A quantitative autonomous bioluminescence reporter system with a wide dynamic range for Plant Synthetic Biology.

Plant Synthetic Biology aims to enhance the capacities of plants by designing and integrating synthetic gene circuits (SGCs). Quantitative reporting solutions that can produce quick, rich datasets affordably are necessary for SGC optimization. In this paper, we present a new, low-cost, and high-throughput reporter system for the quantitative measurement of gene expression in plants based on autonomous bioluminescence. This method eliminates the need for an exogenous supply of luciferase substrate by exploiting the entire Neonothopanus nambi fungal bioluminescence cyclic pathway to build a self-sustained reporter. The HispS gene, the pathway's limiting step, was set up as the reporter's transcriptional entry point as part of the new system's design, which significantly improved the output's dynamic range and brought it on par with that of the gold standard FLuc/RLuc reporter. Additionally, transient ratiometric measurements in N. benthamiana were made possible by the addition of an enhanced GFP as a normalizer. The performance of new NeoLuc/eGFP system was extensively validated with SGCs previously described, including phytohormone and optogenetic sensors. Furthermore, we employed NeoLuc/eGFP in the optimization of challenging SGCs, including new configurations for an agrochemical (copper) switch, a new blue optogenetic sensor, and a dual copper/red-light switch for tight regulation of metabolic pathways.

Tunable control of insect pheromone biosynthesis in Nicotiana benthamiana.

Previous work has demonstrated that plants can be used as production platforms for molecules used in health, medicine, and agriculture. Production has been exemplified in both stable transgenic plants and using transient expression strategies. In particular, species of Nicotiana have been engineered to produce a range of useful molecules, including insect sex pheromones, which are valued for species-specific control of agricultural pests. To date, most studies have relied on strong constitutive expression of all pathway genes. However, work in microbes has demonstrated that yields can be improved by controlling and balancing gene expression. Synthetic regulatory elements that provide control over the timing and levels of gene expression are therefore useful for maximizing yields from heterologous biosynthetic pathways. In this study, we demonstrate the use of pathway engineering and synthetic genetic elements for controlling the timing and levels of production of Lepidopteran sex pheromones in Nicotiana benthamiana. We demonstrate that copper can be used as a low-cost molecule for tightly regulated inducible expression. Further, we show how construct architecture influences relative gene expression and, consequently, product yields in multigene constructs. We compare a number of synthetic orthogonal regulatory elements and demonstrate maximal yields from constructs in which expression is mediated by dCas9-based synthetic transcriptional activators. The approaches demonstrated here provide new insights into the heterologous reconstruction of metabolic pathways in plants.

A copper switch for inducing CRISPR/Cas9-based transcriptional activation tightly regulates gene expression in Nicotiana benthamiana.

BACKGROUND: CRISPR-based programmable transcriptional activators (PTAs) are used in plants for rewiring gene networks. Better tuning of their activity in a time and dose-dependent manner should allow precise control of gene expression. Here, we report the optimization of a Copper Inducible system called CI-switch for conditional gene activation in Nicotiana benthamiana. In the presence of copper, the copper-responsive factor CUP2 undergoes a conformational change and binds a DNA motif named copper-binding site (CBS). RESULTS: In this study, we tested several activation domains fused to CUP2 and found that the non-viral Gal4 domain results in strong activation of a reporter gene equipped with a minimal promoter, offering advantages over previous designs. To connect copper regulation with downstream programmable elements, several copper-dependent configurations of the strong dCasEV2.1 PTA were assayed, aiming at maximizing activation range, while minimizing undesired background expression. The best configuration involved a dual copper regulation of the two protein components of the PTA, namely dCas9:EDLL and MS2:VPR, and a constitutive RNA pol III-driven expression of the third component, a guide RNA with anchoring sites for the MS2 RNA-binding domain. With these optimizations, the CI/dCasEV2.1 system resulted in copper-dependent activation rates of 2,600-fold and 245-fold for the endogenous N. benthamiana DFR and PAL2 genes, respectively, with negligible expression in the absence of the trigger. CONCLUSIONS: The tight regulation of copper over CI/dCasEV2.1 makes this system ideal for the conditional production of plant-derived metabolites and recombinant proteins in the field.

Insect pest management in the age of synthetic biology.

Arthropod crop pests are responsible for 20% of global annual crop losses, a figure predicted to increase in a changing climate where the ranges of numerous species are projected to expand. At the same time, many insect species are beneficial, acting as pollinators and predators of pest species. For thousands of years, humans have used increasingly sophisticated chemical formulations to control insect pests but, as the scale of agriculture expanded to meet the needs of the global population, concerns about the negative impacts of agricultural practices on biodiversity have grown. While biological solutions, such as biological control agents and pheromones, have previously had relatively minor roles in pest management, biotechnology has opened the door to numerous new approaches for controlling insect pests. In this review, we look at how advances in synthetic biology and biotechnology are providing new options for pest control. We discuss emerging technologies for engineering resistant crops and insect populations and examine advances in biomanufacturing that are enabling the production of new products for pest control.

Studies on the biological role of the antifungal protein PeAfpA from Penicillium expansum by functional gene characterization and transcriptomic profiling.

Antifungal proteins (AFPs) from filamentous fungi have enormous potential as novel biomolecules for the control of fungal diseases. However, little is known about the biological roles of AFPs beyond their antifungal action. Penicillium expansum encodes three phylogenetically different AFPs (PeAfpA, PeAfpB and PeAfpC) with diverse profiles of antifungal activity. PeAfpA stands out as a highly active AFP that is naturally produced at high yields. Here, we provide new data about the function of PeAfpA in P. expansum through phenotypical characterization and transcriptomic studies of null mutants of the corresponding afpA gene. Mutation of afpA did not affect axenic growth, conidiation, virulence, stress responses or sensitivity towards P. expansum AFPs. However, RNA sequencing evidenced a massive transcriptomic change linked to the onset of PeAfpA production. We identified two large gene expression clusters putatively involved in PeAfpA function, which correspond to genes induced or repressed with the production of PeAfpA. Functional enrichment analysis unveiled significant changes in genes related to fungal cell wall remodeling, mobilization of carbohydrates and plasma membrane transporters. This study also shows a putative co-regulation between the three afp genes. Overall, our transcriptomic analyses provide valuable insights for further understanding the biological functions of AFPs.

Increased salt concentration promotes competitive block of OmpF channel by protons.

Porins are channel-forming proteins that are located in the outer membranes (OM) of Gram-negative bacteria and allow the influx of hydrophilic nutrients and the extrusion of waste products. The fine regulation of the ion transport through these wide channels could play an important role in the survival of the bacteria in acidic media. We investigate here the mechanism responsible for the pH sensitivity of the trimeric porin OmpF, of Escherichia coli. Planar lipid bilayer electrophysiology and site-directed mutagenesis were used to study the effect of pH on the ion conductive properties of the OmpF channel in its fully open, "nongated" conformation. At low pH we observe a large drop in the OmpF open channel conductance that is accompanied by a substantial increase of the current noise. These channel features are strongly dependent on the salt concentration and we propose that they are originated by competitive binding of cations and protons occurring in the narrow central constriction of the channel. This subtle mechanism reveals to be capital for the channel function because it not only drives the channel sensitivity to pH but is also indispensable for the particularly efficient permeation mechanism of the channel at physiological conditions (~neutral pH).

The European medicines agency review of abiraterone for the treatment of metastatic castration-resistant prostate cancer in adult men after docetaxel chemotherapy and in chemotherapy-naive disease: summary of the scientific assessment of the committee for medicinal products for human use.

On September 5, 2011, abiraterone was approved in the European Union in combination with prednisone or prednisolone for the treatment of metastatic castration-resistant prostate cancer (CRPC) in adult men whose disease has progressed on or after a docetaxel-based chemotherapy regimen. On December 18, 2012, the therapeutic indication was extended to include the use of abiraterone in combination with prednisone or prednisolone for the treatment of metastatic CRPC in adult men who are asymptomatic or mildly symptomatic after failure of androgen deprivation therapy in whom chemotherapy is not yet clinically indicated. Abiraterone is a selective, irreversible inhibitor of cytochrome P450 17α, an enzyme that is key in the production of androgens. Inhibition of androgen biosynthesis deprives prostate cancer cells from important signals for growth, even in cases of resistance to castration. At the time of European Union approval and in a phase III trial in CRPC patients who had failed at least one docetaxel-based chemotherapy regimen, median overall survival for patients treated with abiraterone was 14.8 months versus 10.9 months for those receiving placebo (hazard ratio, 0.65; 95% confidence interval 0.54-0.77; p < .0001). In a subsequent phase III trial in a similar but chemotherapy-naïve patient population, median radiographic progression-free survival was 16.5 months for patients in the abiraterone treatment arm versus 8.3 months for patients in the placebo arm (hazard ratio, 0.53; 95% confidence interval, 0.45-0.62; p < .0001). Abiraterone was most commonly associated with adverse reactions resulting from increased or excessive mineralocorticoid activity. These were generally manageable with basic medical interventions. The most common side effects (affecting more than 10% of patients) were urinary tract infection, hypokalemia, hypertension, and peripheral edema.

FungalBraid 2.0: expanding the synthetic biology toolbox for the biotechnological exploitation of filamentous fungi.

Fungal synthetic biology is a rapidly expanding field that aims to optimize the biotechnological exploitation of fungi through the generation of standard, ready-to-use genetic elements, and universal syntax and rules for contributory use by the fungal research community. Recently, an increasing number of synthetic biology toolkits have been developed and applied to filamentous fungi, which highlights the relevance of these organisms in the biotechnology field. The FungalBraid (FB) modular cloning platform enables interchangeability of DNA parts with the GoldenBraid (GB) platform, which is designed for plants, and other systems that are compatible with the standard Golden Gate cloning and syntax, and uses binary pCAMBIA-derived vectors to allow Agrobacterium tumefaciens-mediated transformation of a wide range of fungal species. In this study, we have expanded the original FB catalog by adding 27 new DNA parts that were functionally validated in vivo. Among these are the resistance selection markers for the antibiotics phleomycin and terbinafine, as well as the uridine-auxotrophic marker pyr4. We also used a normalized luciferase reporter system to validate several promoters, such as PpkiA, P7760, Pef1α, and PafpB constitutive promoters, and PglaA, PamyB, and PxlnA inducible promoters. Additionally, the recently developed dCas9-regulated GB_SynP synthetic promoter collection for orthogonal CRISPR activation (CRISPRa) in plants has been adapted in fungi through the FB system. In general, the expansion of the FB catalog is of great interest to the scientific community since it increases the number of possible modular and interchangeable DNA assemblies, exponentially increasing the possibilities of studying, developing, and exploiting filamentous fungi.

The autologous chondral platelet-rich plasma matrix implantation. A new therapy in cartilage repair and regeneration: macroscopic and biomechanical study in an experimental sheep model.

Introduction: Articular cartilage injuries are a severe problem, and the treatments for these injuries are complex. The present study investigates a treatment for full-thickness cartilage defects called Autologous Chondral Platelet Rich Plasma Matrix Implantation (PACI) in a sheep model. Methods: Chondral defects 8 mm in diameter were surgically induced in the medial femoral condyles of both stifles in eight healthy sheep. Right stifles were treated with PACI and an intraarticular injection with a plasma rich in growth factors (PRGF) solution [treatment group (TRT)], while an intraarticular injection of Ringer's lactate solution was administered in left stifles [Control group (CT)]. The limbs' function was objectively assessed with a force platform to obtain the symmetry index, comparing both groups. After 9 and 18 months, the lesions were macroscopically evaluated using the International Cartilage Repair Society and Goebel scales. Results: Regarding the symmetry index, the TRT group obtained results similar to those of healthy limbs at 9 and 18 months after treatment. Regarding the macroscopic assessment, the values obtained by the TRT group were very close to those of normal cartilage and superior to those obtained by the CT group at 9 months. Conclusion: This new bioregenerative treatment modality can regenerate hyaline articular cartilage. High functional outcomes have been reported, together with a good quality repair tissue in sheep. Therefore, PACI treatment might be a good therapeutic option for full-thickness chondral lesions.

GB_SynP: A Modular dCas9-Regulated Synthetic Promoter Collection for Fine-Tuned Recombinant Gene Expression in Plants.

Programmable transcriptional factors based on the CRISPR architecture are becoming commonly used in plants for endogenous gene regulation. In plants, a potent CRISPR tool for gene induction is the so-called dCasEV2.1 activation system, which has shown remarkable genome-wide specificity combined with a strong activation capacity. To explore the ability of dCasEV2.1 to act as a transactivator for orthogonal synthetic promoters, a collection of DNA parts was created (GB_SynP) for combinatorial synthetic promoter building. The collection includes (i) minimal promoter parts with the TATA box and 5'UTR regions, (ii) proximal parts containing single or multiple copies of the target sequence for the gRNA, thus functioning as regulatory cis boxes, and (iii) sequence-randomized distal parts that ensure the adequate length of the resulting promoter. A total of 35 promoters were assembled using the GB_SynP collection, showing in all cases minimal background and predictable activation levels depending on the proximal parts used. GB_SynP was also employed in a combinatorial expression analysis of an autoluminescence pathway in Nicotiana benthamiana, showing the value of this tool in extracting important biological information such as the determination of the limiting steps in an enzymatic pathway.

Transcriptional deregulation of stress-growth balance in Nicotiana benthamiana biofactories producing insect sex pheromones.

Plant biofactories are a promising platform for sustainable production of high-value compounds, among which are insect sex pheromones, a green alternative to conventional insecticides in agriculture. Recently, we have constructed transgenic Nicotiana benthamiana plants ("Sexy Plants", SxP) that successfully produce a blend of moth (Lepidoptera) sex pheromone compounds (Z)-11-hexadecen-1-ol and (Z)-11-hexadecenyl acetate. However, efficient biosynthesis of sex pheromones resulted in growth and developmental penalty, diminishing the potential for commercial use of SxP in biomanufacturing. To gain insight into the underlying molecular responses, we analysed the whole-genome transcriptome and evaluated it in relation to growth and pheromone production in low- and high-producing transgenic plants of v1.0 and v1.2 SxP lines. In our study, high-producing SxPv1.2 plants accumulated the highest amounts of pheromones but still maintained better growth compared to v1.0 high producers. For an in-depth biological interpretation of the transcriptomic data, we have prepared a comprehensive functional N. benthamiana genome annotation as well as gene translations to Arabidopsis thaliana, enabling functional information transfer by using Arabidopsis knowledge networks. Differential gene expression analysis, contrasting pheromone producers to wild-type plants, revealed that while only a few genes were differentially regulated in low-producing plants, high-producing plants exhibited vast transcriptional reprogramming. They showed signs of stress-like response, manifested as downregulation of photosynthesis-related genes and significant differences in expression of hormonal signalling and secondary metabolism-related genes, the latter presumably leading to previously reported volatilome changes. Further network analyses confirmed stress-like response with activation of jasmonic acid and downregulation of gibberellic acid signalling, illuminating the possibility that the observed growth penalty was not solely a consequence of a higher metabolic burden imposed upon constitutive expression of a heterologous biosynthetic pathway, but rather the result of signalling pathway perturbation. Our work presents an example of comprehensive transcriptomic analyses of disadvantageous stress signalling in N. benthamiana biofactory that could be applied to other bioproduction systems.

Appropriateness Criteria for Ureteral Stent Omission following Ureteroscopy for Urinary Stone Disease.

Objective: To bridge the gap between evidence and clinical judgement, we defined scenarios appropriate for ureteral stent omission after uncomplicated ureteroscopy (URS) using the RAND/UCLA Appropriateness Method (RAM). We retrospectively assessed rates of appropriate stent omission, with the goal to implement these criteria in clinical practice. Methods: A panel of 15 urologists from the Michigan Urological Surgery Improvement Collaborative (MUSIC) met to define uncomplicated URS and the variables that influence stent omission decision-making. Over two rounds, they scored clinical scenarios for Appropriateness Criteria (AC) for stent omission based on a combination of variables. AC were defined by median scores: 1 to 3 (inappropriate), 4 to 6 (uncertain), and 7 to 9 (appropriate). Multivariable analysis determined the association of each variable with AC scores. Uncomplicated URS cases in the MUSIC registry were assigned AC scores and stenting rates assessed. Results: Seven variables affecting stent decision-making were identified. Of the 144 scenarios, 26 (18%) were appropriate, 88 (61%) inappropriate, and 30 (21%) uncertain for stent omission. Most scenarios appropriate for omission were pre-stented (81%). Scenarios with ureteral access sheath or stones >10mm were only appropriate if pre-stented. Stenting rates of 5,181 URS cases correlated with AC scores. Stents were placed in 61% of cases appropriate for omission (practice range, 25% to 98%). Conclusion: We defined objective variables and AC for stent omission following uncomplicated URS. AC scores correlated with stenting rates but there was substantial practice variation. Our findings demonstrate that the appropriate use of stent omission is underutilized.

Development of a FungalBraid Penicillium expansum-based expression system for the production of antifungal proteins in fungal biofactories.

Fungal antifungal proteins (AFPs) have attracted attention as novel biofungicides. Their exploitation requires safe and cost-effective producing biofactories. Previously, Penicillium chrysogenum and Penicillium digitatum produced recombinant AFPs with the use of a P. chrysogenum-based expression system that consisted of the paf gene promoter, signal peptide (SP)-pro sequence and terminator. Here, the regulatory elements of the afpA gene encoding the highly produced PeAfpA from Penicillium expansum were developed as an expression system for AFP production through the FungalBraid platform. The afpA cassette was tested to produce PeAfpA and P. digitatum PdAfpB in P. chrysogenum and P. digitatum, and its efficiency was compared to that of the paf cassette. Recombinant PeAfpA production was only achieved using the afpA cassette, being P. chrysogenum a more efficient biofactory than P. digitatum. Conversely, P. chrysogenum only produced PdAfpB under the control of the paf cassette. In P. digitatum, both expression systems allowed PdAfpB production, with the paf cassette resulting in higher protein yields. Interestingly, these results did not correlate with the performance of both promoters in a luciferase reporter system. In conclusion, AFP production is a complex outcome that depends on the regulatory sequences driving afp expression, the fungal biofactory and the AFP sequence.

In-Depth Characterization of greenflesh Tomato Mutants Obtained by CRISPR/Cas9 Editing: A Case Study With Implications for Breeding and Regulation.

Gene editing has already proved itself as an invaluable tool for the generation of mutants for crop breeding, yet its ultimate impact on agriculture will depend on how crops generated by gene editing technologies are regulated, and on our ability to characterize the impact of mutations on plant phenotype. A starting operational strategy for evaluating gene editing-based approaches to plant breeding might consist of assessing the effect of the induced mutations in a crop- and locus-specific manner: this involves the analysis of editing efficiency in different cultivars of a crop, the assessment of potential off-target mutations, and a phenotypic evaluation of edited lines carrying different mutated alleles. Here, we targeted the GREENFLESH (GF) locus in two tomato cultivars ('MoneyMaker' and 'San Marzano') and evaluated the efficiency, specificity and mutation patterns associated with CRISPR/Cas9 activity for this gene. The GF locus encodes a Mg-dechelatase responsible for initiating chlorophyll degradation; in gf mutants, ripe fruits accumulate both carotenoids and chlorophylls. Phenotypic evaluations were conducted on two transgene-free T2 'MoneyMaker' gf lines with different mutant alleles (a small insertion of 1 nucleotide and a larger deletion of 123 bp). Both lines, in addition to reduced chlorophyll degradation, showed a notable increase in carotenoid and tocopherol levels during fruit ripening. Infection of gf leaves and fruits with Botrytis cinerea resulted in a significant reduction of infected area and pathogen proliferation compared to the wild type (WT). Our data indicates that the CRISPR/Cas9-mediated mutation of the GF locus in tomato is efficient, specific and reproducible and that the resulting phenotype is robust and consistent with previously characterized greenflesh mutants obtained with different breeding techniques, while also shedding light on novel traits such as vitamin E overaccumulation and pathogen resistance. This makes GF an appealing target for breeding tomato cultivars with improved features for cultivation, as well as consumer appreciation and health.

Linearity, saturation and blocking in a large multiionic channel: divalent cation modulation of the OmpF porin conductance.

Measurement of unitary conductance is a fundamental step in the characterization of a protein ion channel permeabilizing a membrane. We study here the effect of salts of divalent cations on the OmpF channel conductance with a particular emphasis in dissecting the role of the electrolyte itself, the role of the counterion accumulation induced by the protein channel charges and other effects not found in salts of monovalent cations. We show that current saturation and blocking are not exclusive properties of narrow (single-file) ion channels but may be observed in large, multiionic channels like bacterial porins. Single-channel conductance measurements performed over a wide range of salt concentrations (up to 3 M) combined with continuum electrodiffusion calculations demonstrate that current saturation cannot be simply ascribed to ion interaction with protein channel residues.

Divalent cations reduce the pH sensitivity of OmpF channel inducing the pK(a) shift of key acidic residues.

In contrast to the highly-selective channels of neurophysiology employing mostly the exclusion mechanism, different factors account for the selectivity of large channels. Elucidation of these factors is essential for understanding the permeation mechanisms in ion channels and their regulation in vivo. The interaction between divalent cations and a protein channel, the bacterial porin OmpF, has been investigated paying attention to the channel selectivity and its dependence on the solution pH. Unlike the experiments performed in salts of monovalent cations, the channel is now practically insensitive to pH, being anion selective all over the pH range considered. Electrostatic calculations based on the available structural data suggest that the binding of divalent cations has two main effects: (i) the pK(a) values of key ionizable groups differ significantly from those of the isolated groups in solution and (ii) the cation binding has a decisive impact on the effective electric charge regulating the channel selectivity. A simple molecular model based on statistical thermodynamics provides additional qualitative explanations to the experimental findings that could also be useful for other related systems like synthetic nanopores, ion exchange membranes, and polyelectrolyte multilayers.

Production of Volatile Moth Sex Pheromones in Transgenic Nicotiana benthamiana Plants.

Plant-based bioproduction of insect sex pheromones has been proposed as an innovative strategy to increase the sustainability of pest control in agriculture. Here, we describe the engineering of transgenic plants producing (Z)-11-hexadecenol (Z11-16OH) and (Z)-11-hexadecenyl acetate (Z11-16OAc), two main volatile components in many Lepidoptera sex pheromone blends. We assembled multigene DNA constructs encoding the pheromone biosynthetic pathway and stably transformed them into Nicotiana benthamiana plants. The constructs contained the Amyelois transitella AtrΔ11 desaturase gene, the Helicoverpa armigera fatty acyl reductase HarFAR gene, and the Euonymus alatus diacylglycerol acetyltransferase EaDAct gene in different configurations. All the pheromone-producing plants showed dwarf phenotypes, the severity of which correlated with pheromone levels. All but one of the recovered lines produced high levels of Z11-16OH, but very low levels of Z11-16OAc, probably as a result of recurrent truncations at the level of the EaDAct gene. Only one plant line (SxPv1.2) was recovered that harboured an intact pheromone pathway and which produced moderate levels of Z11-16OAc (11.8 µg g-1 FW) and high levels of Z11-16OH (111.4 µg g-1). Z11-16OAc production was accompanied in SxPv1.2 by a partial recovery of the dwarf phenotype. SxPv1.2 was used to estimate the rates of volatile pheromone release, which resulted in 8.48 ng g-1 FW per day for Z11-16OH and 9.44 ng g-1 FW per day for Z11-16OAc. Our results suggest that pheromone release acts as a limiting factor in pheromone biodispenser strategies and establish a roadmap for biotechnological improvements.

Laparoendoscopic single site live donor nephrectomy: initial experience.

PURPOSE: We present our initial experience in 40 patients undergoing laparoendoscopic single site donor nephrectomy. MATERIALS AND METHODS: We prospectively collected data on 40 consecutive patients. A single access GelPOINT™ device was inserted into the abdomen through a 4 to 5 cm periumbilical incision. We used a bariatric camera with a right angle attachment for the light cord to maximize triangulation. Parameters analyzed included warm ischemia time, operative time, estimated blood loss, visual analog pain score, time to recipient creatinine less than 3 mg/dl, and recipient creatinine at discharge home, and 3 and 6 months. RESULTS: A total of 38 left and 2 right donor nephrectomies were performed. Complete laparoendoscopic single site donor nephrectomy was successful in 38 cases. One left and 1 right case were converted to a hand assisted approach. Average ± SD body mass index was 26.1 ± 5.2 kg/m(2). Mean operative time to allograft extraction was 93.5 ± 27.5 minutes and mean total operative time was 166.7 ± 33.8 minutes. Average estimated blood loss was 106.7 ± 93.5 cc. Mean warm ischemia time was 3.96 ± 0.72 minutes. Mean hospital stay was 1.77 ± 0.43 days and median time to recipient creatinine less than 3.0 mg/dl was 54.2 ± 110.3 hours. Mean recipient creatinine at discharge home, and at 3 and 6 months was 1.48 ± 0.67, 1.29 ± 0.38 and 1.19 ± 0.34 mg/dl, respectively. Complications included hyponatremia in 1 patient, wound infection in 1, and a grade III laceration in an allograft that was sustained during extraction. CONCLUSIONS: Our initial experience with laparoendoscopic single site donor nephrectomy is encouraging. This approach to kidney donation without an extra-umbilical incision could become particularly relevant to minimize morbidity in young, healthy organ donors.

Diffusion, exclusion, and specific binding in a large channel: a study of OmpF selectivity inversion.

We find that moderate cationic selectivity of the general bacterial porin OmpF in sodium and potassium chloride solutions is inversed to anionic selectivity in concentrated solutions of barium, calcium, nickel, and magnesium chlorides. To understand the origin of this phenomenon, we consider several factors, which include the binding of divalent cations, electrostatic and steric exclusion of differently charged and differently sized ions, size-dependent hydrodynamic hindrance, electrokinetic effects, and significant "anionic" diffusion potential for bulk solutions of chlorides of divalent cations. Though all these factors contribute to the measured selectivity of this large channel, the observed selectivity inversion is mostly due to the following two. First, binding divalent cations compensates, or even slightly overcompensates, for the negative charge of the OmpF protein, which is known to be the main cause of cationic selectivity in sodium and potassium chloride solutions. Second, the higher anionic (versus cationic) transport rate expected for bulk solutions of chloride salts of divalent cations is the leading cause of the measured anionic selectivity of the channel. Interestingly, at high concentrations the binding of cations does not show any pronounced specificity within the divalent series because the reversal potentials measured in the series correlate well with the corresponding bulk diffusion potentials. Thus our study shows that, in contrast to the highly selective channels of neurophysiology that employ mostly the exclusion mechanism, quite different factors account for the selectivity of large channels. The elucidation of these factors is essential for understanding large channel selectivity and its regulation in vivo.

Cutaneous renocolic fistula associated with diverticulitis.

We report a case of a cutaneous renocolic fistula in a patient with staghorn calculus and diverticulitis. The most common origins of renocolic fistula are primary renal diseases including xanthogranulomatous pyelonephritis, trauma, malignancy or tuberculosis. While diverticulitis has rarely been associated with renocolic fistula, previous instances of fistulae have been noted in patients with simultaneous kidney disease. Inflammation resulting from kidney disease may place patients with colonic diverticulitis at higher risk for developing renocolic or cutaneous renocolic fistulas.