Lipids modulate the BH3-independent membrane targeting and activation of BAX and Bcl-xL.

Regulation of apoptosis is tightly linked with the targeting of numerous Bcl-2 proteins to the mitochondrial outer membrane (MOM), where their activation or inhibition dictates cell death or survival. According to the traditional view of apoptotic regulation, BH3-effector proteins are indispensable for the cytosol-to-MOM targeting and activation of proapoptotic and antiapoptotic members of the Bcl-2 protein family. This view is challenged by recent studies showing that these processes can occur in cells lacking BH3 effectors by as yet to be determined mechanism(s). Here, we exploit a model membrane system that recapitulates key features of MOM to demonstrate that the proapoptotic Bcl-2 protein BAX and antiapoptotic Bcl-xL have an inherent ability to interact with membranes in the absence of BH3 effectors, but only in the presence of cellular concentrations of Mg2+/Ca2+ Under these conditions, BAX and Bcl-xL are selectively targeted to membranes, refolded, and activated in the presence of anionic lipids especially the mitochondrial-specific lipid cardiolipin. These results provide a mechanistic explanation for the mitochondrial targeting and activation of Bcl-2 proteins in cells lacking BH3 effectors. At cytosolic Mg2+ levels, the BH3-independent activation of BAX could provide localized amplification of apoptotic signaling at regions enriched in cardiolipin (e.g., contact sites between MOM and mitochondrial inner membrane). Increases in MOM cardiolipin, as well as cytosolic [Ca2+] during apoptosis could further contribute to its MOM targeting and activity. Meanwhile, the BH3-independent targeting and activation of Bcl-xL to the MOM is expected to counter the action of proapoptotic BAX, thereby preventing premature commitment to apoptosis.

Urine shedding patterns of pathogenic Leptospira spp. in dairy cows.

Pathogenic Leptospira spp. are zoonotic bacteria that infect wild and domestic animals. Humans contract leptospirosis directly through contact with infected animals or indirectly from contaminated water or soil. In mammalian reservoirs, the pathogen can colonize renal tubules for lengthy periods and persistently contaminate the environment through urine. Cattle have been reported to shed several serovars; with Hardjo the most common serovar found in cattle. Without clinical manifestations, the infection can spread within a herd, impairing productivity, and putting workers like farmers, abattoir operators and veterinarians at risk. The dynamics of pathogenic Leptospira shedding was studied in six dairy herds in southern Chile. Various intermittent urine shedding patterns were found, with elimination periods between 79 and 259 days and bacterial loads ranging from 3 × 104 to 4.4 × 104 bacteria/mL. The current study was the first to assess the various urine shedding patterns and loads of pathogenic leptospires shed through urine of naturally-infected dairy cows. In addition, the study suggests that vaccination does not prevent cattle infection, although it influences loads of pathogenic leptospires excreted in urine. Our study provides a great awareness of asymptomatic animal carriers in an endemic area and will contribute to improving disease control and designing better prevention strategies.

Long-term oncological and surgical outcomes after Video Endoscopic Inguinal Lymphadenectomy (VEIL) in patients with penile cancer.

OBJECTIVE: To report outcomes from the largest multicenter series of penile cancer patients undergoing video endoscopic inguinal lymphadenectomy (VEIL). MATERIALS AND METHODS: Retrospective multicenter analysis. Authors of 21 centers from the Penile Cancer Collaborative Coalition-Latin America (PeC-LA) were included. All centers performed the procedure following the same previously described standardized technique. Inclusion criteria included penile cancer patients with no palpable lymph nodes and intermediate/high-risk disease and those with non-fixed palpable lymph nodes less than 4 cm in diameter. Categorical variables are shown as percentages and frequencies whereas continuous variables as mean and range. RESULTS: From 2006 to 2020, 210 VEIL procedures were performed in 105 patients. Mean age was 58 (45-68) years old. Mean operative time was 90 minutes (60-120). Mean lymph node yield was 10 nodes (6-16). Complication rate was 15.7%, including severe complications in 1.9% of procedures. Lymphatic and skin complications were noted in 8.6 and 4.8% of patients, respectively. Histopathological analysis revealed lymph node involvement in 26.7% of patients with non-palpable nodes. Inguinal recurrence was observed in 2.8% of patients. 10y- overall survival was 74.2% and 10-y cancer specific survival was 84.8%. CSS for pN0, pN1, pN2 and pN3 were 100%, 82.4%, 72.7% and 9.1%, respectively. CONCLUSION: VEIL seems to offer appropriate long term oncological control with minimal morbidity. In the absence of non-invasive stratification measures such as dynamic sentinel node biopsy, VEIL emerged as the alternative for the management of non-bulky lymph nodes in penile cancer.

Advantages of Quantitative Analysis of Depth-Dependent Fluorescence Quenching: Case Study of BAX.

Dynamic disorder of the lipid bilayer presents a challenge for establishing structure-function relationships in membrane proteins, especially to those that insert by refolding from soluble states, e.g., bacterial toxins and Bcl-2 apoptotic regulators. Because many high-resolution structural techniques cannot be easily applied to such systems, methods like depth-dependent fluorescence quenching gained prominence. Over three decades ago, Prof. Erwin London and his co-workers revolutionized the studies of membrane protein insertion by introducing a quantitative approach to the analysis of membrane quenching data and inspired many researchers to continue this work. Here, we illustrate how the application of the quantitative analysis yields new insights into previously published results. We have used the method of distribution analysis (DA) to calculate the precise immersion depth of NBD fluorophores selectively attached to various single-cysteine mutants of the apoptotic factor BAX from quenching data obtained with a series of spin-labeled lipids. The original qualitative analysis interpreted the higher quenching determined for shallower probes in positions flanking the helix 9 of BAX as evidence of a transmembrane helix 9 topology. The quantitative DA, however, revealed that a transmembrane topology of helix 9 of BAX is unlikely, as it would require labeling sites that are only 15 residues apart in a helical conformation to be separated by a transverse distance of over 45 Å.

Spectroscopic evidence of tetanus toxin translocation domain bilayer-induced refolding and insertion.

Tetanus neurotoxin (TeNT) is an A-B toxin with three functional domains: endopeptidase, translocation (HCT), and receptor binding. Endosomal acidification triggers HCT to interact with and insert into the membrane, translocating the endopeptidase across the bilayer. Although the function of HCT is well defined, the mechanism by which it accomplishes this task is unknown. To gain insight into the HCT membrane interaction on both local and global scales, we utilized an isolated, beltless HCT variant (bHCT), which retained the ability to release potassium ions from vesicles. To examine which bHCT residues interact with the membrane, we widely sampled the surface of bHCT using 47 single-cysteine variants labeled with the environmentally sensitive fluorophore NBD. At neutral pH, no interaction was observed for any variant. In contrast, all NBD-labeled positions reported environmental change in the presence of acidic pH and membranes containing anionic lipids. We then examined the conformation of inserted bHCT using circular dichroism and intrinsic fluorescence. Upon entering the membrane, bHCT retained predominantly α-helical secondary structure, whereas the tertiary structure exhibited substantial refolding. The use of lipid-attached quenchers revealed that at least one of the three tryptophan residues penetrated deep into the hydrocarbon core of the membrane, suggesting formation of a bHCT transmembrane conformation. The possible conformational topology was further explored with the hydropathy analysis webtool MPEx, which identified a large, potential α-helical transmembrane region. Altogether, the spectroscopic evidence supports a model in which, upon acidification, the majority of TeNT bHCT entered the membrane with a concurrent change in tertiary structure.

Expanding MPEx Hydropathy Analysis to Account for Electrostatic Contributions to Protein Interactions with Anionic Membranes.

Hydropathy plots are a crucial tool to guide experimental design, as they generate predictions of protein-membrane interactions and their bilayer topology. The predictions are based on experimentally determined hydrophobicity scales, which provide an estimate for the propensity and stability of these interactions. A significant improvement to the accuracy of hydropathy analyses was provided by the development of the popular Wimley-White interfacial and octanol hydrophobicity scales. These scales have been previously incorporated into the freely available MPEx (Membrane Protein Explorer) online application. Here, we introduce a substantial update to MPEx that allows for the consideration of electrostatic contributions to the bilayer partitioning free energy. This component originates from the Coulombic attraction or repulsion of charges between proteins and membranes. Its inclusion in hydropathy calculations increases the accuracy of hydropathy plot predictions and extends their use to more complex systems (i.e., anionic membranes). We illustrate the application of this analysis to studies on the membrane selectivity of antimicrobial peptides, the membrane partitioning of ion-channel gating modifiers, and the amyloid proteins α-synuclein and Tau, as well as pH-dependent bilayer interactions of diphtheria toxin and apoptotic inhibitor Bcl-xL.

Effects of Cardiolipin on the Conformational Dynamics of Membrane-Anchored Bcl-xL.

The anti-apoptotic protein Bcl-xL regulates apoptosis by preventing the permeation of the mitochondrial outer membrane by pro-apoptotic pore-forming proteins, which release apoptotic factors into the cytosol that ultimately lead to cell death. Two different membrane-integrated Bcl-xL constructs have been identified: a membrane-anchored and a membrane-inserted conformation. Here, we use molecular dynamics simulations to study the effect of the mitochondrial specific lipid cardiolipin and the protein protonation state on the conformational dynamics of membrane-anchored Bcl-xL. The analysis reveals that the protonation state of the protein and cardiolipin content of the membrane modulate the orientation of the soluble head region (helices α1 through α7) and hence the exposure of its BH3-binding groove, which is required for its interaction with pro-apoptotic proteins.

Correction to: Experimental and Computational Characterization of Oxidized and Reduced Protegrin Pores in Lipid Bilayers.

The original version of the article was published without the Graphic Abstract. Graphic Abstract image of the article is given below.

Experimental and Computational Characterization of Oxidized and Reduced Protegrin Pores in Lipid Bilayers.

Protegrin-1 (PG-1), an 18-residue ß-hairpin stabilized by two disulfide bonds, is a member of a family of powerful antimicrobial peptides which are believed to act through membrane permeabilization. Here we used a combination of experimental and computational approaches to characterize possible structural arrangements of PG-1 in lipid bilayers mimicking bacterial membranes. We have measured the dose-response function of the PG-1-induced leakage of markers of various sizes from vesicles and found it to be consistent with the formation of pores of two different sizes. The first one allows the release of small dyes and occurs at peptide:lipid ratios < 0.006. Above this ratio, larger pores are observed through which the smallest of dextrans FD4 can be released. In parallel with pore formation, we observe a general large-scale destabilization of vesicles which is probably related to complete rupture of some vesicles. The population of vesicles that are completely ruptured depends linearly on PG-1:lipid ratio. Neither pore size, nor vesicle rupture are influenced by the formation of disulfide bonds. Previous computational work on oxidized protegrin is complemented here by all-atom MD simulations of PG-1 with reduced disulfide bonds both in solution (monomer) and in a bilayer (dimer and octamer). The simulations provide molecular insights into the influence of disulfide bonds on peptide conformation, aggregation, and oligomeric structure.

Comparison of lipid-dependent bilayer insertion of pHLIP and its P20G variant.

The ability of the pH-Low Insertion Peptide (pHLIP) to insert into lipid membranes in a transbilayer conformation makes it an important tool for targeting acidic diseased tissues. pHLIP can also serve as a model template for thermodynamic studies of membrane insertion. We use intrinsic fluorescence and circular dichroism spectroscopy to examine the effect of replacing pHLIP's central proline on the pH-triggered lipid-dependent conformational switching of the peptide. We find that the P20G variant (pHLIP-P20G) has a higher helical propensity than the native pHLIP (pHLIP-WT), in both water:organic solvent mixtures and in the presence of lipid bilayers. Spectral shifts of tryptophan fluorescence reveal that with both pHLIP-WT and pHLIP-P20G, the deeply penetrating interfacial form (traditionally called State II) is populated only in pure phosphocholine bilayers. The presence of either anionic lipids or phosphatidylethanolamine leads to a much shallower penetration of the peptide (referred to here as State IIS, for "shallow"). This novel state can be differentiated from soluble state by a reduction in accessibility of tryptophans to acrylamide and by FRET to vesicles doped with Dansyl-PE, but not by a spectral shift in fluorescence emission. FRET experiments indicate free energies for interfacial partitioning range from 6.2 to 6.8kcal/mol and are marginally more favorable for pHLIP-P20G. The effective pKa for the insertion of both peptides depends on the lipid composition, but is always higher for pHLIP-P20G than for pHLIP-WT by approximately one pH unit, which corresponds to a difference of 1.3kcal/mol in free energy of protonation favoring insertion of pHLIP-P20G.

Refining Protein Penetration into the Lipid Bilayer Using Fluorescence Quenching and Molecular Dynamics Simulations: The Case of Diphtheria Toxin Translocation Domain.

Dynamic disorder of the lipid bilayer presents a challenge for establishing structure-function relationships in membranous systems. The resulting structural heterogeneity is especially evident for peripheral and spontaneously inserting membrane proteins, which are not constrained by the well-defined transmembrane topology and exert their action in the context of intimate interaction with lipids. Here, we propose a concerted approach combining depth-dependent fluorescence quenching with Molecular Dynamics simulation to decipher dynamic interactions of membrane proteins with the lipid bilayers. We apply this approach to characterize membrane-mediated action of the diphtheria toxin translocation domain. First, we use a combination of the steady-state and time-resolved fluorescence spectroscopy to characterize bilayer penetration of the NBD probe selectively attached to different sites of the protein into membranes containing lipid-attached nitroxyl quenching groups. The constructed quenching profiles are analyzed with the Distribution Analysis methodology allowing for accurate determination of transverse distribution of the probe. The results obtained for 12 NBD-labeled single-Cys mutants are consistent with the so-called Open-Channel topology model. The experimentally determined quenching profiles for labeling sites corresponding to L350, N373, and P378 were used as initial constraints for positioning TH8-9 hairpin into the lipid bilayer for Molecular Dynamics simulation. Finally, we used alchemical free energy calculations to characterize protonation of E362 in soluble translocation domain and membrane-inserted conformation of its TH8-9 fragment. Our results indicate that membrane partitioning of the neutral E362 is more favorable energetically (by ~ 6 kcal/mol), but causes stronger perturbation of the bilayer, than the charged E362.

[Pancreaticoduodenectomy for a solid pseudopapillary tumor of the pancreas in children]. / Pancreatoduodenectomía por tumor sólido pseudopapilar de páncreas en niños.

The solid pseudopapillary tumor of the pancreas (SPT) is a rare neoplasm with low malignant potential in children. We report the case of a 9 years old child with a SPT localized in the pancreatic head. She underwent a pancreaticoduodenectomy (PD) with favorable evolution. The PD in high-volume centers is safe in both adults and children.

Nerve Sparing, Robot-Assisted Radical Cystectomy with Intracorporeal Bladder Substitution in the Male.

PURPOSE: We provide a step-by-step description of our technique of nerve and seminal vesicle sparing robot-assisted radical cystectomy with an orthotopic neobladder. We also present preliminary oncologic and functional outcomes. MATERIALS AND METHODS: Nerve and seminal vesicle sparing robot-assisted radical cystectomy with a modified Y-shaped orthotopic neobladder was performed by the same surgeon in 40 men with clinically localized bladder cancer from January 2011 to September 2014. Operative, perioperative and pathological data as well as continence and erectile function outcomes are presented. RESULTS: Median followup was 26.5 months (range 8 to 52). A soft tissue positive surgical margin was found in a patient with pT3a disease. A global rate of 30% early and 32.5% late complications was observed. However, the grade III or higher complication rate was low in both settings at 2.5% and 5%, respectively. There was 1 cancer related death 23 months after surgery. Of the 40 patients 30 (75%) gained daytime continence (0 pad) within 1 month postoperatively. The 12-month nocturnal continence rate was 72.5% (29 of 40 patients). Mean preoperative IIEF-6 (International Index of Erectile Function-6) score was 24.4. Erectile function returned to normal, defined as an IIEF-6 score greater than 17, in 31 of 40 patients (77.5%) within 3 months while 29 of 40 patients (72.5%) returned to the preoperative IIEF-6 score within 12 months. CONCLUSIONS: In the hands of an experienced surgeon nerve and seminal vesicle sparing robot-assisted radical cystectomy with intracorporeal reconstruction of the neobladder seems feasible and safe. It provides short-term oncologic efficacy and promising functional outcomes. Yet comparative, long-term followup studies with standard open cystectomy are required.

Lipid headgroups modulate membrane insertion of pHLIP peptide.

The pH low insertion peptide (pHLIP) is an important tool for drug delivery and visualization of acidic tissues produced by various maladies, including cancer, inflammation, and ischemia. Numerous studies indicate that pHLIP exists in three states: unfolded and soluble in water at neutral pH (State I), unfolded and bound to the surface of a phosphatidylcholine membrane at neutral pH (State II), and inserted across the membrane as an α-helix at low pH (State III). Here we report how changes in lipid composition modulate this insertion scheme. First, the presence of either anionic lipids, cholesterol, or phosphoethanolamine eliminates membrane binding at neutral pH (State II). Second, the apparent pKa for the insertion transition (State I → State III) is increased with increasing content of anionic lipids, suggesting that electrostatic interactions in the interfacial region modulate protonation of acidic residues of pHLIP responsible for transbilayer insertion. These findings indicate a possibility for triggering protonation-coupled conformational switching in proteins at membrane interfaces through changes in lipid composition.

Mass Spectrometric Analysis of Surface-Exposed Regions in the Hexadecameric Phosphorylase Kinase Complex.

Phosphorylase kinase (PhK) is a 1.3 MDa (αßγδ)4 enzyme complex, in which αßγδ protomers associate in D2 symmetry to form two large octameric lobes that are interconnected by four bridges. The approximate locations of the subunits have been mapped in low-resolution cryo-electron microscopy structures of the complex; however, the disposition of the subunits within the complex remains largely unknown. We have used partial proteolysis and chemical footprinting in combination with high-resolution mass spectrometry to identify surface-exposed regions of the intact nonactivated and phospho-activated conformers. In addition to the known interaction of the γ subunit's C-terminal regulatory domain with the δ subunit (calmodulin), our exposure results indicate that the catalytic core of γ may also anchor to the PhK complex at the bottom backside of its C-terminal lobe facing away from the active site cleft. Exposed loops on the α and ß regulatory subunits within the complex occur at regions overlapping with tissue-specific alternative RNA splice sites and regulatory phosphorylatable domains. Their phosphorylation alters the surface exposure of α and ß, corroborating previous biophysical and biochemical studies that detected phosphorylation-dependent conformational changes in these subunits; however, for the first time, specific affected regions have been identified.

Pentafecta evaluation in robot-assisted radical prostatectomy by risk group.

OBJECTIVE: Radical prostatectomy is a therapeutic option in organ-confined prostate cancer. As the development of robotic systems progresses, the approach with this technology has begun to impact the functional and oncological outcomes of urological patients. The objective is to report the rate of pentafecta in patients undergoing robot-assisted radical prostatectomy (RARP) stratified by risk groups. METHOD: Retrospective, observational, descriptive study from 2013 to 2020 that included 112 patients undergoing RARP. RESULTS: A rate of pentafecta at 12 months of follow-up of 35.7% (n = 40) was obtained. In the subanalysis by risk groups, at 1-year follow-up, was obtained an index of 43% (n = 26), 26% (n = 9) and 22% (n = 4) in low-, intermediate-, and high-risk patients, respectively. CONCLUSIONS: Prostatectomy showed functional and oncological results similar to those reported in the literature with robotic approach, regardless of the risk group for prostate cancer.

OBJETIVO: La prostatectomía radical es la alternativa terapéutica de elección en el cáncer de próstata confinado al órgano. Conforme avanza el desarrollo de los sistemas robóticos, el abordaje con esta tecnología ha comenzado a impactar en los desenlaces funcionales y oncológicos de los pacientes urológicos. El objetivo es reportar el índice de pentafecta en pacientes sometidos a prostatectomía radical asistida por robot (PRRA) estratificados por grupos de riesgo. MÉTODO: Estudio retrospectivo, observacional, descriptivo, de 2013 a 2020, que incluyó 112 pacientes sometidos a PRAR. RESULTADOS: Se obtuvo un índice de pentafecta a 12 meses de seguimiento del 35.7% (n = 40). En el subanálisis por grupos de riesgo, al año de seguimiento, se obtuvieron unos índices del 43% (n = 26), el 26% (n = 9) y el 22% (n = 4) en los pacientes de bajo, intermedio y alto riesgo, respectivamente. CONCLUSIONES: La prostatectomía demostró resultados funcionales y oncológicos similares a lo reportado en la literatura con abordaje robótico independientemente del grupo de riesgo del cáncer de próstata.

Promoting the activity of a receptor tyrosine phosphatase with a novel pH-responsive transmembrane agonist inhibits cancer-associated phenotypes.

Cell signaling by receptor protein tyrosine kinases (RTKs) is tightly controlled by the counterbalancing actions of receptor protein tyrosine phosphatases (RPTPs). Due to their role in attenuating the signal-initiating potency of RTKs, RPTPs have long been viewed as therapeutic targets. However, the development of activators of RPTPs has remained limited. We previously reported that the homodimerization of a representative member of the RPTP family (protein tyrosine phosphatase receptor J or PTPRJ) is regulated by specific transmembrane (TM) residues. Disrupting this interaction by single point mutations promotes PTPRJ access to its RTK substrates (e.g., EGFR and FLT3), reduces RTK's phosphorylation and downstream signaling, and ultimately antagonizes RTK-driven cell phenotypes. Here, we designed and tested a series of first-in-class pH-responsive TM peptide agonists of PTPRJ that are soluble in aqueous solution but insert as a helical TM domain in lipid membranes when the pH is lowered to match that of the acidic microenvironment of tumors. The most promising peptide reduced EGFR's phosphorylation and inhibited cancer cell EGFR-driven migration and proliferation, similar to the PTPRJ's TM point mutations. Developing tumor-selective and TM-targeting peptide binders of critical RPTPs could afford a potentially transformative approach to studying RPTP's selectivity mechanism without requiring less specific inhibitors and represent a novel class of therapeutics against RTK-driven cancers.

[Robot-assisted radical prostatectomy with Retzius-Sparing approach vs. modified Frankfurt: comparison of results at 1 year of follow-up]. / Prostatectomía radical asistida por robot con abordaje Retzius-Sparing vs. Frankfurt modificado: comparación de resultados a 1 año de seguimiento.

Background: Robot-assisted radical prostatectomy has positioned itself as the approach of choice in the treatment of prostate cancer. Objective: To compare the outcomes of robot-assisted radical prostatectomy using the Retzius-Sparing (RS) approach against the modified Frankfurt (MF) technique. Method: To describe the perioperative, functional and oncological outcomes of 13 patients with prostate cancer who underwent RS robotic radical prostatectomy compared to MF, evaluating pathological results, urinary continence, sexual function and oncological control in 1 year of follow-up. Results: The average age was 64 years in RS group vs. 61 years in MF group. The values of total prostate antigen were higher in the RS group (25 ng/dl) vs. MF group (11 ng/dl). The volume of gland in RS group was 40.62 ml vs. 63.33 ml in the RS group. All patients were bilaterally neuropreserved, being statistically significant in favor of MF group (p = 0.016). Positive surgical margins were higher in R-S group (38.4%) vs. MF group (33.3%). Conclusions: With RS the same tendency to urinary continence is observed, with a significant difference in erectile function in favor of MF. This preliminary study shows better impact on erectile function.

Antecedentes: La prostatectomía radical asistida por robot se ha posicionado como el abordaje de elección en el tratamiento del cáncer de próstata. Objetivo: Comparar los resultados de la prostatectomía radical asistida por robot utilizando el abordaje Retzius-Sparing (RS) contra el Frankfurt modificado (FM). Método: Se describen los desenlaces perioperatorios, funcionales y oncológicos de 13 pacientes con cáncer de próstata que fueron llevados a prostatectomía radical robótica con RS, en comparación con FM, y se evalúan los resultados patológicos, continencia urinaria, función sexual y control oncológico a 1 año de seguimiento. Resultados: La media de edad fue de 64 años en el grupo RS y de 61 años en el grupo FM. Los valores de antígeno prostático total fueron mayores en el grupo RS (25 ng/dl) que en el FM (11 ng/dl). El volumen de la glándula fue menor en el grupo RS (40.62 ml) que en el FM (63.33 ml). Todos los pacientes fueron neuropreservados bilateralmente, siendo la diferencia estadísticamente significativa a favor de FM (p = 0.016). Los márgenes quirúrgicos positivos fueron mayores en el grupo RS (38.4%) que en el FM (33.3%). Conclusiones: Con RS se observa la misma tendencia a la continencia urinaria, con diferencia significativa en la función eréctil a favor de FM. Este estudio preliminar muestra mejor impacto en la función eréctil.

Pielonefritis xantogranulomatosa: un reto actual para la laparoscopia.

OBJECTIVE: To present the results of our case series on laparoscopic nephrectomy in xanthogranulomatous pyelonephritis (XGP). METHODS: A retrospective study was conducted that included 143 patients treated with laparoscopic nephrectomy for non-functioning kidney, of whom 15 had XGP, within the time frame of 2011 to 2019. The demographic and clinical data were collected, along with the intraoperative results, complications, and days of hospital stay. RESULTS: Transperitoneal laparoscopic nephrectomy was successfully performed on 15 patients with XGP, with no need for conversion. Mean intraoperative time was 124.4 minutes (range 70-240) and intraoperative blood loss was 148.5 ml (range 30-550), with no blood transfusion required. No intraoperative complications occurred but there was one postoperative complication (6.6%), classified as Clavien-Dindo I (surgical wound infection). Mean hospital stay was 2.85 days (range 2-7). CONCLUSIONS: Nephrectomy is the definitive management for XGP, and the laparoscopic approach should be considered a treatment modality, despite the fact that the pathology involves a severe chronic inflammatory process. Its benefits are reduced surgery duration, less blood loss, a lower complication rate, and fewer days of hospital stay, when performed by a skilled and experienced surgeon.

OBJETIVO: Presentar los resultados de nuestra serie de nefrectomía laparoscópica en pielonefritis xantogranulomatosa (PXG). MÉTODO: Se realizó un estudio retrospectivo que incluyó 143 pacientes tratados con nefrectomía laparoscópica por exclusión renal, de los cuales 15 fueron por PXG, en el periodo comprendido de 2011 a 2019. Se recolectaron datos demográficos y clínicos, resultados transoperatorios, complicaciones y días de estancia hospitalaria. RESULTADOS: Se realizó nefrectomía laparoscópica transperitoneal de forma exitosa en 15 pacientes con PXG, sin necesidad de conversión. El tiempo transoperatorio promedio fue de 124.4 minutos (rango: 70-240). El sangrado transoperatorio fue de 148.5 ml (rango: 30-550), sin requerimiento de transfusión sanguínea. No se reportaron complicaciones transoperatorias; se presentó una complicación en el posoperatorio (6.6%) clasificada como Clavien-Dindo I (infección de la herida quirúrgica). La estancia hospitalaria promedio fue de 2.85 días (rango: 2-7). CONCLUSIONES: El manejo definitivo de la PXG es la nefrectomía, y el abordaje laparoscópico debe ser considerado como una modalidad de tratamiento a pesar de ser una patología que presenta un proceso inflamatorio grave y crónico, obteniéndose beneficios como disminución en el tiempo quirúrgico, menor sangrado, menor tasa de complicaciones y menos días de estancia hospitalaria cuando es realizado por un cirujano experimentado.

Histidine Protonation and Conformational Switching in Diphtheria Toxin Translocation Domain.

Protonation of key histidine residues has been long implicated in the acid-mediated cellular action of the diphtheria toxin translocation (T-) domain, responsible for the delivery of the catalytic domain into the cell. Here, we use a combination of computational (constant-pH Molecular Dynamics simulations) and experimental (NMR, circular dichroism, and fluorescence spectroscopy along with the X-ray crystallography) approaches to characterize the initial stages of conformational change happening in solution in the wild-type T-domain and in the H223Q/H257Q double mutant. This replacement suppresses the acid-induced transition, resulting in the retention of a more stable protein structure in solutions at pH 5.5 and, consequently, in reduced membrane-disrupting activity. Here, for the first time, we report the pKa values of the histidine residues of the T-domain, measured by NMR-monitored pH titrations. Most peaks in the histidine side chain spectral region are titrated with pKas ranging from 6.2 to 6.8. However, the two most up-field peaks display little change down to pH 6, which is a limiting pH for this protein in solution at concentrations required for NMR. These peaks are absent in the double mutant, suggesting they belong to H223 and H257. The constant-pH simulations indicate that for the T-domain in solution, the pKa values for histidine residues range from 3.0 to 6.5, with those most difficult to protonate being H251 and H257. Taken together, our experimental and computational data demonstrate that previously suggested cooperative protonation of all six histidines in the T-domain does not occur.