Shaping the future US bioeconomy through safety, security, sustainability, and social responsibility.

Biomanufacturing practitioners and researchers describe the norms that should govern the growing, global field, to include safety, security, sustainability, and social responsibility. These '4S Principles' should be broadly adopted so that the future of the field may provide the greatest benefits to society.

Improved soluble expression and use of recombinant human renalase.

Electrochemical bioreactor systems have enjoyed significant attention in the past few decades, particularly because of their applications to biobatteries, artificial photosynthetic systems, and microbial electrosynthesis. A key opportunity with electrochemical bioreactors is the ability to employ cofactor regeneration strategies critical in oxidative and reductive enzymatic and cell-based biotransformations. Electrochemical cofactor regeneration presents several advantages over other current cofactor regeneration systems, such as chemoenzymatic multi-enzyme reactions, because there is no need for a sacrificial substrate and a recycling enzyme. Additionally, process monitoring is simpler and downstream processing is less costly. However, the direct electrochemical reduction of NAD(P)+ on a cathode may produce adventitious side products, including isomers of NAD(P)H that can act as potent competitive inhibitors to NAD(P)H-requiring enzymes such as dehydrogenases. To overcome this limitation, we examined how nature addresses the adventitious formation of isomers of NAD(P)H. Specifically, renalases are enzymes that catalyze the oxidation of 1,2- and 1,6-NAD(P)H to NAD(P)+, yielding an effective recycling of unproductive NAD(P)H isomers. We designed several mutants of recombinant human renalase isoform 1 (rhRen1), expressed them in E. coli BL21(DE3) to enhance protein solubility, and evaluated the activity profiles of the renalase variants against NAD(P)H isomers. The potential for rhRen1 to be employed in engineering applications was then assessed in view of the enzyme's stability upon immobilization. Finally, comparative modeling was performed to assess the underlying reasons for the enhanced solubility and activity of the mutant enzymes.

Controlling the Growth of the Skin Commensal Staphylococcus epidermidis Using d-Alanine Auxotrophy.

Using live microbes as therapeutic candidates is a strategy that has gained traction across multiple therapeutic areas. In the skin, commensal microorganisms play a crucial role in maintaining skin barrier function, homeostasis, and cutaneous immunity. Alterations of the homeostatic skin microbiome are associated with a number of skin diseases. Here, we present the design of an engineered commensal organism, Staphylococcus epidermidis, for use as a live biotherapeutic product (LBP) candidate for skin diseases. The development of novel bacterial strains whose growth can be controlled without the use of antibiotics or genetic elements conferring antibiotic resistance enables modulation of therapeutic exposure and improves safety. We therefore constructed an auxotrophic strain of S. epidermidis that requires exogenously supplied d-alanine. The S. epidermidis NRRL B-4268 Δalr1 Δalr2 Δdat strain (SEΔΔΔ) contains deletions of three biosynthetic genes: two alanine racemase genes, alr1 and alr2 (SE1674 and SE1079), and the d-alanine aminotransferase gene, dat (SE1423). These three deletions restricted growth in d-alanine-deficient medium, pooled human blood, and skin. In the presence of d-alanine, SEΔΔΔ colonized and increased expression of human ß-defensin 2 in cultured human skin models in vitro. SEΔΔΔ showed a low propensity to revert to d-alanine prototrophy and did not form biofilms on plastic in vitro. These studies support the potential safety and utility of SEΔΔΔ as a live biotherapeutic strain whose growth can be controlled by d-alanine.IMPORTANCE The skin microbiome is rich in opportunities for novel therapeutics for skin diseases, and synthetic biology offers the advantage of providing novel functionality or therapeutic benefit to live biotherapeutic products. The development of novel bacterial strains whose growth can be controlled without the use of antibiotics or genetic elements conferring antibiotic resistance enables modulation of therapeutic exposure and improves safety. This study presents the design and in vitro evidence of a skin commensal whose growth can be controlled through d-alanine. The basis of this strain will support future clinical studies of this strain in humans.

Dosimetric impact of organ at risk daily variation during prostate stereotactic ablative radiotherapy.

OBJECTIVE: Prostate stereotactic ablative radiotherapy (SABR) delivers large doses using a fast dose rate. This amplifies the effect geometric uncertainties have on normal tissue dose. The aim of this study was to determine whether the treatment dose-volume histogram (DVH) agrees with the planned dose to organs at risk (OAR). METHODS: 41 low-intermediate risk prostate cancer patients were treated with SABR using a linac based technique. Dose prescribed was 35 Gy in five fractions delivered on alternate days, planned using volumetric modulated arc therapy (VMAT) with 10X flattening filter free (FFF). On treatment, prostate was matched to fiducial markers on cone beam CT (CBCT). OAR were retrospectively delineated on 205 pre-treatment CBCT images. Daily CBCT contours were overlaid on the planning CT for dosimetric analysis. Verification plan used to evaluate the daily DVH for each structure. The daily doses received by OAR were recorded using the D%. RESULTS: The median rectum and bladder volumes at planning were 67.1 cm3 (interquartile range 56.4-78.2) and 164.4 cm3 (interquartile range 120.3-213.4) respectively. There was no statistically significant difference in median rectal volume at each of the five treatment scans compared to the planning scan (p = 0.99). This was also the case for median bladder volume (p = 0.79). The median dose received by rectum and bladder at each fraction was higher than planned, at the majority of dose levels. For rectum the increase ranged from 0.78-1.64Gy and for bladder 0.14-1.07Gy. The percentage of patients failing for rectum D35% < 18 Gy (p = 0.016), D10% < 28 Gy (p = 0.004), D5% < 32 Gy (p = 0.0001), D1% < 35 Gy (p = 0.0001) and bladder D1% < 35 Gy (p = 0.001) at treatment were all statistically significant. CONCLUSION: In this cohort of prostate SABR patients, we estimate the OAR treatment DVH was higher than planned. This was due to rectal and bladder organ variation. ADVANCES IN KNOWLEDGE: OAR variation in prostate SABR using a FFF technique, may cause the treatment DVH to be higher than planned.

Electrochemical Bioreactor Technology for Biocatalysis and Microbial Electrosynthesis.

Two seemingly distinct fields, industrial biocatalysis and microbial electrosynthesis, can be viewed together through the lens of electrochemical bioreactor technology in order to highlight the challenges that exist in creating a versatile platform technology for use in chemical and biological applications. Industrial biocatalysis applications requiring NAD(P)H to perform redox transformations often necessitate convoluted coupled-enzyme regeneration systems to regenerate reduced cofactor, NAD(P)H from oxidized cofactor, NAD(P). Renewed interest in continuously recycling the cofactor via electrochemical reduction is motivated by the low cost of performing electrochemical reactions, easy monitoring of the reaction progress, and straightforward product recovery. However, electrochemical cofactor regeneration methods invariably produce adventitious reduced cofactor side products which result in unproductive loss of input NAD(P). Microbial electrosynthesis is a form of microbially driven catalysis in which electricity is supplied to living microorganisms for the production of industrially relevant chemical products at higher carbon efficiencies and yields compared with traditional, nonelectrically driven, fermentations. The fundamental biochemistry of these organisms as related to selected biochemical redox processes will be explored in order to highlight opportunities to devise strategies for taking advantage of these biochemical processes in engineered systems.

Safety and feasibility of prostate stereotactic ablative radiotherapy using multimodality imaging and flattening filter free.

OBJECTIVE: To investigate feasibility and safety of stereotactic ablative radiotherapy in the management of prostate cancer while employing MR/CT fusion for delineation, fiducial marker seeds for positioning and Varian RapidArc with flattening filter free (FFF) delivery. METHODS: 41 patients were treated for low-intermediate risk prostate cancer with initial prostate-specific antigen of ≤20 ng ml-1, Gleason score 6-7. Patients had MR/CT fusion for delineation of prostate ±seminal vesicles. CT/MR fusion images were used for delineation and planned using flattening filter free modality. Verification on treatment was cone beam CT imaging with fiducial markers for matching. Patients had Radiation Therapy Oncology Group scoring for genitourinary and gastointestinal symptoms at baseline, week 4, 10 and 18. RESULTS: Clinically acceptable plans were achieved for all patients, all plans achieved the objective clinical target volume D99% ≥ 95%, and for planning target volume D95% ≥ 95%. Rectum dose constraints were met for 95.1% for V18 Gy ≤ 35%, 80% V28 Gy ≤ 10%. A total of 32 (78.0%) plans achieved all rectum dose constraints. Grade 1 acute genitourinary symptoms were 53.7% of patients at baseline, 90.2% [95% CI (76.8-97.3%)] (p = 0.0005) at treatment 5, falling to 78.0% (62.4-89.4%) at week 4, and 75.0% (58.8-87.3%) by week 10 and 52.5% (36.1-68.5%) (p = 1.00) at week 18. Acute gastrointestinal symptoms were 5% at baseline, 46.3% [95% CI (30.7-62.6%)] at treatment 5, week 4 43.9% [95% CI (28.5-60.3%)], week 10 25.0% (11.1-42.3%), and declined slightly by week 18 [-20.095% CI (12.7-41.2)] p = 0.039. Overall 75.6% (31/41) of patients experienced Grade 1-2 toxicity during or after treatment. CONCLUSION: This planning and delivery technique is feasible, safe and efficient. A homogeneous dose can be delivered to prostate with confidence, whilst limiting high dose to nearby structures. The use of this technology can be applied safely within further randomized study protocols. Advances in knowledge: Multimodality imaging for delineation and linac-based image-guided RT with FFF for the treatment of prostate stereotactic ablative radiotherapy.

Real-world uptake, safety profile and outcomes of docetaxel in newly diagnosed metastatic prostate cancer.

OBJECTIVES: To investigate the uptake, safety and efficacy of docetaxel chemotherapy in hormone-naïve metastatic prostate cancer (mPC) in the first year of use outside of a clinical trial. PATIENTS AND METHODS: Patients in the West of Scotland Cancer Network with newly diagnosed mPC were identified from the regional multidisciplinary team meetings and their treatment details were collected from electronic patient records. The rate of febrile neutropenia, hospitalisations, time to progression, and overall survival were compared between those patients who received docetaxel and androgen-deprivation therapy (ADT), or ADT alone using survival analysis. RESULTS: Of the 270 eligible patients, 103 received docetaxel (38.1%). 35 patients (34%) were hospitalised and there were 17 episodes of febrile neutropenia (16.5%). Two patients (1.9%) died within 30 days of chemotherapy. Patients who received ADT alone had an increased risk of progression (hazard ratio [HR] 2.03, 95% confidence interval [CI] 1.27-3.25; log-rank test, P = 0.002) and had an increased risk of death (HR 5.88, 95% CI: 2.52-13.72; log-rank test, P = 0.001) compared to the docetaxel group. The risk of febrile neutropenia was nine-times greater if chemotherapy was started within 3 weeks of ADT initiation (95% CI: 1.22-77.72; P = 0.032). CONCLUSION: Docetaxel chemotherapy in hormone-naïve mPC has significant toxicities, but has a similar effect on time to progression and overall survival as seen in randomised trials. Chemotherapy should be started at ≥3 weeks after ADT.

Improved strategies for electrochemical 1,4-NAD(P)H2 regeneration: A new era of bioreactors for industrial biocatalysis.

Industrial enzymatic reactions requiring 1,4-NAD(P)H2 to perform redox transformations often require convoluted coupled enzyme regeneration systems to regenerate 1,4-NAD(P)H2 from NAD(P) and recycle the cofactor for as many turnovers as possible. Renewed interest in recycling the cofactor via electrochemical means is motivated by the low cost of performing electrochemical reactions, easy monitoring of the reaction progress, and straightforward product recovery. However, electrochemical cofactor regeneration methods invariably produce adventitious reduced cofactor side products which result in unproductive loss of input NAD(P). We review various literature strategies for mitigating adventitious product formation by electrochemical cofactor regeneration systems, and offer insight as to how a successful electrochemical bioreactor system could be constructed to engineer efficient 1,4-NAD(P)H2-dependent enzyme reactions of interest to the industrial biocatalysis community.

Antibiotic resistance: A current epilogue.

The history of the first commercial antibiotics is briefly reviewed, together with data from the US and WHO, showing the decrease in death due to infectious diseases over the 20th century, from just under half of all deaths, to less than 10%. The second half of the 20th century saw the new use of antibiotics as growth promoters for food animals in the human diet, and the end of the 20th century and beginning of the 21st saw the beginning and rapid rise of advanced microbial resistance to antibiotics.

Chemical- and radiation-induced haemorrhagic cystitis: current treatments and challenges.

• To review the published data on predisposing risk factors for cancer treatment-induced haemorrhagic cystitis (HC) and the evidence for the different preventive and therapeutic measures that have been used in order to help clinicians optimally define and manage this potentially serious condition. • Despite recognition that HC can be a significant complication of cancer treatment, there is currently a lack of UK-led guidelines available on how it should optimally be defined and managed. • A systematic literature review was undertaken to evaluate the evidence for preventative measures and treatment options in the management of cancer treatment-induced HC. • There is a wide range of reported incidence due to several factors including variability in study design and quality, the type of causal agent, the grading of bleeding, and discrepancies in definition criteria. • The most frequently reported causal factors are radiotherapy to the pelvic area, where HC has been reported in up to 20% of patients, and treatment with cyclophosphamide and bacillus Calmette-Guérin, where the incidence has been reported as up to 30%. • Mesna (2-mercaptoethane sodium sulphonate), hyperhydration and bladder irrigation have been the most frequently used prophylactic measures to prevent treatment-related cystitis, but are not always effective. • Cranberry juice is widely cited as a preventative measure and sodium pentosanpolysulphate as a treatment, although the evidence for both is very limited. • The best evidence exists for intravesical hyaluronic acid as an effective preventative and active treatment, and for hyperbaric oxygen as an equally effective treatment option. • The lack of robust data and variability in treatment strategies used highlights the need for further research, as well as best practice guidance and consensus on the management of HC.

Attitudes toward retirement of ophthalmology department chairs.

PURPOSE: To identify common perceptions and ideas about preparation and planning for retirement of chairs of academic departments of ophthalmology, determining areas of particular stress and proposing ways to better prepare for retirement. DESIGN: Cross-sectional study. PARTICIPANTS: One-hundred sixteen chairs of academic departments of ophthalmology in the United States. METHODS: A confidential online survey emailed to ophthalmology chairs. MAIN OUTCOME MEASURES: Surveys assessed demographics; current work schedule; perceptions, preparation, and planning for retirement; and retirement training for faculty and residents. RESULTS: Ninety-six department chairs responded to the survey (82% response rate). Most chairs anticipate retiring around age 70. Significantly, only 9% are looking forward to retirement. Reasons for delaying retirement include keeping active (37%), income/insurance/benefits (20%), and maintaining lifestyle (17%). The most common concern is financing retirement (46%). Forty percent anticipate their reason for retirement will be because of age or health, whereas 20% anticipate fatigue or burnout. Nearly half of the respondents have no specific plan upon retirement. Most respondents anticipate pursuing other interests (43%); 32% intend to spend time with family, vacationing, and travelling. Younger respondents are more concerned with the financial aspects of retirement while more senior respondents appear to delay retirement to keep active or because they enjoy their work. CONCLUSIONS: Retirement is a source of stress for many ophthalmology department chairs and many indicate financial preparation is their major concern. Despite this, the major reason for putting off retirement is a desire to keep active. Developing a retirement plan eases stress and engenders a feeling of confidence about the future.

A two-phase genetic K-means algorithm for placement of radioports in cellular networks.

This paper proposes a two-phase hybrid algorithm for optimal placement of radioports. The placement strategy minimizes the dynamic range of the channel, and thus reduces the peak transmit power needed by a wireless handset. The performance of the proposed algorithm is compared with that of an exhaustive search, as well as with that of a simplex algorithm. Simulation results show that the two-phase hybrid algorithm, although suboptimal, outperforms the simplex algorithm with only a modest increase in computation time. It has the added advantage of being quite insensitive to the seed solution.