An engineered ultrahigh affinity bi-paratopic uPAR targeting agent confers enhanced tumor targeting.

Urokinase-type plasminogen activator receptor (uPAR) is overexpressed on tumor cells in multiple types of cancer and contributes to disease progression and metastasis. In this work, we engineered a novel bi-paratopic uPAR targeting agent by fusing the binding domains of two native uPAR ligands: uPA and vitronectin, with a flexible peptide linker. The linker length was optimized to facilitate simultaneous engagement of both domains to their adjacent epitopes on uPAR, resulting in a high affinity and avid binding interaction. Furthermore, the individual domains were affinity-matured using yeast surface display and directed evolution, resulting in a bi-paratopic protein with affinity in the picomolar to femtomolar range. This engineered uPAR targeting agent demonstrated significantly enhanced tumor localization in mouse tumor models compared to the native uPAR ligand and warrants further investigation as a diagnostic and therapeutic agent for cancer.

Rapid Diagnostic Testing for Response to the Monkeypox Outbreak - Laboratory Response Network, United States, May 17-June 30, 2022.

As part of public health preparedness for infectious disease threats, CDC collaborates with other U.S. public health officials to ensure that the Laboratory Response Network (LRN) has diagnostic tools to detect Orthopoxviruses, the genus that includes Variola virus, the causative agent of smallpox. LRN is a network of state and local public health, federal, U.S. Department of Defense (DOD), veterinary, food, and environmental testing laboratories. CDC developed, and the Food and Drug Administration (FDA) granted 510(k) clearance* for the Non-variola Orthopoxvirus Real-time PCR Primer and Probe Set (non-variola Orthopoxvirus [NVO] assay), a polymerase chain reaction (PCR) diagnostic test to detect NVO. On May 17, 2022, CDC was contacted by the Massachusetts Department of Public Health (DPH) regarding a suspected case of monkeypox, a disease caused by the Orthopoxvirus Monkeypox virus. Specimens were collected and tested by the Massachusetts DPH public health laboratory with LRN testing capability using the NVO assay. Nationwide, 68 LRN laboratories had capacity to test approximately 8,000 NVO tests per week during June. During May 17-June 30, LRN laboratories tested 2,009 specimens from suspected monkeypox cases. Among those, 730 (36.3%) specimens from 395 patients were positive for NVO. NVO-positive specimens from 159 persons were confirmed by CDC to be monkeypox; final characterization is pending for 236. Prompt identification of persons with infection allowed rapid response to the outbreak, including isolation and treatment of patients, administration of vaccines, and other public health action. To further facilitate access to testing and increase convenience for providers and patients by using existing provider-laboratory relationships, CDC and LRN are supporting five large commercial laboratories with a national footprint (Aegis Science, LabCorp, Mayo Clinic Laboratories, Quest Diagnostics, and Sonic Healthcare) to establish NVO testing capacity of 10,000 specimens per week per laboratory. On July 6, 2022, the first commercial laboratory began accepting specimens for NVO testing based on clinician orders.

Complete Reconstitution and Deorphanization of the 3 MDa Nocardiosis-Associated Polyketide Synthase.

Several Nocardia strains associated with nocardiosis, a potentially life-threatening disease, house a nonamodular assembly line polyketide synthase (PKS) that presumably synthesizes an unknown polyketide. Here, we report the discovery and structure elucidation of the NOCAP (nocardiosis-associated polyketide) aglycone by first fully reconstituting the NOCAP synthase in vitro from purified protein components followed by heterologous expression in E. coli and spectroscopic analysis of the purified products. The NOCAP aglycone has an unprecedented structure comprised of a substituted resorcylaldehyde headgroup linked to a 15-carbon tail that harbors two conjugated all-trans trienes separated by a stereogenic hydroxyl group. This report is the first example of reconstituting a trans-acyltransferase assembly line PKS in vitro and of using these approaches to "deorphanize" a complete assembly line PKS identified via genomic sequencing. With the NOCAP aglycone in hand, the stage is set for understanding how this PKS and associated tailoring enzymes confer an advantage to their native hosts during human Nocardia infections.

An engineered NKp46 antibody for construction of multi-specific NK cell engagers.

Recent developments in cancer immunotherapy have highlighted the potential of harnessing natural killer (NK) cells in the treatment of neoplastic malignancies. Of these, bispecific antibodies, and NK cell engager (NKCE) protein therapeutics in particular, have been of interest. Here, we used phage display and yeast surface display to engineer RLN131, a unique cross-reactive antibody that binds to human, mouse, and cynomolgus NKp46, an activating receptor found on NK cells. RLN131 induced proliferation and activation of primary NK cells, and was used to create bispecific NKCE constructs of varying configurations and valency. All NKCEs were able to promote greater NK cell cytotoxicity against tumor cells than an unmodified anti-CD20 monoclonal antibody, and activity was observed irrespective of whether the constructs contained a functional Fc domain. Competition binding and fine epitope mapping studies were used to demonstrate that RLN131 binds to a conserved epitope on NKp46, underlying its species cross-reactivity.

Transmittance ratio constrained retrieval technique for lidar cirrus measurements.

This letter describes a lidar retrieval technique that uses the transmittance ratio as a constraint to determine an average lidar ratio as well as extinction and backscatter coefficients of transparent cirrus clouds. The cloud transmittance ratio is directly obtained from two adjacent elastic lidar backscatter signals. The technique can be applied to cirrus measurements where neither the molecular scattering dominant signals above and below the cloud layer are found nor cloudfree reference profiles are available. The technique has been tested with simulated lidar signals and applied to backscatter lidar measurements at Hampton University, Hampton, Virginia.

Obtaining a ground-based lidar geometric form factor using coincident spaceborne lidar measurements.

We present a method to determine the geometric form factor of a ground-based lidar using simultaneous lidar measurements made from the ground and from space. The theoretical basis is described. The feasibility of the method is demonstrated by applying it to the measurement data acquired by the Cloud Aerosol Lidar Infrared Pathfinder Satellite Observation (CALIPSO) lidar and a ground-based lidar located at the Hampton University (37.02 degrees N, 76.34 degrees W). The geometric factors with different aerosol conditions are retrieved.

An engineered antibody binds a distinct epitope and is a potent inhibitor of murine and human VISTA.

V-domain immunoglobulin (Ig) suppressor of T cell activation (VISTA) is an immune checkpoint that maintains peripheral T cell quiescence and inhibits anti-tumor immune responses. VISTA functions by dampening the interaction between myeloid cells and T cells, orthogonal to PD-1 and other checkpoints of the tumor-T cell signaling axis. Here, we report the use of yeast surface display to engineer an anti-VISTA antibody that binds with high affinity to mouse, human, and cynomolgus monkey VISTA. Our anti-VISTA antibody (SG7) inhibits VISTA function and blocks purported interactions with both PSGL-1 and VSIG3 proteins. SG7 binds a unique epitope on the surface of VISTA, which partially overlaps with other clinically relevant antibodies. As a monotherapy, and to a greater extent as a combination with anti-PD1, SG7 slows tumor growth in multiple syngeneic mouse models. SG7 is a promising clinical candidate that can be tested in fully immunocompetent mouse models and its binding epitope can be used for future campaigns to develop species cross-reactive inhibitors of VISTA.

Thermal compatibility of dental ceramic systems using cylindrical and spherical geometries.

OBJECTIVE: To test the hypothesis that bilayer ceramic cylinders and spheres can provide valid confirmation of thermal incompatibility stresses predicted by finite element analyses. METHODS: A commercial core ceramic and an experimental core ceramic were used to fabricate open-ended cylinders and core ceramic spheres. The core cylinders and spheres were veneered with one of four commercial dental ceramics representing four thermally compatible groups and four thermally incompatible groups. Axisymmetric thermal and viscoelastic elements in the ANSYS finite element program were used to calculate temperatures and stresses for each geometry and ceramic combination. This process required a transient heat transfer analysis for each combination to determine input temperatures for the structural model. RESULTS: After fabrication, each specimen was examined visually using fiberoptic transillumination for evidence of cracking. There were 100% failures of the thermally incompatible cylinders while none of the thermally compatible combinations failed. Among the spheres, 100% of the thermally incompatible systems failed, 16% of one of the thermally compatible systems failed, and none of the remaining compatible combinations failed. The calculated stress values were in general agreement with the experimental observations, i.e., low residual stresses for the specimens that did not fail and high residual stresses for the specimens that did fail. SIGNIFICANCE: Simple screening geometries can be used to identify highly incompatible ceramic combinations, but they do not identify marginally incompatible systems.

Functional replacement of oxygen by other oxidants in articular cartilage.

OBJECTIVE: Articular cartilage chondrocytes consume remarkably little O(2) in comparison with most other animal cells; glycolysis forms the principal source of ATP in this cartilage. Although not lethal for many days, imposition of anoxia immediately lowers intracellular ATP, inhibits rates of glycolysis, and prevents articular chondrocytes from producing extracellular matrix macromolecules. This study was undertaken to investigate the role of O(2) in articular chondrocyte metabolism. METHODS: We examined the effects of oxygen and of several other classes of exogenous oxidants, i.e., 1) the dyes methylene blue and 2,6-dichlorophenol-indophenol, 2) the iron (III) complex ferricyanide, and 3) the keto-acids oxaloacetate and pyruvate (and phosphoenolpyruvate, a metabolic precursor of pyruvate), on rates of glycolysis and of sulfate incorporation by bovine articular cartilage in vitro. RESULTS: Lactate production was lowest under conditions of anoxia and was stimulated severalfold by addition of O(2) (air-saturated medium). Under strict anoxia, other oxidants restored lactate production to rates at least comparable with those seen in aerobic controls; under aerobic conditions, they had little effect. Oxygen and all of the other oxidants examined stimulated sulfate incorporation more strongly than lactate production. The compounds that promoted glycolysis and hence sulfate incorporation in cartilage under anoxia were themselves reduced; that is, they functioned as oxidants in lieu of O(2). CONCLUSION: For normal function, articular cartilage appears to require exogenous oxidants to stimulate glycolysis and produce ATP and extracellular matrix. Under physiologic conditions, oxygen acts as this oxidant, but its role can be adequately assumed by other agents.

Campylobacter jejuni cytolethal distending toxin promotes DNA repair responses in normal human cells.

Cytolethal distending toxin (CDT) is a multisubunit protein found in various gram-negative bacterial pathogens of humans which is thought to cause cell death by direct DNA damage of host cells. We sought to determine if a cellular response to DNA damage could be detected by exogenous addition of the holotoxin. Exogenous addition of the Campylobacter jejuni 81-176 CDT to primary human fibroblasts resulted in formation of Rad50 foci, which are formed around double-stranded-DNA breaks. Moreover, such foci are formed in both proliferating and nonproliferating cells that are treated with C. jejuni CDT. Fibroblasts that were intoxicated and later stimulated to proliferate failed to divide and remained arrested in the G(1) phase of the cell cycle.

Interactions of Campylobacter jejuni cytolethal distending toxin subunits CdtA and CdtC with HeLa cells.

Campylobacter jejuni produces a toxin, called cytolethal distending toxin (CDT), which causes direct DNA damage leading to invocation of DNA damage checkpoint pathways. The affected cells arrest in G(1) or G(2) and eventually die. CDT consists of three protein subunits, CdtA, CdtB, and CdtC, with CdtB recently identified as a nuclease. However, little is known about the functions of CdtA or CdtC. In this work, enzyme-linked immunosorbent assay-based experiments were used to show, for the first time, that both CdtA and CdtC bound with specificity to the surface of HeLa cells, whereas CdtB did not. Varying the order of the addition of subunits for reconstitution of the holotoxin had no effect on activity. In addition, mutants containing deletions of conserved regions of CdtA and CdtC were able to bind to the surface of HeLa cells but were not able to participate in holotoxin assembly. Finally, both Cdt mutant subunits were able to effectively compete with CDT holotoxin in the HeLa cell binding assay.

Patterns of variations in Escherichia coli strains that produce cytolethal distending toxin.

A collection of 20 Escherichia coli strains that produce cytolethal distending toxin (CDT) were analyzed for their virulence-associated genes. All of these strains were serotyped, and multiplex PCR analysis was used to ascertain the presence of genes encoding other virulence factors, including Shiga toxin, intimin, enterohemolysin, cytotoxic necrotizing factor type 1 (CNF1) and CNF2, heat-stable toxin, and heat-labile toxin. These CDT-producing strains possessed various combinations of known virulence genes, some of which have not been noted before. Partial cdtB sequences were obtained from 10 of these strains, and their predicted CdtB sequences were compared to known E. coli CdtB sequences; some of the sequences were identical to known CdtB sequences, but two were not. PCR primers based on sequence differences between the known cdt sequences were tested for their ability to detect CDT producers and to determine CDT type. Correlations between the type of CDT produced, the presence of other virulence properties, and overall strain relatedness revealed that the CDT producers studied here can be divided into three general groups, with distinct differences in CDT type and in their complement of virulence-associated genes.