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Supplementary materials include detailed chemistry, supplementary materials and methods, references, and supplementary figures.

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PDF file - 62K, Table S1. Humanized MAb159 Pharmacokinetics in mouse. Table S2. Toxicology study of humanized MAb159 in mouse.

Deletion of endoplasmic reticulum resident chaperone Grp78 results in activation of the unfolded protein response and causes rapid depletion of the entire intestinal epithelium. Whether modest reduction of Grp78 may affect stem cell fate without compromising intestinal integrity remains unknown. Here, we employ a model of epithelial-specific, heterozygous Grp78 deletion by use of VillinCreERT2-Rosa26ZsGreen/LacZ-Grp78+/fl mice and organoids. We examine models of irradiation and tumorigenesis, both in vitro and in vivo. Although we observed no phenotypic changes in Grp78 heterozygous mice, Grp78 heterozygous organoid growth was markedly reduced. Irradiation of Grp78 heterozygous mice resulted in less frequent regeneration of crypts compared with nonrecombined (wild-type) mice, exposing reduced capacity for self-renewal upon genotoxic insult. We crossed mice to Apc-mutant animals for adenoma studies and found that adenomagenesis in Apc heterozygous-Grp78 heterozygous mice was reduced compared with Apc heterozygous controls (1.43 vs. 3.33; P < 0.01). In conclusion, epithelium-specific Grp78 heterozygosity compromises epithelial fitness under conditions requiring expansive growth such as adenomagenesis or regeneration after γ-irradiation. These results suggest that Grp78 may be a therapeutic target in prevention of intestinal neoplasms without affecting normal tissue.Significance: Heterozygous disruption of chaperone protein Grp78 reduces tissue regeneration and expansive growth and protects from tumor formation without affecting intestinal homeostasis. Cancer Res; 78(21); 6098–106. ©2018 AACR.

PDF file - 736K, Fig. S1. Generation and characterization of MAb159. Fig. S2. MAb159 induces GRP78 endocytosis through clathrin mediated pathway. Fig. S3. MAb159 induces apoptosis of tumor cells in vitro. Fig. S4. Surface GRP78 Forms Complex with PI3K. Fig. S5. Surface GRP78 level in cancer cell lines. Fig. S6. MAb159 treatment of xenograft tumor leads to reduced vessel density and mTOR signaling. Fig. S7. MAb159 shows no toxicity to normal organs in tumor xenograft study. Fig. S8. Inhibition of primary tumor growth and metastasis by MAb159 in 4T1 orthotopic model. Fig. S9. MAb159 inhibits the growth of hormone refractory mouse prostate cancer cell CE1 in vivo.

Purpose: The ER chaperone GRP78 translocates to the surface of tumor cells and promotes survival, metastasis, and resistance to therapy. An oncogenic function of cell surface GRP78 has been attributed to the activation of the phosphoinositide 3-kinase (PI3K) pathway. We intend to use a novel anti-GRP78 monoclonal antibody (MAb159) to attenuate PI3K signaling and inhibit tumor growth and metastasis.Experimental Design: MAb159 was characterized biochemically. Antitumor activity was tested in cancer cell culture, tumor xenograft models, tumor metastasis models, and spontaneous tumor models. Cancer cells and tumor tissues were analyzed for PI3K activity. MAb159 was humanized and validated for diagnostic and therapeutic application.Results: MAb159 specifically recognized surface GRP78, triggered GRP78 endocytosis, and localized to tumors but not to normal organs in vivo. MAb159 inhibited tumor cell proliferation and enhanced tumor cell death both in vitro and in vivo. In MAb159-treated tumors, PI3K signaling was inhibited without compensatory MAPK pathway activation. Furthermore, MAb159 halted or reversed tumor progression in the spontaneous PTEN–loss-driven prostate and leukemia tumor models, and inhibited tumor growth and metastasis in xenograft models. Humanized MAb159, which retains high affinity, tumor specific localization, and the antitumor activity, was nontoxic in mice, and had desirable pharmacokinetics.Conclusions: GRP78-specific antibody MAb159 modulates the PI3K pathway and inhibits tumor growth and metastasis. Humanized MAb159 will enter human trials shortly. Clin Cancer Res; 19(24); 6802–11. ©2013 AACR.

Therapeutic targeting of the tumor vasculature that destroys preexisting blood vessels of the tumor and antiangiogenesis therapy capitalize on the requirement of tumor cells on an intact vascular supply for oxygen and nutrients for growth, expansion and metastasis to the distal organs. Whereas these classes of agents show promise in delaying tumor progression, they also create glucose and oxygen deprivation conditions within the tumor that could trigger unintended prosurvival responses. The glucose-regulated protein GRP78, a major endoplasmic reticulum chaperone, is inducible by severe glucose depletion, anoxia, and acidosis. Here we report that in a xenograft model of human breast cancer, treatment with the vascular targeting agent, combretastatin A4P, or the antiangiogenic agent, contortrostatin, promotes transcriptional activation of the Grp78 promoter and elevation of GRP78 protein in surviving tumor cells. We further show that GRP78 is overexpressed in a panel of human breast cancer cells that has developed resistance to a variety of drug treatment regimens. Suppression of GRP78 through the use of lentiviral vector expressing small interfering RNA sensitizes human breast cancer cells to etoposide-mediated cell death. Our studies imply that antivascular and antiangiogenesis therapy that results in severe glucose and oxygen deprivation will induce GRP78 expression that could lead to drug resistance.

Supplementary Data from Vascular Targeting and Antiangiogenesis Agents Induce Drug Resistance Effector GRP78 within the Tumor Microenvironment

Supplementary Figure 1 from A Critical Role for GRP78/BiP in the Tumor Microenvironment for Neovascularization during Tumor Growth and Metastasis

Supplementary Figure Legend from A Critical Role for GRP78/BiP in the Tumor Microenvironment for Neovascularization during Tumor Growth and Metastasis

Supplementary information, references, figure legend and Tables S1 & S2

Supplementary Figure 1 from GRP78 as a Novel Predictor of Responsiveness to Chemotherapy in Breast Cancer

The discovery of predictive factors for chemoresistance is critical for improving adjuvant therapy for cancer patients. The 78-kDa glucose-regulated protein (GRP78), widely used as an indicator of the unfolded protein response (UPR), is induced in the tumor microenvironment. In vitro studies suggest that GRP78 confers chemoresistance to topoisomerase inhibitors, such as Adriamycin (doxorubicin). Here, we report on a retrospective cohort study of 127 stage II and III breast cancer patients who were treated with Adriamycin-based chemotherapy. Archival tumor specimens were available for analysis and the relationship of GRP78 expression level to “time to recurrence” (TTR), used as a surrogate marker for drug resistance, was examined. Our data show that 67% of the study subjects expressed high level of GRP78 in their tumors before the initiation of chemotherapy and suggest an association between GRP78 positivity and shorter TTR [hazard ratio (HR), 1.78; P = 0.16]. Interestingly, subgroup analysis reveals that the HR for the GRP78-positive group increased significantly among patients who did not receive further taxane treatment (HR, 3.00; P = 0.022) and among mastectomy patients (HR, 3.33; P = 0.027). The HR was even stronger among mastectomy patients who did not receive further taxane treatment (HR, 4.82; P = 0.010). The use of GRP78 as a predictor for chemoresponsiveness and the potential interaction of GRP78 and/or the UPR pathways with taxanes warrant larger studies. (Cancer Res 2006; 66(16): 7849-53)

OBJECTIVE: Addressing firearm access is recommended when patients are identified at risk for suicide. However, the practice of assessing firearm access is controversial and there are no national guidelines to inform practice. This study qualitatively explored patient perspectives on a routine question about firearm access to optimize the patient-centeredness of this practice in the context of suicide risk. METHODS: Electronic health record data was used to identify primary care patients reporting depressive symptoms, including suicidal thoughts, within 2 weeks of sampling. Participants completed a semi-structured telephone interview (recorded and transcribed), which focused broadly on the experience of being screened for suicidality and included specific questions to elicit beliefs/opinions about being asked a standard firearm access question. Directive (deductive) and conventional (inductive) content analysis was used to analyze responses to the portion of the interview focused on firearm assessment and disclosure. RESULTS: A sample of 37 patients in Washington State ages 20–95 completed the qualitative interview by phone. Organizing themes included: 1) Apprehensions about disclosing access to firearms related to privacy, autonomy and firearm ownership rights, 2) Perceptions regarding relevance of the firearm question, informed by experiences with suicidality and common beliefs and misconceptions about the inevitability of suicide, and 3) Suggestions for connecting questions about firearms and other lethal means to suicide risk. CONCLUSION: Clarifying the purpose and use of routine firearm access assessment, contextualizing firearm questions within injury prevention broadly, and addressing misconceptions about suicide prevention may help encourage firearm access disclosure and increase the patient-centeredness of this practice.

Background Most people with opioid use disorder (OUD) never receive treatment. Medication treatment of OUD in primary care is recommended as an approach to increase access to care. The PRimary Care Opioid Use Disorders treatment (PROUD) trial tests whether implementation of a collaborative care model (Massachusetts Model) using a nurse care manager (NCM) to support medication treatment of OUD in primary care increases OUD treatment and improves outcomes. Specifically, it tests whether implementation of collaborative care, compared to usual primary care, increases the number of days of medication for OUD (implementation objective) and reduces acute health care utilization (effectiveness objective). The protocol for the PROUD trial is presented here. Methods PROUD is a hybrid type III cluster-randomized implementation trial in six health care systems. The intervention consists of three implementation strategies: salary for a full-time NCM, training and technical assistance for the NCM, and requiring that three primary care providers have DEA waivers to prescribe buprenorphine. Within each health system, two primary care clinics are randomized: one to the intervention and one to Usual Primary Care. The sample includes all patients age 16–90 who visited the randomized primary care clinics from 3 years before to 2 years after randomization (anticipated to be > 170,000). Quantitative data are derived from existing health system administrative data, electronic medical records, and/or health insurance claims (“electronic health records,” [EHRs]). Anonymous staff surveys, stakeholder debriefs, and observations from site visits, trainings and technical assistance provide qualitative data to assess barriers and facilitators to implementation. The outcome for the implementation objective (primary outcome) is a clinic-level measure of the number of patient days of medication treatment of OUD over the 2 years post-randomization. The patient-level outcome for the effectiveness objective (secondary outcome) is days of acute care utilization [e.g. urgent care, emergency department (ED) and/or hospitalizations] over 2 years post-randomization among patients with documented OUD prior to randomization. Discussion The PROUD trial provides information for clinical leaders and policy makers regarding potential benefits for patients and health systems of a collaborative care model for management of OUD in primary care, tested in real-world diverse primary care settings. Trial registration # NCT03407638 (February 28, 2018); CTN-0074 https://clinicaltrials.gov/ct2/show/NCT03407638?term=CTN-0074&draw=2&rank=1

Voltage-gated Cav1 and Cav2 Ca2+ channels are comprised of a pore-forming α1 subunit (Cav1.1-1.4, Cav2.1-2.3) and auxiliary β (β1-4) and α2δ (α2δ-1-4) subunits. The properties of these channels vary with distinct combinations of Cav subunits and alternative splicing of the encoding transcripts. Therefore, the impact of disease-causing mutations affecting these channels may depend on the identities of Cav subunits and splice variants. Here, we analyzed the effects of a congenital stationary night blindness type 2 (CSNB2)-causing mutation, I745T (IT), in Cav1.4 channels typical of those in human retina: Cav1.4 splice variants with or without exon 47 (Cav1.4+ex47 and Cav1.4Δex47, respectively), and the auxiliary subunits, β2X13 and α2δ-4. We find that IT caused both Cav1.4 splice variants to activate at significantly more negative voltages and with slower deactivation kinetics than the corresponding WT channels. These effects of the IT mutation, along with unexpected alterations in ion selectivity, were generally larger in channels lacking exon 47. The weaker ion selectivity caused by IT led to hyperpolarizing shifts in the reversal potential and large outward currents that were evident in channels containing the auxiliary subunits β2X13 and α2δ-4 but not in those with β2A and α2δ-1. We conclude that the IT mutation stabilizes channel opening and alters ion selectivity of Cav1.4 in a manner that is strengthened by exclusion of exon 47 and inclusion of β2X13 and α2δ-4. Our results reveal complex actions of IT in modifying the properties of Cav1.4 channels, which may influence the pathological consequences of this mutation in retinal photoreceptors.

Background: The authors tested the efficacy of the “I-STAND” intervention for reducing sitting time, a novel and potentially health-promoting approach, in older adults with obesity. Methods: The authors recruited 60 people (mean age = 68 ± 4.9 years, 68% female, 86% White; mean body mass index = 35.4). The participants were randomized to receive the I-STAND sitting reduction intervention (n = 29) or healthy living control group (n = 31) for 12 weeks. At baseline and at 12 weeks, the participants wore activPAL devices to assess sitting time (primary outcome). Secondary outcomes included fasting glucose, blood pressure, and weight. Linear regression models assessed between-group differences in the outcomes. Results: The I-STAND participants significantly reduced their sitting time compared with the controls (–58 min per day; 95% confidence interval [–100.3, –15.6]; p = .007). There were no statistically significant changes in the secondary outcomes. Conclusion: I-STAND was efficacious in reducing sitting time, but not in changing health outcomes in older adults with obesity.

OBJECTIVESelective dorsal rhizotomy (SDR) is effective at permanently reducing spasticity in children with spastic cerebral palsy. The value of intraoperative neurophysiological monitoring in this procedure remains controversial, and its robustness has been questioned. This study describes the authors’ institutional electrophysiological technique (based on the technique of Park et al.), intraoperative findings, robustness, value to the procedure, and occurrence of new motor or sphincter deficits.METHODSThe authors analyzed electrophysiological data of all children who underwent SDR at their center between September 2013 and February 2019. All patients underwent bilateral SDR through a single-level laminotomy at the conus and with transection of about 60% of the L2–S2 afferent rootlets (guided by intraoperative electrophysiology) and about 50% of L1 afferent roots (nonselectively).RESULTSOne hundred forty-five patients underwent SDR (64% male, mean age 6 years and 7 months, range 2 years and 9 months to 14 years and 10 months). Dorsal roots were distinguished from ventral roots anatomically and electrophysiologically, by assessing responses on free-running electromyography (EMG) and determining stimulation thresholds (≥ 0.2 mA in all dorsal rootlets). Root level was determined anatomically and electrophysiologically by assessing electromyographic response to stimulation. Median stimulation threshold was lower in sacral compared to lumbar roots (p < 0.001), and 16% higher on the first operated (right) side (p = 0.023), but unrelated to age, sex, or functional status. Similarly, responses to tetanic stimulation were consistent: 87% were graded 3+ or 4+, with similar distributions between sides. This was also unrelated to age, sex, and functional status. The L2–S2 rootlets were divided (median 60%, range 50%–67%), guided by response to tetanic stimulation at threshold amplitude. No new motor or sphincter deficits were observed, suggesting sparing of ventral roots and sphincteric innervation, respectively.CONCLUSIONSThis electrophysiological technique appears robust and reproducible, allowing reliable identification of afferent nerve roots, definition of root levels, and guidance for rootlet division. Only a direct comparative study will establish whether intraoperative electrophysiology during SDR minimizes risk of new motor or sphincter worsening and/or maximizes functional outcome.

Exosomes are cell-secreted microvesicles that play important roles in epithelial ovarian cancer (EOC) progression, as they are constantly secreted into ascites fluids. While cells spontaneously release exosomes, alterations in intracellular calcium or extracellular pH can release additional exosomes. Yet, little is known about how these exosomes compare to those that are continuously released without stimulation and how they mediate cellular activities important in cancer progression. Here, we demonstrate that chelation of extracellular calcium leads to release of chelation-induced exosomes (CI-exosomes) from OVCAR-3 EOC cells. CI-exosomes display a unique miRNA profile compared to naturally secreted exosomes (SEC-exosomes). Furthermore, treatment with CI- and SEC-exosomes leads to differential biophysical and functional changes including, adhesion and migration in EOC-derived fibroblasts that suggest the development of a malignant tumor microenvironment. This result highlights how tumor environmental factors contribute to heterogeneity in exosome populations and how different exosome populations mediate diversity in stromal cell behavior.

High expression of LIN28B is associated with aggressive malignancy and poor survival. Here, probing MYCN-amplified neuroblastoma as a model system, we showed that LIN28B expression was associated with enhanced cell migration in vitro and invasive and metastatic behavior in murine xenografts. Sequence analysis of the polyribosome fraction of LIN28B-expressing neuroblastoma cells revealed let-7–independent enrichment of transcripts encoding components of the translational and ribosomal apparatus and depletion of transcripts of neuronal developmental programs. We further observed that LIN28B utilizes both its cold shock and zinc finger RNA binding domains to preferentially interact with MYCN-induced transcripts of the ribosomal complex, enhancing their translation. These data demonstrated that LIN28B couples the MYCN-driven transcriptional program to enhanced ribosomal translation, thereby implicating LIN28B as a posttranscriptional driver of the metastatic phenotype.

Klebsiella pneumoniae has been implicated in wide-ranging nosocomial outbreaks, causing severe infections without effective treatments due to antibiotic resistance. Here, we performed genome sequencing of 70 extensively drug resistant clinical isolates, collected from Brasília’s hospitals (Brazil) between 2010 and 2014. The majority of strains (60 out of 70) belonged to a single clonal complex (CC), CC258, which has become distributed worldwide in the last two decades. Of these CC258 strains, 44 strains were classified as sequence type 11 (ST11) and fell into two distinct clades, but no ST258 strains were found. These 70 strains had a pan-genome size of 10 366 genes, with a core-genome size of ~4476 genes found in 95 % of isolates. Analysis of sequences revealed diverse mechanisms of resistance, including production of multidrug efflux pumps, enzymes with the same target function but with reduced or no affinity to the drug, and proteins that protected the drug target or inactivated the drug. β-Lactamase production provided the most notable mechanism associated with K. pneumoniae . Each strain presented two or three different β-lactamase enzymes, including class A (SHV, CTX-M and KPC), class B and class C AmpC enzymes, although no class D β-lactamase was identified. Strains carrying the NDM enzyme involved three different ST types, suggesting that there was no common genetic origin.

Bordetella pertussis has been shown to encode regulatory RNAs, yet the posttranscriptional regulatory circuits on which they act remain to be fully elucidated. We generated mutants lacking the endonucleases RNase III and RNase E and assessed their individual impact on the B. pertussis transcriptome. Transcriptome sequencing (RNA-Seq) analysis showed differential expression of ∼25% of the B. pertussis transcriptome in each mutant, with only 28% overlap between data sets. Both endonucleases exhibited substantial impact on genes involved in amino acid uptake (e.g., ABC transporters) and in virulence (e.g., the type III secretion system and the autotransporters vag8, tcfA, and brkA). Interestingly, mutations in RNase III and RNase E drove the stability of many transcripts, including those involved in virulence, in opposite directions, a result that was validated by qPCR and immunoblotting for tcfA and brkA. Of note, whereas similar mutations to RNase E in Escherichia coli have subtle effects on transcript stability, a striking >20-fold reduction in four gene transcripts, including tcfA and vag8, was observed in B. pertussis. We further compared our data set to the regulon controlled by the RNA chaperone Hfq to identify B. pertussis loci influenced by regulatory RNAs. This analysis identified ∼120 genes and 19 operons potentially regulated at the posttranscriptional level. Thus, our findings revealed how changes in RNase III- and RNase E-mediated RNA turnover influence pathways associated with virulence and cellular homeostasis. Moreover, we highlighted loci potentially influenced by regulatory RNAs, providing insights into the posttranscriptional regulatory networks involved in fine-tuning B. pertussis gene expression. IMPORTANCE Noncoding, regulatory RNAs in bacterial pathogens are critical components required for rapid changes in gene expression profiles. However, little is known about the role of regulatory RNAs in the growth and pathogenesis of Bordetella pertussis. To address this, mutants separately lacking ribonucleases central to regulatory RNA processing, RNase III and RNase E, were analyzed by RNA-Seq. Here, we detail the first transcriptomic analysis of the impact of altered RNA degradation in B. pertussis. Each mutant showed approximately 1,000 differentially expressed genes, with significant changes in the expression of pathways associated with metabolism, bacterial secretion, and virulence factor production. Our analysis suggests an important role for these ribonucleases during host colonization and provides insights into the breadth of posttranscriptional regulation in B. pertussis, further informing our understanding of B. pertussis pathogenesis.

BACKGROUND: Alcohol use disorder (AUD) is highly prevalent but underrecognized and undertreated in primary care settings. Alcohol Symptom Checklists can engage patients and providers in discussions of AUD-related care. However, the performance of Alcohol Symptom Checklists when they are used in routine care and documented in electronic health records (EHRs) remains unevaluated. OBJECTIVE: To evaluate the psychometric performance of an Alcohol Symptom Checklist in routine primary care. DESIGN: Cross-sectional study using item response theory (IRT) and differential item functioning analyses of measurement consistency across age, sex, race, and ethnicity. PATIENTS: Patients seen in primary care in the Kaiser Permanente Washington Healthcare System who reported high-risk drinking on the Alcohol Use Disorder Identification Test Consumption screening measure (AUDIT-C ≥ 7) and subsequently completed an Alcohol Symptom Checklist between October 2015 and February 2020. MAIN MEASURE: Alcohol Symptom Checklists with 11 items assessing AUD criteria defined in the Diagnostic and Statistical Manual for Mental Disorders, 5th edition (DSM-5), completed by patients during routine medical care and documented in EHRs. KEY RESULTS: Among 11,464 patients who screened positive for high-risk drinking and completed an Alcohol Symptom Checklist (mean age 43.6 years, 30.5% female), 54.1% reported ≥ 2 DSM-5 AUD criteria (threshold for AUD diagnosis). IRT analyses demonstrated that checklist items measured a unidimensional continuum of AUD severity. Differential item functioning was observed for some demographic subgroups but had minimal impact on accurate measurement of AUD severity, with differences between demographic subgroups attributable to differential item functioning never exceeding 0.42 points of the total symptom count (of a possible range of 0–11). CONCLUSIONS: Alcohol Symptom Checklists used in routine care discriminated AUD severity consistently with current definitions of AUD and performed equitably across age, sex, race, and ethnicity. Integrating symptom checklists into routine care may help inform clinical decision-making around diagnosing and managing AUD. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s11606-021-07038-3.

Cyclic GMP–AMP synthase (cGAS) is a cytosolic DNA sensor that produces the second messenger cG[2′–5′]pA[3′–5′]p (2′3′-cGAMP) and controls activation of innate immunity in mammalian cells1–5. Animal genomes typically encode multiple proteins with predicted homology to cGAS6–10, but the function of these uncharacterized enzymes is unknown. Here we show that cGAS-like receptors (cGLRs) are innate immune sensors that are capable of recognizing divergent molecular patterns and catalysing synthesis of distinct nucleotide second messenger signals. Crystal structures of human and insect cGLRs reveal a nucleotidyltransferase signalling core shared with cGAS and a diversified primary ligand-binding surface modified with notable insertions and deletions. We demonstrate that surface remodelling of cGLRs enables altered ligand specificity and used a forward biochemical screen to identify cGLR1 as a double-stranded RNA sensor in the model organism Drosophila melanogaster. We show that RNA recognition activates Drosophila cGLR1 to synthesize the novel product cG[3′–5′]pA[2′–5′]p (3′2′-cGAMP). A crystal structure of Drosophila stimulator of interferon genes (dSTING) in complex with 3′2′-cGAMP explains selective isomer recognition, and 3′2′-cGAMP induces an enhanced antiviral state in vivo that protects from viral infection. Similar to radiation of Toll-like receptors in pathogen immunity, our results establish cGLRs as a diverse family of metazoan pattern recognition receptors., The authors identify Drosophila cGLR1 as a double-stranded RNA sensor and 3′2′-cGAMP as a nucleotide second messenger and activator of Drosophila STING signalling.

Background Treponema pallidum ssp. pallidum (Tp), the causative agent of syphilis, is a highly invasive pathogen that moves throughout the body via the bloodstream and invades organs and tissues to cause the serious sequelae associated with sexually transmitted and congenitally acquired syphilis infections. The endothelium represents a crucial barrier for Tp dissemination, and we hypothesized that Tp alters endothelial structure and signalling. In this study, we apply an integrative omics approach to characterize transcriptomic, proteomic, and immune secretomic responses of endothelial cells to Tp. Methods Endothelial cells were grown in vitro and exposed to viable Tp or a background control lacking the pathogen. For transcriptomic analysis, RNA was harvested from the endothelial cells and sequenced via RNAseq. For proteomic analysis, experimental and control samples grown using stable isotope labeling by amino acids in cell culture (SILAC), total protein was harvested and analyzed via orbitrap fusion tandem mass-spectrometry (LC-MS/MS). For immune secretomic analysis, supernatant from endothelial cells co-incubated with Tp was harvested and analyzed for cytokine content via cytometric bead arrays (CBA) using flow-cytometry. Results RNAseq studies identified genes within immune signalling pathways that are differentially regulated in response to Tp. Proteomic analysis identified several pathways involved in cellular structure and modulation, interleukin signalling, and intracellular signalling. Data from CBA experiments further indicate the ability of Tp to alter endothelial cytokine production and secretion. These data give a novel multi-faceted look into the role and response of the endothelium during Tp infection. Conclusion These studies represent a novel systems-level approach to understanding the molecular mechanisms behind the establishment and persistence of Tp infection. Further, they provide insight into how Tp may induce host cellular changes to enable barrier traversal and immune cell recruitment, and in this way provide information that is relevant to syphilis vaccine design.

BACKGROUND: This pilot study evaluated whether use of evidence-based implementation strategies to integrate care for cannabis and other drug use into primary care (PC) as part of Behavioral Health Integration (BHI) increased diagnosis and treatment of substance use disorders (SUDs). METHODS: Patients who visited the three pilot PC sites were eligible. Implementation strategies included practice coaching, electronic health record decision support, and performance feedback (3/2015–4/2016). BHI introduced annual screening for past-year cannabis and other drug use, a Symptom Checklist for DSM-5 SUDs, and shared decision-making about treatment options. Main analyses tested whether the proportions of PC patients diagnosed with, and treated for, new cannabis or other drug use disorders (CUDs and DUDs, respectively), differed significantly pre- and post-implementation. RESULTS: Of 39,599 eligible patients, 57% and 59% were screened for cannabis and other drug use, respectively. Among PC patients reporting daily cannabis use (2%) or any drug use (1%), 51% and 37%, respectively, completed an SUD Symptom Checklist. The proportion of PC patients with newly diagnosed CUD increased significantly post-implementation (5 v 17 per 10,000 patients, p

Translocation of 78-kDa glucose-regulated protein (GRP78) from endoplasmic reticulum (ER) to plasma membrane represents a paradigm shift beyond its traditional function as an ER chaperone protein. Cell surface GRP78 (csGRP78) exerts novel signaling functions, and mechanisms underlying its cell surface expression are just emerging. Acquired tamoxifen resistance of breast cancer cells is accompanied with elevated level of csGRP78. Therefore, the tamoxifen-resistant MCF7 breast cancer cells (MCF7-LR) represents a clinically relevant model to study mechanisms of csGRP78 expression. We discovered that a proline-rich region (PRR) containing three consecutive prolines close to the COOH-terminus of GRP78 is important for its ability to form a complex with the partner protein, CD44v, as demonstrated by in vitro glutathione S-transferase pull-down assay. Proline to alanine mutations at the PRR compromised GRP78 expression level on the cell surface as evidenced by purification of biotinylated cell surface proteins. Reconstitution of MCF7-LR cells with the PRR mutant after knockdown of endogenous GRP78 diminished the capacity of GRP78 to stimulate STAT3 activation. The enforced expression of a short peptide bearing the PRR region of GRP78 led to reduction of CD44v and Cyclin D1 protein levels as well as cell viability, accompanied with increase in apoptotic signaling including cleaved Caspase-3 and PARP. These findings suggest that the COOH-terminal PRR of GRP78 is critical for its interaction with CD44v as well as its cell surface expression, and enforced expression of the short peptide bearing the PRR region may provide a new approach to lower the viability of tamoxifen-resistant breast cancer cells.

BACKGROUND: Although periodic chlamydia and gonorrhea testing is recommended for men who have sex with men (MSM), little is known about testing rates in China. This study examines chlamydia and gonorrhea testing rates and testing correlates among Chinese MSM. METHODS: An online survey of MSM was conducted in August 2017. Men aged 16 years or above who had ever had sex with a man were enrolled through a gay social networking mobile application. We asked men about their sexual behaviors, community engagement in sexual health, and previous testing for chlamydia, gonorrhea and HIV. Multivariable logistic regressions were used to examine the association of testing with community engagement and recent HIV testing. RESULTS: Of 1031 men, 819 (79.5%) were under 30 years of age, and 263 (25.5%) reported condomless sex in past three months. In total, 294 (28.5%) men tested for chlamydia, 315 (30.6%) men tested for gonorrhea, and 817(79.2%) men tested for HIV. One hundred and twenty-five (42.5%) men who received chlamydia testing and 134 (42.5%) men who received gonorrhea testing had substantial community engagement. Compared to men with no/minimal community engagement, men with substantial community engagement had greater odds of chlamydia testing (adjusted odds ratio [AOR] =2.8, 95%CI: 1.9–4.3) and gonorrhea testing (AOR=2.9, 95%CI: 2.0–4.4). Men with recent HIV testing were more likely to have received chlamydia testing (AOR=1.5, 95%CI: 1.1–2.0) and gonorrhea testing (AOR=1.6, 95%CI: 1.2–2.1). CONCLUSIONS: Chlamydia and gonorrhea testing levels are low among Chinese MSM. Integrating chlamydia and gonorrhea test promotion strategies into HIV prevention programs that engage MSM communities may help bridge the gap. SUMMARY: We found low chlamydia and gonorrhea lifetime testing rates among MSM in China and integrated STI/HIV testing programs that engage MSM may improve lifetime chlamydia and gonorrhea test uptake.

Cyclic dinucleotides (CDNs) have central roles in bacterial homeostasis and virulence by acting as nucleotide second messengers. Bacterial CDNs also elicit immune responses during infection when they are detected by pattern-recognition receptors in animal cells. Here we perform a systematic biochemical screen for bacterial signalling nucleotides and discover a large family of cGAS/DncV-like nucleotidyltransferases (CD-NTases) that use both purine and pyrimidine nucleotides to synthesize a diverse range of CDNs. A series of crystal structures establish CD-NTases as a structurally conserved family and reveal key contacts in the enzyme active-site lid that direct purine or pyrimidine selection. CD-NTase products are not restricted to CDNs and also include an unexpected class of cyclic trinucleotide compounds. Biochemical and cellular analyses of CD-NTase signalling nucleotides demonstrate that these cyclic di- and trinucleotides activate distinct host receptors and thus may modulate the interaction of both pathogens and commensal microbiota with their animal and plant hosts.

Significance: Surgery is often paramount in the management of many solid organ malignancies because optimal resection is a major factor in disease-specific survival. Cancer surgery has multiple challenges including localizing small lesions, ensuring negative surgical margins around a tumor, adequately staging patients by discriminating positive lymph nodes, and identifying potential synchronous cancers. Intraoperative molecular imaging (IMI) is an emerging potential tool proposed to address these issues. IMI is the process of injecting patients with fluorescenttargeted contrast agents that highlight cancer cells prior to surgery. Over the last 5 to 7 years, enormous progress has been achieved in tracer development, near-infrared camera approvals, and clinical trials. Therefore, a second biennial conference was organized at the University of Pennsylvania to gather surgical oncologists, scientists, and experts to discuss new investigative findings in the field. Our review summarizes the discussions from the conference and highlights findings in various clinical and scientific trials.Aim: Recent advances in IMI were presented, and the importance of each clinical trial for surgical oncology was critically assessed. A major focus was to elaborate on the clinical endpoints that were being utilized in IMI trials to advance the respective surgical subspecialties.Approach: Principal investigators presenting at the Perelman School of Medicine Abramson Cancer Center's second clinical trials update on IMI were selected to discuss their clinical trials and endpoints.Results: Multiple phase III, II, and I trials were discussed during the conference. Since the approval of 5-ALA for commercial use in neurosurgical malignancies, multiple tracers and devices have been developed to address common challenges faced by cancer surgeons across numerous specialties. Discussants also presented tracers that are being developed for delineation of normal anatomic structures that can serve as an adjunct during surgical procedures.Conclusions: IMI is increasingly being recognized as an improvement to standard oncologic surgical resections and will likely advance the art of cancer surgery in the coming years. The endpoints in each individual surgical subspecialty are varied depending on how IMI helps each specialty solve their clinical challenges. (C) The Authors. Published by SPIE under a Creative Commons Attribution 4.0 Unported License.

Despite new advances on the functions of ER chaperones at the cell surface, the translocation mechanisms whereby these chaperones can escape from the ER to the cell surface are just emerging. Previously we reported that in many cancer types, upon ER stress, IRE1α binds to and triggers SRC activation resulting in KDEL receptor dispersion from the Golgi and suppression of retrograde transport. In this study, using a combination of molecular, biochemical, and imaging approaches, we discovered that in colon and lung cancer, upon ER stress, ER chaperones, such as GRP78 bypass the Golgi and unconventionally traffic to the cell surface via endosomal transport mediated by Rab GTPases (Rab4, 11 and 15). Such unconventional transport is driven by membrane fusion between ER-derived vesicles and endosomes requiring the v-SNARE BET1 and t-SNARE Syntaxin 13. Furthermore, GRP78 loading into ER-derived vesicles requires the co-chaperone DNAJC3 that is regulated by ER-stress induced PERK-AKT-mTOR signaling.

Vaccination to prevent infectious disease is one of the most successful public health interventions ever developed. And yet, variability in individual vaccine effectiveness suggests a better mechanistic understanding of vaccine-induced immune responses could improve vaccine design and efficacy. We have previously shown that protective antibody levels could be elicited in a subset of recipients with only a single dose of the hepatitis B virus (HBV) vaccine. Why some, but not all, recipients responded in this way was not clear. Using single cell RNA sequencing of sorted innate immune cell subsets, we identified two distinct myeloid dendritic cell subsets (NDRG1-expressing mDC2 and CDKN1C-expressing mDC4), the ratio of which at baseline (pre-vaccination) predicted immune response to a single dose of HBV vaccine. Our results suggest that the participants in our vaccine study were in one of two different dendritic cell dispositional states at baseline – an NDRG2-mDC2 state in which the vaccine elicited an antibody response after a single immunization or a CDKN1C-mDC4 state in which the vaccine required two or three doses for induction of antibody responses. Genes expressed in these mDC subsets were used as an approach for feature selection prior to the construction of a predictive model using supervised canonical correlation machine learning. The resulting model showed an improved ability to predict serum antibody titers in response to vaccination. Taken together, these results suggest that the propensity of circulating dendritic cells toward either activation or suppression, their “dispositional endotype”, could dictate response to vaccination. The fact that these mDCs could be modulated via TLR stimulation could guide progress towards design of effective single dose vaccination strategies.

GRP78 (glucose-regulated protein, 78 kDa) is a key regulator of endoplasmic reticulum (ER) stress signaling. Cancer cells are highly proliferative and have high demand for protein synthesis and folding, which results in significant stress on the ER. To respond to ER stress and maintain cellular homeostasis, cells activate the unfolded protein response (UPR) that promotes either survival or apoptotic death. Cancer cells utilize the UPR to promote survival and growth. In this study, we describe the discovery of a series of novel hydroxyquinoline GRP78 inhibitors. A representative analogue, YUM70, inhibited pancreatic cancer cell growth in vitro and showed in vivo efficacy in a pancreatic cancer xenograft model with no toxicity to normal tissues. YUM70 directly bound GRP78 and inactivated its function, resulting in ER stress–mediated apoptosis. A YUM70 analogue conjugated with BODIPY showed colocalization of the compound with GRP78 in the ER. Moreover, a YUM70-PROTAC (proteolysis targeting chimera) was synthesized to force degradation of GRP78 in pancreatic cancer cells. YUM70 showed a strong synergistic cytotoxicity with topotecan and vorinostat. Together, our study demonstrates that YUM70 is a novel inducer of ER stress, with preclinical efficacy as a monotherapy or in combination with topoisomerase and HDAC inhibitors in pancreatic cancer. Significance: This study identifies a novel ER stress inducer that binds GRP78 and inhibits pancreatic cancer cell growth in vitro and in vivo, demonstrating its potential as a therapeutic agent for pancreatic cancer.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the current COVID-19 global pandemic, utilizes the host receptor angiotensin-converting enzyme 2 (ACE2) for viral entry. However, other host factors may also play major roles in viral infection. Here we report that the stress-inducible molecular chaperone GRP78 can form a complex with the SARS-CoV-2 Spike protein and ACE2 intracellularly and on the cell surface, and that the substrate binding domain of GRP78 is critical for this function. Knock-down of GRP78 by siRNA dramatically reduced cell surface ACE2 expression. Treatment of lung epithelial cells with a humanized monoclonal antibody (hMAb159), selected for its ability to cause GRP78 endocytosis and its safe clinical profile in preclinical models, reduces cell surface ACE2 expression, SARS-CoV-2 Spike-driven viral entry, and significantly inhibits SARS-CoV-2 infection in vitro. Our data suggest that GRP78 is an important host auxiliary factor for SARS-CoV-2 entry and infection and a potential target to combat this novel pathogen and other viruses that utilize GRP78.