Effects of glycans and hinge on dynamics in the IgG1 Fc.

The crystallizable fragment (Fc) domain of immunoglobulin subclass IgG1 antibodies is engineered for a wide variety of pharmaceutical applications. Two important structural variables in Fc constructs are the hinge region connecting the Fc to the antigen binding fragments (Fab) and the glycans present in various glycoforms. These components affect receptor binding interactions that mediate immune activation. To design new antibody drugs, a robust in silico method for linking stability to structural changes is necessary. In this work, all-atom simulations were used to compare the dynamic behavior of the four structural variants arising from presence or absence of the hinge and glycans. We expressed the simplest of these constructs, the 'minimal Fc' with no hinge and no glycans, in Escherichia coli and report its crystal structure. The 'maximal Fc' that includes full hinge and G0F/G1F glycans is based on a previously reported structure, Protein Data Bank (PDB) ID: 5VGP. These, along with two intermediate structures (with only the glycans or with only the hinge) were used to independently measure the stability effects of the two structural variables using umbrella sampling simulations. Principal component analysis (PCA) was used to determine free energy effects along the Fc's dominant mode of motion. This work provides a comprehensive picture of the effects of hinge and glycans on Fc dynamics and stability.Communicated by Ramaswamy H. Sarma.

SARS-CoV-2 infection establishes a stable and age-independent CD8+ T cell response against a dominant nucleocapsid epitope using restricted T cell receptors.

The resolution of SARS-CoV-2 replication hinges on cell-mediated immunity, wherein CD8+ T cells play a vital role. Nonetheless, the characterization of the specificity and TCR composition of CD8+ T cells targeting non-spike protein of SARS-CoV-2 before and after infection remains incomplete. Here, we analyzed CD8+ T cells recognizing six epitopes from the SARS-CoV-2 nucleocapsid (N) protein and found that SARS-CoV-2 infection slightly increased the frequencies of N-recognizing CD8+ T cells but significantly enhanced activation-induced proliferation compared to that of the uninfected donors. The frequencies of N-specific CD8+ T cells and their proliferative response to stimulation did not decrease over one year. We identified the N222-230 peptide (LLLDRLNQL, referred to as LLL thereafter) as a dominant epitope that elicited the greatest proliferative response from both convalescent and uninfected donors. Single-cell sequencing of T cell receptors (TCR) from LLL-specific CD8+ T cells revealed highly restricted Vα gene usage (TRAV12-2) with limited CDR3α motifs, supported by structural characterization of the TCR-LLL-HLA-A2 complex. Lastly, transcriptome analysis of LLL-specific CD8+ T cells from donors who had expansion (expanders) or no expansion (non-expanders) after in vitro stimulation identified increased chromatin modification and innate immune functions of CD8+ T cells in non-expanders. These results suggests that SARS-CoV-2 infection induces LLL-specific CD8+ T cell responses with a restricted TCR repertoire.

Inactivation kinetics of a surrogate yield conservative predictions of foodborne pathogen reductions from low water activity foods of varying size and composition during low-temperature steam processing.

There is a growing interest in using models to predict foodborne pathogen inactivation as a way to validate or verify preventive controls. Unlike liquid foods, solid, low water activity foods (LWAF) are heterogenous in composition and structure and do not transfer heat uniformly. Using models constructed from one food to predict pathogen inactivation on another LWAF is complex and may not always be possible, even if the foods have similar composition. Using models constructed from inactivation kinetics of three foodborne pathogens and a surrogate from vacuum-steam-pasteurized (72 and 82 °C) whole macadamia nuts and dried apricot halves, 3-log reductions were predicted for the same pathogens and foods of reduced size. Model fits (First-order, Weibull, and Gompertz) were significantly impacted by the food type regardless of particle size. Despite the foods being identical in composition with particle size as the only altered characteristic, best-fit models accurately predicted the 3-log reductions only 50% of the time, but the surrogate inactivation models provided conservative predictions for pathogen reductions, highlighting that a surrogate's model may be a suitable tool for predicting pathogen reduction on LWAFs.

Monomeric crystal structure of the vaccine carrier protein CRM197 and implications for vaccine development.

CRM197 is a genetically detoxified mutant of diphtheria toxin (DT) that is widely used as a carrier protein in conjugate vaccines. Protective immune responses to several bacterial diseases are obtained by coupling CRM197 to glycans from these pathogens. Wild-type DT has been described in two oligomeric forms: a monomer and a domain-swapped dimer. Their proportions depend on the chemical conditions and especially the pH, with a large kinetic barrier to interconversion. A similar situation occurs in CRM197, where the monomer is preferred for vaccine synthesis. Despite 30 years of research and the increasing application of CRM197 in conjugate vaccines, until now all of its available crystal structures have been dimeric. Here, CRM197 was expressed as a soluble, intracellular protein in an Escherichia coli strain engineered to have an oxidative cytoplasm. The purified product, called EcoCRM, remained monomeric throughout crystallization. The structure of monomeric EcoCRM is reported at 2.0 Šresolution with the domain-swapping hinge loop (residues 379-387) in an extended, exposed conformation, similar to monomeric wild-type DT. The structure enables comparisons across expression systems and across oligomeric states, with implications for monomer-dimer interconversion and for the optimization of conjugation.

Reversible switching between two common protein folds in a designed system using only temperature.

Naturally occurring metamorphic proteins have the ability to interconvert from one folded state to another through either a limited set of mutations or by way of a change in the local environment. Here, we show in a designed system that it is possible to switch reversibly between two of the most common monomeric folds employing only temperature changes. We demonstrate that a latent 3α state can be unmasked from an α/ß-plait topology with a single V90T amino acid substitution, populating both forms simultaneously. The equilibrium between these two states exhibits temperature dependence, such that the 3α state is predominant (>90%) at 5 °C, while the α/ß-plait fold is the major species (>90%) at 30 °C. We describe the structure and dynamics of these topologies, how mutational changes affect the temperature dependence, and the energetics and kinetics of interconversion. Additionally, we demonstrate how ligand-binding function can be tightly regulated by large amplitude changes in protein structure over a relatively narrow temperature range that is relevant to biology. The 3α/αß switch thus represents a potentially useful approach for designing proteins that alter their fold topologies in response to environmental triggers. It may also serve as a model for computational studies of temperature-dependent protein stability and fold switching.

Design and characterization of a protein fold switching network.

To better understand how amino acid sequence encodes protein structure, we engineered mutational pathways that connect three common folds (3α, ß-grasp, and α/ß-plait). The structures of proteins at high sequence-identity intersections in the pathways (nodes) were determined using NMR spectroscopy and analyzed for stability and function. To generate nodes, the amino acid sequence encoding a smaller fold is embedded in the structure of an ~50% larger fold and a new sequence compatible with two sets of native interactions is designed. This generates protein pairs with a 3α or ß-grasp fold in the smaller form but an α/ß-plait fold in the larger form. Further, embedding smaller antagonistic folds creates critical states in the larger folds such that single amino acid substitutions can switch both their fold and function. The results help explain the underlying ambiguity in the protein folding code and show that new protein structures can evolve via abrupt fold switching.

Engineering subtilisin proteases that specifically degrade active RAS.

We describe the design, kinetic properties, and structures of engineered subtilisin proteases that degrade the active form of RAS by cleaving a conserved sequence in switch 2. RAS is a signaling protein that, when mutated, drives a third of human cancers. To generate high specificity for the RAS target sequence, the active site was modified to be dependent on a cofactor (imidazole or nitrite) and protease sub-sites were engineered to create a linkage between substrate and cofactor binding. Selective proteolysis of active RAS arises from a 2-step process wherein sub-site interactions promote productive binding of the cofactor, enabling cleavage. Proteases engineered in this way specifically cleave active RAS in vitro, deplete the level of RAS in a bacterial reporter system, and also degrade RAS in human cell culture. Although these proteases target active RAS, the underlying design principles are fundamental and will be adaptable to many target proteins.

Crystal Structure of a Bivalent Antibody Fab Fragment.

We determined the crystal structure to 1.8 Å resolution of the Fab fragment of an affinity-matured human monoclonal antibody (HC84.26.5D) that recognizes the E2 envelope glycoprotein of hepatitis C virus (HCV). Unlike conventional Fabs, which are monovalent monomers, Fab HC84.26.5D assembles into a bivalent domain-swapped dimer in which the two VL/VH modules are separated by ~25 Å. In solution, Fab HC84.26.5D exists predominantly as a dimer (~80%) in equilibrium with the monomeric form of the Fab (~20%). Dimerization is mediated entirely by deletion of a single residue, VHSer113 (Kabat numbering), in the elbow region linking the VH and CH1 domains. In agreement with the crystal structure, dimeric Fab HC84.26.5D is able to bind two HCV E2 molecules in solution. This is only the second example of a domain-swapped Fab dimer from among >3000 Fab crystal structures determined to date. Moreover, the architecture of the doughnut-shaped Fab HC84.26.5D dimer is completely different from that of the previously reported Fab 2G12 dimer. We demonstrate that the highly identifiable shape of dimeric Fab HC84.26.5D makes it useful as a fiducial marker for single-particle cryoEM analysis of HCV E2. Bivalent domain-swapped Fab dimers engineered on the basis of HC84.26.5D may also serve as a means of doubling the effective size of conventional Fab-protein complexes for cryoEM.

Management of anal intraepithelial neoplasia and anal squamous cell carcinoma at a tertiary referral centre with a dedicated infectious diseases unit: an 18-year review.

INTRODUCTION: Anal squamous cell carcinoma (ASCC) is a rare malignancy with rising incidence rates. Risk factors include human immunodeficiency virus (HIV) infection, high-risk sexual activity and HPV-related genitourinary dysplasia/neoplasia. There is an overlap between high-risk patients and those attending HIV Medicine/Sexual Health (HMSH) services. We hypothesised that HMSH involvement may facilitate earlier referral to colorectal surgeons, with better outcomes. METHODS: Retrospective review of all ASCC and anal intraepithelial neoplasia (AIN) treated at a tertiary-referral hospital with a dedicated HMSH clinic between 2000 and 2018. Comparative analysis was performed of demographics, management and outcomes between HMSH and non-HMSH patients. RESULTS: One hundred and nine patients had anal pathology, eighty-five with ASCC (78%) and twenty-four with AIN (22%). Seventy (64%) were male. Median (range) age at ASCC diagnosis was 51 years (26-88). Thirty-six percent of all patients attended HMSH services, 28% were HIV positive, and 41% of males were men-who-have-sex-with-men (MSM). Eighty-one ASCC patients (97.5%) were treated with curative intent. Sixty-seven (80%) had primary chemoradiation therapy. Fifteen (17.5%) had primary surgical excision. Twelve (14%) developed recurrent disease. Ultimately, seven required salvage APR. Overall 3-year survival (3YS) was 76%. HMSH patients were significantly younger at ASCC diagnosis (p < 0.001), with a higher prevalence of HIV, HPV and MSM. HMSH attenders also tended to be diagnosed at earlier stages, were less likely to develop recurrence and achieved better overall outcomes, with a superior overall 3YS than non-HMSH patients (92% vs 72%, p = 0.037). CONCLUSION: ASCC incidence is increasing worldwide. The HMSH cohort has emerged as a distinct subpopulation of younger, high-risk, male patients. Collaboration between HMSH and colorectal surgeons offers an opportunity for risk reduction strategies and earlier intervention.

Structural basis for oligoclonal T cell recognition of a shared p53 cancer neoantigen.

Adoptive cell therapy (ACT) with tumor-specific T cells can mediate cancer regression. The main target of tumor-specific T cells are neoantigens arising from mutations in self-proteins. Although the majority of cancer neoantigens are unique to each patient, and therefore not broadly useful for ACT, some are shared. We studied oligoclonal T-cell receptors (TCRs) that recognize a shared neoepitope arising from a driver mutation in the p53 oncogene (p53R175H) presented by HLA-A2. Here we report structures of wild-type and mutant p53-HLA-A2 ligands, as well as structures of three tumor-specific TCRs bound to p53R175H-HLA-A2. These structures reveal how a driver mutation in p53 rendered a self-peptide visible to T cells. The TCRs employ structurally distinct strategies that are highly focused on the mutation to discriminate between mutant and wild-type p53. The TCR-p53R175H-HLA-A2 complexes provide a framework for designing TCRs to improve potency for ACT without sacrificing specificity.

Localized Amyloidosis Involving Palatine Tonsils: A Case Report and Literature Review.

Amyloidosis is a localized or systemic process where extracellular insoluble plasma protein fibers are deposited into tissues. Localized amyloidosis is rare and curable by surgical resection. While the head and neck region represents 19% of localized amyloidosis cases, only one other case of bilateral involvement of the pharyngeal tonsils has been published in the international literature. We report a case of asymptomatic amyloidosis isolated to the bilateral palatine tonsils and a cervical lymph node in a 59-year-old male. Systemic amyloidosis was ruled out through multidisciplinary consultation, and resection of the masses was performed. This represents the second reported case of bilateral tonsillar amyloidosis.

Alfvénic velocity spikes and rotational flows in the near-Sun solar wind.

The prediction of a supersonic solar wind1 was first confirmed by spacecraft near Earth2,3 and later by spacecraft at heliocentric distances as small as 62 solar radii4. These missions showed that plasma accelerates as it emerges from the corona, aided by unidentified processes that transport energy outwards from the Sun before depositing it in the wind. Alfvénic fluctuations are a promising candidate for such a process because they are seen in the corona and solar wind and contain considerable energy5-7. Magnetic tension forces the corona to co-rotate with the Sun, but any residual rotation far from the Sun reported until now has been much smaller than the amplitude of waves and deflections from interacting wind streams8. Here we report observations of solar-wind plasma at heliocentric distances of about 35 solar radii9-11, well within the distance at which stream interactions become important. We find that Alfvén waves organize into structured velocity spikes with duration of up to minutes, which are associated with propagating S-like bends in the magnetic-field lines. We detect an increasing rotational component to the flow velocity of the solar wind around the Sun, peaking at 35 to 50 kilometres per second-considerably above the amplitude of the waves. These flows exceed classical velocity predictions of a few kilometres per second, challenging models of circulation in the corona and calling into question our understanding of how stars lose angular momentum and spin down as they age12-14.

Structure and Dynamics of a Site-Specific Labeled Fc Fragment with Altered Effector Functions.

Antibody-drug conjugates (ADCs) are a class of biotherapeutic drugs designed as targeted therapies for the treatment of cancer. Among the challenges in generating an effective ADC is the choice of an effective conjugation site on the IgG. One common method to prepare site-specific ADCs is to engineer solvent-accessible cysteine residues into antibodies. Here, we used X-ray diffraction and hydrogen-deuterium exchange mass spectroscopy to analyze the structure and dynamics of such a construct where a cysteine has been inserted after Ser 239 (Fc-239i) in the antibody heavy chain sequence. The crystal structure of this Fc-C239i variant at 0.23 nm resolution shows that the inserted cysteine structurally replaces Ser 239 and that this causes a domino-like backward shift of the local polypeptide, pushing Pro 238 out into the hinge. Proline is unable to substitute conformationally for the wild-type glycine at this position, providing a structural reason for the previously observed abolition of both FcγR binding and antibody-dependent cellular cytotoxicity. Energy estimates for the both the FcγR interface (7 kcal/mol) and for the differential conformation of proline (20 kcal/mol) are consistent with the observed disruption of FcγR binding, providing a quantifiable case where strain at a single residue appears to disrupt a key biological function. Conversely, the structure of Fc-C239i is relatively unchanged at the intersection of the CH2 and CH3 domains; the site known to be involved in binding of the neonatal Fc receptor (FcRn), and an alignment of the Fc-C239i structure with an Fc structure in a ternary Fc:FcRn:HSA (human serum albumin) complex implies that these favorable contacts would be maintained. Hydrogen deuterium exchange mass spectroscopy (HDX-MS) data further suggest a significant increase in conformational mobility for the Fc-C239i protein relative to Fc that is evident even far from the insertion site but still largely confined to the CH2 domain. Together, the findings provide a detailed structural and dynamic basis for previously observed changes in ADC functional binding to FcγR, which may guide further development of ADC designs.

The ophthalmic diagnosis and management of four siblings with Werner syndrome.

INTRODUCTION: Werner syndrome is a rare autosomal recessive disorder caused by mutations in the Werner syndrome WRN gene, on chromosome 8. Those affected manifest early the features of ageing. DISCUSSION: Cataract surgery is prone to post-operative complications in those with Werner syndrome. The development of cystoid macular oedema (CMO) is likely multifactorial. Patients with WS have diabetes mellitus type 2 which can contribute to macular oedema. There is a deposition of abnormal WRN proteins in the macula which also predisposes to macular oedema. The trauma of cataract surgery appears to be the main stimulus for the development of CMO. CMO may, as a result, be difficult to manage in Werner syndrome patients. CONCLUSION: Further study is needed to elucidate the precise role of retinal WRN protein expression in the development of CMO in those with Werner syndrome. A tailored and more successful approach to the treatment of CMO in such patients may result.

Relationship of BMI z score to fat percent and fat mass in multiethnic prepubertal children.

OBJECTIVE: The objective of the study is to examine the validity of body mass index z score (zBMI) as a measure of percent body fat in prepubertal children. METHODS: One hundred eleven multiethnic, healthy, Tanner 1 children aged 6-12 years had fat percent and fat mass measured by the four-compartment method as part of the Paediatric Rosetta Body Composition Cohort. Multiple regression models were developed with fat percent as the dependent variable and zBMI, age, sex and ethnicity as independent variables. RESULTS: Body mass index z score predicted fat percent, adjusted for age in both girls (P < 0.001, RMSE 5.67 and R2 0.54) and boys (P < 0.001, RMSE 4.71, R2 0.69). The average model percent error was 20.3% in girls and 21.6% in boys. zBMI2 predicted fat mass when adjusted for age and zBMI in both girls (P < 0.001, RMSE 2.27 and R2 0.82) and boys (P < 0.001, RMSE 2.08 and R2 0.81). The average percent error was 7.2% in girls and 8.7% in boys. Age was associated with percentage body fat (P < 0.01), while ethnicity was not (P > 0.05). CONCLUSIONS: Given the relatively large error in the models, zBMI are not a useful indicator of fat mass in healthy, Tanner 1 children. zBMI2 scores are associated with significantly lower absolute percent errors in girls and boys.

Structure of the Fc fragment of the NIST reference antibody RM8671.

As the link between antigen binding and immune activation, the antibody Fc region has received extensive structural study. In this report, the structure of the Fc fragment of the NIST IgG1 mAb (reference material 8671) is described at 2.1 Šresolution in space group P212121, with approximate unit-cell parameters a = 50, b = 80, c = 138 Å. Prior Fc structures with a wide variety of modifications are also surveyed, focusing on those in the same crystal form. To facilitate the analysis of conformations, a reference frame and a two-parameter metric are proposed, considering the CH2 domains as mobile with respect to a fixed dimeric CH3 core. Over several human Fc structures, a significant variation in Fc elbow conformations is observed, which may serve to facilitate the regulation of Fc effector signaling.

Clinical Audit of Sexual Abuse Referral Forms to the Sexual Assault Follow-up and Evaluation Clinic, Nassau, The Bahamas, among Females Aged 13 to 19 Years / Auditoría clínica de los formularios de remisión de abuso sexual a la Clínica de Evaluación y Seguimiento de Agresiones Sexuales, Nassau, Bahamas, entre mujeres de 13 a 19 años

ABSTRACT Objective: To evaluate the adequacy of the documentation of referral forms for sexually abused females aged 13-19 years directed to the Sexual Assault Follow-up and Evaluation (SAFE) Clinic at the Agape Family Medicine Clinic, Nassau, The Bahamas, for interim management. Methods: An approved review was performed on 123 referral forms regarding sexually abused females aged 13-19 years who attended the SAFE Clinic from 2011 to 2015. The exercise focussed on documentation adequacy based on a scoring system developed by the researchers (> 50% was assessed to be adequate; records of the referee's disposition of the patient, the date of the incident and evidence of sexually transmitted infection (STI) screening were considered vital for adequacy). Descriptive and inferential statistics were calculated. Results: The median age of the participants was 14 years (interquartile range: 13-15). Of the 63.4% (78) with documented nationality, 88.5% (69) were Bahamian and 11.5% (9) Haitian. Documentation status did not differ statistically significantly by nationality. Regarding documentation, 74% (91) recorded the name of the patient's school, 59.3% (73) recorded that the patient knew the assailant and 17.9% (22) indicated that the patient did not know the assailant, while 22.8% (28) did not document this latter information. Type of sexual penetration was indicated by 65.9% (81). Of the vital variables, 18.7% (23) recorded the referee's disposition of the patient, 29.8% (36) the date of the incident and 60.2% (74) evidence of STI screening; 7.3% (9) documented all three and 22.8% (28) two. The mean percentage of documentation for vital variables was 49.3% (± 3.6) for the Accident and Emergency (A&E) Department, Princess Margaret Hospital, Nassau, versus 30.5% (± 4.0) for public health clinics (PHCs) (p = 0.001). Overall, 69.9% (86 of 123) of the referral forms were deemed inadequate: 64.7% (33 of 51) from the A&E Department versus 73.4% (47 of 64) from PHCs among the 115 patients who provided referral information. Conclusion: Documentation deficiencies of the sexual abuse referral forms demand reform. Complete and consistent documentation is required.

RESUMEN Objetivo: Evaluar la idoneidad de la documentación de los formularios de remisión para mujeres de 13 a 19 años sexualmente abusadas, dirigidas a la Clínica de Evaluación y Seguimiento de Agresiones Sexuales (ESAS) en la Clínica Ágape de Medicina Familiar, Nassau, Bahamas, para la administración interina. Métodos: Se aprobó una revisión para examinar 123 formularios de remisión con respecto a las mujeres de 13 a 19 años sexualmente abusadas, que asistieron a la clínica de ESAS de 2011 a 2015. El ejercicio se centró en la idoneidad de la documentación basada en un sistema de puntuación desarrollado por los investigadores (50% fue adecuado según la valoración; los registros de la disposición de la paciente en el arbitraje, la fecha del incidente y la evidencia del tamizaje de la infección de transmisión sexual (ITS), fueron todos vitales a la hora de determinar la idoneidad). Se calcularon las estadísticas descriptivas e inferenciales. Resultados: La edad promedio de las participantes fue 14 años (rango intercuartil: 13-15). De 63.4% (78) con nacionalidad documentada, el 88.5% (69) fueron bahameñas y el 11.5% (9) haitianas. El estado de la documentación en término de las estadísticas no difirió significativamente por nacionalidad. Con respecto a la documentación, el 74% (91) registró el nombre de la escuela de la paciente, 59.3% (73) registró que la paciente conocía al agresor, y el 17.9% (22) indicó que la paciente no conocía al agresor, mientras que el 22.8% (28) no documentó esta última información. El tipo de penetración sexual fue indicado por 65.9% (81). De las variables vitales, 18.7% (23) registró la disposición de la paciente en el arbitraje, 29.8% (36) la fecha del incidente, y el 60.2% (74) evidencia del tamizaje de las ITS; 7.3% (9) documentó tres de ellas y 2.8% (28) dos. El porcentaje medio de documentación de las variables vitales fue 49.3% (± 3.6) para el Departamento de Accidentes y Emergencias (A&E), Hospital Princess Margaret, Nassau, frente al 30.5% (± 4.0) de las clínicas de salud pública (CSP) (p = 0.001). En general, el 69.9% (86 de 123) de los formularios de referencia se consideró inadecuado: 64.7% (33 de 51) del Departamento de A&E frente al 73.4% (47 de 64) de las CSP entre las 115 pacientes que proporcionaron la información de la remisión. Conclusión: Las deficiencias de la documentación de los formularios de remisión de abuso sexual exigen reformas. Se requiere una documentación completa y consistente.