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ABSTRACT European river basin authorities are responsible for the implementation of the new river basin management plans in accordance with the European Water Framework Directive. This paper presents a new methodology framework and approach to define and evaluate environmental flow regimes in the realistic complexities that exist with multiple water resource needs at a basin scale. This approach links river basin simulation models and habitat time series analysis to generate ranges of environmental flows (e-flows), which are evaluated by using habitat, hydropower production and reliability of water supply criteria to produce best possible alternatives. With the use of these tools, the effects of the proposed e-flows have been assessed to help in the consultation process. The possible effects analysed are impacts on water supply reliability, hydropower production and aquatic habitat. After public agreements, a heuristic optimization process was applied to maximize e-flows and habitat indicators, while maintaining a legal level of reliability for water resource demands. The final optimal e-flows were considered for the river basin management plans of the Duero river basin. This paper demonstrates the importance of considering quantitative hydrologic and ecological aspects of e-flows at the basin scale in addressing complex water resource systems. This approach merges standard methods such as physical habitat simulations and time series analyses for evaluating alternatives, with recent methods to simulate and optimize water management alternatives in river networks. It can be integrated with or used to complement other frameworks for e-flow assessments such as the In-stream Flow Incremental Methodology and Ecological Limits of Hydrologic Alteration. Copyright © 2011 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

We have computed the pKa’s of the ionizable residues of a protein ion channel, the Staphylococcus aureus toxin $ \alpha$ -hemolysin, by using two types of input structures, namely the crystal structure of the heptameric $ \alpha$ -hemolysin and a set of over four hundred snapshots from a 4.38ns Molecular Dynamics simulation of the protein inserted in a phospholipid planar bilayer. The comparison of the dynamic picture provided by the Molecular Simulation with the static one based on the X-ray crystal structure of the protein embedded in a lipid membrane allows analyzing the influence of the fluctuations in the protein structure on its ionization properties. We find that the use of the dynamic structure provides interesting information about the sensitivity of the computed pKa of a given residue to small changes in the local structure. The calculated pKa are consistent with previous indirect estimations obtained from single-channel conductance and selectivity measurements. [ABSTRACT FROM AUTHOR]

Nonstructural protein 2B of foot-and-mouth disease (FMD) virus (FMDV) is comprised of a small, hydrophobic, 154-amino-acid protein. Structurefunction analyses demonstrated that FMDV 2B is an ion channel-forming protein. Infrared spectroscopy measurements using partially overlapping peptides that spanned regions between amino acids 28 and 147 demonstrated the adoption of helical conformations in two putative transmembrane regions between residues 60 and 78 and between residues 119 and 147 and a third transmembrane region between residues 79 and 106, adopting a mainly extended structure. Using synthetic peptides, ion channel activity measurements in planar lipid bilayers and imaging of single giant unilamellar vesicles (GUVs) revealed the existence of two sequences endowed with membrane-porating activity: one spanning FMDV 2B residues 55 to 82 and the other spanning the C-terminal region of 2B from residues 99 to 147. Mapping the latter sequence identified residues 119 to 147 as being responsible for the activity. Experiments to assess the degree of insertion of the synthetic peptides in bilayers and the inclination angle adopted by each peptide regarding the membrane plane normal confirm that residues 55 to 82 and 119 to 147 of 2B actively insert as transmembrane helices. Using reverse genetics, a panel of 13 FMD recombinant mutant viruses was designed, which harbored nonconservative as well as alanine substitutions in critical amino acid residues in the area between amino acid residues 28 and 147. Alterations to any of these structures interfered with pore channel activity and the capacity of the protein to permeabilize the endoplasmic reticulum (ER) to calcium and were lethal for virus replication. Thus, FMDV 2B emerges as the first member of the viroporin family containing two distinct pore domains. [ABSTRACT FROM AUTHOR]

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) envelope (E) protein forms a pentameric ion channel in the lipid membrane of the endoplasmic reticulum Golgi intermediate compartment (ERGIC) of the infected cell. The cytoplasmic domain of E interacts with host proteins to cause virus pathogenicity and may also mediate virus assembly and budding. To understand the structural basis of these functions, here we investigate the conformation and dynamics of an E protein construct (residues 8-65) that encompasses the transmembrane domain and the majority of the cytoplasmic domain using solid-state NMR. 13 C and 15 N chemical shifts indicate that the cytoplasmic domain adopts a β-sheet-rich conformation that contains three β-strands separated by turns. The five subunits associate into an umbrella-shaped bundle that is attached to the transmembrane helices by a disordered loop. Water-edited NMR spectra indicate that the third β-strand at the C terminus of the protein is well hydrated, indicating that it is at the surface of the β-bundle. The structure of the cytoplasmic domain cannot be uniquely determined from the inter-residue correlations obtained here due to ambiguities in distinguishing intermolecular and intramolecular contacts for a compact pentameric assembly of this small domain. Instead, we present four structural topologies that are consistent with the measured inter-residue contacts. These data indicate that the cytoplasmic domain of the SARS-CoV-2 E protein has a strong propensity to adopt β-sheet conformations when the protein is present at high concentrations in lipid bilayers. The equilibrium between the β-strand conformation and the previously reported α-helical conformation may underlie the multiple functions of E in the host cell and in the virion., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Purpose: We analyzed all patients who underwent local transanal surgery at our institution to determine oncological outcomes and perioperative risk., Methods: In 1997, we developed a prospective protocol for rectal tumors: transanal local full-thickness excision was considered curative in patients with benign adenoma and early cancers. In this analysis, 404 patients were included. To analyze survival, only those patients exposed to the risk of dying for at least 5 years were considered for the study., Results: The final pathological analysis revealed that 262 (64.8%) patients had benign lesions, whereas 142 had malignant lesions. Postoperative complications were recorded in 12.6%. At the median time of 21 months, 14% of the adenomas and 12% of cancers had recurred, half of which were surgically resected. The overall 5-year survival rate was 94%., Conclusion: With similar outcomes and significantly lower morbidity, we found local surgery to be an adequate alternative to radical surgery in selected cases of early rectal cancer., (© 2022. The Author(s).)

Local surgery for rectal tumors has been traditionally limited due to technical issues for lower risk lesions. In the 1980s, Buess described the transanal endoscopic microsurgery (TEM) technique that enabled excision of lesions that were larger and in a higher location. TEM in a short time became the gold standard for the excision of large adenomas and early rectal carcinomas. Since the advent of TEM, and due to advances in minimal invasive technology, new devices and procedures have come to the surgical armamentarium, in many cases with only very slight differences between them. However, local surgery for rectal cancer still remains a challenge. An overview of the current status of minimal transanal invasive techniques is presented here and sheds some light on the controversies that the local management of rectal cancer still raises.

Purpose: Local excision of malignant rectal tumors remains controversial due to the lack of prospective studies. The principal aim of this paper is to analyze survival and recurrence of patients with rectal cancer who were operated by transanal endoscopic microsurgery with curative intention., Methods: In 1997, we started a prospective protocol for patients who had T1/T2 rectal tumors: transanal local full-thickness excision was considered curative in T1 low risk (group A); patients with T1 high-risk and T2 low-risk tumors received postoperative radiotherapy (group B). From 1997 to 2006, 88 patients were enrolled. Sixty eight entered the study after the preoperative workup and 20 patients with an initial diagnosis of adenoma after postoperative definitive pathological assessment., Results: After definitive histological findings, 54 patients were to group A, 28 to group B, and 6 had immediate radical surgery. One patient was lost for follow-up. At a mean follow-up of 71 months, 7 (4 from group A and 3 from group B) out of 81 patients recurred. Five-year overall survival was of 94% and cancer-specific survival of 96%., Conclusions: Our data support that transanal endoscopic microsurgery is an adequate treatment for T1 low-risk tumor, and no additional measures are required. For T2 low-risk lesions, our study showed a higher local recurrence rate than that reported after radical surgery but a similar survival outcome.

Objective: Removing rectal adenomas not only relieves symptoms, but also eradicates the incidence of carcinoma. There are many techniques for local removal of rectal polyps. Transanal endoscopic microsurgery (TEM) is the most recent. The purpose of this study is to present our long-term results using TEM for rectal adenomas, paying special attention to the risk factors of harboring a malignancy., Methods: Data from all patients undergoing TEM from December 1995 to December 2005 were collected prospectively. The selection criteria were benign sessile adenomas below the peritoneal reflection. In the study period, 173 patients were operated on for an apparently benign rectal adenoma. The mean distance of lower tumor was 7.6 cm (range, 1-18 cm), and the mean distance to upper edge was 11 cm (2-20 cm). Full-thickness local excision was performed in all procedures. Patients were followed for a minimum of 1 year., Results: According to the histologic findings, 14% of the specimens were invasive carcinomas. No statistical differences were found when comparing the histologic findings by tumor size, distance to the anal verge, or location.In 10 (5.8%) cases, the dissection was considered uncompleted because of a normal mucosa margin smaller than 1 mm. The mean hospital stay was 4 days (2-30 days). The morbidity rate was 14.5%. There was 1 postoperative death (0.6%). There were 9 (5.4%) histologically proven recurrences. Four of the patients with recurrence had uncompleted microscopic circumferential resection (P = 0.001). At a mean follow-up of 35 months (range, 12-82 months), all carcinoma patients were alive with no evidence of disease., Conclusions: In conclusion, a significant number of adenomas that we assumed preoperatively to be benign were already carcinomas and we were unable to find any reliable predictor to identify them. TEM full-thickness excision provided a low rate of postoperative morbidity and potentially avoided a significant number of major abdominal operations and local recurrences.

Background: Laparoscopic gastric bypass, currently the most popular surgical method for bariatric therapy, have proved to be effective in weight loss, but some matters regarding its long-term efficacy for super-obese patients (BMI >50 kg/m2) have arisen. Biliopancreatic diversion (BPD) is a complex technique that has shown good results in the treatment of the super-obese patient. We analyze our >5 years results, evaluating weight loss, morbidity and mortality of this operation, depending on the length of the common and alimentary limbs., Methods: We studied two series of patients: 150 patients with BPD of Scopinaro (50-200 cm) and 70 patients with modified BPD (75-225 cm). The results have been analyzed in terms of weight loss, co-morbidity improvement, and postoperative morbidity using BAROS., Results: Range of follow-up is 1-12 years. Weight loss was slightly higher for the Scopinaro group than for the Modified group but with no significant difference. There was more prevalence of malnutrition and of iron deficiency in the Scopinaro group (16% and 60%) than in the modified group (2% and 40%), with similar postoperative morbidities., Conclusion: The modified BPD method (75-225 cm) shows long-term effectiveness in weight loss and comorbidity improvement for super-obesity. Proteins, vitamins and oligoelement deficits appear distant in time, and thus it is necessary to maintain strict followup of these patients and supplement against deficiencies for the rest of their lives.

Objective: The endoanal sonography in female patients with faecal incontinence is sometimes difficult and can lead to diagnostic errors. The aim of this study is to evaluate the value of endovaginal sonography in such cases., Material and Method: Thirty female patients complaining of faecal incontinence are included in the study. Anal endosonography was performed in all of them in a single ambulatory session, pictures were taken from all along the anal and results were analyzed afterward. Vaginal endosonography was then performed using the same equipment. Result from both techniques were compared., Results: Endoanal sonography was performed in all 30 patients. In 17 cases no anomalies were found. In 3 patients a simple internal anal sphincter defect was found. One case showed a lateral lesion in both sphincters. Six cases presented anterior external defect and in the rest 3 cases a clear view of the anterior wall was impossible. Vaginal endosonography shows a clear image of the anal canal in 23 out of 30 patients. In two cases changed the results of anal endosonography., Conclusion: In the study of faecal incontinence, despite of its technical limitations, endovaginal ultrasound could be of help when the anterior wall of the anal canal is not properly defined.

Objective: The aim of this study is to clinically test the efficacy of a new approach for patients having symptomatic grade III and IV hemorrhoids., Material and Method: 32 patients (17 females) complaining of grade III or IV hemorrhoids were included in the study. A specially designed proctoscope coupled with a Doppler transducer on its tip was used to identify the hemorrhoidal arteries, which were afterwards suture ligated. Operating time as well as per- and post-operative complications were analyzed. Follow-up was planned following discharge after 1 week, 1 month, 6 months and 1 year., Results: Mean operation time was 27 (range 18-43) minutes, and 5 (range 4-7) arteries were located on average. No patient had severe or moderate postoperative pain, with anal discomfort being the main complaint. Rectal bleeding and tenesmus were the commonest post-operative complications. After one year of follow-up, 19 patients were free of symptoms and 6 of them had significant symptom relief. According to grade, the technique failed in just 3 grade III patients, but in as many as 4 grade IV hemorrhoid cases., Conclusions: Doppler-guided hemorrhoid artery ligation is an easy-to-perform technique that is well accepted by patients and has good results for grade III hemorrhoids.

The binding of a polar macromolecule to a large ion channel is studied theoretically, paying special attention to the influence of external conditions (applied voltage and ion strength of solution). The molecule behavior in bound state is considered as random thermal fluctuations within a limited fraction of its phase space. The mean duration of molecule binding (residence time tau r) is represented as the mean first passage time to reach the boundary of that restricted domain. By invoking the adiabatic approximation we reduce the problem to one dimension with the angle between macromolecule dipole and channel axes being the key variable of the problem. The model accounts for experimental measurements of tau r for the antibiotic Ampicillin within the bacterial porin OmpF of Escherichia coli. By assuming that the electrical interaction between Ampicillin dipole and OmpF ionizable groups affects the fluctuations, we find that the biased residence time-voltage dependence observed in experiments is the result of the strong transversal electric field in OmpF constriction with a tilt approximately 30 degrees aside the cis side.

Synthetic nanopores with fixed charges exhibit ionic equilibrium and transport properties that resemble those displayed by biological ion channels. We present an electrodiffusion model based on the Nernst-Planck flux equations, which allows for a qualitative description of the steady state ionic transport through a nanopore when the membrane fixed charges and all mobile carriers (including the water ions) are properly taken into account. In particular, we study the current-voltage curve, the electrical conductance, the reversal potential (a measure of the nanopore ionic selectivity), as well as the flux inhibition by protons and divalent cations in the nanopore. The model clearly shows how the changes in the ionization state of the fixed charges with pH and salt concentration dictate the electrical properties of the nanopore. The agreement between the model predictions and previous experimental data allows us to identify which are the main characteristics that permit a simple description of this complex system.

The membrane surface charge modifies the conductance of ion channels by changing the electric potential and redistributing the ionic composition in their vicinity. We have studied the effects of lipid charge on the conductance of a multi-state channel formed in planar lipid bilayers by the peptide antibiotic alamethicin. The channel conductance was measured in two lipids: in a neutral dioleoylphosphatidylethanolamine (DOPE) and a negatively charged dioleoylphosphatidylserine (DOPS). The charge state of DOPS was manipulated by the pH of the membrane-bathing solution. We find that at high salt concentrations (e.g., 2 M NaCl) the effect of the lipid charge is below the accuracy of our measurements. However, when the salt concentration in the membrane-bathing solution is decreased, the surface charge manifests itself as an increase in the conductance of the first two channel levels that correspond to the smallest conductive alamethicin aggregates. Our analysis shows that both the salt and pH dependence of the surface charge effect can be rationalized within the nonlinear Poisson-Boltzmann approach. Given channel conductance in neutral lipids, we use different procedures to account for the surface charge (e.g., introduce averaging over the channel aperture and take into account Na+ adsorption to DOPS heads), but only one adjustable parameter: an effective distance from the nearest lipid charge to the channel mouth center. We show that this distance varies by 0.3-0.4 nm upon channel transition from the minimal conducting aggregate (level L0) to the next larger one (level L1). This conclusion is in accord with a simple geometrical model of alamethicin aggregation.

One of the contributions to the surface potential in charged phospholipid monolayers at air-water interfaces is the double layer potential. In this note several misconceptions found in the literature concerning the relationship between the double layer potential and the degree of dissociation of the lipid polar headgroups are critically analyzed. The deviations of the double layer potential measurements from the Gouy-Chapman theory observed by several authors are explained by taking into account the dependence of the degree of dissociation with concentration, area per lipid molecule and pH.

We manipulate lipid bilayer surface charge and gauge its influence on gramicidin A channel conductance by two strategies: titration of the lipid charge through bulk solution pH and dilution of a charged lipid by neutral. Using diphytanoyl phosphatidylserine (PS) bilayers with CsCl aqueous solutions, we show that the effects of lipid charge titration on channel conductance are masked 1) by conductance saturation with Cs+ ions in the neutral pH range and 2) by increased proton concentration when the bathing solution pH is less than 3. A smeared charge model permits us to separate different contributions to the channel conductance and to introduce a new method for "bilayer pKa" determination. We use the Gouy-Chapman expression for the charged surface potential to obtain equilibria of protons and cations with lipid charges. To calculate cation concentration at the channel mouth, we compare different models for the ion distribution, exact and linearized forms of the planar Poisson-Boltzmann equation, as well as the construction of a "Gibbs dividing surface" between salt bath and charged membrane. All approximations yield the intrinsic pKain of PS lipid in 0.1 M CsCl to be in the range 2.5-3.0. By diluting PS surface charge at a fixed pH with admixed neutral diphytanoyl phosphatidylcholine (PC), we obtain a conductance decrease in magnitude greater than expected from the electrostatic model. This observation is in accord with the different conductance saturation values for PS and PC lipids reported earlier (, Biochim. Biophys. Acta. 552:369-378) and verified in the present work for solvent-free membranes. In addition to electrostatic effects of surface charge, gramicidin A channel conductance is also influenced by lipid-dependent structural factors.

We have considered the access resistance (AR) of a single conducting channel placed in a membrane bathed by an electrolyte. The classical expression for AR is due to Hall, who modeled the electrolyte as an ohmic conducting homogeneous medium. This approach is discussed in the present paper and it is shown that it is not valid in all cases, but depends on the ion concentration in solution and the ratio between solution and channel resistivities. To get a new expression for AR, we have combined the use of one-dimensional Nernst-Planck and Poisson (NPP) equations for the mouth of the channel and three-dimensional NPP equations for the outside solution. The influence of ion gradients and the channel itself on AR tums out to be considerable in diluted solutions (and also in the case of small channels in any solution). This influence is weaker in concentrated solutions, for which AR is well described by Hall's expression.

A perturbation analysis is applied to a nonequilibrium double electric layer (NDEL) near a membrane with extended soft permeable interfaces (polar zones) under steady-state ion flux conditions. The membrane has been modeled as composed of three layers: an inner hydrophobic layer and two polar zones with fixed charges and dipoles. Ion flux is described according to Nernst-Planck and Poisson equations. A first order approximation of the nonequilibrium electric potential has been obtained, using the electric current as the small parameter. The influence of the ionic flux, the surface dipole density, and the thickness of the polar zone on the NDEL near the membrane has been analyzed. The electric potential and hence the ion concentration profiles in the NDEL change considerably with surface dipole density. A maximum in the electric potential, which is determined by ion flux and polar zone parameters, is predicted in certain cases. The effects shown can help in the interpretation of permselectivity measurements.