Synthesis and characterization of the N-succinyl-l,l-diaminopimelic acid desuccinylase (DapE) alternate substrate analog N,N-dimethyl-l,l-SDAP.

Growing antibiotic resistance by pathogenic bacteria has led to a global crisis. The bacterial enzyme N-succinyl-l,l-diaminopimelic acid desuccinylase (DapE) provides a very attractive target for the discovery of a new class of antibiotics, as it resides exclusively in many pathogenic bacterial strains and is a key enzyme in the lysine biosynthetic pathway. This pathway is responsible for the production of lysine as well as meso-diaminopimelate (m-DAP), both of which are required for peptidoglycan cell-wall synthesis, and lysine for peptide synthesis. The enzyme DapE catalyzes the hydrolysis of N-succinyl-l,l-diaminopimelic acid (l,l-SDAP) to succinate and l,l-diaminopimelic acid (l,l-DAP), and due to its absence in humans, inhibition of DapE avoids mechanism-based side effects. We have executed the asymmetric synthesis of N,N-dimethyl-SDAP, an l,l-SDAP substrate analog and an analog of the synthetic substrate of our previously described DapE assay. Previous modeling studies advocated that N,N-dimethyl-SDAP might function as an inhibitor, however the compound behaves as a substrate, and we have demonstrated the use of N,N-dimethyl-SDAP as the substrate in a modified ninhydrin-based DapE assay. Thermal shift experiments of DapE in the presence of N,N-dimethyl-SDAP are consistent with a melt temperature (Tm) shifted by succinate, the product of enzymatic hydrolysis.

Basic Salt Additives Modulate the Acidic Microenvironment Around In Situ Forming Implants.

There is a growing number of protein drugs, yet their limited oral bioavailability requires that patients receive frequent, high dose injections. In situ forming implants (ISFIs) for controlled release of biotherapeutics have the potential to greatly reduce the injection frequency and improve patient compliance. However, protein release from ISFIs is a challenge due to their proclivity for instability. Specifically, factors such as the acidic microclimate within ISFIs can lead to protein aggregation and denaturation. Basic salts have been shown in PLGA microparticle and microcylinder formulations to significantly reduce protein instability by neutralizing this acidic environment. The overall objective of the study was to demonstrate that basic salts can be used with an ISFI system to neutralize the implant acidification. To this end, the basic salts MgCO3 and Mg(OH)2 were added to a protein-releasing ISFI and the effect on drug release, pH, implant swelling, implant diffusivity, and implant erosion were evaluated. Either salt added at 3 wt% neutralized the acidic environment surrounding the implants, keeping the pH at 6.64 ± 0.03 (MgCO3) and 6.46 ± 0.11 (Mg(OH)2) after 28 day compared to 3.72 ± 0.05 with no salts added. The salts initially increased solution uptake into the implants but delayed implant degradation and erosion. The 3 wt% Mg(OH)2 formulation also showed slightly improved drug release with a lower burst and increased slope. We showed that salt additives can be an effective way to modulate the pH in the ISFI environment, which can improve protein stability and ultimately improve the capacity of ISFIs for delivering pH-sensitive biomolecules. Such a platform represents a low-cost method of improving overall patient compliance and reducing the overall healthcare burden.

A sensitive and drug tolerant assay for detecting anti-AAV9 antibodies using affinity capture elution.

Adeno-associated virus (AAV) based gene therapies are gaining significant momentum as a novel therapeutic modality. However, a yet unsolved concern for using AAV as a vector is the high potential to elicit humoral and cellular responses, which are often exacerbated by pre-existing immunity due to exposure to wild type AAV. Therefore, characterization of pre-existing and treatment emergent anti-AAV antibodies is of great importance to the development of AAV based gene therapies. In this project, a sensitive and drug tolerant total antibody (TAb) assay was developed using recombinant AAV9-GFP (green fluorescent protein) as a surrogate AAV9. The assay format was affinity capture and elution (ACE) with ruthenium labeled AAV9-GFP as detection. Upon evaluation, three commercial anti-AAV9 monoclonal antibodies (clones HI17, HI35, and HL2374) were chosen and mixed at equal concentrations as positive control material. The assay sensitivity was estimated to be 11.2 ng/mL. Drug tolerance was estimated to be 5.4 × 10E10 DRP/mL AAV9-GFP at 100 ng/mL anti-AAV9 antibodies and to be at least 1 × 10E11 DRP/mL at 500 ng/mL and 250 ng/mL anti-AAV9 antibodies. The assay showed desirable specificity and precision. Using this TAb assay, significant pre-existing antibodies were detected from normal human sera.

The role of liquid rheological properties on the injection process of a spring-driven autoinjector.

Accurate injection time prediction is essential in developing spring-driven autoinjector devices since the drug delivery is expected to finish within seconds to bring convenience, reduce the risk for early lift-off, and provide a consistent experience to users. The Carreau model captures the liquid's shear-dependent viscosity measured in our experiments. Thus, a quasi-steady model, which uses the Carreau model to describe the liquid's viscosity, is developed to predict the injection time of spring-driven autoinjectors. Analytical relations between the flow rate and the pressure drop in the needle are also obtained. The Carreau number in the spring-driven autoinjector is greater than one and smaller than a critical value; in this region, using the power-law model to describe the liquid viscosity accurately predicts the injection time, which agrees with the current literature findings. Additionally, a force threshold is identified for the friction force between the plunger and the syringe barrel, beyond which the injection time is infinite. Appreciation of this force threshold can help avoid device stalling and reduce the risk of underdosing. Moreover, the role of liquid's shear-thinning index on the injection time of spring-driven autoinjectors is quantified. Understanding the shear-thinning index allows formulators to experiment with excipients and pH to enhance confidence in drug/device combination product design and integration. Our experimental and theoretical results can help drug product and device developers with integrated product design and improve the patient experience.

Noninvasive characterization of in situ forming implant diffusivity using diffusion-weighted MRI.

In situ forming implants (ISFIs) form a solid drug-eluting depot, releasing drug for an extended period of time after a minimally-invasive injection. Clinical use of ISFIs has been limited because many factors affect drug release kinetics. The aim of this study was to use diffusion-weighted MRI (DWI) to noninvasively quantify spatial-temporal changes in implant diffusivity in situ. ISFIs were formed using poly(lactic-co-glycolic) acid, with a molecular weight of either 15 kDa or 52 kDa, and fluorescein as the mock drug. Drug release, polymer erosion, polymer degradation, and implant diffusivity were analyzed in vitro over 21 days. DWI was also performed in vivo over 5 days. Spatial diffusivity maps of the implant were generated using DWI data. Results showed constant diffusivity at the implant shell ((1.17 ±â€¯0.13) × 10-3 mm2/s) and increasing diffusivity within the interior over time (from (0.268 ±â€¯0.081) × 10-3 mm2/s during day 1 to (1.88 ±â€¯0.04) × 10-3 mm2/s at 14 d), which correlated with increasing porosity of the implant microstructure. Implants formed in vivo followed the same diffusivity trend as those in vitro. This study validates the use of DWI to provide novel functional information about implant behavior through its ability to noninvasively characterize transport properties within the implant both in vitro and in vivo.

Validation of digital pathology imaging for primary histopathological diagnosis.

AIMS: Digital pathology (DP) offers advantages over glass slide microscopy (GS), but data demonstrating a statistically valid equivalent (i.e. non-inferior) performance of DP against GS are required to permit its use in diagnosis. The aim of this study is to provide evidence of non-inferiority. METHODS AND RESULTS: Seventeen pathologists re-reported 3017 cases by DP. Of these, 1009 were re-reported by the same pathologist, and 2008 by a different pathologist. Re-examination of 10 138 scanned slides (2.22 terabytes) produced 72 variances between GS and DP reports, including 21 clinically significant variances. Ground truth lay with GS in 12 cases and with DP in nine cases. These results are within the 95% confidence interval for existing intraobserver and interobserver variability, proving that DP is non-inferior to GS. In three cases, the digital platform was deemed to be responsible for the variance, including a gastric biopsy, where Helicobacter pylori only became visible on slides scanned at the ×60 setting, and a bronchial biopsy and penile biopsy, where dysplasia was reported on DP but was not present on GS. CONCLUSIONS: This is one of the largest studies proving that DP is equivalent to GS for the diagnosis of histopathology specimens. Error rates are similar in both platforms, although some problems e.g. detection of bacteria, are predictable.

HyMaP: A hybrid magnitude-phase approach to unsupervised segmentation of tumor areas in breast cancer histology images.

BACKGROUND: Segmentation of areas containing tumor cells in standard H&E histopathology images of breast (and several other tissues) is a key task for computer-assisted assessment and grading of histopathology slides. Good segmentation of tumor regions is also vital for automated scoring of immunohistochemical stained slides to restrict the scoring or analysis to areas containing tumor cells only and avoid potentially misleading results from analysis of stromal regions. Furthermore, detection of mitotic cells is critical for calculating key measures such as mitotic index; a key criteria for grading several types of cancers including breast cancer. We show that tumor segmentation can allow detection and quantification of mitotic cells from the standard H&E slides with a high degree of accuracy without need for special stains, in turn making the whole process more cost-effective. METHOD: BASED ON THE TISSUE MORPHOLOGY, BREAST HISTOLOGY IMAGE CONTENTS CAN BE DIVIDED INTO FOUR REGIONS: Tumor, Hypocellular Stroma (HypoCS), Hypercellular Stroma (HyperCS), and tissue fat (Background). Background is removed during the preprocessing stage on the basis of color thresholding, while HypoCS and HyperCS regions are segmented by calculating features using magnitude and phase spectra in the frequency domain, respectively, and performing unsupervised segmentation on these features. RESULTS: All images in the database were hand segmented by two expert pathologists. The algorithms considered here are evaluated on three pixel-wise accuracy measures: precision, recall, and F1-Score. The segmentation results obtained by combining HypoCS and HyperCS yield high F1-Score of 0.86 and 0.89 with re-spect to the ground truth. CONCLUSIONS: In this paper, we show that segmentation of breast histopathology image into hypocellular stroma and hypercellular stroma can be achieved using magnitude and phase spectra in the frequency domain. The segmentation leads to demarcation of tumor margins leading to improved accuracy of mitotic cell detection.

Patient and clinician ethical perspectives on the 2006 Centers for Disease Control and prevention HIV testing methods.

OBJECTIVES: CDC 2006 recommendations for new HIV testing methods in U.S. health-care settings (opt-out approach, general medical consent, and optional prevention counseling) have been the subject of a public ethical debate. Ethical concerns might limit their implementation and affect expanded HIV screening efforts. We compared clinicians' and patients' perspectives on the ethical concerns raised about, justifications provided in support of, and preferences for the 2006 CDC-recommended HIV testing methods for the U.S. health-care setting, in contrast with the 2001 CDC-recommended HIV testing methods (opt-in approach, specific written consent, and mandatory prevention counseling). METHODS: We conducted a non-inferiority trial and survey of 249 clinicians and random samples of 1,013 of their patients at three emergency departments and three ambulatory care clinics at university-affiliated hospitals in Rhode Island from June to December 2007. RESULTS: Clinicians found the 2006 CDC HIV testing methods to be more ethically concerning than the 2001 testing methods (i.e., ethically inferior), while patients had few ethical concerns. In regard to ethical justifications cited for the 2006 CDC HIV testing methods, clinicians were more supportive of the ethical justifications cited for using an opt-out approach and general medical consent, while patients were more supportive of the justifications for optional HIV prevention counseling. Clinicians showed a relatively greater preference for the opt-out approach and use of general medical consent, while patients had a relatively greater preference for optional HIV prevention counseling. CONCLUSIONS: Clinicians and their patients hold divergent ethical perspectives on CDC's 2006 HIV testing methods. The results indicate an opportunity to review not only these but also future HIV testing recommendations, as well as how they are presented for implementation.

Enhanced biomedical scientist cut-up role in colonic cancer reporting.

AIMS: To extend the biomedical scientist (BMS) cut-up role to include gastrointestinal category D colorectal cancer resection specimens, and to address issues of quality and safety by presenting performance data from the first 50 BMS cut-up specimens in comparison with national guidelines and pathologist performance over the same timeframe. METHODS: Close mentoring and consultant supervision was carried out for every case with adherence to standard operating procedures and following colorectal cancer dataset guidelines as published by the Royal College of Pathologists. Performance targets were audited including anticipated spread of Dukes' stage, targets for mean lymph node harvest, percentage extramural vascular invasion and serosal involvement, and mean tumour blocks sampled. Histological pre-reporting of 20 cases was encouraged, and time spent by BMS and consultant at all stages of specimen reporting was noted. RESULTS: Performance targets were all exceeded by the BMS and compared favourably with pathologist performance. A measure of consultant cut-up and histology reporting time saved was identified. CONCLUSIONS: Benefits of extending the BMS role to category D specimens may include BMS professional advancement, efficient use of consultant time and the development of a team approach to cancer reporting. The achievement of colorectal cancer performance targets and favourable comparison with pathologist performance implies there was no perceived detrimental effect on quality or safety and thus patient management.

Barriers and facilitators to HIV testing in primary care among health care providers.

PURPOSE: Human immunodeficiency virus (HIV) is a preventable disease that can have improved outcomes with early diagnosis and treatment. The CDC recommends that HIV testing be incorporated into clinical settings as part of routine medical care. METHODS: Individual, open-ended interviews were conducted with primary care providers and administrators to obtain their views regarding the meaning of routine HIV testing and the barriers and facilitators to implementing routine HIV testing in their respective practices. RESULTS: Most respondents supported routine HIV testing, although their definitions of routine varied. Barriers for providers included time and financial constraints to appropriately conduct HIV counseling and testing and inadequate HIV education and training. Facilitators for implementing routine HIV testing included patients' feelings of empowerment and reduced HIV stigma. CONCLUSIONS: The implementation of routine HIV testing in primary care practices appears to be an acceptable public health intervention. Next steps should include efforts to standardize the definition of routine HIV testing and working with primary care settings to better understand and reduce barriers to routine testing.

Racial distribution of patient population and family physician endorsed importance of screening patients for inherited predisposition to cancer.

Uptake of genetic testing is higher among racial majority versus minority patients for reasons that remain unclear. Primary care physicians represent the front line of screening for inherited cancer risk. We surveyed family physicians enrolled in the Massachusetts Practice Based Research Network to assess whether their attitudes about cancer-predictive genetic testing related to the race of their patients. Among the 65 physicians who responded (91.5% response rate), those whose practices had higher proportions of White patients were more likely to strongly endorse the value of screening for inherited cancer risk (ORadj 3.18, 95% CI 1.05, 9.66). These findings, though limited by use of a small convenience sample, suggest that clinical attention to screening for genetic cancer risk is greater in practices serving fewer racial minority patients. More research is necessary to confirm these findings and to determine whether these factors affect disparities in genetic testing and health outcomes.

Lessons learned from a training collaboration between an Ivy League institution and a historically Black university.

The Miriam Hospital, Brown Medical School, and Jackson State University developed a joint training program for predoctoral, Black psychology students under the auspices of a training grant funded by the National Institutes of Health. The students in the program at Jackson State University had unlimited access to the clinical research resources and mentoring expertise at Brown Medical School. This innovative program began in 2001 and addresses the need for Black leaders in clinical research and academia who will focus on HIV and other infections that disproportionately affect the Black community. This collaboration has served as a bridge between an Ivy League institution and a historically Black university for training in clinical research to develop successful minority academicians.

Racial/ethnic sexual health disparities among incarcerated women.

Rates of sexually transmitted infections (STIs) in women in U.S. corrections facilities are higher than rates in community samples. Research that combines behavioral correlates of STI with STI history by race/ethnicity has not been done in incarcerated women. The purpose of this study was to compare by race/ethnicity self-reported sexual risk behaviors with self-reported history of STI in an incarcerated sample. An interviewer administered a questionnaire to 428 incarcerated women. Blacks were more likely to report consistent condom use in the three months prior to incarceration (47% vs. 28%, p < 0.05), and Hispanics were less likely to report sex work than were whites (16% vs. 39%, p < 0.05). Whites were more likely than blacks to report having had an unplanned pregnancy (88% vs. 67%, p < 0.05). Despite having lower self-reported risk on several measures, Blacks were more likely to report history of STI (65% vs. 40%, p < 0.05). The correctional setting is an opportune place to better understand and address the complex issue of sexual health disparities.

Self-rated breast cancer risk among women reporting a first-degree family history of breast cancer on office screening questionnaires in routine medical care: the role of physician-delivered risk feedback.

PURPOSE: We investigated whether risk-related feedback delivered by one's primary care physician is associated with self-ratings of risk among women found to have a first-degree family history of breast cancer on office screening questionnaires. DESIGN: Mailed survey of women registered with the Cancer Genetics Network having a first-degree family history of breast cancer. Eligibility: Completion of primary care-based family history screening within the past year. INDEPENDENT VARIABLE: presence of physician feedback about breast cancer risk. Dependent variable: self-rated breast cancer risk. Modifying variable: trust in one's doctor. RESULTS: Three hundred one women met eligibility criteria (73% minimum response rate); feedback was associated with rating one's risk to be "high" in both crude and multivariate analysis. (ORadj = 2.38; 95% CI = 1.30, 4.38). Higher levels of trust in the physician were associated in a dose-dependent fashion with the strength of association between feedback and self-rating one's risk to be high. CONCLUSIONS: Physician feedback following the identification of a first-degree family history of breast cancer appears to influence whether or not women categorize themselves to be at high risk and trust is an important modifier of this association.

Routine testing for HIV in the United States: the intersection between recommendations and practice.

The purpose of this study was to examine the current practices of family practice (FP) providers and their allied staff with regard to routine HIV testing in Rhode Island (RI) and Mississippi (MS). Anonymous experimenter-derived surveys were mailed to both groups of providers in 2002. The questionnaire contained five questions about their current practices and attitudes toward HIV testing as well as patient demographics. Five hundred twenty-one questionnaires were sent to American Academy of Family Practitioners (AAFP) members in RI and MS and to FPs with listings in the phone book in RI. The response rate was 52% in RI and 41% in MS. The vast majority of providers (93%) tested their high-risk patients for HIV, but less tested pregnant (57%) and other sexually active (37%) patients. The FPs in this survey wanted HIV testing to be done in the primary care setting, yet only 7% recommended HIV testing to their sexually active patients aged 18-50 in the previous year. In order not to stigmatize any specific risk group, nor to miss any patients who are unable to be identified as being "at high risk," routine testing in the primary care setting should be encouraged.

Acceptance of HIV testing among African-American college students at a historically black university in the south.

PURPOSE: Routine HIV testing on college campuses has the potential to increase students' awareness of their HIV status. Testing targeted only at persons reporting HIV risk behaviors will not identify infected persons who may deny or be unaware of their risk. Thus, this study sought to investigate the acceptability of rapid HIV testing among African-American college students in a nontraditional setting on a historically black college/university (HBCU) campus. METHODS: A cross-sectional survey on risk behaviors, barriers to testing, and HIV testing history was administered to 161 African-American college students at an HBCU. All approached students (both those participating and not) were offered free HIV rapid testing. RESULTS: Eighty-one African-American college students consented to be tested for HIV and all tested negative. Results of the questionnaire indicated that African-American college students engage in risky sexual behaviors (such as unprotected sex) yet perceive themselves as at little or no risk. College students who reported past HIV testing often did so in conjunction with routine exams, such as annual pap smears, rather than specifically seeking HIV testing. CONCLUSIONS: Routine HIV testing on college campuses may be an important public health initiative in reducing the spread of HIV. Specifically, this strategy may provide a model for student access to HIV testing, particularly males and other students who may be less likely to seek HIV testing at traditional medical settings. These data supports expansion of routine testing programs directed at African-American college students.

It is time to implement routine, not risk-based, HIV testing.

Approximately one-quarter of a million persons in the United States who are infected with human immunodeficiency virus (HIV) do not know it. To decrease the number of such persons, primary care providers should make HIV testing a routine component of health care. HIV testing should also be offered routinely in other settings, such as emergency departments, jails, and substance abuse treatment centers. Currently, the Centers for Disease Control and Prevention and the Infectious Diseases Society of America recommend routine HIV testing only in settings where the prevalence of HIV infection is > or =1%; in settings where the prevalence of HIV infection is <1%, testing should be based on risk assessment. Because of the impracticality of strategies for testing that are based on estimates of prevalence, and because of the inaccuracy of risk assessment, we propose that HIV testing be routinely offered to any person who is sexually active. As an adjunct to the implementation of routine testing programs, counseling practices need to be streamlined, and rapid HIV testing needs to be implemented in the appropriate settings.

Racial/ethnic attitudes towards HIV testing in the primary care setting.

PURPOSE: To determine attitudes of patients towards routine HIV testing in the primary care setting. METHODS: Cross-sectional survey on the risk factors, beliefs, attitudes and knowledge of HIV/AIDS administered to 101 individuals present in urban primary care clinics in Providence, RI. RESULTS: Previous HIV testing was done most frequently for those respondents requiring prenatal services and for those who were curious about their HIV status. Patients' perceptions of their personal risk for HIV infection and their self-identified risk factors were frequently discordant. Patients wanted to be tested routinely for HIV by their primary care providers, even when they did not feel that they were at high risk for HIV acquisition. CONCLUSIONS: Patients in this study clearly indicated their desire to be tested for HIV routinely by their primary care providers. Routine HIV testing is a reasonable option to identify HIV infections in the primary care setting, as it is nondiscriminatory, allows increased awareness of actual risk for infection, and provides an opportunity for earlier detection of HIV.