Stroke Patients with Nearly Independent Transfer Ability are at High Risk of Falling.

OBJECTIVES: To examine the relationship between patients' transfer ability and fall risk in stroke patients during hospitalization. MATERIALS AND METHODS: We retrospectively enrolled 237 stroke patients who were transferred to a convalescent rehabilitation ward from acute wards in the same hospital. Using incident reports, we investigated their fall rates and activity status at the falls according to their transfer abilities, which were assessed with Functional Independence Measure (FIM) transfer scores. The bi-weekly time trend of fall rates in all patients and in three subgroups based on FIM transfer scores of 1-3, 4-5, and 6-7, and activity status at the falls, were investigated. In addition, changes of patients' transfer ability on admission, at the first fall, and at discharge were investigated among falling patients. RESULTS: The fall rate was the greatest in patients with a FIM transfer score of 4 (14.3 times/1000 person-days). The majority of falls for patients with a FIM transfer score of 1 occurred at the activity status of "on the bed" and "sitting", while three quarters of patients with a FIM score of 7 had falls during "standing" and "walking". No longitudinal trend in fall rates was found overall; however, the fall rate trends differed depending on the FIM transfer score. The majority of the patients who fell required full assistance for transfers upon admission but required no assistance at discharge. CONCLUSIONS: Fall risk differed among patients with various transfer abilities; the greatest risk was in those who needed minimal assistance for transfers.

Fluorescence Enhancement of a Microbial Rhodopsin via Electronic Reprogramming.

The engineering of microbial rhodopsins with enhanced fluorescence is of great importance in the expanding field of optogenetics. Here we report the discovery of two mutants (W76S/Y179F and L83Q) of a sensory rhodopsin from the cyanobacterium Anabaena PCC7120 with opposite fluorescence behavior. In fact, while W76S/Y179F displays, with respect to the wild-type protein, a nearly 10-fold increase in red-light emission, the second is not emissive. Thus, the W76S/Y179F, L83Q pair offers an unprecedented opportunity for the investigation of fluorescence enhancement in microbial rhodopsins, which is pursued by combining transient absorption spectroscopy and multiconfigurational quantum chemistry. The results of such an investigation point to an isomerization-blocking electronic effect as the direct cause of instantaneous (subpicosecond) fluorescence enhancement.

Spectroscopic Study of Proton-Transfer Mechanism of Inward Proton-Pump Rhodopsin, Parvularcula oceani Xenorhodopsin.

Parvularcula oceani xenorhodopsin is the first light-driven inward proton pump. To understand the mechanism of inward proton transport, comprehensive transient absorption spectroscopy was conducted. Ultrafast pump-probe spectroscopy revealed that the isomerization time of retinal is 1.2 ps, which is considerably slower than those of other microbial rhodopsins (180-770 fs). Following the production of J, the K intermediate was formed at 4 ps. Proton transfer occurred on a slower timescale. Proton release and uptake were observed on the L/M-to-M and M decay, respectively, by monitoring transient absorption changes of pH-indicating dye, pyranine. Although a proton was released from Asp216 into the cytoplasmic medium, no proton-donating residue was identified on the extracellular side in mutation experiments. We revealed that a branched retinal isomerization (from 13-cis-15-anti to 13-cis-15-syn and all-trans-15-anti) occurred simultaneously with proton uptake. Furthermore, although the proton release showed a large kinetic isotope effect (KIE), the KIE of proton uptake was negligible. These results suggest that retinal isomerization is the rate-limiting process in proton uptake and that the regulation of p Ka of the retinal Schiff base by thermal isomerization enables the uptake from extracellular medium. This proton uptake mechanism differs from that of the outward proton pump with an internal proton donor and is important for understanding how the direction of ion transport by membrane proteins is determined.

A natural light-driven inward proton pump.

Light-driven outward H+ pumps are widely distributed in nature, converting sunlight energy into proton motive force. Here we report the characterization of an oppositely directed H+ pump with a similar architecture to outward pumps. A deep-ocean marine bacterium, Parvularcula oceani, contains three rhodopsins, one of which functions as a light-driven inward H+ pump when expressed in Escherichia coli and mouse neural cells. Detailed mechanistic analyses of the purified proteins reveal that small differences in the interactions established at the active centre determine the direction of primary H+ transfer. Outward H+ pumps establish strong electrostatic interactions between the primary H+ donor and the extracellular acceptor. In the inward H+ pump these electrostatic interactions are weaker, inducing a more relaxed chromophore structure that leads to the long-distance transfer of H+ to the cytoplasmic side. These results demonstrate an elaborate molecular design to control the direction of H+ transfers in proteins.

Toward Automatic Rhodopsin Modeling as a Tool for High-Throughput Computational Photobiology.

We report on a prototype protocol for the automatic and fast construction of congruous sets of QM/MM models of rhodopsin-like photoreceptors and of their mutants. In the present implementation the information required for the construction of each model is essentially a crystallographic structure or a comparative model complemented with information on the protonation state of ionizable side chains and distributions of external counterions. Starting with such information, a model formed by a fixed environment system, a flexible cavity system, and a chromophore system is automatically generated. The results of the predicted vertical excitation energy for 27 different rhodopsins including vertebrate, invertebrate, and microbial pigments indicate that such basic models could be employed for predicting trends in spectral changes and/or correlate the spectral changes with structural variations in large sets of proteins.

The photochemistry of sodium ion pump rhodopsin observed by watermarked femto- to submillisecond stimulated Raman spectroscopy.

Krokinobacter rhodopsin 2 (KR2) is a recently discovered light-driven Na(+) pump that holds significant promise for application as a neural silencer in optogenetics. KR2 transports Na(+) (in NaCl solution) or H(+) (in larger cation solution, e.g. in CsCl) during its photocycle. Here, we investigate the photochemistry of KR2 with the recently developed watermarked, baseline-free femto- to submillisecond transient stimulated Raman spectroscopy (TSRS), which enables us to investigate retinal chromophore dynamics in real time with high spectral resolution over a large time range. We propose a new photocycle from femtoseconds to submilliseconds: J (formed in ∼200 fs) → K (∼3 ps) → K/L1 (∼20 ps) → K/L2 (∼30 ns) → L/M (∼20 µs). KR2 binds a Na(+) ion that is not transported on the extracellular side, of which the function is unclear. We demonstrate with TSRS that for the D102N mutant in NaCl (with Na(+) unbound, Na(+) transport) and for WT KR2 in CsCl (with Na(+) unbound, H(+) transport), the extracellular Na(+) binding significantly influences the intermediate K/L/M state equilibrium on the photocycle, while the identity of the transported ion, Na(+) or H(+), does not affect the photocycle. Our findings will contribute to further elucidation of the molecular mechanisms of KR2.

Mutant of a Light-Driven Sodium Ion Pump Can Transport Cesium Ions.

Krokinobacter eikastus rhodopsin 2 (KR2) is a light-driven Na(+) pump found in marine bacterium. KR2 pumps Li(+) and Na(+), but it becomes an H(+) pump in the presence of K(+), Rb(+), and Cs(+). Site-directed mutagenesis of the cytoplasmic surface successfully converted KR2 into a light-driven K(+) pump, suggesting that ion selectivity is determined at the cytoplasmic surface. Here we extended this research and successfully created a light-driven Cs(+) pump. KR2 N61L/G263F pumps Cs(+) as well as other monovalent cations in the presence of a protonophore. Ion-transport activities correlated with the additive volume of the residues at 61 and 263. The result suggests that an ion-selectivity filter is affected by these two residues and functions by strict exclusion of K(+) and larger cations in the wild type (N61/G263). In contrast, introduction of large residues possibly destroys local structures of the ion-selectivity filter, leading to the permeation of K(+) (P61/W263) and Cs(+) (L61/F263).

Kinetic Analysis of H(+)-Na(+) Selectivity in a Light-Driven Na(+)-Pumping Rhodopsin.

Krokinobacter eikastus rhodopsin 2 (KR2) is a recently identified light-driven Na(+) pump from a marine bacterium. KR2 pumps Na(+) in NaCl solution but pumps H(+) in the absence of Na(+) and Li(+). The Na(+) transport mechanism in KR2 has been extensively studied, whereas understanding of the H(+) transport mechanism is very limited. Here we studied ion uptake mechanisms and H(+)-Na(+) selectivity using flash photolysis. The results show that decay of the blue-shifted M intermediate is dependent on both [Na(+)] and [H(+)], indicating that KR2 competitively uptakes Na(+) or H(+) upon M decay. Comprehensive concentration dependence of Na(+) and H(+) revealed that the rate constant of H(+) uptake (kH) was much larger than that of Na(+) uptake (kNa) with a ratio (kH/kNa) of >10(3). Therefore, KR2 pumps only H(+) when Na(+) and H(+) concentrations are similar. On the contrary, KR2 pumps Na(+) exclusively under physiological conditions in which [Na(+)] is much greater than [H(+)].

Structural basis for Na(+) transport mechanism by a light-driven Na(+) pump.

Krokinobacter eikastus rhodopsin 2 (KR2) is the first light-driven Na(+) pump discovered, and is viewed as a potential next-generation optogenetics tool. Since the positively charged Schiff base proton, located within the ion-conducting pathway of all light-driven ion pumps, was thought to prohibit the transport of a non-proton cation, the discovery of KR2 raised the question of how it achieves Na(+) transport. Here we present crystal structures of KR2 under neutral and acidic conditions, which represent the resting and M-like intermediate states, respectively. Structural and spectroscopic analyses revealed the gating mechanism, whereby the flipping of Asp116 sequesters the Schiff base proton from the conducting pathway to facilitate Na(+) transport. Together with the structure-based engineering of the first light-driven K(+) pumps, electrophysiological assays in mammalian neurons and behavioural assays in a nematode, our studies reveal the molecular basis for light-driven non-proton cation pumps and thus provide a framework that may advance the development of next-generation optogenetics.

Light-driven ion-translocating rhodopsins in marine bacteria.

Microbial rhodopsins are the photoreceptive membrane proteins found in diverse microorganisms from within Archaea, Eubacteria, and eukaryotes. They have a hep-tahelical transmembrane structure that binds to an all-trans retinal chromophore. Since 2000, thousands of proteorhodopsins, genes of light-driven proton pump rhodopsins, have been identified from various species of marine bacteria. This suggests that they are used for the conversion of light into chemical energy, contribut-ing to carbon circulation related to ATP synthesis in the ocean. Furthermore, novel types of rhodopsin (sodium and chloride pumps) have recently been discovered. Here, we review recent progress in our understanding of ion-transporting rhodopsins of marine bacteria, based mainly on biophysical and biochemical research.

Spectroscopic study of a light-driven chloride ion pump from marine bacteria.

Thousands of light-driven proton-pumping rhodopsins have been found in marine microbes, and a light-driven sodium-ion pumping rhodopsin was recently discovered, which utilizes sunlight for the energy source of the cell. Similarly, a light-driven chloride-ion pump has been found from marine bacteria, and three eubacterial light-driven pumps possess the DTE (proton pump), NDQ (sodium-ion pump), and NTQ (chloride-ion pump) motifs corresponding to the D85, T89, and D96 positions in bacteriorhodopsin (BR). The corresponding motif of the known haloarchaeal chloride-ion pump, halorhodopsin (HR), is TSA, which is entirely different from the NTQ motif of a eubacterial chloride-ion pump. It is thus intriguing to compare the molecular mechanism of these two chloride-ion pumps. Here we report the spectroscopic study of Fulvimarina rhodopsin (FR), a eubacterial light-driven chloride-ion pump from marine bacterium. FR binds a chloride-ion near the retinal chromophore and chloride-ion binding causes a spectral blue-shift. FR predominantly possesses an all-trans retinal, which is responsible for the light-driven chloride-ion pump. Upon light absorption, the red-shifted K intermediate is formed, followed by the appearance of the L and O intermediates. When the M intermediate does not form, this indicates that the Schiff base remains in the protonated state during the photocycle. These molecular mechanisms are common in HR, and a common mechanism for chloride-ion pumping by evolutionarily distant proteins suggests the importance of the electric quadrupole in the Schiff base region and their changes through hydrogen-bonding alterations. One noticeable difference between FR and HR is the uptake of chloride-ion from the extracellular surface. While the uptake occurs upon decay of the O intermediate in HR, chloride-ion uptake accompanies the rise of the O intermediate in FR. This suggests the presence of a second chloride-ion binding site near the extracellular surface of FR, which is unique to the NTQ rhodopsin.

pH-Dependent photoreaction pathway of the all-trans form of Anabaena sensory rhodopsin.

Anabaena sensory rhodopsin (ASR) is well-known as the only retinal protein that achieves the photochromic reaction between the all-trans form (AT-ASR) and the 13-cis form (C-ASR). Although it is known that the structure of the hydrogen-bonding network of ASR is pH-dependent, it is so far unclear how pH affects the photoreaction of ASR. We investigated the pH dependence of the photoreaction of AT-ASR by means of time-resolved absorption spectroscopy and found it to be extremely dependent on pH. At pH 7 and 9, not only the L intermediate but also the K intermediate consisted of two decay components. The formation ratios of the two distinct L intermediates L(fast):L(slow) at pH 7 and 9 were different from each other, although the K(fast):K(slow) ratio was pH-independent. The photoreaction at pH 5 was entirely different from that at pH 7 and 9. Two K intermediates existed, but their formation ratio and lifetimes were different at pH 7 and 9. Moreover, only one L intermediate exists, with a longer lifetime relative to pH 7 and 9. The final product of the photoreaction of AT-ASR was C-ASR at all pH values. Finally, we successfully determined the pH-related photoreaction pathway of AT-ASR.

Effects of nitrate and oxygen on photoautotrophic lipid production from Chlorococcum littorale.

Effects of oxygen and nitrate on fatty acid/lipid production from a highly CO(2)-tolerant microalgal species Chlorococcum littorale were examined under photoautotrophic conditions of 295 K, a light intensity of 170 µmol-photon m(-2) s(-1), a bubbling CO(2) concentration of 5% (v/v) and bubbling oxygen concentrations to be volumetrically adjusted by mixing oxygen gas with inert nitrogen gas at concentrations ranging from 0% to 95% (v/v). The results showed that maximum fatty acid content reached ca. 34 wt.% under oxygen-freely bubbling conditions and this value decreased to be ca. 20 wt.% when air-like oxygen concentration of 20% was chosen. This means that degree of the accumulation strongly depended on the level of bubbling oxygen concentrations, which can be a crucial factor after nitrogen depletion in the photoautotrophic culture system. TLC-FID/FPD analyses showed that triglycerides were found to be a dominant lipid class for this accumulation.

Carotenoid production from Chlorococcum littorale in photoautotrophic cultures with downstream supercritical fluid processing.

Carotenoid production from highly CO(2 )tolerant microalga Chlorococcum littorale in photoautotrophic cultures with downstream supercritical fluid processing was studied. Increasing temperature, increasing light intensity and decreasing CO(2) and O(2) gas concentrations enhanced growth rate under nitrate-rich conditions. Carotenoid content was insensitive to nitrate concentration, temperature and gas composition, but was greatly promoted by light intensity. Growth rate and carotenoid content had an optimum light intensity of ca. 120 micromol-photon * m(-2)s(-1). Separation of two sample cultures was studied by applying supercritical fluid extraction with CO(2 )and 10 mol% ethanol co-solvent. Extraction yield of carotenoids was 90% with 10 mol% ethanol at 333 K and 30 MPa. Selectivity of a sample with less lipid content (12.9 wt%) was five-fold higher than that with higher lipid content (29.4 w%).

Fatty acid production from a highly CO2 tolerant alga, Chlorocuccum littorale, in the presence of inorganic carbon and nitrate.

Photoautotrophic fatty acid production of a highly CO(2)-tolerant green alga Chlorococcum littorale was investigated in the presence of inorganic carbon and nitrate at 295 K and a light intensity of 170 micromol-photon m(-2) s(-1). CO(2) concentration in the bubbling gas was adjusted by mixing pure gas components of CO(2) and N(2) to avoid photorespiration and beta-oxidation of fatty acids under O(2) atmosphere conditions. Fatty acid content was almost constant for the CO(2) concentrations ranging from 5% to 50% under nitrate-rich conditions corresponding to the logarithmic growth phase. After nitrate depletion, the content drastically increased with a decrease in CO(2) concentration. HCO(3)(-)/CO(2) ratio in the culture media was found to be a controlling factor for fatty acid production after the nitrate limitation phase. For a CO(2) concentration of 5%, the fatty acid content was ca. 34 wt.% at maximum, which is comparable with other land plant seed oils.

Effect of inorganic carbon on photoautotrophic growth of microalga Chlorococcum littorale.

The growth rate of a highly CO(2)-tolerant green alga, Chlorococcum littorale, was investigated in semi-batch cultures at a temperature of 22 degrees C, a light intensity of 170 micromol-photon m(-2) s(-1) and CO(2) concentrations ranging from 1 to 50% (v/v) at atmospheric pressure. In the experiments, solutions were bubbled with CO(2) and N(2) gas mixtures to adjust CO(2) concentrations to minimize the influence of O(2). Growth rate, which was defined in terms of a specific growth rate mu, decreased with increasing CO(2) concentration at the conditions studied. The inhibition of growth by CO(2) gas could be attributed to the concentration of inorganic carbon in the culture medium. A growth model is proposed where key assumptions are the formation of bicarbonate ion HCO(3) (-) as substrate for algal growth and equilibrium between CO(2) inhibitor. The proposed growth model based on the Monod equation agreed with the experimental data to within 5% and provides better correlation than the conventional inhibition model, especially in the high CO(2) concentration region.