how it occurs in the motor cortex remain unclear. This systematic review aims to analyze the neuroplasticity of motor cortex in patients with LGGs, as determined by neuroimaging and functional techniques. METHODS: Following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines, medical subject headings (MeSH) and the following terms related to neuroimaging, LGGs and neuroplasticity were used with the Boolean operators AND and OR to synonymous terms in the PubMed database. Among the 118 results, 19 studies were included in the systematic review. RESULTS: Motor function in patients with LGG was characterized by a compensation in the contralateral and supplementary motor areas and premotor functional networks. Furthermore, ipsilateral activation in these types of gliomas was rarely described. Moreover, some studies did not reveal statistical significance in association between functional reorganization and the postoperative period, which can be explained by the low number of patients. CONCLUSION: Our findings suggest a high pattern of reorganization per different eloquent motor areas and gliomas diagnosis. Understanding this process is useful to guide safe surgical resection and to develop protocols that assess the plasticity, even though functional network rearrangement needs to be better characterized by more studies.

Structure-function analysis of ceTIR-1/hSARM1 explains the lack of Wallerian axonal degeneration in C. elegans.

Wallerian axonal degeneration (WD) does not occur in the nematode C. elegans, in contrast to other model animals. However, WD depends on the NADase activity of SARM1, a protein that is also expressed in C. elegans (ceSARM/ceTIR-1). We hypothesized that differences in SARM between species might exist and account for the divergence in WD. We first show that expression of the human (h)SARM1, but not ceTIR-1, in C. elegans neurons is sufficient to confer axon degeneration after nerve injury. Next, we determined the cryoelectron microscopy structure of ceTIR-1 and found that, unlike hSARM1, which exists as an auto-inhibited ring octamer, ceTIR-1 forms a readily active 9-mer. Enzymatically, the NADase activity of ceTIR-1 is substantially weaker (10-fold higher Km) than that of hSARM1, and even when fully active, it falls short of consuming all cellular NAD+. Our experiments provide insight into the molecular mechanisms and evolution of SARM orthologs and WD across species.

Women Representation in Surgical Specialties: Reflections about Gender Equity after the 34th Brazilian Surgical Conference.

In September 2021, the 34th Brazilian Surgical Conference hosted the "Panel: Women in Surgery" - the only session in the event solely composed of female speakers. Although gender inequities in surgery are well recognized in the international literature, the panel portrayed how distant we are from the desired equity in our country. In addition, the session emphasized the need to broaden the debate and identify the mechanisms for greater inclusion and maintenance of women in the surgical career. In this editorial, we provide a historical overview of gender disparities in the Brazilian surgical ecosystem, highlight the contributing factors to a reduced number of female surgeons, and how the structure of medical societies may influence the rise of women to leadership positions. Accordingly, we discuss the benefits of gender diversity for surgeons, patients, and institutions. Furthermore, we analyze the representation of women in the Brazilian College of Surgeons since its foundation and in the scientific sessions at the conference, demonstrating that more initiatives are required to encourage female representation in the college. Finally, we propose a series of recommendations to foster engagement and contribute to the prosperity of women surgeons in Brazil.

Structural basis for SARM1 inhibition and activation under energetic stress.

SARM1, an executor of axonal degeneration, displays NADase activity that depletes the key cellular metabolite, NAD+, in response to nerve injury. The basis of SARM1 inhibition and its activation under stress conditions are still unknown. Here, we present cryo-EM maps of SARM1 at 2.9 and 2.7 Å resolutions. These indicate that SARM1 homo-octamer avoids premature activation by assuming a packed conformation, with ordered inner and peripheral rings, that prevents dimerization and activation of the catalytic domains. This inactive conformation is stabilized by binding of SARM1's own substrate NAD+ in an allosteric location, away from the catalytic sites. This model was validated by mutagenesis of the allosteric site, which led to constitutively active SARM1. We propose that the reduction of cellular NAD+ concentration contributes to the disassembly of SARM1's peripheral ring, which allows formation of active NADase domain dimers, thereby further depleting NAD+ to cause an energetic catastrophe and cell death.

"My Dog Deafened Me!": Case Report of Capnocytophaga canimorsus Infection and Literature Review.

Capnocytophaga canimorsus infection is an emerging zoonotic disease that could cause meningitis and subsequent sensorineural hearing loss (SNHL), especially in the immunocompromised population. It is a gram-negative rod that belongs to the normal oral flora of dogs and cats and may be transmitted to humans by biting or licking. Our case report and literature review showed that this postmeningitic SNHL has distinct features from common bacterial meningitis-related SNHL. We therefore discuss the diagnostic, therapeutic, and preventive aspects of this uncommon cause of SNHL. Because it is now the second-most common dog-bite transmitted pathogen, otolaryngologists are more likely to encounter this zoonotic disease and should be aware of its characteristics. Laryngoscope, 129:41-43, 2019.

Structural Evidence for an Octameric Ring Arrangement of SARM1.

SARM1 induces axonal degeneration in response to various insults and is therefore considered an attractive drug target for the treatment of neuro-degenerative diseases as well as for brain and spinal cord injuries. SARM1 activity depends on the integrity of the protein's SAM domains, as well as on the enzymatic conversion of NAD+ to ADPR (ADP Ribose) products by the SARM1's TIR domain. Therefore, inhibition of either SAM or TIR functions may constitute an effective therapeutic strategy. However, there is currently no SARM1-directed therapeutic approach available because of an insufficient structural and mechanistic understanding of this protein. In this study, we found that SARM1 assembles into an octameric ring. This arrangement was not described before in other SAM proteins, but is reminiscent of the apoptosome and inflammasome-well-known apoptotic ring-like oligomers. We show that both SARM1 and the isolated tandem SAM1-2 domains form octamers in solution, and electron microscopy analysis reveals an octameric ring of SARM1. We determined the crystal structure of SAM1-2 and found that it also forms a closed octameric ring in the crystal lattice. The SAM1-2 ring interactions are mediated by complementing "lock and key" hydrophobic grooves and inserts and electrostatic charges between the neighboring protomers. We have mutated several interacting SAM1-2 interfaces and measured how these mutations affect SARM1 apoptotic activity in cultured cells, and in this way identified critical oligomerization sites that facilitate cell death. These results highlight the importance of oligomerization for SARM1 function and reveal critical epitopes for future targeted drug development.

Leukocyte Cytoskeleton Polarization Is Initiated by Plasma Membrane Curvature from Cell Attachment.

Cell polarization is important for various biological processes. However, its regulation, particularly initiation, is incompletely understood. Here, we investigated mechanisms by which neutrophils break their symmetry and initiate their cytoskeleton polarization from an apolar state in circulation for their extravasation during inflammation. We show here that a local increase in plasma membrane (PM) curvature resulting from cell contact to a surface triggers the initial breakage of the symmetry of an apolar neutrophil and is required for subsequent polarization events induced by chemical stimulation. This local increase in PM curvature recruits SRGAP2 via its F-BAR domain, which in turn activates PI4KA and results in PM PtdIns4P polarization. Polarized PM PtdIns4P is targeted by RPH3A, which directs PIP5K1C90 and subsequent phosphorylated myosin light chain polarization, and this polarization signaling axis regulates neutrophil firm attachment to endothelium. Thus, this study reveals a mechanism for the initiation of cell cytoskeleton polarization.

Approaching the Roundabout: cis and trans Robo1 Contacts Revealed.

In this issue of Structure,Aleksandrova et al. (2018) present low- and high-resolution structures of Robo1, a key player in axonal guidance. The structures shed light on the arrangement of Robo1 at the plasma membrane and provide evidence for back-to-back trans Robo1 contacts.

Robo Ig4 Is a Dimerization Domain.

Robo receptors play pivotal roles in axonal guidance as well as in neurogenesis, angiogenesis, cell migration, and cancer progression and invasiveness. They are considered to be attractive drug targets for the treatment of cancer, ocular neovascular disorders, chronic kidney diseases, and more. Despite their great importance, the mechanisms by which Robo receptors switch from their "off" to "on" states remain obscure. One possibility involves a monomer-to-dimer or dimer-to-monomer transition that facilitates the recruitment and activation of enzymatic effectors to instigate intracellular signaling. However, it is not known which domains mediate Robo dimerization, or the structural properties of the dimeric interactions. Here, we identify the extracellular Ig4 (D4) as a Robo dimerization domain. We have determined the crystal structure of the tandem Ig4-5 domains (D4-5) of human Robo2 and found that a hydrophobic surface on D4 mediates close homotypic contacts with a reciprocal D4. Analytical ultracentrifugation measurements of intact and mutated D4-5 shows that dimerization through the D4 interface is specific and has a dimerization dissociation constant of 16.9µM in solution. Direct fluorescence resonance energy transfer dimerization measurements in HEK293 cells corroborate the dimerization of transmembrane hRobo2 through D4, and a functional COS-7 cell collapse assay links D4-mediated dimerization with Robo intracellular signaling. The high level of conservation in the D4 dimerization interface throughout all Robo orthologs and paralogs implies that D4-mediated dimerization is a central hallmark in Robo activation and signaling.