Heliorhodopsin-mediated light-modulation of ABC transporter.

Heliorhodopsins (HeRs) have been hypothesized to have widespread functions. Recently, the functions for few HeRs have been revealed; however, the hypothetical functions remain largely unknown. Herein, we investigate light-modulation of heterodimeric multidrug resistance ATP-binding cassette transporters (OmrDE) mediated by Omithinimicrobium cerasi HeR. In this study, we classifiy genes flanking the HeR-encoding genes and identify highly conservative residues for protein-protein interactions. Our results reveal that the interaction between OcHeR and OmrDE shows positive cooperatively sequential binding through thermodynamic parameters. Moreover, light-induced OcHeR upregulates OmrDE drug transportation. Hence, the binding may be crucial to drug resistance in O. cerasi as it survives in a drug-containing habitat. Overall, we unveil a function of HeR as regulatory rhodopsin for multidrug resistance. Our findings suggest potential applications in optogenetic technology.

Real-time Visualization of Transcribed mRNA via Click Chemistry in a Liposomal Space.

We present a CuAAC (Copper-Catalyzed Azide-Alkyne Cycloaddition) reaction protocol designed for the visualization of mRNA. To achieve this, we synthesized stable mRNA molecules incorporating the modified nucleoside analog, EU, a crucial element for fluorophore attachment. Leveraging this modified mRNA, we successfully executed the CuAAC reaction, wherein the pro-fluorophore, coumarin, was conjugated to EU on the mRNA through our meticulously designed CuAAC process. This innovative approach resulted in the emission of fluorescence, enabling both precise quantification and visual observation of mRNA. Furthermore, we demonstrated the feasibility of concurrent mRNA synthesis and visualization by seamlessly integrating the CuAAC reaction mix into the mRNA transcription process. Additionally, our novel methodology opens avenues for prospective real-time monitoring of mRNA transcription within artificial cells. These advancements hold significant promise for expanding our comprehension of fundamental cellular processes and finding applications across diverse biological contexts in the future.

Impact of protein-chromophore interaction on the retinal excited state and photocycle of Gloeobacter rhodopsin: role of conserved tryptophan residues.

The function of microbial as well as mammalian retinal proteins (aka rhodopsins) is associated with a photocycle initiated by light excitation of the retinal chromophore of the protein, covalently bound through a protonated Schiff base linkage. Although electrostatics controls chemical reactions of many organic molecules, attempt to understand its role in controlling excited state reactivity of rhodopsins and, thereby, their photocycle is scarce. Here, we investigate the effect of highly conserved tryptophan residues, between which the all-trans retinal chromophore of the protein is sandwiched in microbial rhodopsins, on the charge distribution along the retinal excited state, quantum yield and nature of the light-induced photocycle and absorption properties of Gloeobacter rhodopsin (GR). Replacement of these tryptophan residues by non-aromatic leucine (W222L and W122L) or phenylalanine (W222F) does not significantly affect the absorption maximum of the protein, while all the mutants showed higher sensitivity to photobleaching, compared to wild-type GR. Flash photolysis studies revealed lower quantum yield of trans-cis photoisomerization in W222L as well as W222F mutants relative to wild-type. The photocycle kinetics are also controlled by these tryptophan residues, resulting in altered accumulation and lifetime of the intermediates in the W222L and W222F mutants. We propose that protein-retinal interactions facilitated by conserved tryptophan residues are crucial for achieving high quantum yield of the light-induced retinal isomerization, and affect the thermal retinal re-isomerization to the resting state.

AT-specific DNA visualization revisits the directionality of bacteriophage λ DNA ejection.

In this study, we specifically visualized DNA molecules at their AT base pairs after in vitro phage ejection. Our AT-specific visualization revealed that either end of the DNA molecule could be ejected first with a nearly 50% probability. This observation challenges the generally accepted theory of Last In First Out (LIFO), which states that the end of the phage λ DNA that enters the capsid last during phage packaging is the first to be ejected, and that both ends of the DNA are unable to move within the extremely condensed phage capsid. To support our observations, we conducted computer simulations that revealed that both ends of the DNA molecule are randomized, resulting in the observed near 50% probability. Additionally, we found that the length of the ejected DNA by LIFO was consistently longer than that by First In First Out (FIFO) during in vitro phage ejection. Our simulations attributed this difference in length to the stiffness difference of the remaining DNA within the phage capsid. In conclusion, this study demonstrates that a DNA molecule within an extremely dense phage capsid exhibits a degree of mobility, allowing it to switch ends during ejection.

A Bibliometric Analysis of the Field of Computer-Assisted Orthopedic Surgery during 2002-2021.

Background: This study aimed to investigate the characteristics of research articles and research trends in computer-assisted orthopedic surgery (CAOS) by conducting bibliometric analyses. Methods: CAOS-related research articles published in international journals from 2002 to 2021 were collected using the PubMed database and analyzed using the bibliometric method. Their publication year, journal name, corresponding author's country name, and the number of citations of all collected articles were noted. Contents of the articles were analyzed to evaluate the time point and anatomical site at which the digital technique was applied. Further, the 20-year period was divided into two halves of 10 years each to analyze the research trends. Results: A total of 639 CAOS-related articles were identified. An average of 32.0 CAOS-related articles were published annually, with an average of 20.6 and 43.3 published in the first half and second half, respectively. Of all articles, 47.6% were published in the top 10 journals, and 81.2% were written in the top 10 countries. The total numbers of citations were 11.7 and 6.3 in the first and second halves, respectively, but the average annual number of citations was higher in the second half than in the first one. Articles on application of digital techniques during surgery were 62.3% and those on pre-surgery application were 36.9%. Further, articles in the knee (39.0%), spine (28.5%), and hip and pelvis (21.5%) fields accounted for 89.0% of the total publications. But the increase in publications in the said period was highest in the fields of the hand and wrist (+1,300.0%), ankle (+466.7%), and shoulder (+366.7%). Conclusions: Over the last 20 years, the publication of CAOS-related research articles in international journals has grown steadily. Although the knee, spine, hip, and pelvis fields account for most CAOS-related research, research in new fields is also increasing. This study analyzed the types of articles and trends in CAOS-related research and provided useful information for future research in the field of CAOS.

Retinal-Carotenoid Interactions in a Sodium-Ion-Pumping Rhodopsin: Implications on Oligomerization and Thermal Stability.

Microbial rhodopsin (also called retinal protein)-carotenoid conjugates represent a unique class of light-harvesting (LH) complexes, but their specific interactions and LH properties are not completely elucidated as only few rhodopsins are known to bind carotenoids. Here, we report a natural sodium-ion (Na+)-pumping Nonlabens (Donghaeana) dokdonensis rhodopsin (DDR2) binding with a carotenoid salinixanthin (Sal) to form a thermally stable rhodopsin-carotenoid complex. Different spectroscopic studies were employed to monitor the retinal-carotenoid interaction as well as the thermal stability of the protein, while size-exclusion chromatography (SEC) and homology modeling are performed to understand the protein oligomerization process. In analogy with that of another Na+-pumping protein Krokinobacter eikastus rhodopsin 2 (KR2), we propose that DDR2 (studied concentration range: 2 × 10-6 to 4 × 10-5 M) remains mainly as a pentamer at room temperature and neutral pH, while heating above 55 °C partially converted it into a thermally less stable oligomeric form of the protein. This process is affected by both the pH and concentration. At high concentrations (4 × 10-5 to 2 × 10-4 M), the protein adopts a pentamer form reflected in the excitonic circular dichroism (CD) spectrum. In the presence of Sal, the thermal stability of DDR2 is increased significantly, and the pigment is stable even at 85 °C. The results presented could have implications in designing stable rhodopsin-carotenoid antenna complexes.

Surgery-related anxiety on geriatric patients undergoing total knee arthroplasty: a retrospective observational study.

BACKGROUND: The prevalence of anxiety in patients undergoing total knee arthroplasty (TKA) and its association with postoperative functions are well known; however, the levels of anxiety or anxiety-related characteristics are unknown. This study aimed to investigate the prevalence of clinically significant state anxiety in geriatric patients undergoing TKA for osteoarthritis (OA) of the knee and to evaluate the anxiety-related characteristics experienced by these patients pre- and post-operatively. METHODS: This retrospective observational study recruited patients who had undergone TKA for knee OA using general anesthesia between February 2020 and August 2021. The study participants were geriatric patients older than 65 years who had moderate or severe OA. We evaluated patient characteristics including age, sex, body mass index, smoking status, hypertension, diabetes, and cancer. We assessed their levels of anxiety status using the STAI-X which comprises 20-item scales. Clinically meaningful state anxiety was defined as a total score of 52 or higher. An independent Student's t-test was used to determine differences of STAI score between subgroups in terms of patient characteristics. And patients were asked to complete questionnaires, which assessed four areas: (1) the main cause of anxiety; (2) the most helpful factor in overcoming anxiety before surgery; (3) the most helpful factor in reducing anxiety after surgery; and (4) the most anxious moment during the entire process. RESULTS: The mean STAI score of patients who underwent TKA was 43.0 points and 16.4% of patients experienced clinically significant state anxiety. The current smoking status affect STAI score and the proportion of patients with clinically meaningful state anxiety. The most common cause of preoperative anxiety was the surgery itself. Overall, 38% of patients reported that they experienced the greatest level of anxiety when the surgeon had recommended TKA in the outpatient clinic. The trust in the medical staff before surgery and the surgeon's explanations after surgery helped the most in reducing anxiety. CONCLUSIONS: One in six patients before TKA experience clinically meaningful state anxiety, and about 40% of patients experience anxiety from the time they are recommended for surgery. Patients tended to overcome anxiety before TKA through trust in the medical staff, and the surgeon's explanations after surgery was found to be helpful in reducing anxiety.

Heliorhodopsin Helps Photolyase to Enhance the DNA Repair Capacity.

Light quality is a significant factor for living organisms that have photosensory systems, such as rhodopsin, a seven alpha-helical transmembrane protein with the retinal chromophore. Here, we report, for the first time, the function of new rhodopsin, which is an inverted 7-transmembrane protein, isolated from Trichococcus flocculiformis. T. flocculiformis heliorhodopsin (TfHeR) works as a regulatory helper rhodopsin that binds with class 2 cyclobutane pyrimidine dimer (CPDII) photolyase to broaden the spectrum and upregulate DNA repair activity. We have confirmed their interaction through isothermal titration calorimetry (dissociation constant of 21.7 µM) and identified the charged residues for the interaction. Based on in vivo and in vitro experiments, we showed that the binding of heliorhodopsin with photolyase improved photolyase activity by about 3-fold to repair UV-caused DNA damage. Also, the DNA repair activity of TfHeR/T. flocculiformis photolyase (TfPHR) was observed in the presence of green light. Our results suggested that heliorhodopsin directly controls the activity of photolyase and coevolves to broaden the activity spectrum by protein-protein interaction. IMPORTANCE This study reports a function for Heliorhodopsin working as a regulatory helper rhodopsin that with CPDII photolyase to broaden the spectrum and upregulating the DNA repair activity. Our results suggested that heliorhodopsin directly controls photolyase activity and coevolves to broaden the DNA repair capacity by protein-protein interaction.

Heliorhodopsin binds and regulates glutamine synthetase activity.

Photoreceptors are light-sensitive proteins found in various organisms that respond to light and relay signals into the cells. Heliorhodopsin, a retinal-binding membrane protein, has been recently discovered, however its function remains unknown. Herein, we investigated the relationship between Actinobacteria bacterium IMCC26103 heliorhodopsin (AbHeR) and an adjacent glutamine synthetase (AbGS) in the same operon. We demonstrate that AbHeR binds to AbGS and regulates AbGS activity. More specifically, the dissociation constant (Kd) value of the binding between AbHeR and AbGS is 6.06 µM. Moreover, the absence of positively charged residues within the intracellular loop of AbHeR impacted Kd value as they serve as critical binding sites for AbGS. We also confirm that AbHeR up-regulates the biosynthetic enzyme activity of AbGS both in vitro and in vivo in the presence of light. GS is a key enzyme involved in nitrogen assimilation that catalyzes the conversion of glutamate and ammonia to glutamine. Hence, the interaction between AbHeR and AbGS may be critical for nitrogen assimilation in Actinobacteria bacterium IMCC26103 as it survives in low-nutrient environments. Overall, the findings of our study describe, for the first time, to the best of our knowledge, a novel function of heliorhodopsin as a regulatory rhodopsin with the capacity to bind and regulate enzyme activity required for nitrogen assimilation.

Characterization of the Interfacial Structures of Core/Shell CdSe/ZnS QDs.

Core/shell quantum dots (QDs) have been extensively studied, yet their optical properties widely vary among studies. Such variation may arise from the variation in interfacial structures induced by the subtle difference in each synthetic procedure. Here, we studied the interfacial structures of CdSe/ZnS QDs using the time-of-flight medium energy ion-scattering spectroscopy (TOF-MEIS), which offers the radial elemental distributions as well as the overall elemental compositions of QDs. The TOF-MEIS spectra provided strong evidence for the existence of an alloyed layer at the interface between CdSe and ZnS in typical CdSe/ZnS QDs. On the basis of the emission and absorption spectra of QDs sampled during the synthesis, we conclude that such interfacial alloying is caused by the dissolution of CdSe seeds during the synthesis steps. Such a dissolution mechanism is further corroborated by the observation that the ligand environment of solvent (X or L type) leads to different shapes of interfaces.

Fabrication of a Tailored, Hybrid Extracellular Matrix Composite.

The extracellular matrix (ECM) is a network of connective fibers that supports cells living in their surroundings. Native ECM, generated by the secretory products of each tissue's resident cells, has a unique architecture with different protein composition depending on the tissue. Therefore, it is very difficult to artificially design in vivo architecture in tissue engineering. In this study, a hybrid ECM scaffold from the basic structure of fibroblast-derived cellular ECMs is fabricated by adding major ECM components of fibronectin (FN) and collagen (COL I) externally. It is confirmed that while maintaining the basic structure of the native ECM, major protein components can be regulated. Then, decellularization is performed to prepare hybrid ECM scaffolds with various protein compositions and it is demonstrated that a liver-mimicking fibronectin (FN)-rich hybrid ECM promoted successful settling of H4IIE rat hepatoma cells. The authors believe that their method holds promise for the fabrication of scaffolds that provide a tailored cellular microenvironment for specific organs and serve as novel pathways for the replacement or regeneration of specific organ tissues.

Does spiked tibial cement spacer reduce spacer-related problems in two-stage revision total knee arthroplasty for infection?

PURPOSE: Articulating cement spacers are frequently used in staged approaches for infected total knee arthroplasty (TKA). This study investigated whether a tibial cement spacer (TCS) with spikes could reduce spacer-related problems in two-stage revision TKA (R-TKA). METHODS: A total of 27 patients (27 knees; 10 men and 17 women) who underwent two-stage R-TKA for infected TKA were retrospectively analyzed. Group A comprised 12 patients who used TCS with spikes added to the bottom surface, whereas group B consisted of 15 patients who used conventional TCS with a flat bottom. For each group, plain radiographs were obtained after cement spacer implantation and before R-TKA to measure mediolateral (ML) translation and TCS's tilting angle. Patients' demographic data, ML translation of the TCS, and changes in the TCS's tilting angle between the groups were analyzed. RESULTS: The mean ML translation was significantly lower in group A than that in group B (1.7 mm vs. 5.4 mm, p = 0.04). The mean change in the tilting angle was significantly lower in group A than that in group B (4.5° vs. 19.4°, p = 0.047). CONCLUSION: The spiked TCS in two-stage R-TKA provides superior stability compared to the TCS with a conventional design.

Carotenoid binding in Gloeobacteria rhodopsin provides insights into divergent evolution of xanthorhodopsin types.

The position of carotenoid in xanthorhodopsin has been elucidated. However, a challenging expression of this opsin and a complex biosynthesis carotenoid in the laboratory hold back the insightful study of this rhodopsin. Here, we demonstrated co-expression of the xanthorhodopsin type isolated from Gloeobacter violaceus PCC 7421-Gloeobacter rhodopsin (GR) with a biosynthesized keto-carotenoid (canthaxanthin) targeting the carotenoid binding site. Direct mutation-induced changes in carotenoid-rhodopsin interaction revealed three crucial features: (1) carotenoid locked motif (CLM), (2) carotenoid aligned motif (CAM), and color tuning serines (CTS). Our single mutation results at 178 position (G178W) confirmed inhibition of carotenoid binding; however, the mutants showed better stability and proton pumping, which was also observed in the case of carotenoid binding characteristics. These effects demonstrated an adaptation of microbial rhodopsin that diverges from carotenoid harboring, along with expression in the dinoflagellate Pyrocystis lunula rhodopsin and the evolutionary substitution model. The study highlights a critical position of the carotenoid binding site, which significantly allows another protein engineering approach in the microbial rhodopsin family.

Predictive Variables for Interventional Angiography among Patients with Knee Hemarthrosis.

Studies regarding the variables that could predict the success of conservative treatment for knee hemarthrosis are lacking. This retrospective study evaluated the laboratory variables of patients who had unsatisfactory results from conservative treatment for knee hemarthrosis. Twenty-nine patients conservatively treated for knee hemarthrosis were included and divided into two groups: group A comprised 14 patients who underwent interventional angiography and selective embolization due to failed conservative treatment, and group B comprised 15 patients with successful results after conservative treatment. The results of the serological and synovial fluid tests were evaluated. The mean number of synovial red blood cells (RBCs) was 1,905,857 cells/µL and 7730 cells/µL in groups A and B, respectively (p = 0.01), while the mean number of RBCs per high-power field (HPF) was 68.9 and 3.2, respectively (p < 0.01). Patients who underwent interventional angiography and selective embolization after failed conservative treatment for knee hemarthrosis had higher synovial RBC counts and RBC counts per HPF than those with successful outcomes after conservative treatment. It is necessary to carefully interpret the results of the synovial fluid analysis in patients with knee hemarthrosis; if the synovial fluid analysis shows a synovial RBC count greater than 81,500 and RBC count per HPF greater than 16.3, we recommend immediate interventional angiography rather than continuing conservative treatment.

Mutational analyses identify a single amino acid critical for colour tuning in proteorhodopsins.

Microbial rhodopsins are light-activated proteins that contain seven transmembrane alpha-helices. Spectral tuning in microbial rhodopsins is a useful optogenetic tool. In this study, we report a new site that controls spectral tuning. In the proteorhodopsins ISR34 and ISR36, a single amino-acid substitution at Cys189 caused an absorption maximum shift of 44 nm, indicating spectral tuning at a specific site. Comparison of single amino acid substitutions was conducted using photochemical and photobiological approaches. The maximum absorption for red-shift was measured for mutations at positions 189 and 105 in ISR34, both residues being equally important. Structural changes resulting from amino acid substitutions are related to pKa values, pumping activity and spectral tuning.

Kinetics of DNA looping by Anabaena sensory rhodopsin transducer (ASRT) by using DNA cyclization assay.

DNA cyclization assay together with single-molecule FRET was employed to monitor protein-mediated bending of a short dsDNA (~ 100 bp). This method provides a simple and easy way to monitor the structural change of DNA in real-time without necessitating prior knowledge of the molecular structures for the optimal dye-labeling. This assay was applied to study how Anabaena sensory rhodopsin transducer (ASRT) facilitates loop formation of DNA as a possible mechanism for gene regulation. The ASRT-induced DNA looping was maximized at 50 mM of Na+, while Mg2+ also played an essential role in the loop formation.

Discovery of a new light-driven Li+/Na+-pumping rhodopsin with DTG motif.

Microbial pumping rhodopsin is a seven-transmembrane retinal binding protein, which is light-driven ion pump with a functional key motif. Ion-pumping with the key motif and charged amino acids in the rhodopsin is biochemically important. The rhodopsins with DTG motif have been discovered in various eubacteria, and they function as H+ pump. Especially, the DTG motif rhodopsins transported H+ despite the replacement of a proton donor by Gly. We investigated Methylobacterium populi rhodopsin (MpR) in one of the DTG motif rhodopsin clades. To determine which ions the MpR transport, we tested with various monovalent ion solutions and determined that MpR transports Li+/Na+. By replacing the three negatively charged residues residues which are located in helix B, Glu32, Glu33, and Asp35, we concluded that the residues play a critical role in the transport of Li+/Na+. The MpR E33Q transported H+ in place of Li+/Na+, suggesting that Glu33 is a Li+/Na+ binding site on the cytoplasmic side. Gly93 in MpR was replaced by Asp to convert from the Li+/Na+ pump to the H+ pump, resulting in MpR G93D transporting H+. Dissociation constant (Kd) values of Na+ for MpR WT and E33Q were determined to be 4.0 and 72.5 mM, respectively. These results indicated the mechanism by which MpR E33Q transports H+. Up to now, various ion-pumping rhodopsins have been discovered, and Li+/Na+-pumping rhodopsins were only found in the NDQ motif in NaR. Here, we report a new light-driven Na+ pump MpR and have determined the important residues required for Li+/Na+-pumping different from previously known NaR.

The role of carotenoids in proton-pumping rhodopsin as a primitive solar energy conversion system.

Rhodopsin and carotenoids are two molecules that certain bacteria use to absorb and utilize light. Type I rhodopsin, the simplest active proton transporter, converts light energy into an electrochemical potential. Light produces a proton gradient, which is known as the proton motive force across the cell membrane. Some carotenoids are involved in light absorbance and transfer of absorbed energy to chlorophyll during photosynthesis. A previous study in Salinibacter ruber has shown that carotenoids act as antennae to harvest light and transfer energy to retinal in xanthorhodopsin (XR). Here, we describe the role of canthaxanthin (CAN), a carotenoid, as an antenna for Gloeobacter rhodopsin (GR). The non-covalent complex formed by the interaction between CAN and GR doubled the proton pumping speed and improved the pumping capacity by 1.5-fold. The complex also tripled the proton pumping speed and improved the pumping capacity by 5-fold in the presence of strong and weak light, respectively. Interestingly, when canthaxanthin was bound to Gloeobacter rhodopsin, it showed a 126-fold increase in heat resistance, and it survived better under drought conditions than Gloeobacter rhodopsin. The results suggest direct complementation of Gloeobacter rhodopsin with a carotenoid for primitive solar energy harvesting in cyanobacteria.

Discovery of a microbial rhodopsin that is the most stable in extreme environments.

Microbial rhodopsin is a retinal protein that functions as an ion pump, channel, and sensory transducer, as well as a light sensor, as in biosensors and biochips. Tara76 rhodopsin is a typical proton-pumping rhodopsin that exhibits strong stability against extreme pH, detergent, temperature, salt stress, and dehydration stress and even under dual and triple conditions. Tara76 rhodopsin has a thermal stability approximately 20 times higher than that of thermal rhodopsin at 80°C and is even stable at 85°C. Tara76 rhodopsin is also stable at pH 0.02 to 13 and exhibits strong resistance in detergent, including Triton X-100 and SDS. We tested the current flow that electrical current flow across dried proteins on the paper at high temperatures using an electrode device, which was measured stably from 25°C up to 120°C. These properties suggest that this Tara76 rhodopsin is suitable for many applications in the fields of bioengineering and biotechnology.

Assembly of Natively Synthesized Dual Chromophores Into Functional Actinorhodopsin.

Microbial rhodopsin is a simple solar energy-capturing molecule compared to the complex photosynthesis apparatus. Light-driven proton pumping across the cell membrane is a crucial mechanism underlying microbial energy production. Actinobacteria is one of the highly abundant bacterial phyla in freshwater habitats, and members of this lineage are considered to boost heterotrophic growth via phototrophy, as indicated by the presence of actino-opsin (ActR) genes in their genome. However, it is difficult to validate their function under laboratory settings because Actinobacteria are not consistently cultivable. Based on the published genome sequence of Candidatus aquiluna sp. strain IMCC13023, actinorhodopsin from the strain (ActR-13023) was isolated and characterized in this study. Notably, ActR-13023 assembled with natively synthesized carotenoid/retinal (used as a dual chromophore) and functioned as a light-driven outward proton pump. The ActR-13023 gene and putative genes involved in the chromophore (retinal/carotenoid) biosynthetic pathway were detected in the genome, indicating the functional expression ActR-13023 under natural conditions for the utilization of solar energy for proton translocation. Heterologous expressed ActR-13023 exhibited maximum absorption at 565 nm with practical proton pumping ability. Purified ActR-13023 could be reconstituted with actinobacterial carotenoids for additional light-harvesting. The existence of actinorhodopsin and its chromophore synthesis machinery in Actinobacteria indicates the inherent photo-energy conversion function of this microorganism. The assembly of ActR-13023 to its synthesized chromophores validated the microbial community's importance in the energy cycle.