Chiari 1: Is decompression always necessary previous to scoliosis surgery?

The association between Chiari 1 malformation and scoliosis is well known in the literature. Prevalence has increased after the advent of magnetic resonance imaging. In children with this association, prophylactic suboccipital decompression prior to scoliosis correction is a common surgical procedure although the rationale for this surgical management and whether not performing it may lead to spinal cord injury has not been clearly elucidated. We conducted a systematic review of the literature with the aim to obtain strong data to support the hypothesis that it is safe to proceed with scoliosis correction without prior prophylactic suboccipital decompression for Chiari 1 in an asymptomatic population. Using the Prisma methodology, we analyzed 3250 studies published between 1972 and 2018. Only four studies met the inclusion criteria. None of the studies had a level of evidence high enough to recommend prophylactic decompression previous to correction of the spinal deformity.

COVA1-18 neutralizing antibody protects against SARS-CoV-2 in three preclinical models.

One year into the Coronavirus Disease 2019 (COVID-19) pandemic caused by Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2), effective treatments are still needed 1-3 . Monoclonal antibodies, given alone or as part of a therapeutic cocktail, have shown promising results in patients, raising the hope that they could play an important role in preventing clinical deterioration in severely ill or in exposed, high risk individuals 4-6 . Here, we evaluated the prophylactic and therapeutic effect of COVA1-18 in vivo , a neutralizing antibody isolated from a convalescent patient 7 and highly potent against the B.1.1.7. isolate 8,9 . In both prophylactic and therapeutic settings, SARS-CoV-2 remained undetectable in the lungs of COVA1-18 treated hACE2 mice. Therapeutic treatment also caused a dramatic reduction in viral loads in the lungs of Syrian hamsters. When administered at 10 mg kg - 1 one day prior to a high dose SARS-CoV-2 challenge in cynomolgus macaques, COVA1-18 had a very strong antiviral activity in the upper respiratory compartments with an estimated reduction in viral infectivity of more than 95%, and prevented lymphopenia and extensive lung lesions. Modelling and experimental findings demonstrate that COVA1-18 has a strong antiviral activity in three different preclinical models and could be a valuable candidate for further clinical evaluation.

Aneurysmal bone cyst and osteoblastoma: an extremely rare combination in the pediatric spine.

STUDY DESIGN: Case report. OBJECTIVE: To report the clinical and imaging findings of a patient with the extremely rare association of aneurysmal bone cyst and osteoblastoma in the cervical spine. To our knowledge, only three cases have been reported in the published literature in children under 16 years of age with this condition in the cervical spine. METHODS: The patient's history, physical examination, imaging findings, and management with a complete 4-year medical history, surgical intervention and radiological follow-up are reported. RESULTS: A 4-year 11-month-old boy was diagnosed with aneurysmal bone cyst in association of osteoblastoma and was treated with CT-guided intralesional injection calcitonin and methylprednisolone. During the course of intralesional therapy, a pathological fracture of C2 was produced. Subsequently, a widened intralesional excision and instrumented fusion from occiput to cervical spine (C0-C4) was performed. CONCLUSION: The association of aneurysmal bone cyst and osteoblastoma in spine is extremely rare. Although both are benign lesions, in the cervical location, complete removal of the tumors is challenging. Wide resection with reconstruction of the segments for stability associated with adjuvant treatment with calcitonin and corticosteroids provides a good option.

Harvesting Environmental Microalgal Blooms for Remediation and Resource Recovery: A Laboratory Scale Investigation with Economic and Microbial Community Impact Assessment.

A laboratory based microflotation rig termed efficient FLOtation of Algae Technology (eFLOAT) was used to optimise parameters for harvesting microalgal biomass from eutrophic water systems. This was performed for the dual objectives of remediation (nutrient removal) and resource recovery. Preliminary experiments demonstrated that chitosan was more efficient than alum for flocculation of biomass and the presence of bacteria could play a positive role and reduce flocculant application rates under the natural conditions tested. Maximum biomass removal from a hyper-eutrophic water retention pond sample was achieved with 5 mg·L-1 chitosan (90% Chlorophyll a removal). Harvesting at maximum rates showed that after 10 days, the bacterial diversity is significantly increased with reduced cyanobacteria, indicating improved ecosystem functioning. The resource potential within the biomass was characterized by 9.02 µg phosphate, 0.36 mg protein, and 103.7 µg lipid per mg of biomass. Fatty acid methyl ester composition was comparable to pure cultures of microalgae, dominated by C16 and C18 chain lengths with saturated, monounsaturated, and polyunsaturated fatty acids. Finally, the laboratory data was translated into a full-size and modular eFLOAT system, with estimated costs as a novel eco-technology for efficient algal bloom harvesting.

The crystal structure of CD8 in complex with YTS156.7.7 Fab and interaction with other CD8 antibodies define the binding mode of CD8 alphabeta to MHC class I.

The CD8alphabeta heterodimer interacts with class I pMHC on antigen-presenting cells as a co-receptor for TCR-mediated activation of cytotoxic T cells. To characterize this immunologically important interaction, we used monoclonal antibodies (mAbs) specific to either CD8alpha or CD8beta to probe the mechanism of CD8alphabeta binding to pMHCI. The YTS156.7 mAb inhibits this interaction and blocks T cell activation. To elucidate the molecular basis for this inhibition, the crystal structure of the CD8alphabeta immunoglobulin-like ectodomains were determined in complex with mAb YTS156.7 Fab at 2.7 A resolution. The YTS156.7 epitope on CD8beta was identified and implies that residues in the CDR1 and CDR2-equivalent loops of CD8beta are occluded upon binding to class I pMHC. To further characterize the pMHCI/CD8alphabeta interaction, binding of class I tetramers to CD8alphabeta on the surface of T cells was assessed in the presence of anti-CD8 mAbs. In contrast to YTS156.7, mAb YTS105.18, which is specific for CD8alpha, does not inhibit binding of CD8alphabeta to class I tetramers, indicating the YTS105.18 epitope is not occluded in the pMHCI/CD8alphabeta complex. Together, these data indicate a model for the pMHCI/CD8alphabeta interaction similar to that observed for CD8alphaalpha in the CD8alphaalpha/pMHCI complex, but in which CD8alpha occupies the lower orientation (membrane proximal to the antigen presenting cell), and CD8beta occupies the upper position (membrane distal). The implication of this molecular assembly for the function of CD8alphabeta in T cell activation is discussed.

Architecture of CD1 proteins.

The CD1 family of glycosylated cell surface receptors binds and presents lipid antigens for T cell recognition and activation. Crystal structures of CD1-lipid complexes reveal differences in the mode of presentation of lipids by CD1 group 1 (CDla, CDlb, and CDlc) and group 2 isoforms (CDld). For group 1, especially CDla and CD1b, the lipid backbone is anchored inside the hydrophobic binding grooves (lipid anchoring), whereas, for group 2 CDld, a precise hydrogen-bonding network positions the polar ligand headgroups in well-defined orientation at the T cell recognition surface (headgroup positioning). In addition, small, but important, structural changes occur on the surface of CDld upon binding of the potent invariant NKT cell agonist alpha-galactosylceramide due to increased polar interaction with the alphal and alpha2 helices. No such ligand-induced, conformational changes have yet been reported for any group 1 CD1 complexes, even upon binding of chemically diverse antigens, such as dual alkyl chain sphingolipids vs single alkyl chain lipopeptides. These structural data have already been successfully translated into the design of enhanced lipid activators of NKT cells and will likely continue for design of other chemotherapeutic agents or immunostimulatory compounds for a variety of immune-mediated diseases.

PET amyloid ligand [11C]PIB uptake is increased in mild cognitive impairment.

BACKGROUND: Patients with mild cognitive impairment (MCI) have increased risk to develop Alzheimer disease (AD). In AD increased brain amyloid burden has been demonstrated in vivo with PET using N-methyl-[(11)C]2-(4'-methylaminophenyl)-6-hydroxybenzothiazole ([(11)C]PIB) as a tracer. OBJECTIVE: To investigate whether patients with amnestic MCI would show increased [(11)C]PIB uptake, indicating early AD process. METHODS: We studied 13 patients with amnestic MCI and 14 control subjects with PET using [(11)C]PIB as tracer. Parametric images were computed by calculating the region-to-cerebellum ratio in each voxel over 60 to 90 minutes. Group differences in [(11)C]PIB uptake were analyzed with statistical parametric mapping (SPM) and automated region-of-interest (ROI) analysis. RESULTS: The SPM analysis showed that patients with MCI had significantly higher [(11)C]PIB uptake vs control subjects in the frontal, parietal, and lateral temporal cortices as well as in the posterior cingulate showing the most prominent differences. These results were supported by the automated ROI analysis in which MCI patients showed in comparison with healthy control subjects increased [(11)C]PIB uptake in the frontal cortex (39% increase from the control mean, p < 0.01), the posterior cingulate (39%, p < 0.01), the parietal (31%, p < 0.01) and lateral temporal (28%, p < 0.001) cortices, putamen (17%, p < 0.05), and caudate (25%, p < 0.05). Individually, in the frontal cortex and posterior cingulate, 8 of 13 patients with MCI had [(11)C]PIB uptake values above 2 SD from the control mean. MCI subjects having at least one APOE epsilon4 allele tended to have higher [(11)C]PIB uptake than MCI subjects without APOE epsilon4. CONCLUSIONS: At group level the elevated N-methyl-[(11)C]2-(4'-methylaminophenyl)-6-hydroxybenzothiazole ([(11)C]PIB) uptake in patients with mild cognitive impairment (MCI) resembled that seen in Alzheimer disease (AD). At the individual level, about half of the MCI patients had [(11)C]PIB uptake in the AD range, suggestive of early AD process.

Voxel-based analysis of PET amyloid ligand [11C]PIB uptake in Alzheimer disease.

BACKGROUND: PET studies with N-methyl-[(11)C]2-(4':-methylaminophenyl)-6-hydroxybenzothiazole ([(11)C]PIB) have revealed an increased tracer uptake in several brain regions in Alzheimer disease (AD). OBJECTIVE: To employ voxel-based analysis method to identify brain regions with significant increases in [(11)C]PIB uptake in AD vs healthy control subjects, indicative of increased amyloid accumulation in these regions. METHODS: We studied 17 patients with AD and 11 control subjects with PET using [(11)C]PIB as tracer. Parametric images were computed by calculating a region-to-cerebellum ratio over 60 to 90 minutes in each voxel. Group differences in [(11)C]PIB uptake were analyzed with statistical parametric mapping (SPM) and automated region-of-interest (ROI) analysis. RESULTS: SPM showed increased uptake (p < 0.001) in the frontal, parietal, and lateral temporal cortices as well as in the posterior cingulate and the striatum. No significant differences in uptake were found in the primary sensory and motor cortices, primary visual cortex, thalamus, and medial temporal lobe. These results were supported by automated ROI analysis, with most prominent increases in AD subjects in the frontal cortex ([(11)C]PIB uptake 163% of the control mean) and posterior cingulate (146%) followed by the parietal (146%) and temporal (145%) cortices and striatum (133%), as well as small increases in the occipital cortex (117%) and thalamus (115%). CONCLUSIONS: Voxel-based analysis revealed widespread distribution of increased [(11)C]PIB uptake in Alzheimer disease (AD). These findings are in accordance with the distribution and phases of amyloid pathology in AD, previously documented in postmortem studies.

Crystal structure of the TCR co-receptor CD8alphaalpha in complex with monoclonal antibody YTS 105.18 Fab fragment at 2.88 A resolution.

The CD8 glycoprotein functions as an essential element in the control of T-cell selection, maturation and the TCR-mediated response to peptide antigen. CD8 is expressed as both heterodimeric CD8alphabeta and homodimeric CD8alphaalpha isoforms, which have distinct physiological roles and exhibit tissue-specific expression patterns. CD8alphaalpha has previously been crystallized in complex with class I pMHC and, more recently, with the mouse class Ib thymic leukemia antigen (TL). Here, we present the crystal structure of a soluble form of mouse CD8alphaalpha in complex with rat monoclonal antibody YTS 105.18 Fab fragment at 2.88 A resolution. YTS 105.18, which is commonly used in the blockade of CD8+ T-cell activation in response to peptide antigen, is specific for mouse CD8alpha. The YTS 105.18 Fab is one of only five rat IgG Fab structures to have been reported to date. Analysis of the YTS 105.18 Fab epitope on CD8alpha reveals that this antibody blocks CD8 activity by hydrogen bonding to residues that are critical for interaction with both class I pMHC and TL. Structural comparison of the liganded and unliganded forms of soluble CD8alphaalpha indicates that the mouse CD8alphaalpha immunoglobulin-domain dimer does not undergo significant structural alteration upon interaction either with class I pMHC or TL.

Improved design of an antigen with enhanced specificity for the broadly HIV-neutralizing antibody b12.

In an attempt to design immunogens that elicit broadly HIV-neutralizing antibodies, we recently engineered monomeric HIV-1 gp120 to bind preferentially b12, a broadly neutralizing antibody to the CD4-binding site (CD4bs) on gp120, by mutating four central residues in the CD4bs to alanine and introducing extra N-glycosylation sites potentially to mask unwanted B-cell epitopes. Despite the favorable antigenicity of this mutant, it harbors two potential caveats that may limit its effectiveness to elicit b12-like antibodies: (i) b12-binding affinity is reduced relative to wild-type gp120 and (ii) binding of some non-neutralizing antibodies to the N-terminal C1 region of gp120 is still observed. Here, we sought to correct these potential limitations. By reverting one of the added N-glycosylation sites on the gp120 core, b12 binding was improved without affecting the epitope-masking properties of the original mutant. Furthermore, truncation of the gp120 N-terminus eliminated binding of the anti-C1 antibodies. Finally, based on the binding profiles of additional non-neutralizing antibodies tested here, further N-glycosylation sites were incorporated to mask their corresponding epitopes. The resulting hyperglycosylated gp120 variants bind b12 and another broadly neutralizing antibody, 2G12, with apparent affinities approaching that of wild-type gp120, but do not bind 21 non- or weakly neutralizing antibodies to seven different epitopes on gp120. These hyperglycosylated variants expand our panel of glycoengineered gp120s that are currently being evaluated for their ability to elicit broadly neutralizing antibodies.

Crystallographic and biochemical analysis of cocaine-degrading antibody 15A10.

Catalytic antibody 15A10 hydrolyzes the benzoyl ester of cocaine to form the nonpsychoactive metabolites benzoic acid and ecgonine methylester. Here, we report biochemical and structural studies that characterize the catalytic mechanism. The crystal structure of the cocaine-hydrolyzing monoclonal antibody (mAb) 15A10 has been determined at 2.35 A resolution. The binding pocket is fairly shallow and mainly hydrophobic but with a cluster of three hydrogen-bond donating residues (TrpL96, AsnH33, and TyrH35). Computational docking of the transition state analogue (TSA) indicates that these residues are appropriately positioned to coordinate the phosphonate moiety of the TSA and, hence, form an oxyanion hole. Tyrosine modification of the antibody with tetranitromethane reduced hydrolytic activity to background level. The contribution from these and other residues to catalysis and TSA binding was explored by site-directed mutagenesis of 15A10 expressed in a single chain fragment variable (scFv) format. The TyrH35Phe mutant had 4-fold reduced activity, and TrpL96Ala, TrpL96His, and AsnH33Ala mutants were all inactive. Comparison with an esterolytic antibody D2.3 revealed a similar arrangement of tryptophan, asparagine, and tyrosine residues in the oxyanion hole that stabilizes the transition state for ester hydrolysis. Furthermore, the crystal structure of the bacterial cocaine esterase (cocE) also showed that the cocE employs a tyrosine hydroxyl in the oxyanion hole. Thus, the biochemical and structural data are consistent with the catalytic antibody providing oxyanion stabilization as its major contribution to catalysis.

Place cell rigidity correlates with impaired spatial learning in aged rats.

In humans and in animals, some aged individuals are severely impaired in learning and memory capacity whereas others perform as well as young adults. In the present study, the spatial memory capacity of young and aged rats was characterized by the Morris water maze task, and then firing patterns of hippocampal "place cells" were assessed as the animals explored a familiar environment and a geometrically-altered version of the environment. Spatial representations of hippocampal cells in young and memory-intact aged rats changed upon exposure to the altered environment. In contrast, spatial representations of many cells in aged, memory-impaired rats were unaffected by the environmental alteration. Furthermore, combining all groups, the extent to which spatial representations distinguished the familiar and altered environments predicted learning capacity in the water maze. These findings suggest that a major component of memory impairment in aging may be the failure of the hippocampus to encode subtle differences in contextual information that differ across multiple experiences, such as the sequence of training trials in the water maze.

Crystal structure of Sar1-GDP at 1.7 A resolution and the role of the NH2 terminus in ER export.

The Sar1 GTPase is an essential component of COPII vesicle coats involved in export of cargo from the ER. We report the 1.7-A structure of Sar1 and find that consistent with the sequence divergence of Sar1 from Arf family GTPases, Sar1 is structurally distinct. In particular, we show that the Sar1 NH2 terminus contains two regions: an NH2-terminal extension containing an evolutionary conserved hydrophobic motif that facilitates membrane recruitment and activation by the mammalian Sec12 guanine nucleotide exchange factor, and an alpha1' amphipathic helix that contributes to interaction with the Sec23/24 complex that is responsible for cargo selection during ER export. We propose that the hydrophobic Sar1 NH2-terminal activation/recruitment motif, in conjunction with the alpha1' helix, mediates the initial steps in COPII coat assembly for export from the ER.

Unexpected formation of an epoxide-derived multisubstrate adduct inhibitor on the active site of GAR transformylase.

Multisubstrate adduct inhibitors (MAI) of glycinamide ribonucleotide transformylase (GAR Tfase), which incorporate key features of the folate cofactor and the beta-GAR substrate, typically exhibit K(i)'s in the picomolar range. However, these compounds have reduced bioavailability due to the incorporation of a negatively charged phosphate moiety that prevents effective cellular uptake. Thus, a folate analogue that is capable of adduct formation with the substrate on the enzyme active site could lead to a potent GAR Tfase inhibitor that takes advantage of the cellular folate transport systems. We synthesized a dibromide folate analogue, 10-bromo-10-bromomethyl-5,8,10-trideazafolic acid, that was an intermediate designed to assemble with the substrate beta-GAR on the enzyme active site. We have now determined the crystal structure of the Escherichia coli GAR Tfase/MAI complex at 1.6 A resolution to ascertain the nature and mechanism of its time-dependent inhibition. The high-resolution crystal structure clearly revealed the existence of a covalent adduct between the substrate beta-GAR and the folate analogue (K(i) = 20 microM). However, the electron density map surprisingly indicated a C10 hydroxyl in the adduct rather than a bromide and suggested that the multisubstrate adduct is not formed directly from the dibromide but proceeds via an epoxide. Subsequently, we demonstrated the in situ conversion of the dibromide to the epoxide. Moreover, synthesis of the authentic epoxide confirmed that its inhibitory, time-dependent, and cytotoxic properties are comparable to those of the dibromide. Further, inhibition was strongest when the dibromide or epoxide is preincubated with both enzyme and substrate, indicating that inhibition occurs via the enzyme-dependent formation of the multisubstrate adduct. Thus, the crystal structure revealed the successful formation of an enzyme-assembled multisubstrate adduct and highlighted a potential application for epoxides, and perhaps aziridines, in the design of efficacious GAR Tfase inhibitors.

Crystal structure of an isolated V(alpha) domain of the 2C T-cell receptor.

The T-cell receptor (TCR) is a heterodimeric cell-surface protein consisting of two chains, alpha and beta, each of which is composed of a variable (V) and a constant (C) domain. Crystals of the isolated V(alpha) domain of the murine TCR 2C were grown by serendipity from a solution containing the extracellular domains of the intact TCR 2C and CD3 gamma epsilon-chains. The V(alpha) crystal structure shows how crystal packing can substitute for another V(alpha) domain in a different fashion from that observed in V(alpha)/V(alpha) homodimer and V(alpha)/V(beta) heterodimer structures. Significant conformational changes occur in the CDR3 and beta(3)beta(4) loops that normally form part of the dimer interface. The monomeric V(alpha) domain provides the unique opportunity to study the effect of dimerization on the conformation of the unliganded complementarity-determining regions (CDR) of a TCR. This structure of an individual V(alpha) module has implications for stability and bioengineering of isolated antibody and immunoglobulin domains.

Structural basis for a disfavored elimination reaction in catalytic antibody 1D4.

Murine antibody 1D4 selectively catalyzes a highly disfavored beta-elimination reaction. Crystal structures of unliganded 1D4 and 1D4 in complex with a transition-state analog (TSA) have elucidated a possible general base mode of catalysis. The structures of the unliganded and liganded Fabs were determined to 1.80 and 1.85 A resolution, respectively. The structure of the complex reveals a binding pocket with high shape complementarity to the TSA, which is recruited to coerce the substrate into the sterically demanding, eclipsed conformation that is required for catalysis. A histidine residue and two water molecules are likely involved in the catalysis. The structure supports either a concerted E2 or stepwise E1cB-like mechanism for elimination. Finally, the liganded 1D4 structure shows minor conformational rearrangements in CDR H2, indicative of induced-fit binding of the hapten. 1D4 has pushed the boundaries of antibody-mediated catalysis into the realm of disfavored reactions and, hence, represents an important milestone in the development of this technology.

Getting into the groove: unusual features of peptide binding to MHC class I molecules and implications in vaccine design.

The major histocompatibility complex presents antigenic peptides on the surface of antigen presenting cells to T cell receptors. Recognition of peptide-MHC by T cells initiates a cascade of signals in T cells which maintains a T cell dependent immune response. An understanding of the how peptides bind to MHC class I molecules is an important prerequisite in the design of vaccines. Herein, we will discuss, with special emphasis on MUC1, unusual features of MUC1 peptide binding to MHC class I, obtained from vaccine studies including a MUC1 peptide mimic and the crystal structures of low and high affinity peptides lacking canonical anchor motifs in complex with H-2Kb.

Observation of covalent intermediates in an enzyme mechanism at atomic resolution.

In classical enzymology, intermediates and transition states in a catalytic mechanism are usually inferred from a series of biochemical experiments. Here, we derive an enzyme mechanism from true atomic-resolution x-ray structures of reaction intermediates. Two ultra-high resolution structures of wild-type and mutant d-2-deoxyribose-5-phosphate (DRP) aldolase complexes with DRP at 1.05 and 1.10 angstroms unambiguously identify the postulated covalent carbinolamine and Schiff base intermediates in the aldolase mechanism. In combination with site-directed mutagenesis and (1)H nuclear magnetic resonance, we can now propose how the heretofore elusive C-2 proton abstraction step and the overall stereochemical course are accomplished. A proton relay system appears to activate a conserved active-site water that functions as the critical mediator for proton transfer.

Global efforts in structural genomics.

A worldwide initiative in structural genomics aims to capitalize on the recent successes of the genome projects. Substantial new investments in structural genomics in the past 2 years indicate the high level of support for these international efforts. Already, enormous progress has been made on high-throughput methodologies and technologies that will speed up macromolecular structure determinations. Recent international meetings have resulted in the formation of an International Structural Genomics Organization to formulate policy and foster cooperation between the public and private efforts.

Antibody catalysis of the oxidation of water.

Recently we reported that antibodies can generate hydrogen peroxide (H2O2) from singlet molecular oxygen (1O2*). We now show that this process is catalytic, and we identify the electron source for a quasi-unlimited generation of H2O2. Antibodies produce up to 500 mole equivalents of H2O2 from 1O2*, without a reduction in rate, and we have excluded metals or Cl- as the electron source. On the basis of isotope incorporation experiments and kinetic data, we propose that antibodies use H2O as an electron source, facilitating its addition to 1O2* to form H2O3 as the first intermediate in a reaction cascade that eventually leads to H2O2. X-ray crystallographic studies with xenon point to putative conserved oxygen binding sites within the antibody fold where this chemistry could be initiated. Our findings suggest a protective function of immunoglobulins against 1O2* and raise the question of whether the need to detoxify 1O2* has played a decisive role in the evolution of the immunoglobulin fold.