Electrochromic shift supports the membrane destabilization model of Tat-mediated transport and shows ion leakage during Sec transport

The mechanism and pore architecture of the Tat complex during transport of folded substrates remain a mystery, partly due to rapid dissociation after translocation. In contrast, the proteinaceous SecY pore is a persistent structure that needs only to undergo conformational shifts between “closed” and “opened” states when translocating unfolded substrate chains. Where the proteinaceous pore model describes the SecY pore well, the toroidal pore model better accounts for the high-energy barrier that must be overcome when transporting a folded substrate through the hydrophobic bilayer in Tat transport. Membrane conductance behavior can, in principle, be used to distinguish between toroidal and proteinaceous pores, as illustrated in the examination of many antimicrobial peptides as well as mitochondrial Bax and Bid. Here, we measure the electrochromic shift (ECS) decay as a proxy for conductance in isolated thylakoids, both during protein transport and with constitutively assembled translocons. We find that membranes with the constitutively assembled Tat complex and those undergoing Tat transport display conductance characteristics similar to those of resting membranes. Membranes undergoing Sec transport and those with the substrate-engaged SecY pore result in significantly more rapid electric field decay. The responsiveness of the ECS signal in membranes with active SecY recalls the steep relationship between applied voltage and conductance in a proteinaceous pore, while the nonaccelerated electric field decay with both Tat transport and the constitutive Tat complex under the same electric field is consistent with the behavior of a toroidal pore.

The twin arginine translocation pathway is uniquely able to transport fully folded substrates in an ATP-independent manner, relying instead on an electrochemical gradient (i.e., the proton motive force, or pmf) across the transporting membrane. It is crucial to the transport of substrates requiring various cofactors and hetero-oligomeric complexes in prokaryotes and of substrates vital to photosynthetic machinery in thylakoids (1). In plant mitochondria, the Rieske Fe/S protein required for the biogenesis of complex III is transported by the Tat pathway (2–6). It is implicated in both the virulence and antibiotic resistance of various infectious bacteria (7–12) and has been studied for its potential in biotechnology applications (13–15). The uniqueness of Tat functionality and its appearance across the kingdoms of life make it a valuable research target for crop modification, biotechnology, and pathogenesis. Unfortunately, much of the knowledge about its mechanism has been hard won, and the pore structure remains a mystery, likely due to the transient nature of the active complex.The active Tat complex in thylakoids consists of three core subunits, Tha4, Hcf106, and cpTatC, which are homologous to the bacterial TatA, TatB, and TatC, respectively (1, 16). An N-terminal signal peptide with a twin-arginine motif inserts into the cis-side leaflet at the TatBC receptor complex (17–19). Subsequent oligomerization of TatA subunits (20–22) at the TatBC receptor complex results in rings of varying sizes (22, 23), but it is unclear whether these structures represent transport pores. Of particular note is the short TatA transmembrane helix (TMH). Composed of only 16 residues, the solid-state NMR solution suggests that the TMH must tilt and draw a portion of the cis-side amphipathic helix (APH) into the membrane in order to cross the bilayer (24), establishing a resting state of hydrophobic mismatch. During transport, a conformational shift increasing the angle between the TMH and APH results in exacerbated hydrophobic mismatch, as the APH is moved radially away from the center and the TMH is pulled up toward the cis-side in the active state (25, 26). For both native and foreign substrates, the Tat-targeted signal peptide and the pmf are sufficient to cause assembly of the active translocon and achieve transport (27–31). After the translocation event, the complex dissociates into TatA and TatBC components (1, 15, 16) with the exception of some residual TatA bound to the receptor complex in a nonactive state and a spectrum of smaller TatA oligomers (32).Within the thylakoid membrane, it is useful to compare the Tat complex with the general secretory translocon (Sec) because they both function in the same membrane environment (1, 33). Sec translocation first requires recruitment of the substrate to the soluble SecA ATPase to form the substrate–SecA complex, which is then recruited to the SecY pore (1). In the inactive state, the proteinaceous SecY pore prevents ion leakage through a combination of a trans-side plug domain and an internal array of hydrophobic residues (34). Following substrate–SecA docking, a conformational shift in SecY allows substrate movement through the open pore in an ATP-dependent process driven by SecA (35). In the mammalian homolog Sec61, leakage of NAD⁺ ions is recorded during ribosome-bound nascent chain transport in a fluorescence quenching study, suggesting the pore can reach 4 to 6 nm in diameter (36). However, X-ray structures of substrate-fused SecA complexed with SecY (35), conductance studies in ribosome-bound substrates engaged with SecY (37), and SecY plug deletion mutants (38) in Escherichia coli have estimated the open SecY pore diameter to range from 7.3 to 8.8 Å, almost 10-fold lower. This small diameter likely contributes to the restriction of ion movement during Sec transport (39).While the Sec machinery only transports unfolded substrates (40), the Tat pore accommodates substrates ranging from a single unfolded chain in an engineered system (13) to a folded substrate with an average diameter of 70 Å (41). This extended size range raises an interesting question about the pore architecture. In the Sec translocon, X-ray crystallography of the SecY channel in Methanocaldococcus jannaschii (42) and Thermotoga maritima (43) reveals that the SecY pore channel excludes lipids in both the resting state and when engaged with its SecA partner. Further structural work on the E. coli substrate-engaged SecA–SecY complex shows that the SecY channel excludes lipids during transport as well (35). No such structural information about the Tat pore exists, but functional data suggest that TatA plays an important role in the pore (20, 44, 45) and cryogenic electron microscopy structures of TatA oligomers reveal rings of an internal diameter ranging from 30 to 70 Å (23). During transport of the 17-kDa subunit of the oxygen-evolving complex (OE23), the Tat pathway exhibits very low ion leakage (46), estimated to be less than 1 pS. This is despite the exchange of 80,000 protons per substrate (47). Extensive mapping of subunit–subunit and subunit–substrate contacts has revealed no putative plug domain (20, 48–51) that could be compared to that in the SecY protein.Pore architecture can be characterized by membrane conductance behavior. Conductance measured through proteinaceous pores representing the barrel-stave model has a very steep dependence on the voltage applied, whereas conductance in toroidal pores requires a larger voltage to be detected and increases more slowly in response to increasing voltage (52–54). Performing similar experiments on the Tat and Sec translocons would require functional reconstitution of both complexes into an in vitro setting. However, decay of the electrochromic shift (ECS) signal can be used as a measure of ion conductance (46). A transient absorption peak at ∼515 nm arising from carotenoid pigments in response to the native electric field generated by charge separation in the photosynthetic reaction centers (55) can be measured by delivering a single-saturating flash. The decay rate of such a signal is a direct measurement of how quickly the electric field is dissipated by ion movement across the membrane.In the experiments reported herein, ECS signal decay rates revealing the conductance states of resting isolated membranes and those engaged in ongoing transport and in the presence of a constitutively assembled and/or substrate-engaged translocon are used to probe the pore architecture in the Tat and Sec complexes. Increased conductance across the thylakoid membrane is indicated by a more rapidly decaying ECS signal. We find that conductance in thylakoid membranes during Sec-mediated transport and substrate-engaged SecY is consistently higher than that during Tat-mediated transport and with the constitutively assembled Tat complex, respectively, despite the much larger Tat pore required to transport a fully folded substrate. This points to a difference not only in mechanism but in pore architecture between the two. Conductance behavior of membranes undergoing Sec-mediated transport is consistent with that of a proteinaceous pore, while the resistance demonstrated by membranes undergoing Tat-mediated transport is more reminiscent of toroidal pores.     

Factors associated with poor outcomes in patients with lupus nephritis

The objective of this study was to identify the factors associated with important clinical outcomes in a case-control study of 213 patients with lupus nephritis. Included were 47% Hispanics, 44% African Americans and 9% Caucasians with a mean age of 28 years. Fifty-four (25%) patients reached the primary composite outcome of doubling serum creatinine, end-stage renal disease or death during a mean follow-up of 37 months. Thirty-four percent African Americans, 20% Hispanics and 10% Caucasians reached the primary composite outcome (P < 0.05). Patients reaching the composite outcome had predominantly proliferative lupus nephritis (WHO classes: 30% III, 32% IV, 18% V and 5% II, P < 0.025) with higher activity index score (7 +/- 6 versus 5 +/- 5, P < 0.05), chronicity index (CI) score (4 +/- 3 versus 2 +/- 2 unit, P < 0.025), higher baseline mean arterial pressure (MAP) (111 +/- 21 versus 102 +/- 14 mmHg, P < 0.025) and serum creatinine (1.9 +/- 1.3 versus 1.3 +/- 1.0 mg/dL, P < 0.025), but lower baseline hematocrit (29 +/- 6 versus 31 + 5%, P < 0.025) and complement C3 (54 +/- 26 versus 65 + 33 mg/dL, P < 0.025) compared to controls. More patients reaching the composite outcome had nephrotic range proteinuria compared to controls (74% versus 56%, P < 0.025). By multivariate analysis, CI (hazard ratio [95% CI] 1.18 [1.07-1.30] per point), MAP (HR 1.02 [1.00-1.03] per mmHg), and baseline serum creatinine (HR 1.26 [1.04-1.54] per mg/dL) were independently associated with the composite outcome. We concluded that hypertension and elevated serum creatinine at the time of the kidney biopsy as well as a high CI are associated with an increased the risk for chronic renal failure or death in patients with lupus nephritis.     

Impact of physician specialty on the cost of nonvariceal upper GI bleeding care

OBJECTIVE: Upper GI bleeding (UGIB) is a common medical emergency that leads to a high consumption of medical resources and costs. We aimed to analyze the influence of physician specialty on the costs of nonvariceal UGIB care. METHODS: We retrospectively assessed 350 nonvariceal UGIB episodes that were primarily cared for by gastroenterologists (n = 142), internists (n = 67), or surgeons (n = 141). Gastroenterologists followed evidence-based clinical protocols that included early endoscopy and early hospital discharge for uncomplicated bleeding. A risk score system was used to control for severity of illness. Linear regression analyses were performed to find out predictors of costs and the influence of specialist care on length of stay (LOS). RESULTS: The overall mean hospital cost was significantly lower in patients cared for by gastroenterologists (EUR 1,630) than in those managed by internists (EUR 3,745, p < 0.001) or surgeons (EUR 2,513, p < 0.05). The mean LOS was the variable with highest influence on total cost. Patients cared for by gastroenterologists had a mean LOS significantly shorter (7.3 days) than that of those treated by internists (16.2 days, p < 0.001) or surgeons (11 days, p < 0.001). Hospital costs and LOS differences were maintained when adjusting for severity of illness. In caring for low risk patients, nongastroenterologists had a higher probability of having a hospital stay longer than 4 days (odds ratio = 18.4, Cl = 4.6-73.6, p < 0.001). CONCLUSION: The implementation of specific evidence-based protocols by gastroenterologists reduces length of hospital stay and saves medical costs in patients with nonvariceal UGIB, especially those at low risk.     

Documento de posicionamiento de la AEG,la SEED y la SEAP sobre calidad de la endoscopia digestiva alta para la detección y vigilancia de las lesiones precursoras de cáncer gástrico

This position paper, sponsored by the Asociación Española de Gastroenterología [Spanish Association of Gastroenterology], the Sociedad Española de Endoscopia Digestiva [Spanish Gastrointestinal Endoscopy Society] and the Sociedad Española de Anatomía Patológica [Spanish Anatomical Pathology Society], aims to establish recommendations for performing an high quality upper gastrointestinal endoscopy for the screening of gastric cancer precursor lesions (GCPL) in low-incidence populations, such as the Spanish population. To establish the quality of the evidence and the levels of recommendation, we used the methodology based on the GRADE system (Grading of Recommendations Assessment, Development and Evaluation). We obtained a consensus among experts using a Delphi method. The document evaluates different measures to improve the quality of upper gastrointestinal endoscopy in this setting and makes recommendations on how to evaluate and treat the identified lesions. We recommend that upper gastrointestinal endoscopy for surveillance of GCPL should be performed by endoscopists with adequate training, administering oral premedication and use of sedation. To improve the identification of GCPL, we recommend the use of high definition endoscopes and conventional or digital chromoendoscopy and, for biopsies, NBI should be used to target the most suspicious areas of intestinal metaplasia. Regarding the evaluation of visible lesions, the risk of submucosal invasion should be evaluated with magnifying endoscopes and endoscopic ultrasound should be reserved for those with suspected deep invasion. In lesions amenable to endoscopic resection, submucosal endoscopic dissection is considered the technique of choice.     

Aspirin withdrawal in patients treated with ticagrelor presenting with non‐ST elevation myocardial infarction

Essentials

• Strong P2Y₁₂ blockade may cause platelet inhibition that is only minimally enhanced by aspirin.
• We evaluated aspirin withdrawal on platelet reactivity in ticagrelor treated patients.
• Aspirin withdrawal resulted in increased platelet reactivity to arachidonic acid.
• Aspirin withdrawal caused little difference in adenosine diphosphate‐induced platelet aggregation.

Summary

Background

Recent studies have shown that the thromboxane A₂‐dependent pathway is dependent on the ADP–P2Y₁₂ pathway, and that strong P2Y₁₂ receptor blockade alone causes inhibition of platelet aggregation that is minimally enhanced by aspirin. Data from the PLATO trial suggested that, among ticagrelor‐treated patients, high‐dose versus low‐dose (< 100 mg day^?1) aspirin is associated with an increased risk fof ischemic events.

Objectives

To evaluate the impact of aspirin withdrawal on platelet reactivity in acute coronary syndrome (ACS) patients treated with a potent P2Y₁₂ blocker.

Patients/Methods

This was a current prospective, randomized, placebo‐controlled, double‐blind, cross‐over study. The study population comprised 22 consecutive ACS patients who underwent percutaneous coronary intervention and were treated with aspirin (100 mg day^?1) and ticagrelor. Thirty days post‐ACS, open‐label aspirin was stopped, and patients were randomized to either blinded aspirin or placebo for 2 weeks, with each patient crossing over to the other arm for an additional 2 weeks. Platelet reactivity to arachidonic acid and ADP determined with light‐transmission aggregometry (LTA) and VerifyNow was evaluated at baseline, and 2 weeks and 4 weeks later.

Results

Aspirin withdrawal resulted in an increase in arachidonic‐acid induced platelet reactivity as determined with both LTA (77.0% ± 11.3% versus 20.8% ± 4.4%) and VerifyNow (607.7 ± 10.6 aspirin reaction units [ARU] versus 408.5 ± 14.4 ARU). Platelet response to ADP, as determined with both LTA and VerifyNow, did not differ with either aspirin or placebo (32.9% ± 2.6% versus 35.8% ± 3.6%, and 33.5 ± 6.4 P2Y₁₂ reaction units (PRU) versus 29.6 ± 5.7 PRU, respectively).

Conclusions

Aspirin withdrawal early post‐ACS results in increased platelet reactivity in response to arachidonic acid, despite concomitant treatment with the potent P2Y₁₂ blocker ticagrelor.

     

CHADS2 and CHA2DS2-VASc scores as predictors of platelet reactivity in acute coronary syndrome

BackgroundPlatelet function testing (PFT) in patients treated with P2Y₁₂ inhibitors has been widely evaluated for the prediction of stent thrombosis, myocardial infarction, and bleeding events following percutaneous coronary intervention (PCI) in acute coronary syndrome (ACS). Thus, PFT-guided treatment could positively affect patient outcomes. Data regarding clinical parameters for predicting platelet reactivity in ACS patients are limited. Therefore, our study aims to evaluate CHADS2 and CHA2DS2-VASc scores as predictors for platelet reactivity in ACS patients.MethodsTwo hundred and ninety-one consecutive patients who underwent PCI and were treated with aspirin and clopidogrel due to ACS were tested for their CHADS2, CHA2DS2-VASc scores and platelet reactivity using adenosine diphosphate (ADP)-induced aggregation (conventional aggregometry). Patients were classified into groups according to their CHADS2 and CHA2DS2-VASc scores. Low-risk group (0–1 score) for CHADS2 and CHA2DS2-VASc scores and high-risk group (2–6, 2–9) for CHADS2 and CHA2DS2-VASc scores, respectively. Furthermore, platelet reactivity in each group were compared (low CHADS2 group vs high CHADS2 group, and low CHA2DS2-VASc vs high CHA2DS2-VASc). Platelet reactivity was defined as low platelet reactivity (<19 U), optimal platelet reactivity [(OPR); 19–46 U], and high on-treatment platelet reactivity [(HPR); >46 U]. Thereafter receiver operating characteristic curve analysis was conducted to verify whether CHADS2 and CHA2DS2-VASc scores could predict platelet reactivity.ResultsLow CHADS2 and CHA2DS2-VASc scores were significantly correlated with lower mean platelet ADP-induced aggregation as compared with high CHADS2 and CHA2DS2-VASc scores [45.5 U (± 16) vs. 54.8 U (±15) and 44.2 U (±16) vs. 51.0 U (±17), respectively, p = 0.01 for both].ConclusionIn ACS patients treated with clopidogrel following PCI, high CHADS2 and CHA2DS2-VASc scores correlated with HPR and lower scores correlated with OPR. Further studies are needed to evaluate our findings’ clinical implications.