Targeting apoptosis for optical imaging of infection

PURPOSE: Infection is ubiquitous and a major cause of morbidity and mortality. The most reliable method for localizing infection requires radiolabeling autologous white blood cells ex vivo. A compound that can be injected directly into a patient and can selectively image infectious foci will eliminate the drawbacks. The resolution of infection is associated with neutrophil apoptosis and necrosis presenting phosphatidylserine (PS) on the neutrophil outer leaflet. Targeting PS with intravenous administration of a PS-specific, near-infrared (NIR) fluorophore will permit localization of infectious foci by optical imaging. METHODS: Bacterial infection and sterile inflammation were induced in separate groups (n = 5) of mice. PS was targeted with a NIR fluorophore, PSVue(®)794 (2.7 pmol). Imaging was performed (ex = 730 nm, em = 830 nm) using Kodak Multispectral FX-Pro system. The contralateral normal thigh served as an individualized control. Confocal microscopy of normal and apoptotic neutrophils and bacteria confirmed PS specificity. RESULTS: Lesions, with a 10-s image acquisition, were unequivocally visible at 5 min post-injection. At 3 h post-injection, the lesion to background intensity ratios in the foci of infection (6.6 ± 0.2) were greater than those in inflammation (3.2 ± 0.5). Image fusions confirmed anatomical locations of the lesions. Confocal microscopy determined the fluorophore specificity for PS. CONCLUSIONS: Targeting PS presented on the outer leaflet of apoptotic or necrotic neutrophils as well as gram-positive microorganism with PS-specific NIR fluorophore provides a sensitive means of imaging infection. Literature indicates that NIR fluorophores can be detected 7-14 cm deep in tissue. This observation together with the excellent results and the continued development of versatile imaging devices could make optical imaging a simple, specific, and rapid modality for imaging infection

Are Canadian General Internal Medicine training program graduates well prepared for their future careers?

BACKGROUND: At a time of increased need and demand for general internists in Canada, the attractiveness of generalist careers (including general internal medicine, GIM) has been falling as evidenced by the low number of residents choosing this specialty. One hypothesis for the lack of interest in a generalist career is lack of comfort with the skills needed to practice after training, and the mismatch between the tertiary care, inpatient training environment and "real life". This project was designed to determine perceived effectiveness of training for 10 years of graduates of Canadian GIM programs to assist in the development of curriculum and objectives for general internists that will meet the needs of graduates and ultimately society. METHODS: Mailed survey designed to explore perceived importance of training for and preparation for various aspects of Canadian GIM practice. After extensive piloting of the survey, including a pilot survey of two universities to improve the questionnaire, all graduates of the 16 universities over the previous ten years were surveyed. RESULTS: Gaps (difference between importance and preparation) were demonstrated in many of the CanMEDS 2000/2005(® )competencies. Medical problems of pregnancy, perioperative care, pain management, chronic care, ambulatory care and community GIM rotations were the medical expert areas with the largest gaps. Exposure to procedural skills was perceived to be lacking. Some procedural skills valued as important for current GIM trainees and performed frequently (example ambulatory ECG interpretation) had low preparation ratings by trainees. Other areas of perceived discrepancy between training and practice included: manager role (set up of an office), health advocate (counseling for prevention, for example smoking cessation), and professional (end of life issues, ethics). CONCLUSION: Graduates of Canadian GIM training programs over the last ten years have identified perceived gaps between training and important areas for practice. They have identified competencies that should be emphasized in Canadian GIM programs. Ongoing review of graduate's perceptions of training programs as it applies to their current practice is important to ensure ongoing appropriateness of training programs. This information will be used to strengthen GIM training programs in Canada

Distinguishing Binders from False Positives by Free Energy Calculations: Fragment Screening Against the Flap Site of HIV Protease

Molecular docking is a powerful tool used in drug discovery and structural biology for predicting the structures of ligand–receptor complexes. However, the accuracy of docking calculations can be limited by factors such as the neglect of protein reorganization in the scoring function; as a result, ligand screening can produce a high rate of false positive hits. Although absolute binding free energy methods still have difficulty in accurately rank-ordering binders, we believe that they can be fruitfully employed to distinguish binders from nonbinders and reduce the false positive rate. Here we study a set of ligands that dock favorably to a newly discovered, potentially allosteric site on the flap of HIV-1 protease. Fragment binding to this site stabilizes a closed form of protease, which could be exploited for the design of allosteric inhibitors. Twenty-three top-ranked protein–ligand complexes from AutoDock were subject to the free energy screening using two methods, the recently developed binding energy analysis method (BEDAM) and the standard double decoupling method (DDM). Free energy calculations correctly identified most of the false positives (≥83%) and recovered all the confirmed binders. The results show a gap averaging ≥3.7 kcal/mol, separating the binders and the false positives. We present a formula that decomposes the binding free energy into contributions from the receptor conformational macrostates, which provides insights into the roles of different binding modes. Our binding free energy component analysis further suggests that improving the treatment for the desolvation penalty associated with the unfulfilled polar groups could reduce the rate of false positive hits in docking. The current study demonstrates that the combination of docking with free energy methods can be very useful for more accurate ligand screening against valuable drug targets

Updated consensus guidelines on the management of Phelan–McDermid syndrome

Phelan–McDermid syndrome (PMS) is a genetic condition caused by SHANK3 haploinsufficiency and characterized by a wide range of neurodevelopmental and systemic manifestations. The first practice parameters for assessment and monitoring in individuals with PMS were published in 2014; recently, knowledge about PMS has grown significantly based on data from longitudinal phenotyping studies and large-scale genotype–phenotype investigations. The objective of these updated clinical management guidelines was to: (1) reflect the latest in knowledge in PMS and (2) provide guidance for clinicians, researchers, and the general community. A taskforce was established with clinical experts in PMS and representatives from the parent community. Experts joined subgroups based on their areas of specialty, including genetics, neurology, neurodevelopment, gastroenterology, primary care, physiatry, nephrology, endocrinology, cardiology, gynecology, and dentistry. Taskforce members convened regularly between 2021 and 2022 and produced specialty-specific guidelines based on iterative feedback and discussion. Taskforce leaders then established consensus within their respective specialty group and harmonized the guidelines. The knowledge gained over the past decade allows for improved guidelines to assess and monitor individuals with PMS. Since there is limited evidence specific to PMS, intervention mostly follows general guidelines for treating individuals with developmental disorders. Significant evidence has been amassed to guide the management of comorbid neuropsychiatric conditions in PMS, albeit mainly from caregiver report and the experience of clinical experts. These updated consensus guidelines on the management of PMS represent an advance for the field and will improve care in the community. Several areas for future research are also highlighted and will contribute to subsequent updates with more refined and specific recommendations as new knowledge accumulates

Supervising the Supervisors—Procedural Training and Supervision in Internal Medicine Residency

At teaching hospitals, bedside procedures (paracentesis, thoracentesis, lumbar puncture, arthrocentesis and central venous catheter insertion) are performed by junior residents and supervised by senior peers. Residents’ perceptions about supervision or how often peer supervision produces unsafe clinical situations are unknown. To examine the experience and practice patterns of residents performing bedside procedures. Cross-sectional e-mail survey of 653 internal medicine (IM) residents at seven California teaching hospitals. Surveys asked questions in three areas: (1) resident experience performing procedures: numbers of procedures performed and whether they received other (e.g., simulator) training; (2) resident comfort performing and supervising procedures; (3) resident reports of their current level of supervision doing procedures, experience with complications as well as perceptions of factors that may have contributed to complications. Three hundred sixty-seven (56%) of the residents responded. Most PGY1 residents had performed fewer than five of any of the procedures, but most PGY-3 residents had performed at least ten by the end of their training. Resident comfort for each procedure increased with the number of procedures performed (p < 0.001). Although residents reported that peer supervision happened often, they also reported high rates of supervising a procedure before feeling comfortable with proper technique. The majority of residents (64%) reported at least one complication and did not feel supervision would have prevented complications, even though many reported complications represented technique- or preparation-related problems. Residents report low levels of comfort and experience with procedures, and frequently report supervising prior to feeling comfortable. Our findings suggest a need to examine best practices for procedural supervision of trainees

Disordered Structural Ensembles of Vasopressin and Oxytocin and Their Mutants

Vasopressin and oxytocin are intrinsically disordered cyclic nonapeptides belonging to a family of neurohypophysial hormones. Although unique in their functions, these peptides differ only by two residues and both feature a tocin ring formed by the disulfide bridge between first and sixth cysteine residues. This sequence and structural similarity are experimentally linked to oxytocin agonism at vasopressin receptors and vasopressin antagonism at oxytocin receptors. Yet single- or double-residue mutations in both peptides have been shown to have drastic impacts on their activities at either receptor, and possibly the ability to bind to their neurophysin carrier protein. In this study we perform molecular dynamics simulations of the unbound native and mutant sequences of the oxytocin and vasopressin hormones to characterize their structural ensembles. We classify the subpopulations of these structural ensembles on the basis of the distributions of radius of gyration and secondary structure and hydrogen-bonding features of the canonical tocin ring and disordered tail region. We then relate the structural changes observed in the unbound form of the different hormone sequences to experimental information about peptide receptor binding, and more indirectly, carrier protein binding affinity, receptor activity, and protease degradation. This study supports the hypothesis that the structural characteristics of the unbound form of an IDP can be used to predict structural or functional preferences of its functional bound form

Evaluating Molecular Mechanical Potentials for Helical Peptides and Proteins

Multiple variants of the AMBER all-atom force field were quantitatively evaluated with respect to their ability to accurately characterize helix-coil equilibria in explicit solvent simulations. Using a global distributed computing network, absolute conformational convergence was achieved for large ensembles of the capped A21 and Fs helical peptides. Further assessment of these AMBER variants was conducted via simulations of a flexible 164-residue five-helix-bundle protein, apolipophorin-III, on the 100 ns timescale. Of the contemporary potentials that had not been assessed previously, the AMBER-99SB force field showed significant helix-destabilizing tendencies, with beta bridge formation occurring in helical peptides, and unfolding of apolipophorin-III occurring on the tens of nanoseconds timescale. The AMBER-03 force field, while showing adequate helical propensities for both peptides and stabilizing apolipophorin-III, (i) predicts an unexpected decrease in helicity with ALA→ARG+ substitution, (ii) lacks experimentally observed 310 helical content, and (iii) deviates strongly from average apolipophorin-III NMR structural properties. As is observed for AMBER-99SB, AMBER-03 significantly overweighs the contribution of extended and polyproline backbone configurations to the conformational equilibrium. In contrast, the AMBER-99φ force field, which was previously shown to best reproduce experimental measurements of the helix-coil transition in model helical peptides, adequately stabilizes apolipophorin-III and yields both an average gyration radius and polar solvent exposed surface area that are in excellent agreement with the NMR ensemble

Unintended pregnancy: magnitude and correlates in six urban sites in Senegal

BACKGROUND: In Senegal, unintended pregnancy has become a growing concern in public health circles. It has often been described through the press as a sensational subject with emphasis on the multiple infanticide cases as a main consequence, especially among young unmarried girls. Less scientific evidence is known on this topic, as fertility issues are rarely discussed within couples. In a context where urbanization is strong, economic insecurity is persistent and the population is globalizing, it is important to assess the magnitude of unintended pregnancy among urban women and to identify its main determinants. METHODS: Data were collected in 2011 from a representative sample of 9614 women aged 15–49 years in six urban sites in Senegal. For this analysis, we include 5769 women who have ever been pregnant or were pregnant at the time of the survey. These women were asked if their last pregnancy in the last two years was ‘wanted ’then’, ‘wanted later’ or ‘not wanted’. Pregnancy was considered as unintended if the woman responded ‘wanted later’ or ‘not wanted’. Descriptive analyses were performed to measure the magnitude of unintended pregnancies, while multinomial logistic regression models were used to identify factors associated with the occurrence of unintended pregnancy. The analyses were performed using Stata version 12. All results were weighted. RESULTS: The results show that 14.3% of ever pregnant women reported having a recent unintended pregnancy. The study demonstrates important distinctions between women whose last pregnancy was intended and those whose last pregnancy was unintended. Indeed, this last group is more likely to be poor, from a young age (< 25 years) and multiparous. In addition, it appears that low participation of married women in decision-making within the couple (management of financial resources) and the lack of discussion on family planning issues are associated with greater experience of unintended pregnancy. CONCLUSION: This study suggests a need to implement more targeted programs that guarantee access to family planning for all women in need. In urban areas that are characterized by economic insecurity, as in Senegal, it is important to consider strategies for promoting communication within couples on fertility issues

Newly established tumourigenic primary human colon cancer cell lines are sensitive to TRAIL-induced apoptosis in vitro and in vivo

Most data on the therapeutic potential of tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) as well as resistance to FAS ligand (FASL) in colorectal cancer have come from in vitro studies using cell lines. To gain a clearer understanding about the susceptibility of patient tumours to TRAIL and FASL, we derived primary human cancer epithelial cells from colon cancer patients. Characterisation of primary cultures PAP60 and MIH55 determined their highly proliferating advantage, transforming capability and tumorigenicity in vitro and in vivo. Although FASL treatment appeared to cause little apoptosis only in the PAP60 primary culture, increased apoptosis independent of p53 was observed in both primary PAP60 and MIH55 and control cell lines Caco-2, HT29 and DLD-1 after treatment with SuperKiller TRAIL. Expression analysis of death receptors (DR) in the original parental tumours, the primary cultures before and after engraftment as well as the mouse xenografts, revealed a significant upregulation of both DR4 and DR5, which correlated to differences in sensitivity of the cells to TRAIL-induced apoptosis. Treating patient tumour xenograft/SCID mouse models with Killer TRAIL in vivo suppressed tumour growth. This is the first demonstration of TRAIL-induced apoptosis in characterised tumorigenic primary human cultures (in vitro) and antitumour activity in xenograft models (in vivo)