Early-stage dynamics of chloride ion–pumping rhodopsin revealed by a femtosecond X-ray laser

Chloride ion–pumping rhodopsin (ClR) in some marine bacteria utilizes light energy to actively transport Cl⁻ into cells. How the ClR initiates the transport is elusive. Here, we show the dynamics of ion transport observed with time-resolved serial femtosecond (fs) crystallography using the Linac Coherent Light Source. X-ray pulses captured structural changes in ClR upon flash illumination with a 550 nm fs-pumping laser. High-resolution structures for five time points (dark to 100 ps after flashing) reveal complex and coordinated dynamics comprising retinal isomerization, water molecule rearrangement, and conformational changes of various residues. Combining data from time-resolved spectroscopy experiments and molecular dynamics simulations, this study reveals that the chloride ion close to the Schiff base undergoes a dissociation–diffusion process upon light-triggered retinal isomerization.

Chloride ion (Cl⁻) concentration in some bacterial cells is regulated by rhodopsin proteins, generally known as halorhodopsin, or hR. These proteins use light energy to pump Cl⁻ into cells (1, 2). Light is harvested by a molecule of retinal, covalently linked to an essential lysine residue in the seventh transmembrane helix of GPCR-like (G protein–coupled receptor) proteins. Light activation causes retinal to isomerize from the all-trans to the 13-cis configuration. This change triggers subsequent conformational changes throughout the rhodopsin molecule and releases chloride into the cytoplasm. Retinal thermally relaxes to the all-trans configuration within milliseconds and is then ready for the next photocycle. Cl⁻ ions are transported from the extracellular (EC) side to the cytoplasmic (CP) side during each photocycle (3, 4).Light-driven ion-pumping rhodopsin can be used to develop artificial solar energy harvesting and optogenetics (5–8), but the molecular mechanism must be understood in detail for such applications. Despite the importance of hR, our current experimental data concerning the structure and dynamics of the protein remain very limited. A related protein, proton (H⁺)-pumping bacteriorhodopsin (bR) discovered in the early 1970s, has been extensively studied by multiple methods, including time-resolved spectroscopy, crystallography, mutagenesis, and computer simulation (9–12). In particular, recent studies using time-resolved serial femtosecond crystallography (TR-SFX) methods performed at X-ray free-electron laser (XFEL) facilities allow three-dimensional (3D) visualization of retinal isomerization and associated local conformational changes. These changes are accompanied by movement of protons from a donor aspartate group to an acceptor aspartate (13–15). However, the central component of this process, the transported H⁺, is difficult to observe by X-ray crystallography and could not be directly traced in bR TR-SFX studies. Recently, a breakthrough was reported in a study on the sodium-pumping rhodopsin KR2 (K. eikastus rhodopsin 2), in which electron density signals of Na⁺ uptake were observed at Δt = 1 ms after laser illumination (16).Cl⁻, a strong X-ray scatterer, can be directly observed from electron density maps. These maps provide first-hand information on the movement of ions as being transported within short timescales after light activation. Furthermore, hR and bR presumably share a common molecular mechanism despite transporting ions in opposite directions. A close relationship is strongly implied by the interconversion of the function of two rhodopsins. Outward H⁺-pumping bR can be converted to an inward Cl⁻ pump by changing a single residue (D85T) (17), while hR from the cyanobacterium, Mastigocladopsis repens, is reported to pump protons after a single mutation (T74D) (18). The chloride pump can therefore serve as a system analogous to the proton transporter and provide valuable information that is difficult to obtain directly from bR.In this study, we focus on chloride ion–pumping rhodopsin (ClR) from the marine flavobacterium Nonlabens marinus S1-08T (19). The conserved DTD motif (Asp85-Thr89-Asp96) of the bR family, residues 85, 89, and 96, is replaced by an NTQ motif (Asn98- Thr102-Gln109) in ClR (Fig. 1). The sequence identity of ClR and canonical bR from Halobacterium salinarum is only 27%, but the two proteins, nevertheless, have highly similar structures, including the disposition of the retinal chromophore. ClR structures at cryogenic and room temperatures clearly reveal an architecture composed of seven transmembrane helices (TM A to G) (2, 20, 21). The retinal is covalently linked to the Nζ atom of the Lys235 located on TM-G. Anomalous diffraction signals of the Br⁻ identify a stable binding site near the protonated Schiff base (PSB) and a plausible exit site on the CP side (Fig. 1A). Buried water molecules and locations of cavities inside ClR suggest a pathway for Cl⁻ uptake on the EC side, but the molecular mechanism for light-triggered Cl⁻ pumping remains obscure. Upon light activation, the Cl⁻ tightly held near the PSB must break free from its hydrogen bonding network (Fig. 1B). It then passes through a hydrophobic region to reach the CP side (Fig. 1C). Crystal structures of ClR were previously determined with crystals under continuous illumination of visible laser light. Intriguingly, these steady-state models revealed unexpected movement of the retinal, without indication of photo-isomerization (22). Steady-state measurements, which show averages of mixed states, are thus of limited use in deciphering the molecular mechanism of light-driven Cl⁻ pumping.Open in a separate windowFig. 1.Structure of ClR and a plausible pathway of Cl⁻ transport. (A) Cross-sections of ClR with the backbone structure shown in cartoon representation. Transmembrane helices are marked using letters A through G, and the C-terminal helix H in the cytoplasm is also indicated. Surfaces are clipped to show the cross-section colored in yellow and the model being sliced and then opened about the axis near the helix E. Water molecules and Cl⁻ ions are shown as red- and green-colored spheres. Blue curves indicate the path of ion entering ClR and the principal pumping direction after passing retinal. (B) Key residues near the Cl⁻ ion and retinal, together with the NTQ motif shown in stick representation. (C) Residues that form a hydrophobic region between the retinal and the cytoplasm are highlighted in ball-and-stick representation. The red arrow points to a major barrier that Cl⁻ needs to overcome. ClR backbone is shown in cartoon representation, with residues colored based on hydrophobicity (the blue to red spectrum corresponds to the hydrophobicity scale from hydrophilic to hydrophobic).     

Trapped renal arteries: functional renal artery stenosis due to occlusion of the aorta in the arch and below the kidneys

Acute renal failure is a well recognized complication from the use of angiotensin-converting enzyme inhibitors in patients with severe bilateral renovascular disease. A 54-year-old woman presented with acute pulmonary edema with intractable hypertension and a history of lower limb claudication. The addition of lisinopril to her antihypertensive regimen resulted, within 48 h, in the development of acute renal failure that remitted with cessation of the drug. She was found to have a heavily calcified occlusion of her aortic arch and another occlusion of the aorta below the renal arteries. Angiography and Doppler ultrasonography showed normal renal arteries. This is the first reported case of angiotensin-converting enzyme inhibitor-induced renal failure occurring in a patient with atherosclerotic occlusion of the aorta. The literature on suprarenal aortic occlusion is reviewed to determine the manner of presentation, prevalent risk factors and physical findings that typify this unique clinical entity.     

Is there an athlete’s artery? A comparison of brachial and femoral artery structure and function in male strength,power and endurance athletes

ObjectivesExercise places physiological demands upon the cardiovascular system, subsequently leading to adaptations in structure and function. Different exercise modalities (endurance, strength and power) lead to distinct hemodynamic demands and, possibly, different patterns of adaptation. Our aim was to assess and compare brachial and femoral artery function and structure in elite level athletes engaged in endurance, strength and power sports.Designcross sectional comparison.Methods30 male elite athletes (runners n = 10, powerlifters n = 11, weightlifters n = 9) and 23 healthy controls were recruited. Brachial and femoral arterial diameters were assessed using ultrasound. Arterial function (brachial and femoral arteries) was determined using the flow mediated dilation (FMD) technique and body composition using body mass index (BMI) and body surface area (BSA).ResultsWeightlifters had significantly larger brachial arterial diameters compared to controls (4.39 ± 0.34 vs 3.86 ± 0.42 mm, p < 0.01). As weightlifter and power athletes had significantly higher body mass, BMI and BSA, we adjusted diameter for BSA. BSA-correction ameliorated differences in brachial artery resting diameters between athletes and controls. However, BSA-corrected femoral artery diameter was significantly larger in runners compared to controls (3.51 ± 0.28 vs 3.25 ± 0.34 mm, p < 0.05). There were no differences in brachial FMD between groups. Femoral artery FMD was significantly higher in runners and weightlifters compared to controls (p < 0.05 for both groups).ConclusionsHeterogeneous, limb-specific structural and functional vascular adaptation is evident in athletes, which may be influenced by exercise modality. Further, vascular remodelling relates to differences in body shape, specifically body composition, which should be accounted for when comparing athletes.     

Zinc Status Alters Alzheimer's Disease Progression through NLRP3-Dependent Inflammation

Alzheimer''s disease is a devastating neurodegenerative disease with a dramatically increasing prevalence and no disease-modifying treatment. Inflammatory lifestyle factors increase the risk of developing Alzheimer''s disease. Zinc deficiency is the most prevalent malnutrition in the world and may be a risk factor for Alzheimer''s disease potentially through enhanced inflammation, although evidence for this is limited. Here we provide epidemiological evidence suggesting that zinc supplementation was associated with reduced risk and slower cognitive decline, in people with Alzheimer''s disease and mild cognitive impairment. Using the APP/PS1 mouse model of Alzheimer''s disease fed a control (35 mg/kg zinc) or diet deficient in zinc (3 mg/kg zinc), we determined that zinc deficiency accelerated Alzheimer''s-like memory deficits without modifying amyloid β plaque burden in the brains of male mice. The NLRP3-inflammasome complex is one of the most important regulators of inflammation, and we show here that zinc deficiency in immune cells, including microglia, potentiated NLRP3 responses to inflammatory stimuli in vitro, including amyloid oligomers, while zinc supplementation inhibited NLRP3 activation. APP/PS1 mice deficient in NLRP3 were protected against the accelerated cognitive decline with zinc deficiency. Collectively, this research suggests that zinc status is linked to inflammatory reactivity and may be modified in people to reduce the risk and slow the progression of Alzheimer''s disease.SIGNIFICANCE STATEMENT Alzheimer''s disease is a common condition mostly affecting the elderly. Zinc deficiency is also a global problem, especially in the elderly and also in people with Alzheimer''s disease. Zinc deficiency contributes to many clinical disorders, including immune dysfunction. Inflammation is known to contribute to the risk and progression of Alzheimer''s disease; thus, we hypothesized that zinc status would affect Alzheimer''s disease progression. Here we show that zinc supplementation reduced the prevalence and symptomatic decline in people with Alzheimer''s disease. In an animal model of Alzheimer''s disease, zinc deficiency worsened cognitive decline because of an enhancement in NLRP3-driven inflammation. Overall, our data suggest that zinc status affects Alzheimer''s disease progression, and that zinc supplementation could slow the rate of cognitive decline.     

A novel approach to improve undergraduate surgical teaching

Background:

Undergraduate surgery is at an important crossroads. Many departments report significant difficulties delivering effective teaching. Our student feedback indicated a dated surgical curriculum lacking structure, quality and uniformity. We report on a new ”blended” approach employing a combination of professional DVDs, case based discussions, online material and traditional bedside teaching designed to provide structure, standardization, and equality of learning .

Methods:

Year 4 students who had undertaken the new course and year 5 students who had participated in the traditional teaching programme were compared. Students completed a 20 item questionnaire about their experiences of the surgical teaching programme.

Results:

One hundred and seventy-one year 4 (70%) and148 year 5 students (66%) responded. Domains relating to “Overall Satisfaction with the course”, “Approval of innovative teaching methods and interactivity” and “Satisfaction with the clarity of course information” showed improvements when comparing the new and old programmes. However bedside teaching was not rated as highly in the new programme (p<0.05).

Conclusion:

This blended approach has resulted in improved student understanding and engagement. The apparent compromise of bedside teaching may be a reflection of higher expectations. We believe that a similar blended approach has the potential to re-invigorate surgical teaching elsewhere.     

DNA densitometry of colorectal cancer.

DNA analysis was assessed by densitometry for 281 cases of colorectal adenocarcinoma. Detection of aneuploidy in a single case rose from 65% if one, to 92.5% when three or more sections, were analysed. Although aneuploid tumours had significantly larger nuclear areas than near diploid tumours (p = 0.009), densitometric measurements showed no association with clinicopathological variables. DNA content determined by densitometry was compared with that from flow cytometry on 465 tissue sections from 241 cases. Aneuploidy assessed by flow cytometry was significantly associated with that determined by densitometry (p < 0.01 for all comparisons), ploidy state being similar in 381 sections (82%, kappa = 0.63, p < 0.001), and 187 cases (77.6%, kappa = 0.57, p < 0.001). Univariate survival analysis showed that DNA densitometric variables had no significant association with survival in (a) all cases, (b) cases without lymph node metastases, or (c) cases without distant metastases. Multivariate regression analysis of densitometric and clinicopathological variables identified Dukes's stage, patient age, and tumour differentiation as the combination of variables most closely related to survival. Densitometric measurement of DNA content could not significantly improve on the prognostic model containing these three variables. It is concluded that, although the assessment of DNA content by densitometry is comparable with that of flow cytometry, conventional histological variables remain the best predictors of prognosis in colorectal cancer.