Targeting Diseased Tissues by pHLIP Insertion at Low Cell Surface pH

The discovery of the pH Low Insertion Peptides (pHLIPs®) provides an opportunity to develop imaging and drug delivery agents targeting extracellular acidity. Extracellular acidity is associated with many pathological states, such as those in cancer, ischemic stroke, neurotrauma, infection, lacerations, and others. The metabolism of cells in injured or diseased tissues often results in the acidification of the extracellular environment, so acidosis might be useful as a general marker for the imaging and treatment of diseased states if an effective targeting method can be developed. The molecular mechanism of a pHLIP peptide is based on pH-dependent membrane-associated folding. pHLIPs, being moderately hydrophobic peptides, have high affinities for cellular membranes at normal pH, but fold and insert across membranes at low pH, allowing them to sense pH at the surfaces of cells in diseased tissues, where it is the lowest. Here we discuss the main principles of pHLIP interactions with membrane lipid bilayers at neutral and low pHs, the possibility of tuning the folding and insertion pH by peptide sequence variation, and potential applications of pHLIPs for imaging, therapy and image-guided interventions

pH-sensitive membrane peptides (pHLIPs) as a novel class of delivery agents

Here we review a novel class of delivery vehicles based on pH-sensitive, moderately polar membrane peptides, which we call pH (Low) Insertion Peptides (pHLIPs), that target cells located in the acidic environment found in many diseased tissues, including tumours. Acidity targeting by pHLIPs is achieved as a result of helix formation and transmembrane insertion. In contrast to the earlier technologies based on cell-penetrating peptides, pHLIPs act as monomeric membrane-inserting peptides that translocate one terminus across a membrane into the cytoplasm, while the other terminus remains in the extracellular space, locating the peptide in the membrane lipid bilayer. Therefore pHLIP has a dual delivery capability: it can tether cargo molecules or nanoparticles to the surfaces of cells in diseased tissues and/or it can move a cell-impermeable cargo molecule across the membrane into the cytoplasm. The source of energy for moving polar molecules attached to pHLIP through the hydrophobic layer of a membrane bilayer is the membrane-associated folding of the polypeptide. A drop in pH leads to the protonation of negatively charged residues (Asp or Glu), which enhances peptide hydrophobicity, increasing the affinity of the peptide for the lipid bilayer and triggering peptide folding and subsequent membrane insertion. The process is accompanied by the release of energy that can be utilized to move cell-impermeable cargo across a membrane. That the mechanism is now understood, and that targeting of tumours in mice has been shown, suggest a number of future applications of the pHLIP technology in the diagnosis and treatment of disease

Chromatinized templates reveal the requirement for the LEDGF/p75 PWWP domain during HIV-1 integration in vitro

Integration is an essential step in the retroviral lifecycle, and the lentiviral integrase binding protein lens epithelium-derived growth factor (LEDGF)/p75 plays a crucial role during human immunodeficiency virus type 1 (HIV-1) cDNA integration. In vitro, LEDGF/p75 stimulates HIV-1 integrase activity into naked target DNAs. Here, we demonstrate that this chromatin-associated protein also stimulates HIV-1 integration into reconstituted polynucleosome templates. Activation of integration depended on the LEDGF/p75-integrase interaction with either type of template. A differential requirement for the dominant DNA and chromatin-binding elements of LEDGF/p75 was however observed when using naked DNA versus polynucleosomes. With naked DNA, the complete removal of these N-terminal elements was required to abate cofactor function. With polynucleosomes, activation mainly depended on the PWWP domain, and to a lesser extent on nearby AT-hook DNA-binding motifs. GST pull-down assays furthermore revealed a role for the PWWP domain in binding to nucleosomes. These results are completely consistent with recent ex vivo studies that characterized the PWWP and integrase-binding domains of LEDGF/p75 as crucial for restoring HIV-1 infection to LEDGF-depleted cells. Our studies therefore establish novel in vitro conditions, highlighting chromatinized DNA as target acceptor templates, for physiologically relevant studies of LEDGF/p75 in lentiviral cDNA integration

Health status among black African-born women in Kansas City: a preliminary assessment

A grant from the One-University Open Access Fund at the University of Kansas was used to defray the author’s publication fees in this Open Access journal. The Open Access Fund, administered by librarians from the KU, KU Law, and KUMC libraries, is made possible by contributions from the offices of KU Provost, KU Vice Chancellor for Research & Graduate Studies, and KUMC Vice Chancellor for Research. For more information about the Open Access Fund, please see http://library.kumc.edu/authors-fund.xml.Background Health information and statistics for Black foreign-born women in the United States are under-reported or not available. Black foreign-born women typically are classified under the general category of African American, ignoring the heterogeneity that exists in the United States Black population. It is important to identify health issues and behaviors of African-born women to effectively address health disparities. Methods Black African-born women (N = 29), 20 years or older completed a survey about general and women’s health, health history, acculturation, lifestyle, social and health challenges, beliefs about breast cancer. Data were analyzed using SPSS 14.0 software. Categorical variables were summarized with frequencies and percentages and continuous variables were summarized with means and standard variation. A Likert scale (strongly agree, agree, disagree, and strongly disagree) was used to assess beliefs about breast cancer. Results Most (71.4 %) participants had a high school education or more, 70 % were employed, and 50 % had health insurance. Two-thirds received health care from primary care doctors, 20.7 % from health departments, and 39.3 % got annual checkups. Lack of jobs, healthcare cost, language barrier, discrimination, and child care were the top social issues faced by participants. High blood pressure, obesity, oral health, HIV/AIDS, and diabetes were indicated as the most common health problems. The percent of participants (60 %) that had not had a mammogram within the previous 2 years was more than the state average (24 %) for women 40 years and older reported by the Kansas Department of Health and Environment. The percent of participants (40 %) that had a mammogram within the previous 2 years was lower than the national average (73.2 %) for African American women. Conclusions Study provides a snapshot of social concerns and health issues in an African population residing in Midwestern United States. Understanding the socio-cultural characteristics of this population is necessary to address health disparities

Tuning the insertion properties of pHLIP

The pH (low) insertion peptide (pHLIP) has exceptional characteristics: at neutral pH it is an unstructured monomer in solution or when bound to lipid bilayer surfaces, and it inserts across a lipid bilayer as a monomeric alpha-helix at acidic pH. The peptide targets acidic tissues in vivo and may be useful in cancer biology for delivery of imaging or therapeutic molecules to acidic tumors. To find ways to vary its useful properties, we have designed and analyzed pHLIP sequence variants. We find that each of the Asp residues in the transmembrane segment is critical for solubility and pH-dependent membrane insertion of the peptide. Changing both of the Asp residues in the transmembrane segment to Glu, inserting an additional Asp into the transmembrane segment, or replacing either of the Asp residues with Ala leads to aggregation and/or loss of pH-dependent membrane insertion of the peptide. However, variants with either of the Asp residues changed to Glu remained soluble in an aqueous environment and inserted into the membrane at acidic pH with a higher pKapp of membrane insertion

Roles of Carboxyl Groups in the Transmembrane Insertion of Peptides

We have used pHLIP® [pH (low) insertion peptide] to study the roles of carboxyl groups in transmembrane (TM) peptide insertion. pHLIP binds to the surface of a lipid bilayer as a disordered peptide at neutral pH; when the pH is lowered, it inserts across the membrane to form a TM helix. Peptide insertion is reversed when the pH is raised above the characteristic pKa (6.0). A key event that facilitates membrane insertion is the protonation of aspartic acid (Asp) and/or glutamic acid (Glu) residues, since their negatively charged side chains hinder membrane insertion at neutral pH. In order to gain mechanistic understanding, we studied the membrane insertion and exit of a series of pHLIP variants where the four Asp residues were sequentially mutated to nonacidic residues, including histidine (His). Our results show that the presence of His residues does not prevent the pH-dependent peptide membrane insertion at ∼ pH 4 driven by the protonation of carboxyl groups at the inserting end of the peptide. A further pH drop leads to the protonation of His residues in the TM part of the peptide, which induces peptide exit from the bilayer. We also find that the number of ionizable residues that undergo a change in protonation during membrane insertion correlates with the pH-dependent insertion into the lipid bilayer and exit from the lipid bilayer, and that cooperativity increases with their number. We expect that our understanding will be used to improve the targeting of acidic diseased tissue by pHLIP

A computerized intervention to promote colorectal cancer screening for underserved populations: Theoretical background and algorithm development

Objective The aim of this exploratory study was to assess factors deemed by patients as “important” as they planned and considered undergoing colorectal cancer (CRC) screening, and to use this data to design a computer-delivered intervention to promote screening. Methods Fifty participants 50 years or older, not up-to-date with current recommended CRC screening guidelines, were recruited from a primary care clinic. A semi-structured interview focused on aspects of preparing for colorectal cancer screening was administered; after transcription, researchers used triangulation and consensus to identify relevant themes and concepts. Results Four main themes were identified that dealt with issues important for both FOBT and colonoscopy planning: personal concerns, reminders, communication with healthcare providers and obtaining test results. FOBT specific themes included: sample collection and return. For colonoscopy screening, themes included: scheduling, intervention questions, colonoscopy preparation, and transportation. These can be classified as barrier, process and accessory themes. The developed computer-administered implementation intentions algorithm addressed all the identified concerns in a planned and sequential manner, in order to facilitate planning for CRC screening. Conclusions The results of this study suggest that appropriate reminders, explanations of procedures, and patient understanding of temporary life disruptions, help patients develop and accept a detailed screening plan

Dental-related Emergency Department Visits and Community Dental Care Resources for Emergency Room Patients

BACKGROUND: The number and cost of dental-related visits to Emergency Departments (ED) is a significant issue nationwide. A better understanding of the treatment provided to ED patients presenting with dental complaints and community dental resources is needed. METHODS: A three-tiered approach included: 1) a 12-month retrospective chart review for dental-related ICD-9 visit codes at an urban academic ED in Kansas City; 2) surveys of 30 providers at the same ED regarding the dental patient process and treatment; and 3) telephone surveys of 16 Kansas City area safety net clinics regarding service access. RESULTS: Out of 49,276 ED visits, 676 were related to dental conditions (70 were repeat dental ED visits). Most patients were female (54%), white (45%), age 20-39 (65%), and self-pay (56%). The most prevalent codes utilized were dental disorder not otherwise specified (NOS; 57%), periapical abscess (22%), and dental caries NOS (15%). Nearly all providers (97%) felt comfortable seeing patients with dental complaints. Chart review indicated that patients received a dental screen/exam during 80% of the encounters, with medication provided to 90% of the patients. Over two-thirds of the providers (N = 23/30) regularly prescribed antibiotics and pain medications for their ED dental patients. ED providers performed dental procedures in 63% of the patient cases. The most common procedures included dental blocks (N = 16 providers) and incision and drainage (N = 4 providers). Only two of the 16 safety net clinics provided comprehensive dental care, almost all (94%) clinics required patients to call to schedule an appointment, and there was a two to six month waiting period for 31% of the clinics. CONCLUSION: The limited scope of dental treatment in the ED, coupled with poor availability of safety-net dental resources, may result in dental exacerbations and suboptimal patient clinical outcomes. The enhancement of safety-net dental service accessibility is crucial to reducing dental ED visits and improving dental health, particularly among low-income, self-pay populations

Population-based assessment of cardiovascular complications of rheumatic heart disease in Fiji: a record-linkage analysis

Objective: To determine population-based rates of nonfatal complications of rheumatic heart disease (RHD). Design: Retrospective cohort study based on multiple sources of routine clinical and administrative data amalgamated by probabilistic record-linkage. Setting: Fiji, an upper-middle-income country, where most of the population has access to government-funded health care services. Participants: National cohort of 2,116 patients with clinically apparent RHD aged 5-69 years during 2008-2012. Primary and secondary outcome measures: The primary outcome was hospitalisation for any of heart failure, atrial fibrillation, ischaemic stroke and infective endocarditis. Secondary outcomes were first hospitalisation for each of the complications individually in the national cohort as well as in hospital (n=1300) and maternity (n=210) subsets. Information on outcomes was obtained from discharge diagnoses coded in the hospital patient information system. Population-based rates were obtained using relative survival methods with census data as the denominator. Results: Among 2,116 patients in the national cohort (median age, 23.3 years; 57.7% female), 546 (25.8%) were hospitalised for an RHD complication, a substantial proportion of all cardiovascular admissions in the country during this period in those aged 0–40 years (heart failure, 210/454, 46.2%; ischaemic stroke 31/134, 23.3%). Absolute numbers of RHD complications peaked during the third decade of life with higher population-based rates in women compared to men (incidence rate ratio 1.4, 95% CI 1.3–1.6, P<0.001). Hospitalisation for any RHD complication was associated with substantially increased risk of death (hazard ratio, 5.4, 95% CI, 3.4–8.8, P<0.001), especially after the onset of heart failure (hazard ratio, 6.6, 95% CI, 4.8–9.1, P<0.001). Conclusions: Our study defines the burden of RHD-attributable morbidity in the general population of Fiji, potentially reflecting the situation in low- and middle-income countries worldwide. Hospitalisation for an RHD complication is associated with markedly increased risk of death, re- emphasising the importance of effective early prevention