Diya - A universal light illumination platform for multiwell plate cultures.

Recent progress in protein engineering has established optogenetics as one of the leading external non-invasive stimulation strategies, with many optogenetic tools being designed for in vivo operation. Characterization and optimization of these tools require a high-throughput and versatile light delivery system targeting micro-titer culture volumes. Here, we present a universal light illumination platform - Diya, compatible with a wide range of cell culture plates and dishes. Diya hosts specially designed features ensuring active thermal management, homogeneous illumination, and minimal light bleedthrough. It offers light induction programming via a user-friendly custom-designed GUI. Through extensive characterization experiments with multiple optogenetic tools in diverse model organisms (bacteria, yeast, and human cell lines), we show that Diya maintains viable conditions for cell cultures undergoing light induction. Finally, we demonstrate an optogenetic strategy for in vivo biomolecular controller operation. With a custom-designed antithetic integral feedback circuit, we exhibit robust perfect adaptation and light-controlled set-point variation using Diya.

An electrogenetic interface to program mammalian gene expression by direct current.

Wearable electronic devices are playing a rapidly expanding role in the acquisition of individuals' health data for personalized medical interventions; however, wearables cannot yet directly program gene-based therapies because of the lack of a direct electrogenetic interface. Here we provide the missing link by developing an electrogenetic interface that we call direct current (DC)-actuated regulation technology (DART), which enables electrode-mediated, time- and voltage-dependent transgene expression in human cells using DC from batteries. DART utilizes a DC supply to generate non-toxic levels of reactive oxygen species that act via a biosensor to reversibly fine-tune synthetic promoters. In a proof-of-concept study in a type 1 diabetic male mouse model, a once-daily transdermal stimulation of subcutaneously implanted microencapsulated engineered human cells by energized acupuncture needles (4.5 V DC for 10 s) stimulated insulin release and restored normoglycemia. We believe this technology will enable wearable electronic devices to directly program metabolic interventions.

Blood-Glucose-Powered Metabolic Fuel Cell for Self-Sufficient Bioelectronics.

Currently available bioelectronic devices consume too much power to be continuously operated on rechargeable batteries, and are often powered wirelessly, with attendant issues regarding reliability, convenience, and mobility. Thus, the availability of a robust, self-sufficient, implantable electrical power generator that works under physiological conditions would be transformative for many applications, from driving bioelectronic implants and prostheses to programing cellular behavior and patients' metabolism. Here, capitalizing on a new copper-containing, conductively tuned 3D carbon nanotube composite, an implantable blood-glucose-powered metabolic fuel cell is designed that continuously monitors blood-glucose levels, converts excess glucose into electrical power during hyperglycemia, and produces sufficient energy (0.7 mW cm-2 , 0.9 V, 50 mm glucose) to drive opto- and electro-genetic regulation of vesicular insulin release from engineered beta cells. It is shown that this integration of blood-glucose monitoring with elimination of excessive blood glucose by combined electro-metabolic conversion and insulin-release-mediated cellular consumption enables the metabolic fuel cell to restore blood-glucose homeostasis in an automatic, self-sufficient, and closed-loop manner in an experimental model of type-1 diabetes.

Bioelectronic cell-based device provides a strategy for the treatment of the experimental model of multiple sclerosis.

Wireless powered optogenetic cell-based implant provides a strategy to deliver subcutaneously therapeutic proteins. Immortalize Human Mesenchymal Stem Cells (hMSC-TERT) expressing the bacteriophytochrome diguanylate cyclase (DGCL) were validated for optogenetic controlled interferon-ß delivery (Optoferon cells) in a bioelectronic cell-based implant. Optoferon cells transcriptomic profiling was used to elaborate an in-silico model of the recombinant interferon-ß production. Wireless optoelectronic device integration was developed using additive manufacturing and injection molding. Implant cell-based optoelectronic interface manufacturing was established to integrate industrial flexible compact low-resistance screen-printed Near Field Communication (NFC) coil antenna. Optogenetic cell-based implant biocompatibility, and device performances were evaluated in the Experimental Autoimmune Encephalomyelitis (EAE) mouse model of multiple sclerosis.

Smart-watch-programmed green-light-operated percutaneous control of therapeutic transgenes.

Wearable smart electronic devices, such as smart watches, are generally equipped with green-light-emitting diodes, which are used for photoplethysmography to monitor a panoply of physical health parameters. Here, we present a traceless, green-light-operated, smart-watch-controlled mammalian gene switch (Glow Control), composed of an engineered membrane-tethered green-light-sensitive cobalamin-binding domain of Thermus thermophilus (TtCBD) CarH protein in combination with a synthetic cytosolic TtCBD-transactivator fusion protein, which manage translocation of TtCBD-transactivator into the nucleus to trigger expression of transgenes upon illumination. We show that Apple-Watch-programmed percutaneous remote control of implanted Glow-controlled engineered human cells can effectively treat experimental type-2 diabetes by producing and releasing human glucagon-like peptide-1 on demand. Directly interfacing wearable smart electronic devices with therapeutic gene expression will advance next-generation personalized therapies by linking biopharmaceutical interventions to the internet of things.

Construction of a Multiwell Light-Induction Platform for Traceless Control of Gene Expression in Mammalian Cells.

Mammalian cells can be engineered to incorporate light-responsive elements that reliably sense stimulation by light and activate endogenous pathways, such as the cAMP or Ca2+ pathway, to control gene expression. Light-inducible gene expression systems offer high spatiotemporal resolution, and are also traceless, reversible, tunable, and inexpensive. Melanopsin, a well-known representative of the animal opsins, is a G-protein-coupled receptor that triggers a Gαq-dependent signaling cascade upon activation with blue light (≈470 nm). Here, we describe how to rewire melanopsin activation by blue light to transgene expression in mammalian cells, with detailed instructions for constructing a 96-LED array platform with multiple tunable parameters for illumination of the engineered cells in multiwell plates.

Electrogenetic cellular insulin release for real-time glycemic control in type 1 diabetic mice.

Sophisticated devices for remote-controlled medical interventions require an electrogenetic interface that uses digital electronic input to directly program cellular behavior. We present a cofactor-free bioelectronic interface that directly links wireless-powered electrical stimulation of human cells to either synthetic promoter-driven transgene expression or rapid secretion of constitutively expressed protein therapeutics from vesicular stores. Electrogenetic control was achieved by coupling ectopic expression of the L-type voltage-gated channel CaV1.2 and the inwardly rectifying potassium channel Kir2.1 to the desired output through endogenous calcium signaling. Focusing on type 1 diabetes, we engineered electrosensitive human ß cells (Electroß cells). Wireless electrical stimulation of Electroß cells inside a custom-built bioelectronic device provided real-time control of vesicular insulin release; insulin levels peaked within 10 minutes. When subcutaneously implanted, this electrotriggered vesicular release system restored normoglycemia in type 1 diabetic mice.

Prospective multicenter registration study of colorectal cancer: significant variations in radicality and oncosurgical quality-Swiss Group for Clinical Cancer Research Protocol SAKK 40/00.

PURPOSE: This study aimed to investigate in a multicenter cohort study the radicality of colorectal cancer resections, to assess the oncosurgical quality of colorectal specimens, and to compare the performance between centers. METHODS: One German and nine Swiss hospitals agreed to prospectively register all patients with primary colorectal cancer resected between September 2001 and June 2005. The median number of eligible patients with one primary tumor included per center was 95 (range 12-204). RESULTS: The following variations of median values or percentages between centers were found: length of bowel specimen 20-39 cm (25.8 cm), maximum height of mesocolon 6.5-12.5 cm (9.0 cm), number of examined lymph nodes 9-24 (16), distance to nearer bowel resection margin in colon cancer 4.8-12 cm (7 cm), and in rectal cancer 2-3 cm (2.5 cm), central ligation of major artery 40-97 % (71 %), blood loss 200-500 ml (300 ml), need for perioperative blood transfusion 5-40 % (19 %), tumor opened during mobilization 0-11 % (5 %), T4-tumors not en-bloc resected 0-33 % (4 %), inadvertent perforation of mesocolon/mesorectum 0-8 % (4 %), no-touch isolation technique 36-86 % (67 %), abdominoperineal resection for rectal cancer 0-30 % (17 %), rectal cancer specimen with circumferential margin ≤1 mm 0-19 % (10 %), in-hospital mortality 0-6 % (2 %), anastomotic leak or intra-abdominal abscess 0-17 % (7 %), re-operation 0-17 % (8 %). CONCLUSION: In colorectal cancer, surgery considerable variations between different centers were found with regard to radicality and oncosurgical quality, suggesting a potential for targeted improvement of surgical technique.

Automated optogenetic feedback control for precise and robust regulation of gene expression and cell growth.

Dynamic control of gene expression can have far-reaching implications for biotechnological applications and biological discovery. Thanks to the advantages of light, optogenetics has emerged as an ideal technology for this task. Current state-of-the-art methods for optical expression control fail to combine precision with repeatability and cannot withstand changing operating culture conditions. Here, we present a novel fully automatic experimental platform for the robust and precise long-term optogenetic regulation of protein production in liquid Escherichia coli cultures. Using a computer-controlled light-responsive two-component system, we accurately track prescribed dynamic green fluorescent protein expression profiles through the application of feedback control, and show that the system adapts to global perturbations such as nutrient and temperature changes. We demonstrate the efficacy and potential utility of our approach by placing a key metabolic enzyme under optogenetic control, thus enabling dynamic regulation of the culture growth rate with potential applications in bacterial physiology studies and biotechnology.

Mind-controlled transgene expression by a wireless-powered optogenetic designer cell implant.

Synthetic devices for traceless remote control of gene expression may provide new treatment opportunities in future gene- and cell-based therapies. Here we report the design of a synthetic mind-controlled gene switch that enables human brain activities and mental states to wirelessly programme the transgene expression in human cells. An electroencephalography (EEG)-based brain-computer interface (BCI) processing mental state-specific brain waves programs an inductively linked wireless-powered optogenetic implant containing designer cells engineered for near-infrared (NIR) light-adjustable expression of the human glycoprotein SEAP (secreted alkaline phosphatase). The synthetic optogenetic signalling pathway interfacing the BCI with target gene expression consists of an engineered NIR light-activated bacterial diguanylate cyclase (DGCL) producing the orthogonal second messenger cyclic diguanosine monophosphate (c-di-GMP), which triggers the stimulator of interferon genes (STING)-dependent induction of synthetic interferon-ß promoters. Humans generating different mental states (biofeedback control, concentration, meditation) can differentially control SEAP production of the designer cells in culture and of subcutaneous wireless-powered optogenetic implants in mice.

Two-photon calcium imaging in mice navigating a virtual reality environment.

In recent years, two-photon imaging has become an invaluable tool in neuroscience, as it allows for chronic measurement of the activity of genetically identified cells during behavior(1-6). Here we describe methods to perform two-photon imaging in mouse cortex while the animal navigates a virtual reality environment. We focus on the aspects of the experimental procedures that are key to imaging in a behaving animal in a brightly lit virtual environment. The key problems that arise in this experimental setup that we here address are: minimizing brain motion related artifacts, minimizing light leak from the virtual reality projection system, and minimizing laser induced tissue damage. We also provide sample software to control the virtual reality environment and to do pupil tracking. With these procedures and resources it should be possible to convert a conventional two-photon microscope for use in behaving mice.

Predictive risk factors for intra- and postoperative complications in 526 laparoscopic sigmoid resections due to recurrent diverticulitis: a multivariate analysis.

BACKGROUND: Laparoscopic sigmoid resection is a feasible and frequent operation for patients who suffer from recurrent diverticulitis. There is still an ongoing debate about the optimal timing for surgery in patients who suffer from recurrent diverticulitis episodes. In elective situations the complication rate for this procedure is moderate, but there are patients at high risk for perioperative complications. The few identified risk factors so far refer to open surgery. Data for the elective laparoscopic approach is rare. The objective of this study was to identify potential predictive risk factors for intra- and postoperative complications in patients who underwent laparoscopic sigmoid resection due to diverticular disease. METHODS: Uni- and multivariate analyses of a prospectively gathered database (1993-2006) were performed on a consecutive series of 526 patients who underwent laparoscopic sigmoid resection due to recurrent diverticulitis in a single institution. Patients were assessed for demographic data, operative indications, and intra- and postoperative complications. Altogether, we analyzed 17 potential risk factors to identify significant influence on the intra- and postoperative outcome, including timing of surgery. RESULTS: Statistical analysis of specific medical and surgical complications revealed anemia, previous myocardial infarction, heart failure, experience of the surgeon, and male gender, as independent predictive risk factors for postoperative complications. Patients older than age 75 years was the only independent risk factor for intraoperative complications in a multiple logistic regression model. Early elective surgery led to increased conversion rate but did not influence the postoperative complication rate. CONCLUSIONS: This large, single-center study provides first evidence of the significance of specific predictive risk factors for intra- and postoperative complications in laparoscopic sigmoid resection for diverticular disease.

A multivariate analysis of potential risk factors for intra- and postoperative complications in 1316 elective laparoscopic colorectal procedures.

OBJECTIVE: To identify predictive risk factors for intra- and postoperative complications in patients undergoing laparoscopic colorectal surgery. BACKGROUND DATA: In emergency situations or in elective open and laparoscopic colorectal surgery, there are many risk factors that should be recognized by the surgeon to reduce complications and initiate adequate treatment. Most available data, thus far, refer to open colorectal surgery and literature that focuses mainly on a laparoscopic approach is still rare. METHODS: Univariate and multivariate analyses of a prospectively gathered database (1993-2006) were performed on a consecutive series of patients (1316) undergoing laparoscopic colorectal surgery who were operated at a single institution (first referral center). Patients were assessed for demographic data, operative indications, type of resection, and intra- and postoperative complications. Altogether, we analyzed 20 potential risk factors to identify significant influence on the intra- and postoperative outcome. RESULTS: Significant risk factors that led to intraoperative complications consisted of age > or = 75 years and malignant neoplasia. Increased postoperative rate of surgical complications was significantly influenced by male gender, age > or = 75 years, American Society of Anesthesiology class > or = III, malignant neoplasia, and the experience of the surgeon. The analysis of specific medical postoperative complications revealed even more significant predictive risk factors. In addition, our analysis showed that specific risk factors predict specific complications such as postoperative bleeding, anastomotic leakage, and surgical site infections. The type of surgical procedure performed also influenced patient outcome. CONCLUSION: This large single center study provides the first evidence of the significance of predictive risk factors for intra- and postoperative complications in laparoscopic colorectal surgery.

Safety and efficiency of the Ottawa Ankle Rule in a Swiss population with ankle sprains.

OBJECTIVES: We examined the accuracy of the Ottawa Ankle Rule (OAR) to rule out ankle and mid-foot fractures in patients presenting with acute ankle sprain and differences of accuracy between surgeons and non-surgeons. DESIGN: Prospective cohort study. SETTING: Swiss urban secondary care centre. PARTICIPANTS: Between September 2001 and October 2002 359 patients presented with a case of ankle sprain. Of these, 251 patients both met recruitment criteria and provided data for this study. A group of surgeons and non-surgeons assessed the OAR and all patients underwent blinded radiographic assessment. MAIN OUTCOME MEASURES: Sensitivity, specificity of the OAR. RESULTS: Of the 251 patients with ankle sprains 33 had an ankle fracture (13%) and none had a mid-foot fracture. All cases with a fracture had a positive OAR result (sensitivity 100% 95% CI; 89-100) and of 218 patients without a fracture, the OAR was negative in 45 cases (specificity 21%; 16-27). In the subgroup of patients assessed by surgeons, sensitivity was 100% (77-100) and specificity was 32% (20-46). In the non-surgical group, sensitivity was also 100% (82-100) but specificity was lower (17% (11-23). CONCLUSIONS: This validation study of the OAR in a Swiss setting produced similar results than those published previously in various other settings. We found differences in the performance of the rule between surgical and non-surgical staff indicating that the OAR has its interpretation component which is more difficult to judge properly by well-instructed non-surgical assessors.

Predictors of intra- and postoperative complications in laparoscopic colorectal surgery: results of an expert survey.

BACKGROUND: The decision which patient should undergo laparoscopic rather than open colorectal surgery depends on weighing its benefits against its complications. We explored which criteria prognosticate complications in a laparoscopic intervention by assembling experienced visceral surgeons' beliefs. METHODS: A two-round postal survey was conducted: 21 experts in laparoscopic surgery were contacted and asked to list (first round) and weigh (second round) indicators (scale 1-10) they believed predicted intra- or postoperative complications in patients undergoing laparoscopic colorectal surgery. Median ratings and interquartile ranges (IQRs) were calculated. Rates >or=6 and IQRs

Multidimensional analysis of learning curves in laparoscopic sigmoid resection: eight-year results.

PURPOSE: The number of operations to reach a plateau phase in colorectal laparoscopic surgery is still under debate. There are few publications reporting on multidimensional assessment of the learning curve, including operation time and complication and conversion rates. The purpose of this study was to define a multidimensional learning curve for sigmoid resection performed by two surgeons with experience in laparoscopic surgery. METHODS: Between 1993 and 2001 from a total of 715 laparoscopic colorectal procedures, two surgeons performed self-educated 338 sigmoid resections. Demographic data, indications for surgery, procedure performed, operation time, frequency and kind of complications, conversion rate, and days to discharge were recorded. The moving average method was used to demonstrate the change of the operation time. The cumulative sum technique was used to chart the changes in the conversion and complication rates. RESULTS: Surgeon A performed 199 and Surgeon B 139 sigmoid resections. The operation time decreased from 225 minutes to 169 minutes after approximately 90 operations for Surgeon A and from 270 minutes to 223 minutes after 110 operations for Surgeon B. Based on a decline in intraoperative complications and conversion rate, the steady state was reached after approximately 70 to 80 interventions for both surgeons. CONCLUSIONS: The assessment of a learning curve should not be limited to measurement of a decrease in operation time but should also include the conversion and complication rates. The cumulative sum technique and moving average method as proposed in this study seem appropriate to evaluate the learning curve in this clinical domain. Our findings might be especially useful for those planning training programs in laparoscopic surgery.