Molecular MRD is strongly prognostic in patients with NPM1-mutated AML receiving venetoclax-based nonintensive therapy.

ABSTRACT: Assessment of measurable residual disease (MRD) by quantitative reverse transcription polymerase chain reaction is strongly prognostic in patients with NPM1-mutated acute myeloid leukemia (AML) treated with intensive chemotherapy; however, there are no data regarding its utility in venetoclax-based nonintensive therapy, despite high efficacy in this genotype. We analyzed the prognostic impact of NPM1 MRD in an international real-world cohort of 76 previously untreated patients with NPM1-mutated AML who achieved complete remission (CR)/CR with incomplete hematological recovery following treatment with venetoclax and hypomethylating agents (HMAs) or low-dose cytarabine (LDAC). A total of 44 patients (58%) achieved bone marrow (BM) MRD negativity, and a further 14 (18%) achieved a reduction of ≥4 log10 from baseline as their best response, with no difference between HMAs and LDAC. The cumulative rates of BM MRD negativity by the end of cycles 2, 4, and 6 were 25%, 47%, and 50%, respectively. Patients achieving BM MRD negativity by the end of cycle 4 had 2-year overall of 84% compared with 46% if MRD was positive. On multivariable analyses, MRD negativity was the strongest prognostic factor. A total of 22 patients electively stopped therapy in BM MRD-negative remission after a median of 8 cycles, with 2-year treatment-free remission of 88%. In patients with NPM1-mutated AML attaining remission with venetoclax combination therapies, NPM1 MRD provides valuable prognostic information.

Optogenetic ion pumps differ with respect to the secondary pattern of K+ redistribution.

We recently reported that strong activation of the optogenetic chloride pump, halorhodopsin leads to a secondary redistribution of K+ ions into the cell, through tonically open, "leak" K+ channels. Here we show that this effect is not unique to halorhodopsin but is also seen with activation of another electrogenic ion pump, archaerhodopsin. The two opsins differ however in the size of the rebound rise in extracellular potassium, [K+ ]o , after the end of activation, which is far larger with halorhodopsin than for archaerhodopsin activation. Multiple linear regression modeling indicates that the variance in the postillumination surge in [K+ ]o was explained both by the size of the preceding, illumination-induced drop in [K+ ]o and also by the type of opsin. These data provide additional support for the hypothesis that intense chloride-loading of cells, as occurs naturally following intense bursts of GABAergic synaptic bombardment, or artificially following halorhodopsin activation, is followed by extrusion of both Cl- and K+ coupled together. We discuss this with respect to the pattern of [K+ ]o rise that occurs at the onset of seizure-like events.

FLT3 inhibitors as MRD-guided salvage treatment for molecular failure in FLT3 mutated AML.

Patients with FLT3-mutated AML have a high relapse rate and suboptimal outcomes. Many have co-mutations suitable for measurable residual disease (MRD) monitoring by RT-qPCR and those destined to relapse can be identified by high or rising levels of MRD, called molecular failure.  This provides a window for pre-emptive intervention, but there is little evidence to guide treatment. The use of FLT3 inhibitors (FLT3i) appears attractive but their use has not yet been evaluated.  We identified 56 patients treated with FLT3i at molecular failure.  The FLT3 mutation was an ITD in 52, TKD in 7 and both in 3. Over half of patients had previously received midostaurin. Molecular failure occurred at a median 9.2 months from diagnosis and was treated with gilteritinib (n = 38), quizartinib (n = 7) or sorafenib (n = 11). 60% achieved a molecular response, with 45% reaching MRD negativity. Haematological toxicity was low, and 22 patients were bridged directly to allogeneic transplant with another 6 to donor lymphocyte infusion. 2-year overall survival was 80% (95%CI 69-93) and molecular event-free survival 56% (95%CI 44-72). High-sensitivity next-generation sequencing for FLT3-ITD at molecular failure identified patients more likely to benefit. FLT3i monotherapy for molecular failure is a promising strategy which merits evaluation in prospective studies.

Indirect Effects of Halorhodopsin Activation: Potassium Redistribution, Nonspecific Inhibition, and Spreading Depolarization.

The movement of ions in and out of neurons can exert significant effects on neighboring cells. Here we report several experimentally important consequences of activation of the optogenetic chloride pump, halorhodopsin. We recorded extracellular K+ concentration ([K+]extra) in neocortical brain slices prepared from young adult mice (both sexes) which express halorhodopsin in pyramidal cells. Strong halorhodopsin activation induced a pronounced drop in [K+]extra that persisted for the duration of illumination. Pharmacological blockade of K+ channels reduced the amplitude of this drop, indicating that it represents K+ redistribution into cells during the period of hyperpolarization. Halorhodopsin thus drives the inward movement of both Cl- directly, and K+ secondarily. When the illumination period ended, a rebound surge in extracellular [K+] developed over tens of seconds, partly reflecting the previous inward redistribution of K+, but additionally driven by clearance of Cl- coupled to K+ by the potassium-chloride cotransporter, KCC2. The drop in [K+]extra during light activation leads to a small (2-3 mV) hyperpolarization also of other cells that do not express halorhodopsin. Its activation therefore has both direct and indirect inhibitory effects. Finally, we show that persistent strong activation of halorhodopsin causes cortical spreading depolarizations (CSDs), both in vitro and in vivo This novel means of triggering CSDs is unusual, in that the events can arise during the actual period of illumination, when neurons are being hyperpolarized and [K+]extra is low. We suggest that this fundamentally different experimental model of CSDs will open up new avenues of research to explain how they occur naturally.SIGNIFICANCE STATEMENT Halorhodopsin is a light-activated electrogenic chloride pump, which has been widely used to inhibit neurons optogenetically. Here, we demonstrate three previously unrecognized consequences of its use: (1) intense activation leads to secondary movement of K+ ions into the cells; (2) the resultant drop in extracellular [K+] reduces excitability also in other, nonexpressing cells; and (3) intense persistent halorhodopsin activation can trigger cortical spreading depolarization (CSD). Halorhodopsin-induced CSDs can occur when neurons are hyperpolarized and extracellular [K+] is low. This contrasts with the most widely used experimental models that trigger CSDs with high [K+]. Both models, however, are consistent with the hypothesis that CSDs arise following net inward ionic movement into the principal neuron population.

Cold Rolling Texture Prediction Using Finite Element Simulation with Zooming Analysis.

Cold rolling is widely employed in the manufacturing industry for the production of metal plates. In the cold rolling process, the thickness reduction of the metal plate under the recrystallization temperature generates severe anisotropy; this influences the subsequent forming processes. Therefore, the generation and prediction of metal plate anisotropy during cold rolling is a highly interesting research topic involving upstream studies of sheet metal forming. In this study, using the finite element method with zooming analysis, we established an efficient elastic-plastic analysis method to predict the metal plate texture after cold rolling. This method for cold rolling texture prediction was confirmed by comparing the experimental and simulation results of cold rolling for an S45C plate with a body-centered cubic lattice. Further, the numerical analysis method proposed in this study can contribute to the study of anisotropy as an alternative to experimental approaches.

Influence of implant neck and abutment characteristics on peri-implant tissue health and stability. Oral reconstruction foundation consensus report.

OBJECTIVE: The aim of this consensus meeting was to assess the influence of implant neck and abutment characteristics on peri-implant tissue health and stability. MATERIAL AND METHODS: Group and plenary discussions were based on two systematic reviews focusing on the effect of titanium implants with different collar designs/surface modifications and the abutment material on the stability of marginal bone levels (MBLs), peri-implant health, and survival rates. RESULTS: The changes in MBLs were not influenced by the abutment material and were also similar at one- and two-piece implants after one year of loading. Rough collar implants improved MBLs in comparison to machined collar implants. Additional modifications of the collar had no beneficial clinical effect on MBLs. Titanium abutments were associated with significantly higher increases in bleeding on probing when compared with zirconia abutments. CONCLUSION: MBLs are mainly influenced by the microstructure (i.e., rough surfaced) of the implant neck. Consensus statements and specific recommendations for future research were elaborated during the consensus meeting.

The Effects of a Single Whole-Body Cryotherapy Exposure on Physiological, Performance, and Perceptual Responses of Professional Academy Soccer Players After Repeated Sprint Exercise.

Russell, M, Birch, J, Love, T, Cook, CJ, Bracken, RM, Taylor, T, Swift, E, Cockburn, E, Finn, C, Cunningham, D, Wilson, L, and Kilduff, LP. The effects of a single whole-body cryotherapy exposure on physiological, performance, and perceptual responses of professional academy soccer players after repeated sprint exercise. J Strength Cond Res 31(2): 415-421, 2017-In professional youth soccer players, the physiological, performance, and perceptual effects of a single whole-body cryotherapy (WBC) session performed shortly after repeated sprint exercise were investigated. In a randomized, counterbalanced, and crossover design, 14 habituated English Premier League academy soccer players performed 15 × 30 m sprints (each followed by a 10 m forced deceleration) on 2 occasions. Within 20 minutes of exercise cessation, players entered a WBC chamber (Cryo: 30 seconds at -60° C, 120 seconds at -135° C) or remained seated (Con) indoors in temperate conditions (∼25° C). Blood and saliva samples, peak power output (countermovement jump), and perceptual indices of recovery and soreness were assessed pre-exercise and immediately, 2-hour and 24-hour postexercise. When compared with Con, a greater testosterone response was observed at 2-hour (+32.5 ± 32.3 pg·ml, +21%) and 24-hour (+50.4 ± 48.9 pg·ml, +28%) postexercise (both P = 0.002) in Cryo (trial × treatment interaction: P = 0.001). No between-trial differences were observed for other salivary (cortisol and testosterone/cortisol ratio), blood (lactate and creatine kinase), performance (peak power output), or perceptual (recovery or soreness) markers (all trial × treatment interactions: P > 0.05); all of which were influenced by exercise (time effects: all P ≤ 0.05). A single session of WBC performed within 20 minutes of repeated sprint exercise elevated testosterone concentrations for 24 hours but did not affect any other performance, physiological, or perceptual measurements taken. Although unclear, WBC may be efficacious for professional soccer players during congested fixture periods.

In vivo γ-aminobutyric acid measurement in rats with spectral editing at 4.7T.

PURPOSE: To evaluate the feasibility of spectral editing for quantification of γ-aminobutyric acid (GABA) in the rat brain and to determine whether altered GABA concentration in the ventral striatum is a neural endophenotype associated with trait-like impulsive behavior. MATERIALS AND METHODS: Spectra were acquired at 4.7T for 23 male Lister-hooded rats that had been previously screened for extremely low and high impulsivity phenotypes on an automated behavioral task (n = 11 low-impulsive; n = 12 high-impulsive). Voxels of 3 × 7 × 4 mm(3) (84 µL) centered bilaterally across the ventral striatum were used to evaluate GABA concentration ratios. RESULTS: Quantifiable GABA signals in the ventral striatum were obtained for all rats. Mean-edited GABA to n-acetyl aspartate (NAA) ratios in the ventral striatum were 0.22 (95% confidence interval [CI] [0.18, 0.25]). Mean GABA/NAA ratios in this region were significantly decreased by 28% in high-impulsive rats compared to low-impulsive rats (P = 0.02; 95% CI [-53%, -2%]). CONCLUSION: These findings demonstrate that spectral editing at 4.7T is a feasible method to assess in vivo GABA concentrations in the rat brain. The results show that diminished GABA content in the ventral striatum may be a neural endophenotype associated with impulsivity. J. Magn. Reson. Imaging 2016;43:1308-1312.

Theoretical limitations of quantification for noncompetitive sandwich immunoassays.

Immunoassays exploit the highly selective interaction between antibodies and antigens to provide a vital method for biomolecule detection at low concentrations. Developers and practitioners of immunoassays have long known that non-specific binding often restricts immunoassay limits of quantification (LOQs). Aside from non-specific binding, most efforts by analytical chemists to reduce the LOQ for these techniques have focused on improving the signal amplification methods and minimizing the limitations of the detection system. However, with detection technology now capable of sensing single-fluorescence molecules, this approach is unlikely to lead to dramatic improvements in the future. Here, fundamental interactions based on the law of mass action are analytically connected to signal generation, replacing the four- and five-parameter fittings commercially used to approximate sigmoidal immunoassay curves and allowing quantitative consideration of non-specific binding and statistical limitations in order to understand the ultimate detection capabilities of immunoassays. The restrictions imposed on limits of quantification by instrumental noise, non-specific binding, and counting statistics are discussed based on equilibrium relations for a sandwich immunoassay. Understanding the maximal capabilities of immunoassays for each of these regimes can greatly assist in the development and evaluation of immunoassay platforms. While many studies suggest that single molecule detection is possible through immunoassay techniques, here, it is demonstrated that the fundamental limit of quantification (precision of 10 % or better) for an immunoassay is approximately 131 molecules and this limit is based on fundamental and unavoidable statistical limitations.

Influenza vaccination rates for hospitalised patients: a multiyear quality improvement effort.

BACKGROUND: Influenza vaccination is the most effective method for preventing influenza virus infection. Adult hospitalised patients form a particularly high-risk group for severe influenza given their advanced age and comorbidities. We sought to improve the influenza vaccination rates of hospitalised patients at the White River Junction Veterans Affairs Medical Center. METHODS: The improvement effort started in 2007 when our baseline vaccination rate was about 60%. An interprofessional team analysed the influenza vaccination process for hospitalised patients. During the course of six influenza seasons, eight Plan-Do-Study-Act cycles were used including a hospital-wide flu campaign, embedded orders in the electronic medical record (EMR) to facilitate ordering vaccinations by providers, daily reminders from ward clerks and standing orders for influenza vaccination on discharge. The measure was the monthly percentage of patients discharged from the hospital with an up-to-date influenza vaccination. RESULTS: The percentage of veterans discharged with an up-to-date influenza vaccination increased to over 80% in February 2009 and has remained high. CONCLUSIONS: Although we are confident that our local efforts helped to improve the vaccination rate, external factors such as the 2009 H1N1 pandemic and universal vaccination may have primed our system to respond more readily to the implemented changes. Understanding all of the relevant factors that lead to vaccination uptake can be applied to future hospital influenza vaccination campaigns. In addition, our work demonstrates that an interprofessional approach is still required to apply the functionality of the EMR effectively.

Sedentary behaviour among elite professional footballers: health and performance implications.

BACKGROUND: Elite athletes should have little concern about meeting recommended guidelines on physical activity. However, sedentary behaviour is considered a health risk independent of physical activity, and is recognised in public health guidelines advising against prolonged sedentary time. There has been very little research on athletes' physical activity behaviour outside elite sport. METHODS: Given health and performance links, we investigated in-season post-training activity levels in 28 elite professional footballers during the English Premiership season. Players volunteered to wear a triaxial wrist accelerometer for 1 week, removing it only for training and matches. In total, 25 players met the inclusion criteria for analysis. Players recorded on average 632.6 min wear time p/day during the post-training period (SD±52.9) for a mean of 3.8 days (SD±1.5). RESULTS: On average, players recorded 76.2 min p/day (SD±28.8) of moderate or vigorous activity post-training. The majority (79%) of post-training time was spent in sedentary activities (500.6 min per day±59.0). CONCLUSIONS: Professional footballers are alarmingly sedentary in their leisure time, and comparatively more so than non-athletic groups of a similar age and older. This raises questions over optimum recovery and performance, as well as long-term health and cardiovascular risk. Worryingly, retirement from elite sport is likely to further imbalance activity and sedentary behaviour. Promoting regular periodic light to moderate leisure time activity could be beneficial. Further research and provision of education and support for players is required in this area.

The impact of neuromuscular electrical stimulation on recovery after intensive, muscle damaging, maximal speed training in professional team sports players.

OBJECTIVES: During congested fixture periods in team sports, limited recovery time and increased travel hinder the implementation of many recovery strategies; thus alternative methods are required. We examined the impact of a neuromuscular electrical stimulation device on 24-h recovery from an intensive training session in professional players. DESIGN: Twenty-eight professional rugby and football academy players completed this randomised and counter-balanced study, on 2 occasions, separated by 7 days. METHODS: After baseline perceived soreness, blood (lactate and creatine kinase) and saliva (testosterone and cortisol) samples were collected, players completed a standardised warm-up and baseline countermovement jumps (jump height). Players then completed 60 m × 50 m maximal sprints, with 5 min recovery between efforts. After completing the sprint session, players wore a neuromuscular electrical stimulation device or remained in normal attire (CON) for 8 h. All measures were repeated immediately, 2 and 24-h post-sprint. RESULTS: Player jump height was reduced from baseline at all time points under both conditions; however, at 24-h neuromuscular electrical stimulation was significantly more recovered (mean±SD; neuromuscular electrical stimulation -3.2±3.2 vs. CON -7.2±3.7%; P<0.001). Creatine kinase concentrations increased at all time points under both conditions, but at 24-h was lower under neuromuscular electrical stimulation (P<0.001). At 24-h, perceived soreness was significantly lower under neuromuscular electrical stimulation, when compared to CON (P=0.02). There was no effect of condition on blood lactate, or saliva testosterone and cortisol responses (P>0.05). CONCLUSIONS: Neuromuscular electrical stimulation improves recovery from intensive training in professional team sports players. This strategy offers an easily applied recovery strategy which may have particular application during sleep and travel.

Spatio-temporal image analysis of particle streaks in micro-channels for low-cost electro-hydrodynamic flow characterization.

Flow characterization is a primary analytical method for performance evaluation of microfluidic devices. With the increasing prevalence of microfluidic devices in recent years, there is a growing need for simple methods of automated flow estimation. In this work, a novel flow diagnostic technique based on image analysis of particle streaks is introduced, to characterize local flow velocities. While 1D velocimetry using particle tracks has occasionally been discussed for macro-scale environments, the use of particle streaks for 2-D flow characterization in micro-channels has not been explored. The proposed technique is qualitatively validated against electrokinetic experiment and numerically validated with simulated flows.

Primary care access to computed tomography for chronic headache.

BACKGROUND: The diagnostic yield of neuroimaging in chronic headache is low, but can reduce the use of health services. AIM: To determine whether primary care access to brain computed tomography (CT) referral for chronic headache reduces referral to secondary care. DESIGN OF STUDY: Prospective observational analysis of GP referrals to an open access CT brain scanning service. SETTING: Primary care, and outpatient radiology and neurology departments. METHOD: GPs in Tayside and North East Fife, Scotland were given access to brain CT for patients with chronic headache. All referrals were analysed prospectively over 1 year, and questionnaires were sent to referrers to establish whether imaging had resulted in or stopped a referral to secondary care. The Tayside outpatient clinic database identified scanned patients referred to the neurology clinic for headache from the start of the study period to at least 1 year after their scan. RESULTS: There were 232 referrals (55.1/100 000/year, 95% confidence interval = 50.4 to 59.9) from GPs in 59 (82%) of 72 primary care practices. CT was performed on 215 patients. Significant abnormalities were noted in 3 (1.4%) patients; there were 22 (10.2%) non-significant findings, and 190 (88.4%) normal scans. Questionnaires of the referring GPs reported that 167 (88%) scans stopped a referral to secondary care. GPs referred 30 (14%) scanned patients to a neurologist because of headache. It is estimated that imaging reduced referrals to secondary care by 86% in the follow-up period. CONCLUSION: An open access brain CT service for patients with chronic headache was used by most GP practices in Tayside, and reduced the number of referrals to secondary care.

Demonstration of sandwich and competitive modulated supraparticle fluoroimmunoassay applied to cardiac protein biomarker myoglobin.

Modulated supraparticle structures are used to improve sandwich and competitive fluoroimmunoassays. The improved methods are demonstrated on myoglobin, a key diagnostic protein for detection of heart damage. The resulting method uses microliter volumes with bovine serum samples doped with varying concentrations of equine myoglobin. These immunoassays use micron-diameter iron oxide particles as a solid phase for antibody anchoring. Introduction of a magnetic field creates dipole moments on the particles, which attracts them to each other to form rod-like supraparticle structures. These structures can rotate within an alternating magnetic field generating convective flow and a periodic signal that can be analyzed with lock-in amplification enabling more sensitive detection. The system is demonstrated on a target associated with acute myocardial infarction (AMI). This disease causes decreased oxygen delivery to the heart resulting in tissue death and the release of cardiac myoglobin into the bloodstream. Studies have shown that the assessment and monitoring of serum myoglobin concentrations is important when making an early diagnosis of AMI. Early diagnosis is crucial since treatment is most effective when done within the first two hours of symptoms. The modulated assay is rapid, accurate, and sensitive for myoglobin assessment of small-volume serum samples. Using a cut-off value of 5.0 nM (85 ng/mL) for AMI induced myoglobin, the modulated competitive assay was able to diagnose AMI-like conditions in serum doped with myoglobin after an incubation time of only 10 min. The standard curve developed for the modulated sandwich assay was linear over a range of zero to 1 nM (17 ng/mL) with a lower limit of detection at 50 pM (0.85 ng/mL).

Determining risk factors for candidemia among newborn infants from population-based surveillance: Baltimore, Maryland, 1998-2000.

BACKGROUND: Our objective was to determine risks factors for late onset candidemia, independent of birth weight, in newborn infants. METHODS: We performed a matched case-control study. Cases were identified through active, population-based surveillance for candidemia, conducted in Baltimore City and County during 1998-2000, and were defined as the incident isolation of any Candida species from the bloodstream of an infant 3 months old or younger. Four controls, matched by age, hospital, birth weight category, hospital stay and admission date, were selected for each case. Potential risk factors included clinical, demographic and maternal prenatal data. RESULTS: Of the 35 cases, 19 (54%) infections were with Candida albicans, 9 (26%) were with Candida parapsilosis and 5 (14%) were with Candida glabrata. Cases had a median birth weight of 680 g (range, 430-3200 g); median gestational ages of cases and controls were 25 and 27 weeks, respectively. Compared with controls, cases had significant higher mortality (20% versus 4%; P = 0.004). No maternal factors were associated with increased risk of disease; cases were as likely as controls to be of black race. Multivariable conditional logistic regression analysis revealed that gestational age younger than 26 weeks [adjusted odds ratio, 6.5; 95% confidence interval (95% CI), 1.3-32], vaginal delivery (adjusted odds ratio, 4.3; 95% CI 1.3-14.2) and abdominal surgery (adjusted odds ratio, 10.9; 95% CI 1.9-62) were independently associated with increased risk of candidemia. CONCLUSIONS: Independent of birth weight, infants born at <26 weeks or those who have had abdominal surgery are at a significantly increased risk of candidemia. This study helps define a subgroup of preterm infants at high risk of developing bloodstream infections with Candida species.

Antimicrobial resistance incidence and risk factors among Helicobacter pylori-infected persons, United States.

Helicobacter pylori is the primary cause of peptic ulcer disease and an etiologic agent in the development of gastric cancer. H. pylori infection is curable with regimens of multiple antimicrobial agents, and antimicrobial resistance is a leading cause of treatment failure. The Helicobacter pylori Antimicrobial Resistance Monitoring Program (HARP) is a prospective, multicenter U.S. network that tracks national incidence rates of H. pylori antimicrobial resistance. Of 347 clinical H. pylori isolates collected from December 1998 through 2002, 101 (29.1%) were resistant to one antimicrobial agent, and 17 (5%) were resistant to two or more antimicrobial agents. Eighty-seven (25.1%) isolates were resistant to metronidazole, 45 (12.9%) to clarithromycin, and 3 (0.9%) to amoxicillin. On multivariate analysis, black race was the only significant risk factor (p < 0.01, hazard ratio 2.04) for infection with a resistant H. pylori strain. Formulating pretreatment screening strategies or providing alternative therapeutic regimens for high-risk populations may be important for future clinical practice.

Deformation of helix C in the low temperature L-intermediate of bacteriorhodopsin.

X-ray and electron diffraction studies of specific reaction intermediates, or reaction intermediate analogues, have produced a consistent picture of the structural mechanism of light-driven proton pumping by bacteriorhodopsin. Of central importance within this picture is the structure of the L-intermediate, which follows the retinal all-trans to 13-cis photoisomerization step of the K-intermediate and sets the stage for the primary proton transfer event from the positively charged Schiff base to the negatively charged Asp-85. Here we report the structural changes in bacteriorhodopsin following red light illumination at 150 K. Single crystal microspectrophotometry showed that only the L-intermediate is populated in three-dimensional crystals under these conditions. The experimental difference Fourier electron density map and refined crystallographic structure were consistent with those previously presented (Royant, A., Edman, K., Ursby, T., Pebay-Peyroula, E., Landau, E. M., and Neutze, R. (2000) Nature 406, 645-648; Royant, A., Edman, K., Ursby, T., Pebay-Peyroula, E., Landau, E. M., and Neutze, R. (2001) Photochem. Photobiol. 74, 794-804). Based on the refined crystallographic structures, molecular dynamic simulations were used to examine the influence of the conformational change of the protein that is associated with the K-to-L transition on retinal dynamics. Implications regarding the structural mechanism for proton pumping by bacteriorhodopsin are discussed.