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Philipp Diebolder,1 Miguel Vazquez-Pufleau,2 Nilantha Bandara,1 Cedric Mpoy,1 Ramesh Raliya,2 Elijah Thimsen,2 Pratim Biswas,2 Buck E Rogers1 1Department of Radiation Oncology, Washington University School of Medicine, St Louis, MO, USA; 2Department of Energy, Environmental and Chemical Engineering, Washington University in St Louis, St Louis, MO, USA Introduction: Siliceous nanoparticles (NPs) have been extensively studied in nanomedicine due to their high biocompatibility and immense biomedical potential. Although numerous technologies have been developed, the synthesis of siliceous NPs for biomedical applications mainly relies on a few core technologies predominantly intended to produce spherical-shaped NPs. Methods: In this context, the impact of different morphologies of siliceous NPs on biodistribution in vivo is limited. In the present study, we developed a novel technique based on an aerosol silane reactor to produce sintered silicon NPs of similar size but different surface areas due to distinct spherical subunits. Silica-converted particles were functionalized for radiolabeling with copper-64 (64Cu) to systematically analyze their behavior in the passive targeting of A431 tumor xenografts in mice after intravenous injection.Results: While low nonspecific uptake was observed in most organs, the majority of particles were accumulated in the liver, spleen, and lung. Depending on the morphologies and functionalization, significant differences in the uptake profiles of the particles were observed. In terms of tumor uptake, spherical shapes with lower surface areas showed the highest accumulation and tumor-to-blood ratios of all investigated particles.Conclusion: This study highlights the importance of shape and fuctionalization of siliceous NPs on organ and tumor accumulation as significant factors for biomedical applications. Keywords: silicon, silica, human tumor xenograft, PEGylation, 64Cu

Abstract Background Infections with Herpes simplex virus (HSV)-1 or -2 usually present as mild chronic recurrent disease, however in rare cases can result in life-threatening conditions with a large spectrum of pathology. Monoclonal antibody therapy has great potential especially to treat infections with virus resistant to standard therapies. HDIT101, a humanized IgG targeting HSV-1/2 gB was previously investigated in phase 2 clinical trials. The aim of this study was to develop a next-generation therapy by combining different antiviral monoclonal antibodies. Methods A lymph-node derived phage display library (LYNDAL) was screened against recombinant gB from Herpes simplex virus (HSV) -1 and HDIT102 scFv was selected for its binding characteristics using bio-layer interferometry. HDIT102 was further developed as fully human IgG and tested alone or in combination with HDIT101, a clinically tested humanized anti-HSV IgG, in vitro and in vivo. T-cell stimulating activities by antigen-presenting cells treated with IgG-HSV immune complexes were analyzed using primary human cells. To determine the epitopes, the cryo-EM structures of HDIT101 or HDIT102 Fab bound to HSV-1F as well as HSV-2G gB protein were solved at resolutions

Abstract Structural studies of translating ribosomes traditionally rely on in vitro assembly and stalling of ribosomes in defined states. To comprehensively visualize bacterial translation, we reactivated ex vivo-derived E. coli polysomes in the PURE in vitro translation system and analyzed the actively elongating polysomes by cryo-EM. We find that 31% of 70S ribosomes assemble into disome complexes that represent eight distinct functional states including decoding and termination intermediates, and a pre-nucleophilic attack state. The functional diversity of disome complexes together with RNase digest experiments suggests that paused disome complexes transiently form during ongoing elongation. Structural analysis revealed five disome interfaces between leading and queueing ribosomes that undergo rearrangements as the leading ribosome traverses through the elongation cycle. Our findings reveal at the molecular level how bL9’s CTD obstructs the factor binding site of queueing ribosomes to thwart harmful collisions and illustrate how translation dynamics reshape inter-ribosomal contacts.

With the recent technological and computational advancements, structural biology has begun to tackle more and more difficult questions, including complex biochemical pathways and transient interactions among macromolecules. This has demonstrated that, to approach the complexity of biology, one single technique is largely insufficient and unable to yield thorough answers, whereas integrated approaches have been more and more adopted with successful results. Traditional structural techniques (X-ray crystallography and Nuclear Magnetic Resonance (NMR)) and the emerging ones (cryo-electron microscopy (cryo-EM), Small Angle X-ray Scattering (SAXS)), together with molecular modeling, have pros and cons which very nicely complement one another. In this review, three examples of synergistic approaches chosen from our previous research will be revisited. The first shows how the joint use of both solution and solid-state NMR (SSNMR), X-ray crystallography, and cryo-EM is crucial to elucidate the structure of polyethylene glycol (PEG)ylated asparaginase, which would not be obtainable through any of the techniques taken alone. The second deals with the integrated use of NMR, X-ray crystallography, and SAXS in order to elucidate the catalytic mechanism of an enzyme that is based on the flexibility of the enzyme itself. The third one shows how it is possible to put together experimental data from X-ray crystallography and NMR restraints in order to refine a protein model in order to obtain a structure which simultaneously satisfies both experimental datasets and is therefore closer to the ‘real structure’.

Hereditary epithelial ovarian cancers (EOCs) not expressing functional BRCA1 protein are characterized by defects in homologous recombination DNA repair, rendering such tumors more sensitive to DNA damaging agents and synthetic lethality, that is, poly-ADP-ribose-polymerase inhibitor treatment. The aim of this study was to evaluate the use of BRCA1 immunohistochemistry (IHC) for EOC prognosis and identification of features of the BRCAness phenotype.Twenty-seven patients who were treated for advanced EOC by macroscopic complete surgical tumor resection and first-line carboplatin/paclitaxel treatment were included. Time to recurrence and overall survival time after initial surgery were determined, and patients' samples were evaluated for BRCA1 expression by IHC. BRCA1 messenger RNA expression and promoter methylation was analyzed to elucidate regulatory mechanisms involved in BRCA1 protein loss.BRCA1 IHC-negative patients had a significantly longer overall survival (crude rate, 1537 days) compared to the BRCA1 IHC-positive group (crude rate, 827 days; P = 0.01). The patients in the BRCA1 IHC-negative group were significantly younger (51 years) compared to BRCA1 IHC-positive patients (61 years; P < 0.01). Importantly, both transcriptional and posttranscriptional mechanisms regulate BRCA1 protein expression. Only protein but not messenger RNA level were associated with longer overall survival.Epithelial ovarian cancers with negative BRCA1 protein expression were identified in younger patients, showed a significantly better overall survival, prolonged treatment intervals and a tendency for an extended progression free time interval. BRCA1 IHC negativity of sporadic EOC may be predictive of sensitivity to platinum-based chemotherapy and the poly-ADP-ribose-polymerase inhibitor-sensitive BRCAness phenotype. [ABSTRACT FROM AUTHOR]

Assessment of adnexal masses focuses on the accurate discrimination between benign and malignant lesions. In our tertiary referral center, we evaluated the discriminative power of expert sonography, serum CA-125 measurement, risk malignancy index (RMI) by Jacobs, and 2 preoperative triage strategies (combination of CA-125 measurement and RMI assessment with expert sonography).From 2002 to 2008, a total of 1362 surgical explorations with indication of an adnexal mass from our department were included in this study. Preoperative workup in all patients comprised a gynecologic examination, expert sonography, and serum CA-125 measurement. We calculated sensitivity, specificity, positive and negative predictive value (PPV and NPV), and Cohen κ (prevalence-adjusted measurement) to evaluate the discriminative power of each diagnostic test.Discriminative power of the evaluated tests differed depending on patients' menopausal state. In the premenopause, expert sonography reached the highest discriminative power with a κ value of 0.53, a PPV of 0.45, and an NPV of 0.99. In the postmemopause, the combinations of expert sonography with CA-125 serum measurement or RMI assessment achieved the highest discriminative power: The combination of CA-125 and expert sonography reached a PPV of 0.89 and an NPV of 0.97; κ yielded 0.84. The RMI combined with expert sonography as a triage strategy showed comparable results with a PPV of 0.89, an NPV of 0.96, and a κ value of 0.82.Preoperative assessment of an adnexal mass may be guided by the patient's menopausal state. In premenopausal patients, expert sonography is helpful for preoperative differentiation between benign and malignant lesions; in postmenopausal patients, the use of triage strategies of either CA-125 serum measurement or RMI combined with expert sonography can be recommended. [ABSTRACT FROM AUTHOR]

The article discusses a pilot study which aims to assess the feasibility, efficacy and accuracy of the procedure of sentinel lymph (SLN) node biopsy in vulvar cancer. The subjects of the study were stage one and stage two vulvar cancer patients who underwent SLN detection followed by inguinal-femoral lymphadenectomy. The study found that the incidence of lymph node metastases for stage one patients is less that ten percent, and concluded that SLNB is feasible and safe for vulvar cancer patients.

Methylomirabilis bacteria perform anaerobic methane oxidation coupled to nitrite reduction via an intra-aerobic pathway, producing carbon dioxide and dinitrogen gas. These diderm bacteria possess an unusual polygonal cell shape with sharp ridges that run along the cell body. Previously, a putative surface protein layer (S-layer) was observed as the outermost cell layer of these bacteria. We hypothesized that this S-layer is the determining factor for their polygonal cell shape. Therefore, we enriched the S-layer from M. lanthanidiphila cells and through LC-MS/MS identified a 31 kDa candidate S-layer protein, mela_00855, which had no homology to any other known protein. Antibodies were generated against a synthesized peptide derived from the mela_00855 protein sequence and used in immunogold localization to verify its identity and location. Both on thin sections of M. lanthanidiphila cells and in negative-stained enriched S-layer patches, the immunogold localization identified mela_00855 as the S-layer protein. Using electron cryo-tomography and sub-tomogram averaging of S-layer patches, we observed that the S-layer has a hexagonal symmetry. Cryo-tomography of whole cells showed that the S-layer and the outer membrane, but not the peptidoglycan layer and the cytoplasmic membrane, exhibited the polygonal shape. Moreover, the S-layer consisted of multiple rigid sheets that partially overlapped, most likely giving rise to the unique polygonal cell shape. These characteristics make the S-layer of M. lanthanidiphila a distinctive and intriguing case to study.

ABSTRACT Tsr, the serine chemoreceptor in Escherichia coli, transduces signals from a periplasmic ligand-binding site to its cytoplasmic tip, where it controls the activity of the CheA kinase. To function, Tsr forms trimers of homodimers (TODs), which associate in vivo with the CheA kinase and CheW coupling protein. Together, these proteins assemble into extended hexagonal arrays. Here, we use cryo-electron tomography and molecular dynamics simulation to study Tsr in the context of a near-native array, characterizing its signaling-related conformational changes at both the individual dimer and the trimer level. In particular, we show that individual Tsr dimers within a trimer exhibit asymmetric flexibilities that are a function of the signaling state, highlighting the effect of their different protein interactions at the receptor tips. We further reveal that the dimer compactness of the Tsr trimer changes between signaling states, transitioning at the glycine hinge from a compact conformation in the kinase-OFF state to an expanded conformation in the kinase-ON state. Hence, our results support a crucial role for the glycine hinge: to allow the receptor flexibility necessary to achieve different signaling states while also maintaining structural constraints imposed by the membrane and extended array architecture. IMPORTANCE In Escherichia coli, membrane-bound chemoreceptors, the histidine kinase CheA, and coupling protein CheW form highly ordered chemosensory arrays. In core signaling complexes, chemoreceptor trimers of dimers undergo conformational changes, induced by ligand binding and sensory adaptation, which regulate kinase activation. Here, we characterize by cryo-electron tomography the kinase-ON and kinase-OFF conformations of the E. coli serine receptor in its native array context. We found distinctive structural differences between the members of a receptor trimer, which contact different partners in the signaling unit, and structural differences between the ON and OFF signaling complexes. Our results provide new insights into the signaling mechanism of chemoreceptor arrays and suggest an important functional role for a previously postulated flexible region and glycine hinge in the receptor molecule.

Purpose: Tax-interacting protein 1 (TIP1) is a cancer-specific radiation-inducible cell surface antigen that plays a role in cancer progression and resistance to therapy. This study aimed to develop a novel anti-TIP1 human antibody for noninvasive PET imaging in patients with cancer., Experimental Design: A phage-displayed single-chain variable fragment (scFv) library was created from healthy donors' blood. High-affinity anti-TIP1 scFvs were selected from the library and engineered to human IgG1. Purified Abs were characterized by size exclusion chromatography high-performance liquid chromatography (SEC-HPLC), native mass spectrometry (native MS), ELISA, BIAcore, and flow cytometry. The labeling of positron emitter [89Zr]Zr to the lead Ab, L111, was optimized using deferoxamine (DFO) chelator. The stability of [89Zr]Zr-DFO-L111 was assessed in human serum. Small animal PET studies were performed in lung cancer tumor models (A549 and H460)., Results: We obtained 95% pure L111 by SEC-HPLC. Native MS confirmed the intact mass and glycosylation pattern of L111. Conjugation of three molar equivalents of DFO led to the optimal DFO-to-L111 ratio of 1.05. Radiochemical purity of 99.9% and specific activity of 0.37 MBq/μg was obtained for [89Zr]Zr-DFO-L111. [89Zr]Zr-DFO-L111 was stable in human serum over 7 days. The immunoreactive fraction in cell surface binding studies was 96%. In PET, preinjection with 4 mg/kg cold L111 before [89Zr]Zr-DFO-L111 (7.4 MBq; 20 μg) significantly (P < 0.01) enhanced the tumor-to-muscle standard uptake values (SUVmax) ratios on day 5 compared with day 2 postinjection., Conclusions: L111 Ab targets lung cancer cells in vitro and in vivo. [89Zr]Zr-DFO-L111 is a human antibody that will be evaluated in the first in-human study of safety and PET imaging., (©2024 American Association for Cancer Research.)

Glycoprotein CD44 and alternative splice variants are overexpressed in many cancers and cancer stem cells. Binding of hyaluronic acid to CD44 activates cell signaling pathways, inducing cell proliferation, cell survival, and invasion. As such, CD44 is regarded as an excellent target for cancer therapy when this interaction can be blocked. In this study, we developed a CD44-specific antibody fragment and evaluated it for imaging CD44-positive cancers using PET. Methods: A human single-chain fragment variable (scFv) was generated by phage display, using the extracellular domain of recombinant human CD44. The specificity and affinity of the scFv-CD44 were evaluated using recombinant and tumor cell-expressed CD44. Epitope mapping of the putative CD44 binding site was performed via overlapping peptide microarray. The scFv-CD44 was reformatted into a bivalent scFv-Fc-CD44, based on human IgG 1 -fragment crystallizable (Fc). The scFv-Fc-CD44 was radiolabeled with 64 Cu and 89 Zr. The purified reagents were injected into athymic nude mice bearing CD44-positive human tumors (MDA-MB-231, breast cancer, triple-negative). Biodistribution studies were performed at different times after injection of [ 64 Cu]Cu-NOTA-scFv-Fc-CD44 or [ 89 Zr]Zr-DFO-scFv-Fc-CD44. PET/CT imaging was conducted with [ 89 Zr]Zr-DFO-scFv-Fc-CD44 on days 1 and 7 after injection and compared with a scFv-Fc control antibody construct targeting glycophorin A. Results: Epitope mapping of the scFv binding site revealed a linear epitope within the extracellular domain of human CD44, capable of blocking binding to native hyaluronic acid. Switching from a monovalent scFv to a bivalent scFv-Fc format improved its binding affinity toward native CD44 on human breast cancer cells by nearly 200-fold. In vivo biodistribution data showed the highest tumor uptake and tumor-to-blood ratios for [ 89 Zr]Zr-DFO-scFv-Fc-CD44 between days 5 and 7. PET imaging confirmed excellent tumor specificity for [ 89 Zr]Zr-DFO-scFv-Fc-CD44 when compared with the control scFv-Fc. Conclusion: We developed a CD44-specific scFv-Fc construct that binds with nanomolar affinity to human CD44. When radiolabeled with 64 Cu or 89 Zr, it demonstrated specific uptake in CD44-expressing MDA-MB-231 tumors. The high tumor uptake (∼56% injected dose/g) warrants clinical investigation of [ 89 Zr]Zr-DFO-scFv-Fc-CD44 as a versatile PET imaging agent for patients with CD44-positive tumors., (© 2021 by the Society of Nuclear Medicine and Molecular Imaging.)

Responses to bacterial infections may be manifest systemically without evidence of the location of the infection site. A rapid means of pinpointing infection sites would be useful in providing effective and possibly localized treatment. Successful means of identifying infection sites would require two components: (1) a molecule capable of recognizing bacteria and (2) a means of communicating recognition. For the recognition element, we used a ceragenin, a small molecule with affinity for bacterial membranes that was designed as a mimic of endogenous antimicrobial peptides. For the communication element, we used 64 Cu, which is a positron emitter. By conjugating a copper chelating group to the ceragenin, the two elements were combined. Chelation of 64 Cu by the conjugate was effective and provided a stable complex that allowed in vivo imaging. When administered to mice in a thigh infection model, the 64 Cu-labeled conjugate accumulated at the site of infection (right thigh) without accumulation at the complementary site (left thigh). This conjugate may provide a means of identifying infection sites in patients presenting general signs of infection without localized symptoms., Competing Interests: Conflicts of interest PBS is a paid consultant for CSA Biotechnologies, Inc. Other authors declare no competing financial interest.

Purpose: To evaluate whether tumor uptake of [ 89 Zr]trastuzumab can distinguish HER2-positive from HER2-negative breast cancer., Methods: Women with HER2-positive (n = 34) and HER2-negative (n = 16) breast cancer underwent PET/CT 5 ± 2 days following [ 89 Zr]trastuzumab administration. HER2 status was determined based on immunohistochemistry and/or fluorescence in situ hybridization of primary or metastatic/recurrent tumor. Tumor [ 89 Zr]trastuzumab uptake was assessed qualitatively and semiquantitatively as maximum standardized uptake value (SUV max ), and correlated with HER2 status. Additionally, intrapatient heterogeneity of [ 89 Zr]trastuzumab uptake was evaluated., Results: On a per-patient basis, [ 89 Zr]trastuzumab-PET/CT was positive in 30/34 (88.2%) HER2-positive and negative in 15/16 (93.7%) HER2-negative patients. Considering all lesions, the SUV max was not significantly different in patients with HER2-positive versus HER2-negative disease (p = 0.06). The same was true of when only hepatic lesions were evaluated (p = 0.42). However, after excluding hepatic lesions, tumor SUV max was significantly higher in HER2-positive compared to HER2-negative patients (p = 0.003). A cutoff SUV max of 3.2, determined by ROC analysis, demonstrated positive-predictive value of 83.3% (95% CI 65.3%, 94.4%), sensitivity of 75.8% (57.7%, 88.9%), negative-predictive value of 50% (24.7%, 75.3%), and specificity of 61.5% (95% 31.6%, 86.1%) for differentiating HER2-positive from HER2-negative lesions. There was intrapatient heterogeneity of [ 89 Zr]trastuzumab uptake in 20% of patients with multiple lesions., Conclusions: [ 89 Zr]trastuzumab has the potential to characterize the HER2 status of the complete tumor burden in patients with breast cancer, thus obviating repeat or multiple tissue sampling to assess intrapatient heterogeneity of HER2 status.

Initiation of a tumor-directed immune response and appropriate modulation of its progress are key issues in cancer immunotherapy. Combinatorial strategies addressing both aspects might therefore be especially suitable. Here, we report a targeted approach combining a bispecific antibody with 2 costimulatory antibody-ligand fusion proteins. According to the concept, the bispecific antibody (scDbFAP×CD3) retargets T cells in a MHC-independent manner to tumor cells, providing an artificial first signal that allows the costimulatory antibody-ligand fusion proteins (B7.2-Db and scFv-4-1BBL) likewise targeted to the tumor cells to modulate the T-cell response. In our model system, the target cells coexpress the fibroblast activation protein (FAP) and endoglin as antigens. ScDbFAPCD3 and B7.2-Db are targeted to FAP although by different antibody moieties, whereas scFv-4-1BBL is directed against endoglin. ScDbFAPCD3-induced T-cell stimulation could be enhanced by the addition of either B7.2-Db or scFv-4-1BBL and even further by the combination of both as shown in terms of cytokine release (interleukin-2/interferon γ), proliferation and activation marker expression (CD25). By combined costimulation, overall T-cell population strongly increased in activation-experienced memory phenotype accompanied by a decrease in naive phenotype. ScFv-4-1BBL-mediated costimulation of naive CD8+ T cells promoted the expansion and development of cytotoxic T cells with strong effector potential. Thus, combining a bispecific antibody with antibody-ligand fusion protein-mediated CD28 and 4-1BB costimulation in a targeted approach shows great potential to generate and shape an immune response at the tumor site. Therefore, the adaptation of this approach to other immune modulatory ligands and tumor-relevant targets seems to be promising.