Multi-compartmental diversification of neutralizing antibody lineages dissected in SARS-CoV-2 spike-immunized macaques.

The continued evolution of SARS-CoV-2 underscores the need to understand qualitative aspects of the humoral immune response elicited by spike immunization. Here, we combine monoclonal antibody (mAb) isolation with deep B cell receptor (BCR) repertoire sequencing of rhesus macaques immunized with prefusion-stabilized spike glycoprotein. Longitudinal tracing of spike-sorted B cell lineages in multiple immune compartments demonstrates increasing somatic hypermutation and broad dissemination of vaccine-elicited B cells in draining and non-draining lymphoid compartments, including the bone marrow, spleen and, most notably, periaortic lymph nodes. Phylogenetic analysis of spike-specific monoclonal antibody lineages identified through deep repertoire sequencing delineates extensive intra-clonal diversification that shaped neutralizing activity. Structural analysis of the spike in complex with a broadly neutralizing mAb provides a molecular basis for the observed differences in neutralization breadth between clonally related antibodies. Our findings highlight that immunization leads to extensive intra-clonal B cell evolution where members of the same lineage can both retain the original epitope specificity and evolve to recognize additional spike variants not previously encountered.

Structural basis of broad SARS-CoV-2 cross-neutralization by affinity-matured public antibodies.

Descendants of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant now account for almost all SARS-CoV-2 infections. The Omicron variant and its sublineages have spike glycoproteins that are highly diverged from the pandemic founder and first-generation vaccine strain, resulting in significant evasion from monoclonal antibody therapeutics and vaccines. Understanding how commonly elicited antibodies can broaden to cross-neutralize escape variants is crucial. We isolate IGHV3-53, using "public" monoclonal antibodies (mAbs) from an individual 7 months post infection with the ancestral virus and identify antibodies that exhibit potent and broad cross-neutralization, extending to the BA.1, BA.2, and BA.4/BA.5 sublineages of Omicron. Deep mutational scanning reveals these mAbs' high resistance to viral escape. Structural analysis via cryoelectron microscopy of a representative broadly neutralizing antibody, CAB-A17, in complex with the Omicron BA.1 spike highlights the structural underpinnings of this broad neutralization. By reintroducing somatic hypermutations into a germline-reverted CAB-A17, we delineate the role of affinity maturation in the development of cross-neutralization by a public class of antibodies.

A review on the surface modification of materials for 3D-printed diagnostic devices.

Three-dimensional (3D) printing in tissue engineering and biosensing of analytes by using biocompatible materials or modifying surface structures is an upcoming area of study. This review discusses three common surface modification techniques, viz. alkaline hydrolysis, UV light photografting, and plasma treatment. Alkaline hydrolysis involves the reaction of an alkaline solution with the surface of a material, causing the surface to develop carboxyl and hydroxyl groups. This technique can enhance the biocompatibility, surface wettability, adhesion, printability, and dyeability of materials, such as acrylonitrile butadiene styrene (ABS), polycarbonate, and polylactic acid (PLA). This review also mentions details about some of the surface-modified 3D-printed diagnostic devices. Although most of the devices are modified using chemical processes, there are always multiple techniques involved while designing a diagnostic device. We have, therefore, mentioned some of the devices based on the materials used instead of categorising them as per modification techniques. 3D printing helps in the design of sophisticated shapes and structures using multiple materials. They can, therefore be used even in the design of microfluidic devices that are very useful for biosensing. We have also mentioned a few materials for printing microfluidic devices.

Volumetric Analysis of Renal Masses as Predictors of Partial Nephrectomy Outcomes.

Objective: To examine the role of endophytic tumor volume (TV) assessment (endophycity) on perioperative partial nephrectomy (PN) outcomes. Patients and Methods: Retrospective review of 212 consecutive laparoscopic and open partial nephrectomies from single institution using preoperative imaging and 1-year follow-up. Demographics, comorbidities, RENAL nephrometry scores, and all peri- and postoperative outcomes were recorded. Volumetric analysis performed using imaging software, independently assessed by two blinded radiologists. Univariate and multivariate statistical analysis were completed to assess predictive value of endophycity for all clinically meaningful outcomes. Results: Among those undergoing minimally invasive surgery (MIS), lower tumor endophycity was associated with higher likelihood of trifecta outcome (negative surgical margin, <10% decline in estimated glomerular filtration rate, the absence of complications) irrespective of max tumor size. For MIS, estimated blood loss increased with greater tumor endophycity regardless of tumor size. Among those who underwent open partial nephrectomy, lower tumor endophycity was associated with trifecta outcomes for tumors >4 cm only. On multivariate analysis with log-scaled odds ratios (OR), tumor endophycity and total kidney volume had the strongest correlation with tumor-related complications (OR = 3.23, 2.66). The analysis identified that tumor endophycity and TV on imaging were inversely correlated with of trifecta outcomes (OR = 0.53 for both covariates). Conclusions: Volumetric assessment of tumor endophycity performed well in identifying PN outcomes. As automated imaging software improves, volumetric analysis may prove to be a useful adjunct in preoperative planning and patient counseling.

Immunoglobulin germline gene polymorphisms influence the function of SARS-CoV-2 neutralizing antibodies.

The human immunoglobulin heavy-chain (IGH) locus is exceptionally polymorphic, with high levels of allelic and structural variation. Thus, germline IGH genotypes are personal, which may influence responses to infection and vaccination. For an improved understanding of inter-individual differences in antibody responses, we isolated SARS-CoV-2 spike-specific monoclonal antibodies from convalescent health care workers, focusing on the IGHV1-69 gene, which has the highest level of allelic variation of all IGHV genes. The IGHV1-69∗20-using CAB-I47 antibody and two similar antibodies isolated from an independent donor were critically dependent on allele usage. Neutralization was retained when reverting the V region to the germline IGHV1-69∗20 allele but lost when reverting to other IGHV1-69 alleles. Structural data confirmed that two germline-encoded polymorphisms, R50 and F55, in the IGHV1-69 gene were required for high-affinity receptor-binding domain interaction. These results demonstrate that polymorphisms in IGH genes can influence the function of SARS-CoV-2 neutralizing antibodies.

Non-coding 7S RNA inhibits transcription via mitochondrial RNA polymerase dimerization.

The mitochondrial genome encodes 13 components of the oxidative phosphorylation system, and altered mitochondrial transcription drives various human pathologies. A polyadenylated, non-coding RNA molecule known as 7S RNA is transcribed from a region immediately downstream of the light strand promoter in mammalian cells, and its levels change rapidly in response to physiological conditions. Here, we report that 7S RNA has a regulatory function, as it controls levels of mitochondrial transcription both in vitro and in cultured human cells. Using cryo-EM, we show that POLRMT dimerization is induced by interactions with 7S RNA. The resulting POLRMT dimer interface sequesters domains necessary for promoter recognition and unwinding, thereby preventing transcription initiation. We propose that the non-coding 7S RNA molecule is a component of a negative feedback loop that regulates mitochondrial transcription in mammalian cells.

A bispecific monomeric nanobody induces spike trimer dimers and neutralizes SARS-CoV-2 in vivo.

Antibodies binding to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike have therapeutic promise, but emerging variants show the potential for virus escape. This emphasizes the need for therapeutic molecules with distinct and novel neutralization mechanisms. Here we describe the isolation of a nanobody that interacts simultaneously with two RBDs from different spike trimers of SARS-CoV-2, rapidly inducing the formation of spike trimer-dimers leading to the loss of their ability to attach to the host cell receptor, ACE2. We show that this nanobody potently neutralizes SARS-CoV-2, including the beta and delta variants, and cross-neutralizes SARS-CoV. Furthermore, we demonstrate the therapeutic potential of the nanobody against SARS-CoV-2 and the beta variant in a human ACE2 transgenic mouse model. This naturally elicited bispecific monomeric nanobody establishes an uncommon strategy for potent inactivation of viral antigens and represents a promising antiviral against emerging SARS-CoV-2 variants.

Comparison of Pterygium Recurrence Rates Between Attending Physicians and Supervised Trainee Residents.

PURPOSE: To compare the recurrence rates after pterygium surgery performed by supervised trainee residents and attending physicians. METHODS: This retrospective study included pterygium surgeries performed by trainee residents and attending physicians in an academic institution in South Texas in the years 2008 to 2019. All residents performed surgeries under direct supervision of an attending physician. Only primary pterygium cases with a minimum postoperative follow-up of 6 months were included. Patients' demographics, primary surgeon, use of conjunctival autograft (CAU) or amniotic membrane graft (AMG), recurrence of pterygium, follow-up length, and complications were recorded. RESULTS: This study included 240 eyes of 229 patients with a mean age of 55.6 ± 12.3 years (range, 28-91 years). Of these eyes, 100 surgeries were performed by attending physicians (including 87 with CAU and 13 with AMG) and 140 surgeries by trainee residents (including 119 with CAU and 21 with AMG). There were no significant differences between the 2 groups of patients regarding age, sex, and surgical technique (CAU vs. AMG). Patients were followed up for an average of 19.8 ± 15.2 months. No statistically significant differences were found in comparing the rate of pterygium recurrence between attending physicians and residents when using CAU (6.8% vs. 10.0%, respectively; P = 0.42) and AMG (69.2% vs. 47.6%, respectively; P = 0.22). Moreover, there were no significant differences in other postoperative complications between the groups. CONCLUSIONS: Pterygium recurrence rates were similar between attending physicians and supervised trainee residents. Thus, acceptable outcomes can be expected when pterygium surgery is performed by a supervised ophthalmology resident.

Interfacial reaction during friction stir assisted scribe welding of immiscible Fe and Mg alloy system.

We report on interfacial characteristics and chemistry of bonded Mg-Fe interfaces welded using friction stir assisted scribe technique (FaST). Two pairs of dissimilar joints: (AZ31-DP590) and (Pure Mg-DP590) were studied to shed light on joining mechanisms responsible for bonding of "immiscible" pairs of Mg and Fe. We present first direct experimental evidence of presence of oxide layer, Al segregation by atom probe tomography and nano steel grains close to interface by transmission electron microscopy study.

Structure-guided multivalent nanobodies block SARS-CoV-2 infection and suppress mutational escape.

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to spread, with devastating consequences. For passive immunization efforts, nanobodies have size and cost advantages over conventional antibodies. In this study, we generated four neutralizing nanobodies that target the receptor binding domain of the SARS-CoV-2 spike protein. We used x-ray crystallography and cryo-electron microscopy to define two distinct binding epitopes. On the basis of these structures, we engineered multivalent nanobodies with more than 100 times the neutralizing activity of monovalent nanobodies. Biparatopic nanobody fusions suppressed the emergence of escape mutants. Several nanobody constructs neutralized through receptor binding competition, whereas other monovalent and biparatopic nanobodies triggered aberrant activation of the spike fusion machinery. These premature conformational changes in the spike protein forestalled productive fusion and rendered the virions noninfectious.

Selection, biophysical and structural analysis of synthetic nanobodies that effectively neutralize SARS-CoV-2.

The coronavirus SARS-CoV-2 is the cause of the ongoing COVID-19 pandemic. Therapeutic neutralizing antibodies constitute a key short-to-medium term approach to tackle COVID-19. However, traditional antibody production is hampered by long development times and costly production. Here, we report the rapid isolation and characterization of nanobodies from a synthetic library, known as sybodies (Sb), that target the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein. Several binders with low nanomolar affinities and efficient neutralization activity were identified of which Sb23 displayed high affinity and neutralized pseudovirus with an IC50 of 0.6 µg/ml. A cryo-EM structure of the spike bound to Sb23 showed that Sb23 binds competitively in the ACE2 binding site. Furthermore, the cryo-EM reconstruction revealed an unusual conformation of the spike where two RBDs are in the 'up' ACE2-binding conformation. The combined approach represents an alternative, fast workflow to select binders with neutralizing activity against newly emerging viruses.

An alpaca nanobody neutralizes SARS-CoV-2 by blocking receptor interaction.

SARS-CoV-2 enters host cells through an interaction between the spike glycoprotein and the angiotensin converting enzyme 2 (ACE2) receptor. Directly preventing this interaction presents an attractive possibility for suppressing SARS-CoV-2 replication. Here, we report the isolation and characterization of an alpaca-derived single domain antibody fragment, Ty1, that specifically targets the receptor binding domain (RBD) of the SARS-CoV-2 spike, directly preventing ACE2 engagement. Ty1 binds the RBD with high affinity, occluding ACE2. A cryo-electron microscopy structure of the bound complex at 2.9 Å resolution reveals that Ty1 binds to an epitope on the RBD accessible in both the 'up' and 'down' conformations, sterically hindering RBD-ACE2 binding. While fusion to an Fc domain renders Ty1 extremely potent, Ty1 neutralizes SARS-CoV-2 spike pseudovirus as a 12.8 kDa nanobody, which can be expressed in high quantities in bacteria, presenting opportunities for manufacturing at scale. Ty1 is therefore an excellent candidate as an intervention against COVID-19.

Evolution of lattice distortions in 4H-SiC wafers with varying doping.

Lattice distortions (LD) in 4H-silicon carbide (SiC) wafers were quantified using synchrotron X-ray rocking curve mapping (RCM), and were resolved into their two components of lattice strain (Δd/d) and lattice plane curvature (LPC) for 150 mm diameter wafers. The evolution of these LDs were investigated for three sequential substrates from the same boule, one of which was the substrate reference, and the other two had a 10 µm thick, 1 × 1017 and 4 × 1014 cm-3 n-type doped epitaxial layer. The lattice strain, Δd/d, was highest for the lowest doped wafer due to higher mismatch with the substrate wafer. After epitaxial layer growth, the LPC variation across the wafer increases by a factor of 2, irrespective of doping. The LPC maps indicate presence of a twist in the lattice planes that increases after epitaxial growth. The LPC component has higher influence on wafer shape change, which can reduce device yields. The lattice strain component predominantly affects the glide of basal plane dislocations (BPDs), thereby reducing device reliability. From analysis of peak widths, it was determined that threading dislocations in the top 6 microns of the wafer increase after epitaxial layer growth.

Health Care Disparities in Urologic Oncology: A Systematic Review.

For prostate cancer, we review racial differences in incidence, androgen pathways, growth factors, tumor location, rate of definitive treatment, and outcomes. We review the effect of race on risk-stratification and discuss studies of active surveillance in the African American population. For bladder cancer, race- and gender- associated differences in incidence, sex hormone pathways. For renal cell carcinoma, disparities in incidence, genetic factors, tumor pathology, time to presentation, and disease specific survival have been observed. We evaluate the impact of race and ethnicity on tumor pathology and discuss gaps in our current understanding of renal cell carcinoma pathogenesis.

Abnormal bone mineral content and density in people with tetrasomy 18p.

Tetrasomy 18p is a rare chromosomal abnormality, resulting from an additional iso-chromosome composed of two copies of the short arm. It is characterized by craniofacial abnormalities, neuromuscular dysfunction, and developmental delay. The Chromosome 18 Clinical Research Center has established the largest cohort of individuals with this rare genetic condition. Here, we describe a case series of 21 individuals with tetrasomy 18p who have a previously unreported clinical finding: low bone mineral density. Most individuals met criteria for low bone density despite being relatively young (mean age of 21 years). Clinicians providing care to individuals affected by Tetrasomy 18p should be aware of their increased risk for decreased bone density and pathological fractures.

Acute Life-Threatening Hemorrhage in Neonates With Severe Hemophilia A: A Report of 3 Cases.

Hemorrhagic shock is a rare, emergent condition that is often fatal in newborns. In this article, we report cases of 3 neonates presenting with acute, life-threatening hemorrhage who were subsequently diagnosed with severe hemophilia (<1% factor VIII). The first infant was tachycardic, pale, and had a precipitous drop in his hemoglobin secondary to a subgaleal hemorrhage. The second patient sustained a splenic rupture, a sequela that has been reported in only 4 other neonatal cases. The last infant presented with tonic-clonic seizures and respiratory distress. Head imaging demonstrated extracranial and intracranial hemorrhage, complications that can result in 20% mortality. All 3 patients were successfully treated with clotting factor concentrate and blood products. After normalization of factor VIII levels, the newborns did not develop any new hemorrhages and were discharged home within 3 weeks of birth. Pediatric providers should be aware that these signs and symptoms may be potentially lethal complications in neonates with severe factor VIII deficiency.

Reconstitution of the Recombinant RanBP2 SUMO E3 Ligase Complex.

One of the few proteins that have SUMO E3 ligase activity is the 358 kDa nucleoporin RanBP2 (Nup358). While small fragments of RanBP2 can stimulate SUMOylation in vitro, the physiologically relevant E3 ligase is a stable multi-subunit complex comprised of RanBP2, SUMOylated RanGAP1, and Ubc9. Here, we provide a detailed protocol to in vitro reconstitute the RanBP2 SUMO E3 ligase complex. With the exception of RanBP2, reconstitution involves untagged full-length proteins. We describe the bacterial expression and purification of all complex components, namely an 86 kDa His-tagged RanBP2 fragment, the SUMO E2-conjugating enzyme Ubc9, RanGAP1, and SUMO1, and we provide a protocol for quantitative SUMOylation of RanGAP1. Finally, we present details for the assembly and final purification of the catalytically active RanBP2/RanGAP1*SUMO1/Ubc9 complex.

The RanBP2/RanGAP1*SUMO1/Ubc9 SUMO E3 ligase is a disassembly machine for Crm1-dependent nuclear export complexes.

Continuous cycles of nucleocytoplasmic transport require disassembly of transport receptor/Ran-GTP complexes in the cytoplasm. A basic disassembly mechanism in all eukaryotes depends on soluble RanGAP and RanBP1. In vertebrates, a significant fraction of RanGAP1 stably interacts with the nucleoporin RanBP2 at a binding site that is flanked by FG-repeats and Ran-binding domains, and overlaps with RanBP2's SUMO E3 ligase region. Here, we show that the RanBP2/RanGAP1*SUMO1/Ubc9 complex functions as an autonomous disassembly machine with a preference for the export receptor Crm1. We describe three in vitro reconstituted disassembly intermediates, which show binding of a Crm1 export complex via two FG-repeat patches, cargo-release by RanBP2's Ran-binding domains and retention of free Crm1 at RanBP2 after Ran-GTP hydrolysis. Intriguingly, all intermediates are compatible with SUMO E3 ligase activity, suggesting that the RanBP2/RanGAP1*SUMO1/Ubc9 complex may link Crm1- and SUMO-dependent functions.

Effect of topical mitomycin C on corneal endothelium.

PURPOSE: To evaluate patients with ocular surface squamous neoplasia (OSSN) on treatment with topical mitomycin C (MMC) with regard to changes in corneal thickness and endothelial count. DESIGN: Prospective, nonrandomized, interventional series. METHODS: In this prospective evaluation, 25 patients with OSSN with different clock hours of involvement (range, one to eight), age varying from 19 to 76 years, were treated with topical MMC 0.04% in a cyclic manner. Preoperative corneal thickness measurement and endothelial cell density were obtained, and the tests were repeated after one, three, and six months of complete resolution. The fellow eyes of the same patients served as controls. RESULTS: Mean +/- standard deviation (SD) pachymetry and endothelial cell counts before and after instillation were 504.48 +/- 5.45 microm and 2325.76 +/- 192.52 cells/mm2 and 503.52 +/- 5.75 microm and 2297.60 +/- 192.08 cells/mm2, respectively, suggesting no significant difference in the above two parameters. Similarly, in the fellow control eyes, mean +/- SD pachymetry and endothelial cell counts before and after instillation were 502.80 +/- 4.34 microm and 2326.72 +/- 191.93 cells/mm2 and 504.64 +/- 5.19 microm and 2313.60 +/- 192.03 cells/mm2, respectively. CONCLUSIONS: Topical 0.04% MMC drops do not significantly affect the corneal endothelium. However, its judicious use and long-term follow-up are mandatory.