Family of pH (low) insertion peptides for tumor targeting.

Cancer is a complex disease with a range of genetic and biochemical markers within and among tumors, but a general tumor characteristic is extracellular acidity, which is associated with tumor growth and development. Acidosis could be a universal marker for cancer imaging and the delivery of therapeutic molecules, but its promise as a cancer biomarker has not been fully realized in the clinic. We have discovered a unique approach for the targeting of acidic tissue using the pH-sensitive folding and transmembrane insertion of pH (low) insertion peptide (pHLIP). The essence of the molecular mechanism has been elucidated, but the principles of design need to be understood for optimal clinical applications. Here, we report on a library of 16 rationally designed pHLIP variants. We show how the tuning of the biophysical properties of peptide-lipid bilayer interactions alters tumor targeting, distribution in organs, and blood clearance. Lead compounds for PET/single photon emission computed tomography and fluorescence imaging/MRI were identified, and targeting specificity was shown by use of noninserting variants. Finally, we present our current understanding of the main principles of pHLIP design.

pHLIP peptide targets nanogold particles to tumors.

Progress in nanomedicine depends on the development of nanomaterials and targeted delivery methods. In this work, we describe a method for the preferential targeting of gold nanoparticles to a tumor in a mouse model. The method is based on the use of the pH Low Insertion Peptide (pHLIP), which targets various imaging agents to acidic tumors. We compare tumor targeting by nonfunctionalized nanogold particles with nanogold-pHLIP conjugates, where nanogold is covalently attached to the N terminus of pHLIP. Our most important finding is that both intratumoral and i.v. administration demonstrated a significant enhancement of tumor uptake of gold nanoparticles conjugated with pHLIP. Statistically significant reduction of gold accumulation was observed in acidic tumors and kidney when pH-insensitive K-pHLIP was used as a vehicle, suggesting an important role of pH in the pHLIP-mediated targeting of gold nanoparticles. The pHLIP technology can substantially improve the delivery of gold nanoparticles to tumors by providing specificity of targeting, enhancing local concentration in tumors, and distributing nanoparticles throughout the entire tumor mass where they remain for an extended period (several days), which is beneficial for radiation oncology and imaging.

Propensity weighted analysis of chemical venous thromboembolism prophylaxis agents in isolated severe traumatic brain injury: An EAST sponsored multicenter study.

BACKGROUND: In patients with severe traumatic brain injury (TBI), clinicians must balance preventing venous thromboembolism (VTE) with the risk of intracranial hemorrhagic expansion (ICHE). We hypothesized that low molecular weight heparin (LMWH) would not increase risk of ICHE or VTE as compared to unfractionated heparin (UH) in patients with severe TBI. METHODS: Patients ≥ 18 years of age with isolated severe TBI (AIS ≥ 3), admitted to 24 level I and II trauma centers between January 1, 2014 to December 31, 2020 and who received subcutaneous UH and LMWH injections for chemical venous thromboembolism prophylaxis (VTEP) were included. Primary outcomes were VTE and ICHE after VTEP initiation. Secondary outcomes were mortality and neurosurgical interventions. Entropy balancing (EBAL) weighted competing risk or logistic regression models were estimated for all outcomes with chemical VTEP agent as the predictor of interest. RESULTS: 984 patients received chemical VTEP, 482 UH and 502 LMWH. Patients on LMWH more often had pre-existing conditions such as liver disease (UH vs LMWH 1.7 % vs. 4.4 %, p = 0.01), and coagulopathy (UH vs LMWH 0.4 % vs. 4.2 %, p < 0.001). There were no differences in VTE or ICHE after VTEP initiation. There were no differences in neurosurgical interventions performed. There were a total of 29 VTE events (3 %) in the cohort who received VTEP. A Cox proportional hazards model with a random effect for facility demonstrated no statistically significant differences in time to VTE across the two agents (p = 0.44). The LMWH group had a 43 % lower risk of overall ICHE compared to the UH group (HR = 0.57: 95 % CI = 0.32-1.03, p = 0.062), however was not statistically significant. CONCLUSION: In this multi-center analysis, patients who received LMWH had a decreased risk of ICHE, with no differences in VTE, ICHE after VTEP initiation and neurosurgical interventions compared to those who received UH. There were no safety concerns when using LMWH compared to UH. LEVEL OF EVIDENCE: Level III, Therapeutic Care Management.

Effectiveness and Clinical Usefulness of Electronic Agenda-setting in Psychiatric Practices: A South Texas Psychiatric PBRN Study.

There is a general consensus that the doctor-patient interview should be as productive and efficient as possible. This is becoming increasingly difficult in a health care insurance system that demands shorter appointment times. Clinicians must therefore find ways to condense the clinical encounter without sacrificing quality. The purposes of this study were: (1) to facilitate shared decision-making between psychiatrist and patient via pre-visit patient agenda-setting, (2) to evaluate the effectiveness and ease of use of the agenda-setting tool, and (3) to determine patient and clinician satisfaction with the clinical encounter. Patients completed questionnaires to assist in agenda-setting via an electronic tablet while in the waiting area before seeing the psychiatrist. Both patients and psychiatrists then completed post-visit questionnaires to assess their satisfaction with the encounter. We measured patient satisfaction and the extent to which the psychiatrist addressed concerns before and after the visit, as well as ease of use for the patient, psychiatrist satisfaction, and clinical helpfulness to the treating psychiatrist. Additional analyses also indicated that there was a significant increase in patient satisfaction scores, compared with an average of all previous visits, and a significant increase in the number of concerns addressed during the current visit when compared with the average number of previous concerns addressed. Patients reported little difficulty using the tablet. Similarly, psychiatrists reported that the device was helpful in the clinical setting and they expressed high levels of satisfaction with the visit. We hope our work will encourage others to use this agenda-setting tool in their practices to facilitate better patient care.

Early VTE prophylaxis in severe traumatic brain injury: A propensity score weighted EAST multicenter study.

BACKGROUND: Patients with traumatic brain injury (TBI) are at high risk of venous thromboembolism events (VTE). We hypothesized that early chemical VTE prophylaxis initiation (≤24 hours of a stable head CT) in severe TBI would reduce VTE without increasing risk of intracranial hemorrhage expansion (ICHE). METHODS: A retrospective review of adult patients 18 years or older with isolated severe TBI (Abbreviated Injury Scale score, ≥ 3) who were admitted to 24 Level I and Level II trauma centers from January 1, 2014 to December 31 2020 was conducted. Patients were divided into those who did not receive any VTE prophylaxis (NO VTEP), who received VTE prophylaxis ≤24 hours after stable head CT (VTEP ≤24) and who received VTE prophylaxis >24 hours after stable head CT (VTEP>24). Primary outcomes were VTE and ICHE. Covariate balancing propensity score weighting was utilized to balance demographic and clinical characteristics across three groups. Weighted univariate logistic regression models were estimated for VTE and ICHE with patient group as predictor of interest. RESULTS: Of 3,936 patients, 1,784 met inclusion criteria. Incidences of VTE was significantly higher in the VTEP>24 group, with higher incidences of DVT in the group. Higher incidences of ICHE were observed in the VTEP≤24 and VTEP>24 groups. After propensity score weighting, there was a higher risk of VTE in patients in VTEP >24 compared with those in VTEP≤24 (odds ratio, 1.51; 95% confidence interval, 0.69-3.30; p = 0.307), however was not significant. Although, the No VTEP group had decreased odds of having ICHE compared with VTEP≤24 (odds ratio, 0.75; 95% confidence interval, 0.55-1.02, p = 0.070), the result was not statistically significant. CONCLUSION: In this large multi-center analysis, there were no significant differences in VTE based on timing of initiation of VTE prophylaxis. Patients who never received VTE prophylaxis had decreased odds of ICHE. Further evaluation of VTE prophylaxis in larger randomized studies will be necessary for definitive conclusions. LEVEL OF EVIDENCE: Therapeutic Care Management; Level III.

Microparticle RSV Vaccines Presenting the G Protein CX3C Chemokine Motif in the Context of TLR Signaling Induce Protective Th1 Immune Responses and Prevent Pulmonary Eosinophilia Post-Challenge.

Layer-by-layer microparticle (LbL-MP) fabrication was used to produce synthetic vaccines presenting a fusion peptide containing RSV G protein CX3C chemokine motif and a CD8 epitope of the RSV matrix protein 2 (GM2) with or without a covalently linked TLR2 agonist (Pam3.GM2). Immunization of BALB/c mice with either GM2 or Pam3.GM2 LbL-MP in the absence of adjuvant elicited G-specific antibody responses and M2-specific CD8+ T-cell responses. Following challenge with RSV, mice immunized with the GM2 LbL-MP vaccine developed a Th2-biased immune response in the lungs with elevated levels of IL-4, IL-5, IL-13, and eotaxin in the bronchoalveolar lavage (BAL) fluid and a pulmonary influx of eosinophils. By comparison, mice immunized with the Pam3.GM2 LbL-MP vaccine had considerably lower to non-detectable levels of the Th2 cytokines and chemokines and very low numbers of eosinophils in the BAL fluid post-RSV challenge. In addition, mice immunized with the Pam3.GM2 LbL-MP also had higher levels of RSV G-specific IgG2a and IgG2b in the post-challenge BAL fluid compared to those immunized with the GM2 LbL-MP vaccine. While both candidates protected mice from infection following challenge, as evidenced by the reduction or elimination of RSV plaques, the inclusion of the TLR2 agonist yielded a more potent antibody response, greater protection, and a clear shift away from Th2/eosinophil responses. Since the failure of formalin-inactivated RSV (FI-RSV) vaccines tested in the 1960s has been hypothesized to be partly due to the ablation of host TLR engagement by the vaccine and inappropriate Th2 responses upon subsequent viral infection, these findings stress the importance of appropriate engagement of the innate immune response during initial exposure to RSV G CX3C.

Genome Sequences of Two Gordonia rubripertincta Cluster DJ Bacteriophages, Pherobrine and Burley.

Pherobrine and Burley are siphoviruses infecting Gordonia rubripertincta. Pherobrine has a 60,305-bp genome with 89 predicted protein-coding genes, and Burley has a 60,111-bp genome with 90 predicted protein-coding genes. Both phages are assigned to cluster DJ, where they share 78% gene content similarity with each other.

Family metaphors and moral intuitions: how conservatives and liberals narrate their lives.

This research examines life-narrative interviews obtained from 128 highly religious and politically active adults to test differences between political conservatives and liberals on (a) implicit family metaphors (G. Lakoff, 2002) and (b) moral intuitions (J. Haidt & C. Joseph, 2004). Content analysis of 12 key scenes in life stories showed that conservatives, as predicted, tended to depict authority figures as strict enforcers of moral rules and to identify lessons in self-discipline. By contrast, liberals were more likely to identify lessons learned regarding empathy and openness, even though (contrary to prediction) they were no more likely than conservatives to describe nurturant authority figures. Analysis of extended discourse on the development of religious faith and personal morality showed that conservatives emphasized moral intuitions regarding respect for social hierarchy, allegiance to in-groups, and the purity or sanctity of the self, whereas liberals invested more significance in moral intuitions regarding harm and fairness. The results are discussed in terms of the recent upsurge of interest among psychologists in political ideology and the value of using life-narrative methods and concepts to explore how politically active adults attempt to construct meaningful lives.

Psychometric properties of seven self-report measures of posttraumatic stress disorder in college students with mixed civilian trauma exposure.

In this study psychometric properties of seven self-report measures of posttraumatic stress disorder (PTSD) were compared. The seven scales evaluated were the Davidson Trauma Scale (DTS), the PTSD Checklist (PCL), the Posttraumatic Stress Diagnostic Scale (PDS), the Civilian Mississippi Scale (CMS), the Impact of Event Scale-Revised (IES-R), the Penn Inventory for Posttraumatic Stress Disorder (Penn), and the PK scale of the MMPI-2 (PK). Participants were 239 (79 male and 160 female) trauma-exposed undergraduates. All seven measures exhibited good test-retest reliability and internal consistency. The PDS, PCL and DTS demonstrated the best convergent validity; the IES-R, PDS, and PCL demonstrated the best discriminant validity; and the PDS, PCL, and IES-R demonstrated the best diagnostic utility. Overall, results most strongly support the use of the PDS and the PCL for the assessment of PTSD in this population.

Synthesis and characterization of pHLIP® coated gold nanoparticles.

Novel approaches in synthesis of spherical and multispiked gold nanoparticles coated with polyethylene glycol (PEG) and pH Low Insertion Peptide (pHLIP®) were introduced. The presence of a tumor-targeting pHLIP® peptide in the nanoparticle coating enhances the stability of particles in solution and promotes a pH-dependent cellular uptake. The spherical particles were prepared with sodium citrate as a gold reducing agent to form particles of 7.0±2.5 nm in mean metallic core diameter and ∼43 nm in mean hydrodynamic diameter. The particles that were injected into tumors in mice (21 µg of gold) were homogeneously distributed within a tumor mass with no staining of the muscle tissue adjacent to the tumor. Up to 30% of the injected gold dose remained within the tumor one hour post-injection. The multispiked gold nanoparticles with a mean metallic core diameter of 146.0±50.4 nm and a mean hydrodynamic size of ~161 nm were prepared using ascorbic acid as a reducing agent and disk-like bicelles as a template. Only the presence of a soft template, like bicelles, ensured the appearance of spiked nanoparticles with resonance in the near infrared region. The irradiation of spiked gold nanoparticles by an 805 nm laser led to the time- and concentration-dependent increase of temperature. Both pHLIP® and PEG coated gold spherical and multispiked nanoparticles might find application in radiation and thermal therapies of tumors.

c-Cbl-dependent EphA2 protein degradation is induced by ligand binding.

The EphA2 receptor protein tyrosine kinase is overexpressed and functionally altered in a large number of human carcinomas. Despite its elevated levels in cancer, the EphA2 on the surface of malignant cells demonstrates lower levels of ligand binding and tyrosine phosphorylation than the EphA2 on non-transformed epithelial cells. In our present study, we demonstrate that ligand-mediated stimulation causes EphA2 to be internalized and degraded. The mechanism of this response involves ligand-mediated autophosphorylation of EphA2, which promotes an association between EphA2 and the c-Cbl adaptor protein. We also show that c-Cbl promotes stimulation-dependent EphA2 degradation. These findings are important for understanding the causes of EphA2 overexpression in malignant cells and provide a foundation for investigating EphA2 as a potential target for therapeutic intervention.

pHLIP®-mediated delivery of PEGylated liposomes to cancer cells.

We develop a method for pH-dependent fusion between liposomes and cellular membranes using pHLIP® (pH Low Insertion Peptide), which inserts into lipid bilayer of membrane only at low pH. Previously we establish the molecular mechanism of peptide action and show that pHLIP can target acidic diseased tissue. Here we investigate how coating of PEGylated liposomes with pHLIP might affect liposomal uptake by cells. The presence of pHLIP on the surface of PEGylated-liposomes enhanced membrane fusion and lipid exchange in a pH dependent fashion, leading to increase of cellular uptake and payload release, and inhibition of cell proliferation by liposomes containing ceramide. A novel type of pH-sensitive, "fusogenic" pHLIP-liposomes was developed, which could be used to selectively deliver various diagnostic and therapeutic agents to acidic diseased cells.

Rocking to the beat: effects of music and partner's movements on spontaneous interpersonal coordination.

People move to music and coordinate their movements with others spontaneously. Does music enhance spontaneous coordination? We compared the influence of visual information (seeing or not seeing another person) and auditory information (hearing movement or music or hearing no sound) on spontaneous coordination. Pairs of participants were seated side by side in rocking chairs, told a cover story, and asked to rock at a comfortable rate. Both seeing and hearing the other person rock elicited spontaneous coordination, and effects of hearing amplified those of seeing. Coupling with the music was weaker than with the partner, and the music competed with the partner's influence, reducing coordination. Music did, however, function as a kind of social glue: participants who synchronized more with the music felt more connected.

Inflammation in response to n3 fatty acids in a porcine obesity model.

Fatty acids have distinct cellular effects related to inflammation and insulin sensitivity. Dietary saturated fat activates toll-like receptor 4, which in turn can lead to chronic inflammation, insulin resistance, and adipose tissue macrophage infiltration. Conversely, n3 fatty acids are generally antiinflammatory and promote insulin sensitivity, in part via peroxisome proliferator-activated receptor γ. Ossabaw swine are a useful biomedical model of obesity. We fed Ossabaw pigs either a low-fat control diet or a diet containing high-fat palm oil with or without additional n3 fatty acids for 30 wk to investigate the effect of saturated fats and n3 fatty acids on obesity-linked inflammatory markers. The diet did not influence the inflammatory markers C-reactive protein, TNFα, IL6, or IL12. In addition, n3 fatty acids attenuated the increase in inflammatory adipose tissue CD16(-)CD14(+) macrophages induced by high palm oil. High-fat diets with and without n3 fatty acids both induced hyperglycemia without hyperinsulinemia. The high-fat only group but not the high-fat group with n3 fatty acids showed reduced insulin sensitivity in response to insulin challenge. This effect was not mediated by decreased phosphorylation of protein kinase B. Therefore, in obese Ossabaw swine, n3 fatty acids partially attenuate insulin resistance but only marginally change inflammatory status and macrophage phenotype in adipose tissue.

Absence of Tlr2 protects against high-fat diet-induced inflammation and results in greater insulin-stimulated glucose transport in cultured adipocytes.

We have previously shown that toll-like receptor-4 (Tlr4) is involved in obesity-induced inflammation in adipose tissue (AT). However, less is known about the role of Tlr2 in this process. To determine the involvement of this receptor in obesity-induced inflammation, we utilized male Tlr2(-/-) mice that were backcrossed onto a mouse model of diet-induced obesity (DIO). Mice were fed either low-fat control (LFD) or high-fat diet (HFD) ad libitum for 16 weeks. Despite negligible differences in body weight or energy intake, Tlr2(-/-) mice were protected from HFD-induced adiposity as was evident by reduced epididymal fat pad weight and carcass lipid content. Corresponding with these effects was a blunted accumulation of F4/80-positive macrophages in AT of Tlr2(-/-) mice. Furthermore, transcript abundance of proinflammatory mediators, including monocyte chemotactic protein-1 (MCP-1), tumor necrosis factor-α (TNFα) and nitric oxide synthase-2 (NOS2) in AT of Tlr2(-/-) mice, was lower or less responsive to DIO. There were no significant differences in serum markers of insulin sensitivity (data not shown). However, adipocytes derived from stromal vascular cells (SVCs) isolated from AT of Tlr2(-/-) mice had considerably greater basal and insulin-stimulated glucose uptake as compared with those obtained from Tlr2(+/+) mice. Furthermore, the absence of Tlr2(-/-) precluded the induction of insulin resistance by zymosan A (ZymA) but not by palmitate. These data indicate that Tlr2 may be directly involved in HFD-induced inflammation and may also regulate basal and insulin-stimulated glucose uptake in adipocytes.

Tlr-4 deficiency selectively protects against obesity induced by diets high in saturated fat.

Toll-like receptor-4 (Tlr-4), a key pattern recognition receptor involved in innate immune response, is activated by saturated fatty acids (SFAs). To investigate the involvement of this receptor in obesity caused by consumption of diets high in fat, we utilized male Tlr-4-deficient 10ScN mice and 10J controls. Mice were fed either low fat (low-fat control (LFC)), high unsaturated fat (high-fat control (HFC)), or high saturated fat + palmitate (HFP) diets ad libitum for 16 weeks. Relative to the LFC diet, the HFC diet resulted in greater epididymal fat pad weights and adipocyte hypertrophy in both Tlr-4-deficient and normal mice. However, the 10ScN mice were completely protected against the obesigenic effects of the HFP diet. Moreover, macrophage infiltration and monocyte chemotactic protein-1 (MCP-1) transcript abundance were lower in adipose tissue of 10ScN mice fed the HFP diet, and the hyperinsulinemic response was negated. Tlr-4-deficient mice also had markedly lower circulating concentrations of MCP-1 and much less nuclear factor-kappaB (NFkappaB) protein in nuclear extracts prepared from adipose tissue, irrespective of diet. In contrast, Tlr-4 deficiency did not attenuate the induction of tumor necrosis factor-alpha (TNF-alpha) or interleukin-6 (IL-6) expression in adipose tissue. These data indicate that Tlr-4 deficiency selectively protects against the obesigenic effects of SFA and alters obesity-related inflammatory responses in adipose tissue.

Estrogen and Myc negatively regulate expression of the EphA2 tyrosine kinase.

Estrogen receptor and c-Myc are frequently overexpressed during breast cancer progression but are downregulated in many aggressive forms of the disease. High levels of the EphA2 tyrosine kinase are consistently found in the most aggressive breast cancer cells, and EphA2 overexpression can increase metastatic potential. We demonstrate, herein, that estrogen and Myc negatively regulate EphA2 expression in mammary epithelial cells. These data reveal EphA2 as a downstream target of estrogen and Myc and suggest a mechanism by which estrogen and Myc may regulate breast cancer.