Characterization of a novel method for the production of single-span membrane proteins in Escherichia coli.

The large-scale production and isolation of recombinant protein is a central element of the biotechnology industry and many of the products have proved extremely beneficial for therapeutic medicine. Escherichia coli is the microorganism of choice for the expression of heterologous proteins for therapeutic application, and a range of high-value proteins have been targeted to the periplasm using the well characterized Sec protein export pathway. More recently, the ability of the second mainstream protein export system, the twin-arginine translocase, to transport fully-folded proteins into the periplasm of not only E. coli, but also other Gram-negative bacteria, has captured the interest of the biotechnology industry. In this study, we have used a novel approach to block the export of a heterologous Tat substrate in the later stages of the export process, and thereby generate a single-span membrane protein with the soluble domain positioned on the periplasmic side of the inner membrane. Biochemical and immuno-electron microscopy approaches were used to investigate the export of human growth hormone by the twin-arginine translocase, and the generation of a single-span membrane-embedded variant. This is the first time that a bonafide biotechnologically relevant protein has been exported by this machinery and visualized directly in this manner. The data presented here demonstrate a novel method for the production of single-span membrane proteins in E. coli.

The Bacillus subtilis TatAdCd system exhibits an extreme level of substrate selectivity.

The Tat system preferentially transports correctly folded proteins across the bacterial membrane although little is known of the proofreading mechanism. Most research has focused on TatABC systems from Gram-negative bacteria, especially Escherichia coli, and much less is known of the TatAC-type systems from Gram-positive organisms. We have previously shown that the Bacillus subtilis TatAdCd system is functional in an E. coli tat null background and able to transport TorA-GFP and native TorA (TMAO reductase); here, we examined its ability to transport other proteins bearing a TorA signal sequence. We show that whereas E. coli TatABC transports a wide range of biotherapeutics including human growth hormone, interferon α2b, a VH domain protein and 2 different scFvs, TatAdCd transports the scFvs but completely rejects the other proteins. The system also rejects two native E. coli substrates, NrfC and FhuD. Moreover, we have shown that TatABC will transport a wide range of folded scFv variants with the surface altered to incorporate multiple salt bridges, charged residues (5 glutamate, lysine or arginine), or hydrophobic residues (up to 6 leucines). In contrast, TatAdCd completely rejects many of these variants including those with 5 or 6 added Leu residues. The combined data show that the TatABC and TatAdCd systems have very different substrate selectivities, with the TatAdCd system displaying an extreme level of selectivity when compared to the E. coli system. The data also provide a preliminary suggestion that TatAdCd may not tolerate substrates that contain surface domains with a level of hydrophobicity above a certain threshold.

'Something in the way she moves': The functional significance of flexibility in the multiple roles of protein disulfide isomerase (PDI).

Protein disulfide isomerase (PDI) has diverse functions in the endoplasmic reticulum as catalyst of redox transfer, disulfide isomerization and oxidative protein folding, as molecular chaperone and in multi-subunit complexes. It interacts with an extraordinarily wide range of substrate and partner proteins, but there is only limited structural information on these interactions. Extensive evidence on the flexibility of PDI in solution is not matched by any detailed picture of the scope of its motion. A new rapid method for simulating the motion of large proteins provides detailed molecular trajectories for PDI demonstrating extensive changes in the relative orientation of its four domains, great variation in the distances between key sites and internal motion within the core ligand-binding domain. The review shows that these simulations are consistent with experimental evidence and provide insight into the functional capabilities conferred by the extensive flexible motion of PDI.

Efficient export of human growth hormone, interferon α2b and antibody fragments to the periplasm by the Escherichia coli Tat pathway in the absence of prior disulfide bond formation.

Numerous therapeutic proteins are expressed in Escherichia coli and targeted to the periplasm in order to facilitate purification and enable disulfide bond formation. Export is normally achieved by the Sec pathway, which transports proteins through the plasma membrane in a reduced, unfolded state. The Tat pathway is a promising alternative means of export, because it preferentially exports correctly folded proteins; however, the reducing cytoplasm of standard strains has been predicted to preclude export by Tat of proteins that contain disulfide bonds in the native state because, in the reduced state, they are sensed as misfolded and rejected. Here, we have tested a series of disulfide-bond containing biopharmaceuticals for export by the Tat pathway in CyDisCo strains that do enable disulfide bond formation in the cytoplasm. We show that interferon α2b, human growth hormone (hGH) and two antibody fragments are exported with high efficiency; surprisingly, however, they are efficiently exported even in the absence of cytoplasmic disulfide formation. The exported proteins acquire disulfide bonds in the periplasm, indicating that the normal disulfide oxidation machinery is able to act on the proteins. Tat-dependent export of hGH proceeds even when the disulfide bonds are removed by substitution of the Cys residues involved, suggesting that these substrates adopt tertiary structures that are accepted as fully-folded by the Tat machinery.

Pathological and Biochemical Outcomes among African-American and Caucasian Men with Low Risk Prostate Cancer in the SEARCH Database: Implications for Active Surveillance Candidacy.

PURPOSE: Racial disparities in the incidence and risk profile of prostate cancer at diagnosis among African-American men are well reported. However, it remains unclear whether African-American race is independently associated with adverse outcomes in men with clinical low risk disease. MATERIALS AND METHODS: We retrospectively analyzed the records of 895 men in the SEARCH (Shared Equal Access Regional Cancer Hospital) database in whom clinical low risk prostate cancer was treated with radical prostatectomy. Associations of African-American and Caucasian race with pathological biochemical recurrence outcomes were examined using chi-square, logistic regression, log rank and Cox proportional hazards analyses. RESULTS: We identified 355 African-American and 540 Caucasian men with low risk tumors in the SEARCH cohort who were followed a median of 6.3 years. Following adjustment for relevant covariates African-American race was not significantly associated with pathological upgrading (OR 1.33, p = 0.12), major upgrading (OR 0.58, p = 0.10), up-staging (OR 1.09, p = 0.73) or positive surgical margins (OR 1.04, p = 0.81). Five-year recurrence-free survival rates were 73.4% in African-American men and 78.4% in Caucasian men (log rank p = 0.18). In a Cox proportional hazards analysis model African-American race was not significantly associated with biochemical recurrence (HR 1.11, p = 0.52). CONCLUSIONS: In a cohort of patients at clinical low risk who were treated with prostatectomy in an equal access health system with a high representation of African-American men we observed no significant differences in the rates of pathological upgrading, up-staging or biochemical recurrence. These data support continued use of active surveillance in African-American men. Upgrading and up-staging remain concerning possibilities for all men regardless of race.

Lichen sclerosus and isolated bulbar urethral stricture disease.

PURPOSE: Lichen sclerosus is a chronic inflammatory genital skin condition that can cause destructive urethral scarring. To our knowledge no prior study has described lichen sclerosus in isolated bulbar urethral stricture segments without progressive disease originating from the penile urethra. We report the incidence of lichen sclerosus in isolated bulbar urethral stricture segments. MATERIALS AND METHODS: We retrospectively reviewed the records of 70 patients after urethroplasty for isolated bulbar stricture disease was performed from 2007 to 2013. Stricture specimens were re-reviewed by a single uropathologist. Cases were evaluated using common histological features of lichen sclerosus, including hyperkeratosis or epithelial atrophy, basal cell vacuolar degeneration, lichenoid lymphocytic infiltrate and superepithelial sclerosis. RESULTS: Average patient age was 46.5 years (range 19 to 77) and average stricture length was 3.5 cm (range 1 to 7). Of the patients 51 (73.0%) underwent excision and primary anastomosis, and 19 (27.1%) underwent buccal mucosal onlay. In 6 patients (8.6%) stricture recurred during a median followup of 22 months (IQR 14, 44). Three of those patients had lichen sclerosus. Initial pathology assessment revealed lichen sclerosus in 5 patients (7.1%, 95% CI 1.0-13.3). On re-review of specimens using pathology criteria specific to lichen sclerosus 31 patients (44.3%, 95% CI 32.4-56.2) showed pathology findings highly suggestive of (13) or diagnostic for (18) lichen sclerosus (p = 0.0001). On pathological re-review lichen sclerosus was associated with recurrent stricture. CONCLUSIONS: On re-review of surgical specimens we noted a significant incidence of lichen sclerosus in isolated bulbar strictures in men undergoing urethroplasty. The incidence of lichen sclerosus may be higher than reported in isolated bulbar urethral segments without evidence of distal to proximal progressive urethral disease.

Trauma-Informed Pedagogy: Instructional Strategies to Support Student Success.

Over the past several years, the ongoing coronavirus disease 2019 pandemic has contributed to challenging working and life conditions. As a result, the midwifery and health care workforce has faced significant shortages due to burnout. Increased societal awareness of historical trauma and systemic racism embedded within US culture has also led to increased anxiety and signs of trauma among midwifery and health profession students. Now more than ever, innovative teaching strategies are needed to support students, reduce the risks of burnout, and increase diversity in the workforce. One strategy is to adopt a trauma-informed pedagogy within midwifery education. Trauma-informed pedagogy is founded on core assumptions of trauma-informed care and thus supports student success by recognizing that the student cannot be separated from their own life experiences. Faculty and preceptors can develop empathetic, flexible supports that communicate care and concern regarding students' personal and social situations, and emotions. Empathetic behavior from teachers also increases student learning motivation, making it easier for students to actively engage in learning thereby reducing their distress. The purpose of this State of the Science review, therefore, was to describe the literature surrounding trauma-informed pedagogy and to offer concrete educational strategies that faculty members and educational programs can employ to increase the success of a diverse student body. This can be accomplished through flexibility in curriculum design and outcome measurement to ensure attainment of end of program learning outcomes. Institutional and administrative support are essential to develop a faculty who realize the benefit and value of trauma-informed pedagogy underpinning student success.

Improved retention associated with community-based accompaniment for antiretroviral therapy delivery in rural Rwanda.

BACKGROUND: Minimizing death and ensuring high retention and good adherence remain ongoing challenges for human immunodeficiency virus (HIV) treatment programs. We examined whether the addition of community-based accompaniment (characterized by daily home visits from a community health worker, directly observed treatment, nutritional support, transportation stipends, and other support as needed) to the Rwanda national model for antiretroviral therapy (ART) delivery would improve retention in care, viral load suppression, and change in CD4 count, relative to the national model alone. METHODS: We conducted a prospective observational cohort study among 610 HIV-infected adults initiating ART in 1 of 2 programs in rural Rwanda. Psychosocial and clinical characteristics were recorded at ART initiation. Death, treatment retention, and plasma viral load were assessed at 1 year. CD4 count was evaluated at 6-month intervals. Multivariable regression models were used to adjust for baseline differences between the 2 populations. RESULTS: Eighty-five percent and 79% of participants in the community-based and clinic-based programs, respectively, were retained with viral load suppression at 1 year. After adjusting for CD4 count, depression, physical health quality of life, and food insecurity, community-based accompaniment was protective against death or loss to follow-up during the first year of ART (hazard ratio, 0.17; 95% confidence interval [CI], .09-.35; P < .0001). In a second multivariable analysis, individuals receiving accompaniment were more likely to be retained with a suppressed viral load at 1 year (risk ratio: 1.15; 95% CI, 1.03-1.27; P = .01). CONCLUSIONS: These findings indicate that community-based accompaniment is effective in improving retention, when added to a clinic-based program with fewer patient support mechanisms.

Smoking and prostate cancer in a multi-ethnic cohort.

BACKGROUND: Prostate cancer (PCa) and smoking-related morbidity disproportionately burdens African American (AA) men. Smoking is associated with high-grade PCa and incidence, but few studies have focused on AA men. This study aims to determine the effect of tobacco-use on odds of PCa and of high-grade PCa in a population of predominantly AA men. METHODS: This is a cross-sectional study evaluating smoking and PCa status in men with incident PCa and screened healthy controls. Altogether, 1,085 men (527 cases and 558 controls), age ≥ 40 years were enrolled through outpatient urology clinics in two US cities from 2001 to 2012. Validated questionnaires were used to gather clinical and socioeconomic data. RESULTS: The cases and controls were predominantly AA (79.9% and 71.3%, respectively, P = 0.01). AA men smoked more frequently (53.4% vs. 47.9%, P < 0.001) and quit less frequently than European American (EA) men (31.5% vs. 40.4%, P = 0.01). AA heavy smokers had increased odds of PCa diagnosis (OR 2.57, 95% CI 1.09, 6.10) and high-grade cancer (OR 1.89, 95% CI 1.03, 3.48) relative to never smokers and light smokers. Among AAs, heavy smokers had lower odds of NCCN low PCa recurrence risk stratification. AA former smokers had a trend for increased odds of high-grade cancer compared to never smokers. The associations between smokings, cancer diagnosis and cancer grade did not reach statistical significance in EA men. CONCLUSION: We found ethnic differences in smoking behavior. Heavy smoking is associated with increased odds of PCa and of higher Gleason grade in AA men.

An analysis of outcomes after use of the Maxwell-Brancheau Arthroereisis implant.

The authors present a retrospective study of 35 consecutive patients (60 feet) treated with the Maxwell-Brancheau Arthroereisis (MBA) implant. The mean age of the cohort at the time of surgery was 14.3 (range 5 to 46) years, and 22 (62.86%) men and 13 (37.14%) women were included. Preoperative and postoperative anteroposterior and lateral foot radiographs were compared at a mean of 36 (range 18 to 48) months postoperatively, and the following mean changes were reported: talocalcaneal angle 24.15° ± 7.97° to 18.53° ± 8.23°, calcaneocuboid angle 18.67° ± 8.72° to 11.76° ± 8.49°, first to second intermetatarsal angle 9.42° ± 2.67° to 7.61° ± 2.69°, calcaneal inclination angle 11.93° ± 6° to 14.93° ± 5.85°, and talar declination angle 34.0° ± 8.59° to 28.02° ± 6.85°; all of these differences were statistically significant (p < .0001). A subgroup of 24 (68.57%) patients also answered a subjective questionnaire at a mean of 33 (range 12 to 55) months postoperatively. The presenting chief complaints were resolved in 23 patients (95.83%) of the subgroup, and 21 patients (87.5%) returned postoperatively to either the same or a greater activity level in sports. Twenty-three (95.83% of the subgroup) patients said they were 75% to 100% satisfied with their surgical outcome, and that they would recommend the surgery to a friend or family member with the same condition, whereas 1 (4.17%) claimed 0% satisfaction after placement of inappropriately sized implants (which were later replaced to the patient's clinical satisfaction) in both feet.

Clinical Implications for Women of a Low-Carbohydrate or Ketogenic Diet With Intermittent Fasting.

Obesity in the United States is pervasive and associated with morbidity, mortality, and increased health care costs. For women, obesity may affect stages of life including early menarche, fertility, pregnancy, and menopause as a result of hormonal imbalances and insulin resistance. The insulin-carbohydrate model of obesity has been proposed as an explanation for growing obesity rates and can be used to target weight loss strategies by increasing insulin sensitivity. Together, low-carbohydrate dietary patterns along with intermittent fasting may help individuals with insulin resistance not only lose weight but also increase their insulin sensitivity. The purpose of this article is to review the epidemiology and physiology of obesity and the indicators for health while outlining strategies for nurses and other clinicians to use when counseling women who are following a low-carbohydrate or ketogenic diet with intermittent fasting for weight management.

Rapid Curricular Innovations During COVID-19 Clinical Suspension: Maintaining Student Engagement with Simulation Experiences.

Health care education programs were faced with the need to quickly adapt to a new reality during the coronavirus disease 2019 pandemic. Students were temporarily suspended from campus and clinical sites, requiring prompt changes in structure to their didactic and clinical learning. This article describes the rapid adjustments that one midwifery and women's health nurse practitioner education program created using both synchronous and asynchronous simulation experiences to promote student learning and ongoing engagement. Flexibility and reflexivity were needed by faculty and students alike in the face of the multiple changes wrought by the pandemic. Curricular changes were made simultaneously in many courses. Objective structured clinical examinations simulate telehealth experiences that assess knowledge, clinical reasoning, and professional behaviors via a scripted scenario and an actor patient. On-call simulations mimic telephone triage and provide students the opportunity to build listening, assessment, and management skills for prenatal and intrapartum scenarios. Students are provided equipment and virtual instruction in an intrauterine device insertion session, which promotes skill acquisition and self-confidence. Trigger films are used to visualize real-life or scripted clinical encounters, leading to discussion and decision-making, particularly in the affective domain. Bilateral learning tools, similar to case studies, provide students an opportunity to demonstrate their knowledge and critical thinking with a mechanism for faculty feedback. Web-based virtual clinical encounter learning tools using patient avatars prompt additional student learning. Suturing skills introduced in live remote group sessions are augmented with video-guided individual practice. This article describes each of these adapted and innovative simulation methods and shares lessons learned during their development and implementation.

Consolidation of Guidelines of Postpartum Care Recommendations to Address Maternal Morbidity and Mortality.

Rates of maternal morbidity and mortality in the United States represent an urgent crisis. The purpose of this article is to consolidate current postpartum care guidelines to provide a comprehensive approach to care in the postpartum period. We include a critical examination of the reasons for some women's lack of attendance at postpartum visits, the current state of postpartum care, and the unmet needs of women. We review several postpartum care programs and suggest possible solutions for the postpartum period, including clinical implications for continuity of care for women with comorbidities including gestational diabetes, hypertension, and depression.

Effect of a family-based intervention on electronic media use and body composition among boys aged 8--11 years: a pilot study.

This study measured the effect of a 20-week, family-centered electronic media intervention on electronic media use, body composition (dual-energy X-ray absorptiometry; DXA), physical activity and dietary behaviors in boys. Twenty-two boys were assigned and 21 were analyzed in an experimental or control group. Boys in the experimental group set electronic media use goals and used TV Allowance device and ENUFF software to reduce electronic media use. Data were collected at baseline, 10 weeks and 20 weeks. Interactions were found in daily electronic media use and DXA. At 10 weeks, step counts increased by 543 steps per day in the experimental group and decreased by 340 steps per day in the controls. Steps in both groups were higher at 20 weeks. Meals or snacks eaten while using electronic media decreased in the experimental group only. In conclusion, a family-centered electronic media intervention may reduce electronic media use and contribute to desirable changes in body composition.

High throughput automated microbial bioreactor system used for clone selection and rapid scale-down process optimization.

High throughput automated fermentation systems have become a useful tool in early bioprocess development. In this study, we investigated a 24 x 15 mL single use microbioreactor system, ambr 15f, designed for microbial culture. We compared the fed-batch growth and production capabilities of this system for two Escherichia coli strains, BL21 (DE3) and MC4100, and two industrially relevant molecules, hGH and scFv. In addition, different carbon sources were tested using bolus, linear or exponential feeding strategies, showing the capacity of the ambr 15f system to handle automated feeding. We used power per unit volume (P/V) as a scale criterion to compare the ambr 15f with 1 L stirred bioreactors which were previously scaled-up to 20 L with a different biological system, thus showing a potential 1,300 fold scale comparability in terms of both growth and product yield. By exposing the cells grown in the ambr 15f system to a level of shear expected in an industrial centrifuge, we determined that the cells are as robust as those from a bench scale bioreactor. These results provide evidence that the ambr 15f system is an efficient high throughput microbial system that can be used for strain and molecule selection as well as rapid scale-up. © 2017 The Authors Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers Biotechnol. Prog., 34:58-68, 2018.

Educational Strategies to Help Students Provide Respectful Sexual and Reproductive Health Care for Lesbian, Gay, Bisexual, and Transgender Persons.

Graduate medical, nursing, and midwifery curricula often have limited amounts of time to focus on issues related to cultural competency in clinical practice, and respectful sexual and reproductive health care for all individuals in particular. Respectful health care that addresses sexual and reproductive concerns is a right for everyone, including those who self-identify as lesbian, gay, bisexual, or transgender (LGBT). LGBT persons have unique reproductive health care needs as well as increased risks for poor health outcomes. Both the World Health Organization and Healthy People 2020 identified the poor health of LGBT persons as an area for improvement. A lack of educational resources as well as few student clinical experiences with an LGBT population may be barriers to providing respectful sexual and reproductive health care to LGBT persons. This article offers didactic educational strategies for midwifery and graduate nursing education programs that may result in reducing barriers to the provision of respectful sexual and reproductive health care for LGBT clients. Specific ideas for implementation are discussed in detail. In addition to what is presented here, other educational strategies and clinical experiences may help to support students for caring for LGBT persons prior to entrance into clinical practice.

Charge filling factors in clean and disordered arrays of tunnel junctions.

We simulate one-dimensional arrays of tunnel junctions using the kinetic Monte Carlo method to study charge filling behaviour in the large charging energy limit. By applying a small fixed voltage bias and varying the offset voltage, we investigate this behaviour in clean and disordered arrays (both weak and strong disorder effects). The offset voltage dependent modulation of the current is highly sensitive to background charge disorder and exhibits substantial variation depending on the strength of the disorder. We show that while small fractional charge filling factors are likely to be washed out in experimental devices due to strong background charge disorder, larger factors may be observable.

How Important is Orbital Choice in Single-Determinant Diffusion Quantum Monte Carlo Calculations?

The accuracy of total electronic energies obtained using the fixed-node diffusion quantum Monte Carlo (FN-DMC) method is determined by the choice of the many-body nodal surface. Here, we perform a systematic comparison of the quality of FN-DMC energies for a selection of atoms and diatomic molecules using nodal surfaces defined by single determinants of Hartree-Fock, B3LYP, and LDA orbitals. Through comparison with experimental results, we show that the use of Kohn-Sham orbitals results in significantly improved FN-DMC atomization energies over those obtained using Hartree-Fock orbitals. We also discuss the effect of spin contamination in the orbitals.

Quantum dots for multiplexed detection and characterisation of prostate cancer cells using a scanning near-field optical microscope.

In this study scanning near-field optical microscopy (SNOM) has been utilised in conjunction with quantum dot labelling to interrogate the biomolecular composition of cell membranes. The technique overcomes the limits of optical diffraction found in standard fluorescence microscopy and also yields vital topographic information. The technique has been applied to investigate cell-cell adhesion in human epithelial cells. This has been realised through immunofluorescence labelling of the cell-cell adhesion protein E-cadherin. Moreover, a dual labelling protocol has been optimised to facilitate a comparative study of the adhesion mechanisms and the effect of aberrant adhesion protein expression in both healthy and cancerous epithelial cells. This study reports clear differences in the morphology and phenotype of healthy and cancerous cells. In healthy prostate epithelial cells (PNT2), E-cadherin was predominantly located around the cell periphery and within filopodial extensions. The presence of E-cadherin appeared to be enhanced when cell-cell contact was established. In contrast, examination of metastatic prostate adenocarcinoma cells (PC-3) revealed no E-cadherin labelling around the periphery of the cells. This lack of functional E-cadherin in PC-3 cells coincided with a markedly different morphology and PC-3 cells were not found to form close cell-cell associations with their neighbours. We have demonstrated that with a fully optimised sample preparation methodology, multiplexed quantum dot labelling in conjunction with SNOM imaging can be successfully applied to interrogate biomolecular localisation within delicate cellular membranes.

Feasibility of chronic disease patient navigation in an urban primary care practice.

The purpose of this study was to evaluate the feasibility of incorporating chronic disease navigation using lay health care workers trained in motivational interviewing (MI) into an existing mammography navigation program. Primary-care patient navigators implemented MI-based telephone conversations around mammography, smoking, depression, and obesity. We conducted a small-scale demonstration, using mixed methods to assess patient outcomes and provider satisfaction. One hundred nine patients participated. Ninety-four percent scheduled and 73% completed a mammography appointment. Seventy-one percent agreed to schedule a primary care appointment and 54% completed that appointment. Patients and providers responded positively. Incorporating telephone-based chronic disease navigation supported by MI into existing disease-specific navigation is efficacious and acceptable to those enrolled.