The Impact of Physician Race and Sex on Patient Ranking of Physician Competence and Perception of Leadership Ability.

Background Biases affect patient perceptions of their physician and influence the physician-patient relationship. While racial disparities in care and inequities in the healthcare workforce are well-documented, the impact of physician race on patient perceptions remains unclear. We aimed to investigate the association of physician race and sex on patient perceptions during simulated preoperative encounters. Methods Three hundred patients recruited consecutively in the Preanesthesia Evaluation and Testing Center viewed pictures of 4 anesthesiologists (black male, white male, black female, white female) in random order while listening to a set of paired audio recordings describing general anesthesia. Participants ranked each anesthesiologist on confidence, intelligence, and likelihood of choosing the anesthesiologist to care for their family member, and chose the one anesthesiologist most like a leader. Results Compared to white anesthesiologists, black anesthesiologists had greater odds of being ranked more confident (OR, 1.45; 95% CI, 1.10 to 1.89; P=0.008) and being considered a leader (OR, 2.06; 95% CI, 1.50 to 2.84; P<0.0001). Among white participants, black anesthesiologists had greater odds of being ranked more intelligent (OR, 2.08; 95% CI, 1.54 to 2.81; P<0.0001) and were more likely to be chosen to care for a family member (OR, 2.26; 95% CI, 1.66 to 3.08; P<0.0001). Female anesthesiologists had greater odds of being ranked more intelligent (OR, 1.36; 95% CI, 1.08 to 1.71; P=0.009) and were more likely to be chosen to care for a family member (OR, 1.58; 95% CI, 1.27 to 1.97; P<0.001) compared with male anesthesiologists. Conclusions Contrary to our hypothesis, patients ranked black physicians more highly on multiple competence and leadership quality metrics. Our data likely highlight the role social desirability bias may play in studies of racial disparities within medicine.

Preclinical Assessment of a MUC12-Targeted BiTE (Bispecific T-cell Engager) Molecule.

MUC12 is a transmembrane mucin that is highly expressed in >50% of primary and metastatic colorectal tumors. MUC12 is also expressed by normal epithelial cells of the colon and small intestine. Although MUC12 localization in normal epithelial cells is restricted to the apical membrane, expression in tumors is depolarized and shows broad membrane localization. The differential localization of MUC12 in tumor cells as compared with normal cells makes it a potential therapeutic target. Here, we evaluated targeting of MUC12 with a BiTE (bispecific T-cell engager) molecule. We generated a panel of proof-of-concept half-life extended (HLE) BiTE molecules that bind MUC12 on tumor cells and CD3 on T cells. We prioritized one molecule based on in vitro activity for further characterization in vivo In vitro, the MUC12 HLE BiTE molecule mediated T-cell-redirected lysis of MUC12-expressing cells with half-maximal lysis of 4.4 ± 0.9 to 117 ± 78 pmol/L. In an exploratory cynomolgus monkey toxicology study, the MUC12 HLE BiTE molecule administered at 200 µg/kg with a step dose to 1,000 µg/kg was tolerated with minimal clinical observations. However, higher doses were not tolerated, and there was evidence of damage in the gastrointestinal tract, suggesting dose levels projected to be required for antitumor activity may be associated with on-target toxicity. Together, these data demonstrate that the apically restricted expression of MUC12 in normal tissues is accessible to BiTE molecule target engagement and highlight the difficult challenge of identifying tumor-selective antigens for solid tumor T-cell engagers.

Cell-Selective Cytotoxicity of a Fluorescent Rhodium Metalloinsertor Conjugate Results from Irreversible DNA Damage at Base Pair Mismatches.

Up to 20% of solid tumors are characterized by DNA mismatch repair (MMR) deficiency and microsatellite instability that confer resistance to standard of care chemotherapy. MMR-deficient cancers have an increased mutation rate, and DNA mismatches accumulate as part of these cancers. We previously described a class of compounds, rhodium metalloinsertors, that bind DNA mismatches with high specificity and selectivity and have potential as targeted therapy. [Rh(chrysi)(phen)(PPO)]2+ (RhPPO) is the most potent, selective compound in this class and acts by targeting DNA mismatches, resulting in preferential cytotoxicity to MMR-deficient cancers. To explore further the cellular mechanism of action of RhPPO, we conjugated the metal complex to a fluorescent probe, cyanine 3 (Cy3). RhPPO-Cy3 binds DNA mismatches and retains the selectivity and potent cytotoxic activity of RhPPO for MMR-deficient cell lines. RhPPO-Cy3 forms discrete foci in the cell nucleus that overlap with sites of DNA damage, suggesting that the lesions occur at or near DNA mismatch sites. RhPPO-Cy3 foci persist over time, despite initial processing of the lesion and recruitment of repair proteins, consistent with the idea that the complex binding to a mismatch prevents repair. RhPPO-Cy3 binding does not lead to activation of p53 and the apoptotic pathway. Together, these findings support the idea that RhPPO-Cy3 binding leads to irreversible DNA damage at DNA mismatches that enables selective cytotoxicity to MMR-deficient cells.

Laboratory validation of a clinical metagenomic sequencing assay for pathogen detection in cerebrospinal fluid.

Metagenomic next-generation sequencing (mNGS) for pan-pathogen detection has been successfully tested in proof-of-concept case studies in patients with acute illness of unknown etiology but to date has been largely confined to research settings. Here, we developed and validated a clinical mNGS assay for diagnosis of infectious causes of meningitis and encephalitis from cerebrospinal fluid (CSF) in a licensed microbiology laboratory. A customized bioinformatics pipeline, SURPI+, was developed to rapidly analyze mNGS data, generate an automated summary of detected pathogens, and provide a graphical user interface for evaluating and interpreting results. We established quality metrics, threshold values, and limits of detection of 0.2-313 genomic copies or colony forming units per milliliter for each representative organism type. Gross hemolysis and excess host nucleic acid reduced assay sensitivity; however, spiked phages used as internal controls were reliable indicators of sensitivity loss. Diagnostic test accuracy was evaluated by blinded mNGS testing of 95 patient samples, revealing 73% sensitivity and 99% specificity compared to original clinical test results, and 81% positive percent agreement and 99% negative percent agreement after discrepancy analysis. Subsequent mNGS challenge testing of 20 positive CSF samples prospectively collected from a cohort of pediatric patients hospitalized with meningitis, encephalitis, and/or myelitis showed 92% sensitivity and 96% specificity relative to conventional microbiological testing of CSF in identifying the causative pathogen. These results demonstrate the analytic performance of a laboratory-validated mNGS assay for pan-pathogen detection, to be used clinically for diagnosis of neurological infections from CSF.

Pre- and post-operative anti-PD-L1 plus anti-angiogenic therapies in mouse breast or renal cancer models of micro- or macro-metastatic disease.

BACKGROUND: There are phase 3 clinical trials underway evaluating anti-PD-L1 antibodies as adjuvant (postoperative) monotherapies for resectable renal cell carcinoma (RCC) and triple-negative breast cancer (TNBC); in combination with antiangiogenic VEGF/VEGFR2 inhibitors (e.g., bevacizumab and sunitinib) for metastatic RCC; and in combination with chemotherapeutics as neoadjuvant (preoperative) therapies for resectable TNBC. METHODS: This study investigated these and similar clinically relevant drug combinations in highly translational preclinical models of micro- and macro-metastatic disease that spontaneously develop after surgical resection of primary kidney or breast tumours derived from orthotopic implantation of murine cancer cell lines (RENCAluc or EMT-6/CDDP, respectively). RESULTS: In the RENCAluc model, adjuvant sunitinib plus anti-PD-L1 improved overall survival compared to either drug alone, while the same combination was ineffective as early therapy for unresected primary tumours or late-stage therapy for advanced metastatic disease. In the EMT-6/CDDP model, anti-PD-L1 was highly effective as an adjuvant monotherapy, while its combination with paclitaxel chemotherapy (with or without anti-VEGF) was most effective as a neoadjuvant therapy. CONCLUSIONS: Our preclinical data suggest that anti-PD-L1 plus sunitinib may warrant further investigation as an adjuvant therapy for RCC, while anti-PD-L1 may be improved by combining with chemotherapy in the neoadjuvant but not the adjuvant setting of treating breast cancer.

Making Conjugation-induced Fluorescent PEGylated Virus-like Particles by Dibromomaleimide-disulfide Chemistry.

The recent rise in virus-like particles (VLPs) in biomedical and materials research can be attributed to their ease of biosynthesis, discrete size, genetic programmability, and biodegradability. While they're highly amenable to bioconjugation reactions for adding synthetic ligands onto their surface, the range in bioconjugation methodologies on these aqueous born capsids is relatively limited. To facilitate the direction of functional biomaterials research, non-traditional bioconjugation reactions must be considered. The reaction described in this protocol uses dibromomaleimides to introduce new functionality in the solvent exposed disulfide bonds of a VLP based upon Bacteriophage Qß. Furthermore, the final product is fluorescent, which has the added benefit of generating a trackable in vitro probe using a commercially available filter set.

Neurobrucellosis: Unexpected Answer From Metagenomic Next-Generation Sequencing.

A diagnosis of brucellosis can be difficult because routine culture and serological methods exhibit variable sensitivity and specificity. We present the use of a metagenomic next- generation sequencing assay to diagnose a case of neurobrucellosis from cerebrospinal fluid, resulting in the institution of appropriate antibiotic treatment and a favorable clinical outcome.

Evidence Implicating Immunological Host Effects in the Efficacy of Metronomic Low-Dose Chemotherapy.

Conventional chemotherapy drugs administered at a maximum tolerated dose (MTD) remains the backbone for treating most cancers. Low-dose metronomic (LDM) chemotherapy, which utilizes lower, less toxic, doses given on a close regular basis over prolonged periods, is an alternative and better tolerated potential strategy to improve chemotherapy. LDM chemotherapy has been evaluated preclinically and clinically and has shown therapeutic benefit, in both early and advanced stage metastatic disease, especially when used as a maintenance therapy. However, knowledge about the antitumor mechanisms by which LDM chemotherapy acts remain limited. Here we characterized the effects of LDM and MTD capecitabine therapy on tumor and host cells using high-throughput systems approaches involving mass spectrometry flow cytometry and automated cell imaging followed by in vivo analyses of such therapies. An increase in myeloid and T regulatory cells and a decrease in NK and T cytotoxic cells were found in MTD-capecitabine-treated tumors compared with LDM-capecitbine-treated tumors. Plasma from MTD capecitabine-treated mice induced a more tumorigenic and metastatic profile in both breast and colon carcinoma cells than plasma from mice treated with LDM capecitabine. These results correlated, in part, with in vivo studies using models of human or mouse advanced metastatic disease, where the therapeutic advantage of MTD capecitabine was limited despite a substantial initial antitumor activity found in the primary tumor setting. Overall these results implicate a possible contribution of immunologic host effects in accounting for the therapeutic limitations of MTD compared with LDM capecitabine. Cancer Res; 76(20); 5983-93. ©2016 AACR.

Preclinical Efficacy of Bevacizumab with CRLX101, an Investigational Nanoparticle-Drug Conjugate, in Treatment of Metastatic Triple-Negative Breast Cancer.

VEGF pathway-targeting antiangiogenic drugs, such as bevacizumab, when combined with chemotherapy have changed clinical practice for the treatment of a broad spectrum of human cancers. However, adaptive resistance often develops, and one major mechanism is elevated tumor hypoxia and upregulated hypoxia-inducible factor-1α (HIF1α) caused by antiangiogenic treatment. Reduced tumor vessel numbers and function following antiangiogenic therapy may also affect intratumoral delivery of concurrently administered chemotherapy. Nonetheless, combining chemotherapy and bevacizumab can lead to improved response rates, progression-free survival, and sometimes, overall survival, the extent of which can partly depend on the chemotherapy backbone. A rational, complementing chemotherapy partner for combination with bevacizumab would not only reduce HIF1α to overcome hypoxia-induced resistance, but also improve tumor perfusion to maintain intratumoral drug delivery. Here, we evaluated bevacizumab and CRLX101, an investigational nanoparticle-drug conjugate containing camptothecin, in preclinical mouse models of orthotopic primary triple-negative breast tumor xenografts, including a patient-derived xenograft. We also evaluated long-term efficacy of CRLX101 and bevacizumab to treat postsurgical, advanced metastatic breast cancer in mice. CRLX101 alone and combined with bevacizumab was highly efficacious, leading to complete tumor regressions, reduced metastasis, and greatly extended survival of mice with metastatic disease. Moreover, CRLX101 led to improved tumor perfusion and reduced hypoxia, as measured by contrast-enhanced ultrasound and photoacoustic imaging. CRLX101 durably suppressed HIF1α, thus potentially counteracting undesirable effects of elevated tumor hypoxia caused by bevacizumab. Our preclinical results show pairing a potent cytotoxic nanoparticle chemotherapeutic that complements and improves concurrent antiangiogenic therapy may be a promising treatment strategy for metastatic breast cancer. Cancer Res; 76(15); 4493-503. ©2016 AACR.

A miR-192-EGR1-HOXB9 regulatory network controls the angiogenic switch in cancer.

A deeper mechanistic understanding of tumour angiogenesis regulation is needed to improve current anti-angiogenic therapies. Here we present evidence from systems-based miRNA analyses of large-scale patient data sets along with in vitro and in vivo experiments that miR-192 is a key regulator of angiogenesis. The potent anti-angiogenic effect of miR-192 stems from its ability to globally downregulate angiogenic pathways in cancer cells through regulation of EGR1 and HOXB9. Low miR-192 expression in human tumours is predictive of poor clinical outcome in several cancer types. Using 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) nanoliposomes, we show that miR-192 delivery leads to inhibition of tumour angiogenesis in multiple ovarian and renal tumour models, resulting in tumour regression and growth inhibition. This anti-angiogenic and anti-tumour effect is more robust than that observed with an anti-VEGF antibody. Collectively, these data identify miR-192 as a central node in tumour angiogenesis and support the use of miR-192 in an anti-angiogenesis therapy.

Translational impact of nanoparticle-drug conjugate CRLX101 with or without bevacizumab in advanced ovarian cancer.

PURPOSE: Increased tumor hypoxia and hence elevated hypoxia-inducible factor-1α (HIF1α) is thought to limit the efficacy of vascular endothelial growth factor (VEGF) pathway-targeting drugs by upregulating adaptive resistance genes. One strategy to counteract this is to combine antiangiogenic drugs with agents able to suppress HIF1α. One such possibility is the investigational drug CRLX101, a nanoparticle-drug conjugate (NDC) containing the payload camptothecin, a known topoisomerase-I poison. EXPERIMENTAL DESIGN: CRLX101 was evaluated both as a monotherapy and combination with bevacizumab in a preclinical mouse model of advanced metastatic ovarian cancer. These preclinical studies contributed to the rationale for undertaking a phase II clinical study to evaluate CRLX101 monotherapy in patients with advanced platinum-resistant ovarian cancer. RESULTS: Preclinically, CRLX101 is highly efficacious as a monotherapy when administered at maximum-tolerated doses. Furthermore, chronic low-dose CRLX101 with bevacizumab reduced bevacizumab-induced HIF1α upregulation and resulted in synergistic efficacy, with minimal toxicity in mice. In parallel, initial data reported here from an ongoing phase II clinical study of CRLX101 monotherapy shows measurable tumor reductions in 74% of patients and a 16% RECIST response rate to date. CONCLUSIONS: Given these preclinical and initial clinical results, further clinical studies are currently evaluating CRLX101 in combination with bevacizumab in ovarian cancer and warrant the evaluation of this therapy combination in other cancer types where HIF1α is implicated in pathogenesis, as it may potentially be able to improve the efficacy of antiangiogenic drugs.

Engineered regulation of lysozyme by the SH3-CB1 binding interaction.

The ability to design proteins with desired properties by using protein structural information will allow us to create high-value therapeutic and diagnostic products. Using the protein structures of lambda lysozyme and the SH3 domain of human Crk, we designed a synthetic protein switch that controls the activity of lysozyme by sterically hindering its active cleft through the binding of SH3 to its CB1 peptide-binding partner. First, several fusion protein designs with lysozyme and CB1 were modeled to determine the one with greatest steric effect in the presence of SH3. Next, the selected fusion protein was created and tested in vitro. In the absence of SH3, the lysozyme-CB1 fusion protein functioned normally. In the presence of SH3, the lysozyme activity was inhibited and with the addition of excess CB1 peptides to compete for SH3 binding, the lysozyme activity was restored. Lastly, this structure-based strategy can be used to engineer synthetic regulation by peptide-domain-binding interfaces into a variety of proteins.

Effects of rapamycin-induced oligomerization of parvalbumin, Stim1 and Orai1 in puncta formation.

Elevations of cytosolic Ca2+ from the endoplasmic reticulum (ER) regulate a diverse range of cellular processes. When these luminal stores become depleted, the transmembrane ER protein Stim1 oligomerizes and translocates within the ER membrane to puncta junctions to couple with Orai1 channels, activating store-operated calcium entry (SOCE). Stim1 oligomerization and puncta formation have generally been associated with its luminal domains, however, studies have implicated that the cytoplasmic domains may contribute to this oligomerization. Studies have also suggested that intermediate or regulating elements may be required to fine-tune puncta formation and activation of SOCE. Here we made fusion proteins of Stim1 and Orai1 with FRB and FKBP12 domains that associate in the presence of rapamycin. Rapamycin-induced coupling of Stim1 to Stim1, Orai1 to Orai1 and Stim1 to Orai1 was found to be insufficient for puncta formation. Rapamycin was then used to recruit the cytosolic Ca2+ buffer protein parvalbumin (Pav) to Stim1 in order to buffer the local cytosolic Ca2+ near the ER membrane. Interestingly, Pav buffering near the ER caused puncta formation that was indistinguishable from those caused by thapsigargin. Our results suggest that Stim1 oligomerization and puncta formation may be additionally regulated either by local Ca2+ levels near the ER membrane or by as yet unidentified Ca2+-dependent proteins interacting with the cytoplasmic domains of Stim1.

Engineering a photoactivated caspase-7 for rapid induction of apoptosis.

Apoptosis is a cell death program involved in the development of multicellular organisms, immunity, and pathologies ranging from cancer to HIV/AIDS. We present an engineered protein that causes rapid apoptosis of targeted cells in monolayer culture after stimulation with blue light. Cells transfected with the protein switch L57V, a tandem fusion of the light-sensing LOV2 domain and the apoptosis-executing domain from caspase-7, rapidly undergo apoptosis within 60 min after light stimulation. Constant illumination of under 5 min or oscillating with 1 min exposure had no effect, suggesting that cells have natural tolerance to a short duration of caspase-7 activity. Furthermore, the overexpression of Bcl-2 prevented L57V-mediated apoptosis, suggesting that although caspase-7 activation is sufficient to start apoptosis, it requires mitochondrial contribution to fully commit.

Engineered networks of synthetic and natural proteins to control cell migration.

Mammalian cells reprogrammed with engineered transgenes have the potential to be useful therapeutic platforms because they can support large genetic networks, can be taken from a host or patient, and perform useful functions such as migration and secretion. Successful engineering of mammalian cells will require the development of modules that can perform well-defined, reliable functions, such as directed cell migration toward a chemical or physical signal. One inherently modular cellular pathway is the Ca(2+) signaling pathway: protein modules that mobilize and respond to Ca(2+) are combined across cell types to create complexity. We have designed a chimera of Rac1, a GTPase that controls cell morphology and migration, and calmodulin (CaM), a Ca(2+)-responsive protein, to control cell migration. The Rac1-CaM chimera (named RACer) controlled lamellipodia growth in response to Ca(2+). RACer was combined with LOVS1K (a previously engineered light-sensitive Ca(2+)-mobilizing module) and cytokine receptors to create protein networks where blue light and growth factors regulated cell morphology and, thereby, cell migration. To show the generalizability of our design, we created a Cdc42-CaM chimera that controls filopodia growth in response to Ca(2+). The insights that have been gained into Ca(2+) signaling and cell migration will allow future work to combine engineered protein systems to enable reprogrammed cell sensing of relevant therapeutic targets in vivo.

A synthetic photoactivated protein to generate local or global Ca(2+) signals.

Ca(2+) signals regulate diverse physiological processes through tightly regulated fluxes varying in location, time, frequency, and amplitude. Here, we developed LOVS1K, a genetically encoded and photoactivated synthetic protein to generate local or global Ca(2+) signals. With 300 ms blue light exposure, LOVS1K translocated to Orai1, a plasma membrane Ca(2+) channel, within seconds, generating a local Ca(2+) signal on the plasma membrane, and returning to the cytoplasm after tens of seconds. With repeated photoactivation, global Ca(2+) signals in the cytoplasm were generated to modulate engineered Ca(2+)-inducible proteins. Although Orai1 is typically associated with global store-operated Ca(2+) entry, we demonstrate that Orai1 can also generate local Ca(2+) influx on the plasma membrane. Our photoactivation system can be used to generate spatially and temporally precise Ca(2+) signals and to engineer synthetic proteins that respond to specific Ca(2+) signals.

Structure based design of a Ca2+-sensitive RhoA protein that controls cell morphology.

The Rho proteins are important regulators of cell morphology, and the prototypical protein RhoA is known to regulate contraction, blebbing and bleb retraction. We have identified and experimentally confirmed that RhoA has a binding site for calmodulin, a ubiquitous transducer of the Ca(2+) second messenger. Using structural modeling, a fusion protein was designed wherein RhoA activity was controlled by Ca(2+) via calmodulin. Living cells transfected with this synthetic protein underwent Ca(2+) sensitive and calmodulin-dependent bleb retraction within minutes. Further, the modularity of Ca(2+) signaling was exploited to induce bleb retraction in response to blue light (using channelrhodopsin-2) or exogenous chemicals (with acetylcholine receptor), showing input signal versatility. The widespread use of Ca(2+) signaling in nature suggests that fully exploring its signaling potential may allow powerful applications to other synthetic biological systems.

TEV protease-facilitated stoichiometric delivery of multiple genes using a single expression vector.

Delivery and expression of multiple genes is an important requirement in a range of applications such as the engineering of synthetic signaling pathways and the induction of pluripotent stem cells. However, conventional approaches are often inefficient, nonstoichiometric and may limit the maximum number of genes that can be simultaneously expressed. We here describe a versatile approach for multiple gene delivery using a single expression vector by mimicking the protein expression strategy of RNA viruses. This was accomplished by first expressing the genes together with TEV protease as a single fusion protein, then proteolytically self-cleaving the fusion protein into functional components. To demonstrate this method in E. coli cells, we analyzed the translation products using SDS-PAGE and showed that the fusion protein was efficiently cleaved into its components, which can then be purified individually or as a binding complex. To demonstrate this method in mammalian cells, we designed a differential localization scheme and used live cell imaging to observe the distinctive subcellular targeting of the processed products. We also showed that the stoichiometry of the processed products was consistent and corresponded with the frequency of appearance of their genes on the expression vector. In summary, the efficient expression and separation of up to three genes was achieved in both E. coli and mammalian cells using a single TEV protease self-processing vector.

Design of fluorescent fusion protein probes.

Many fluorescent probes depend on the fluorescence resonance energy transfer (FRET) between fluorescent protein pairs. The efficiency of energy transfer becomes altered by conformational changes of a fused sensory protein in response to a cellular event. A structure-based approach can be taken to design probes better with improved dynamic ranges by computationally modeling conformational changes and predicting FRET efficiency changes of candidate biosensor constructs. FRET biosensors consist of at least three domains fused together: the donor protein, the sensory domain, and the acceptor protein. To more efficiently subclone fusion proteins containing multiple domains, a cassette-based system can be used. Generating a cassette library of commonly used domains facilitates the rapid subcloning of future fusion biosensor proteins. FRET biosensors can then be used with fluorescence microscopy for real-time monitoring of cellular events within live cells by tracking changes in FRET efficiency. Stimulants can be used to trigger a range of cellular events including Ca(2+) signaling, apoptosis, and subcellular translocations.