Patient reported outcome and quality of life measured by a simple questionnaire in patients with symptomatic benign prostate hyperplasia treated by holmium laser enucleation of the prostate (HoLEP).

Introduction: Holmium Laser Enucleation of the Prostate (HoLEP) is established as an effective transurethral treatment option for LUTS due to BPH with improved postoperative outcome. The aim of this study was to evaluate the medium-term results by patient reported outcome measurement and to detect potential risk factors for postoperative complications or impaired outcome. Methods: We performed a retrospective single-center cohort study including all patients undergoing HoLEP in the study center between April 2019 and December 2021. Therefore, perioperative parameters and postoperative outcome was documented and all patients were asked for their outcome (PROM), complications, IPSS, QoL and changes in sexual and continence function by a questionnaire at a single time point. Results: In the study period, a total of 541 patients with a mean age of 72.5 ± 8.4 years were treated by HoLEP in the study center. 71.7% of the questionnaires were returned after a mean observation period of 14.9 ± 6.3 month. 91% of the patients reported to the single-timepoint questionnaire reporting a good satisfaction with the procedure and a low postoperative complication rate. The international prostate symptom score could be reduced significantly to 6.2 ± 5.7 (preoperative 19.0 ± 7.2; p < 0.001). Patients with an ASA score ≥ 3, prostate volume > 80 ml, medication with platelet inhibitors or DOAK or preoperative need of an indwelling catheter didn't show an increased complication rate. Conclusion: The overall satisfaction with the procedure and its results are high. We could not identify any independent risk factors for postoperative complications after HoLEP. The used questionnaire is a simple tool for postoperative patient reported outcome measurement with a good correlation to clinical parameters.

Bottom-up reconstitution of focal adhesion complexes.

Focal adhesions (FA) are large macromolecular assemblies relevant for various cellular and pathological events such as migration, polarization, and metastatic cancer formation. At FA sites at the migrating periphery of a cell, hundreds of players gather and form a network to respond to extra cellular stimuli transmitted by the integrin receptor, the most upstream component within a cell, initiating the FA signaling pathway. Numerous cellular experiments have been performed to understand the FA architecture and functions; however, their intricate network formation hampers unraveling the precise molecular actions of individual players. Here, in vitro bottom-up reconstitution presents an advantageous approach for elucidating the FA machinery and the hierarchical crosstalk of involved cellular players.

Structural insights into integrin α5ß1 opening by fibronectin ligand.

Integrin α5ß1 is a major fibronectin receptor critical for cell migration. Upon complex formation, fibronectin and α5ß1 undergo conformational changes. While this is key for cell-tissue connections, its mechanism is unknown. Here, we report cryo-electron microscopy structures of native human α5ß1 with fibronectin to 3.1-angstrom resolution, and in its resting state to 4.6-angstrom resolution. The α5ß1-fibronectin complex revealed simultaneous interactions at the arginine-glycine-aspartate loop, the synergy site, and a newly identified binding site proximal to adjacent to metal ion-dependent adhesion site, inducing the translocation of helix α1 to secure integrin opening. Resting α5ß1 adopts an incompletely bent conformation, challenging the model of integrin sharp bending inhibiting ligand binding. Our biochemical and structural analyses showed that affinity of α5ß1 for fibronectin is increased with manganese ions (Mn2+) while adopting the half-bent conformation, indicating that ligand-binding affinity does not depend on conformation, and α5ß1 opening is induced by ligand-binding.

Phosphoinositides regulate force-independent interactions between talin, vinculin, and actin.

Focal adhesions (FA) are large macromolecular assemblies which help transmit mechanical forces and regulatory signals between the extracellular matrix and an interacting cell. Two key proteins talin and vinculin connecting integrin to actomyosin networks in the cell. Both proteins bind to F-actin and each other, providing a foundation for network formation within FAs. However, the underlying mechanisms regulating their engagement remain unclear. Here, we report on the results of in vitro reconstitution of talin-vinculin-actin assemblies using synthetic membrane systems. We find that neither talin nor vinculin alone recruit actin filaments to the membrane. In contrast, phosphoinositide-rich membranes recruit and activate talin, and the membrane-bound talin then activates vinculin. Together, the two proteins then link actin to the membrane. Encapsulation of these components within vesicles reorganized actin into higher-order networks. Notably, these observations were made in the absence of applied force, whereby we infer that the initial assembly stage of FAs is force independent. Our findings demonstrate that the local membrane composition plays a key role in controlling the stepwise recruitment, activation, and engagement of proteins within FAs.

The Architecture of Talin1 Reveals an Autoinhibition Mechanism.

Focal adhesions (FAs) are protein machineries essential for cell adhesion, migration, and differentiation. Talin is an integrin-activating and tension-sensing FA component directly connecting integrins in the plasma membrane with the actomyosin cytoskeleton. To understand how talin function is regulated, we determined a cryoelectron microscopy (cryo-EM) structure of full-length talin1 revealing a two-way mode of autoinhibition. The actin-binding rod domains fold into a 15-nm globular arrangement that is interlocked by the integrin-binding FERM head. In turn, the rod domains R9 and R12 shield access of the FERM domain to integrin and the phospholipid PIP2 at the membrane. This mechanism likely ensures synchronous inhibition of integrin, membrane, and cytoskeleton binding. We also demonstrate that compacted talin1 reversibly unfolds to an ∼60-nm string-like conformation, revealing interaction sites for vinculin and actin. Our data explain how fast switching between active and inactive conformations of talin could regulate FA turnover, a process critical for cell adhesion and signaling.

Autodisplay of functional CYP106A2 in Escherichia coli.

Cytochrome P450 enzymes catalyse a wide variety of reactions, including the hydroxylation and epoxidation of CC bonds, and dealkylation reactions. There is high interest in these reactions for biotechnology and pharmaceutical processes. Many P450s require membrane surroundings and have substrates that do not cross biological membranes. To circumvent these obstacles, CYP106A2 from Bacillus megaterium was expressed on the outer membrane of Escherichia coli cells by Autodisplay. Exposure on the surface was confirmed by a protease accessibility test and flow cytometry after immunolabelling. HPLC assays showed that 0.5 ml of cells displaying the enzyme (OD578 = 6) converted 9.13 µmol of deoxycorticosterone to 15ß-OH-deoxycorticosterone within 1h. Imipramine and abietic acid were also accepted as substrates. The number of active enzyme molecules per cell was calculated to be 20,000. Surprisingly, surface-exposed CYP106A2 was active in E. coli BL21 without the external addition of the heme group. However, when CYP106A2 was expressed on the surface of an E. coli strain lacking the TolC channel protein (JW5503), enzymatic activity was almost completely abolished. The activity of CYP106A2 on the surface of E. coli JW5503 could be restored by the external addition of the heme group. This suggests, as has been reported before, that E. coli uses a TolC-dependent mechanism to export heme into the growth media, where it can be scavenged by a surface-displayed apoenzyme. Our results indicate that Autodisplay enables the functional surface display of P450 enzymes and provides a new platform to access their synthetic potential.

Expression of active human P450 3A4 on the cell surface of Escherichia coli by Autodisplay.

The cytochrome P450 enzyme system comprises a large group of enzymes catalyzing a broad diversity of reactions and an extensive substrate specificity, which makes them the most versatile known catalysts. CYP3A4 is one of the important human P450 enzymes and involved in the oxidation of a large range of substrates including toxins and pharmaceuticals. Bottlenecks in studying this enzyme include the difficulty in expressing it in a bacterial host, its need for membrane surroundings and the limited substrate accessibility of enzymes expressed within the cell. To circumvent these difficulties, human CYP3A4 was expressed on the outer membrane of Escherichia coli using Autodisplay. Transport of CYP3A4 to the cell surface was monitored by SDS-PAGE and Western blot analysis of outer membrane proteins. Localization on the cell envelope was determined by flow cytometry after immunolabeling, a whole cell ELISA and a protease accessibility assay. A HPLC assay confirmed the catalytic activity of displayed CYP3A4, using testosterone as a substrate. This activity required the external addition of electron supplying enzymes, however surprisingly, we found that the external addition of a heme group was not necessary. Our results indicate that human CYP3A4 can be recombinantly expressed by surface display in a gram-negative bacterium.

First X-ray structure analyses of rhodium(III) eta1-allyl complexes and a mechanism for allylic isomerization reactions.

Metal-to-metal allyl transfer: Using the first structurally characterized rhodium eta(1)-allyl complexes it is shown that the sigma-bound allyl substituent can be transferred from the Rh(III) complex to a Rh(I) complex in a fast equilibrium. This process may account for the decrease in regioselectivity observed in allylic alkylation reactions in which complex 1 is used as a catalyst.

Obstructive cholestasis induces TNF-alpha- and IL-1 -mediated periportal downregulation of Bsep and zonal regulation of Ntcp, Oatp1a4, and Oatp1b2.

Inverse acinar regulation of Mrp2 and 3 represents an adaptive response to hepatocellular cholestatic injury. We studied whether obstructive cholestasis (bile duct ligation) and LPS treatment affect the zonal expression of Bsep (Abcb11), Mrp4 (Abcc4), Ntcp (Slc10a1), and Oatp isoforms (Slco1a1, Slco1a4, and slco1b2) in rat liver, as analyzed by semiquantitative immunofluorescence. Contribution of TNF-alpha and IL-1beta to transporter zonation in obstructive cholestasis was studied by cytokine inactivation. In normal liver Bsep, Mrp4, Ntcp, and Oatp1a1 were homogeneously distributed in the acinus, whereas Oatp1a4 and Oatp1b2 expression increased from zone 1 to 3. Glutamine synthetase-positive pericentral hepatocytes exhibited markedly lower Oatp1a4 expression than the remaining zone 3 hepatocytes. In cholestatic liver Bsep and Ntcp immunofluorescence in periportal hepatocytes significantly decreased to 66 +/- 4% (P < 0.01) and 67 +/- 7% (P < 0.05), whereas it was not altered in pericentral hepatocytes. Oatp1a4 was significantly induced in hepatocytes with a primarily low expression, i.e., in periportal hepatocytes and in glutamine synthetase-positive pericentral hepatocytes. Likewise, Oatp1b2 was upregulated in periportal hepatocytes. Mrp4 zonal induction was homogeneous. Inactivation of TNF-alpha and IL-1beta prevented periportal downregulation of Bsep. Recruitment of neutrophils and polymorphonuclear cells mainly occurred in the periportal zone. Likewise, IL-1beta induction was largely found periportally. No significant transporter zonation was seen following LPS treatment. In conclusion, zonal downregulation of Bsep in obstructive cholestasis is associated with portal inflammation and is mediated by TNF-alpha and IL-1beta. Periportal downregulation of Ntcp and induction of Oatp1a4 and Oatp1b2 may represent adaptive mechanisms to reduce cholestatic injury in hepatocytes with profound downregulation of Bsep and Mrp2.