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.

Time-resolved serial femtosecond crystallography at the European XFEL.

The European XFEL (EuXFEL) is a 3.4-km long X-ray source, which produces femtosecond, ultrabrilliant and spatially coherent X-ray pulses at megahertz (MHz) repetition rates. This X-ray source has been designed to enable the observation of ultrafast processes with near-atomic spatial resolution. Time-resolved crystallographic investigations on biological macromolecules belong to an important class of experiments that explore fundamental and functional structural displacements in these molecules. Due to the unusual MHz X-ray pulse structure at the EuXFEL, these experiments are challenging. Here, we demonstrate how a biological reaction can be followed on ultrafast timescales at the EuXFEL. We investigate the picosecond time range in the photocycle of photoactive yellow protein (PYP) with MHz X-ray pulse rates. We show that difference electron density maps of excellent quality can be obtained. The results connect the previously explored femtosecond PYP dynamics to timescales accessible at synchrotrons. This opens the door to a wide range of time-resolved studies at the EuXFEL.

Enzyme intermediates captured "on the fly" by mix-and-inject serial crystallography.

BACKGROUND: Ever since the first atomic structure of an enzyme was solved, the discovery of the mechanism and dynamics of reactions catalyzed by biomolecules has been the key goal for the understanding of the molecular processes that drive life on earth. Despite a large number of successful methods for trapping reaction intermediates, the direct observation of an ongoing reaction has been possible only in rare and exceptional cases. RESULTS: Here, we demonstrate a general method for capturing enzyme catalysis "in action" by mix-and-inject serial crystallography (MISC). Specifically, we follow the catalytic reaction of the Mycobacterium tuberculosis ß-lactamase with the third-generation antibiotic ceftriaxone by time-resolved serial femtosecond crystallography. The results reveal, in near atomic detail, antibiotic cleavage and inactivation from 30 ms to 2 s. CONCLUSIONS: MISC is a versatile and generally applicable method to investigate reactions of biological macromolecules, some of which are of immense biological significance and might be, in addition, important targets for structure-based drug design. With megahertz X-ray pulse rates expected at the Linac Coherent Light Source II and the European X-ray free-electron laser, multiple, finely spaced time delays can be collected rapidly, allowing a comprehensive description of biomolecular reactions in terms of structure and kinetics from the same set of X-ray data.

Heterogeneity in M. tuberculosis ß-lactamase inhibition by Sulbactam.

For decades, researchers have elucidated essential enzymatic functions on the atomic length scale by tracing atomic positions in real-time. Our work builds on possibilities unleashed by mix-and-inject serial crystallography (MISC) at X-ray free electron laser facilities. In this approach, enzymatic reactions are triggered by mixing substrate or ligand solutions with enzyme microcrystals. Here, we report in atomic detail (between 2.2 and 2.7 Å resolution) by room-temperature, time-resolved crystallography with millisecond time-resolution (with timepoints between 3 ms and 700 ms) how the Mycobacterium tuberculosis enzyme BlaC is inhibited by sulbactam (SUB). Our results reveal ligand binding heterogeneity, ligand gating, cooperativity, induced fit, and conformational selection all from the same set of MISC data, detailing how SUB approaches the catalytic clefts and binds to the enzyme noncovalently before reacting to a trans-enamine. This was made possible in part by the application of singular value decomposition to the MISC data using a program that remains functional even if unit cell parameters change up to 3 Å during the reaction.

Heterogeneity in the M. tuberculosis ß-Lactamase Inhibition by Sulbactam.

For decades, researchers have been determined to elucidate essential enzymatic functions on the atomic lengths scale by tracing atomic positions in real time. Our work builds on new possibilities unleashed by mix-and-inject serial crystallography (MISC) 1-5 at X-ray free electron laser facilities. In this approach, enzymatic reactions are triggered by mixing substrate or ligand solutions with enzyme microcrystals 6 . Here, we report in atomic detail and with millisecond time-resolution how the Mycobacterium tuberculosis enzyme BlaC is inhibited by sulbactam (SUB). Our results reveal ligand binding heterogeneity, ligand gating 7-9 , cooperativity, induced fit 10,11 and conformational selection 11-13 all from the same set of MISC data, detailing how SUB approaches the catalytic clefts and binds to the enzyme non-covalently before reacting to a trans- enamine. This was made possible in part by the application of the singular value decomposition 14 to the MISC data using a newly developed program that remains functional even if unit cell parameters change during the reaction.

Efficacy and safety evaluation of alcohol-containing and alcohol-free mouth rinses: A clinicocytological study.

CONTEXT: Whether the alcohol-based mouth rinses are as good as nonalcoholic mouth rinses as far as oral mucosal safety is concerned? AIMS: The aim of the study was to investigate the oral mucosal safety of widely used alcohol- and nonalcohol-based mouth rinses at their recommended doses. SETTINGS AND DESIGN: The clinical and cytological investigations were carried out by enrolling 120 systemically healthy volunteers fulfilling the inclusion criteria. The volunteers were subjected to a repeated mouth rinse for 60 days to either alcohol-based or alcohol-free mouth rinses at their recommended dosages. A comparative analysis for any clinical adverse response on the oral mucosa and efficacy, i.e., reduction of plaque and gingival index was done at the terminal of the exposure. The studies were also carried out to investigate the cytotoxicity and genotoxicity potential of alcohol-based and alcohol-free mouth rinses in the exposed mucosal cells. SUBJECTS AND METHODS: The data have been presented in comparative account between alcohol-based and alcohol-free mouth rinses in the volunteers at day 0 and day 60. The cytotoxicity and genotoxicity potential of prescribed doses of alcohol- and alcohol-free mouth rinses have also been evaluated using tetrazolium bromide salt 3-(4, 5-dimethylthiazol-2-yl)-2,-diphenyl tetrazolium bromide, neutral red uptake, and trypan blue dye, micronucleus and chromosomal aberrations. RESULTS: The study findings reveal no statistically as well as biologically significant adverse responses of both alcohol-based and alcohol-free mouth rinses at clinical and cytological level. CONCLUSIONS: Under cytological observation, repeated dose exposure up to 60 days of the mouth rinses (alcohol-based and alcohol-free) used in the study was found to be effective and safe at their prescribed dosages.

The three-dimensional structure of Drosophila melanogaster (6-4) photolyase at room temperature.

(6-4) photolyases are flavoproteins that belong to the photolyase/cryptochrome family. Their function is to repair DNA lesions using visible light. Here, crystal structures of Drosophila melanogaster (6-4) photolyase [Dm(6-4)photolyase] at room and cryogenic temperatures are reported. The room-temperature structure was solved to 2.27 Šresolution and was obtained by serial femtosecond crystallography (SFX) using an X-ray free-electron laser. The crystallization and preparation conditions are also reported. The cryogenic structure was solved to 1.79 Šresolution using conventional X-ray crystallography. The structures agree with each other, indicating that the structural information obtained from crystallography at cryogenic temperature also applies at room temperature. Furthermore, UV-Vis absorption spectroscopy confirms that Dm(6-4)photolyase is photoactive in the crystals, giving a green light to time-resolved SFX studies on the protein, which can reveal the structural mechanism of the photoactivated protein in DNA repair.

High-resolution crystal structures of transient intermediates in the phytochrome photocycle.

Phytochromes are red/far-red light photoreceptors in bacteria to plants, which elicit a variety of important physiological responses. They display a reversible photocycle between the resting Pr state and the light-activated Pfr state. Light signals are transduced as structural change through the entire protein to modulate its activity. It is unknown how the Pr-to-Pfr interconversion occurs, as the structure of intermediates remains notoriously elusive. Here, we present short-lived crystal structures of the photosensory core modules of the bacteriophytochrome from myxobacterium Stigmatella aurantiaca captured by an X-ray free electron laser 5 ns and 33 ms after light illumination of the Pr state. We observe large structural displacements of the covalently bound bilin chromophore, which trigger a bifurcated signaling pathway that extends through the entire protein. The snapshots show with atomic precision how the signal progresses from the chromophore, explaining how plants, bacteria, and fungi sense red light.

Synchronous RNA conformational changes trigger ordered phase transitions in crystals.

Time-resolved studies of biomacromolecular crystals have been limited to systems involving only minute conformational changes within the same lattice. Ligand-induced changes greater than several angstroms, however, are likely to result in solid-solid phase transitions, which require a detailed understanding of the mechanistic interplay between conformational and lattice transitions. Here we report the synchronous behavior of the adenine riboswitch aptamer RNA in crystal during ligand-triggered isothermal phase transitions. Direct visualization using polarized video microscopy and atomic force microscopy shows that the RNA molecules undergo cooperative rearrangements that maintain lattice order, whose cell parameters change distinctly as a function of time. The bulk lattice order throughout the transition is further supported by time-resolved diffraction data from crystals using an X-ray free electron laser. The synchronous molecular rearrangements in crystal provide the physical basis for studying large conformational changes using time-resolved crystallography and micro/nanocrystals.

Observation of substrate diffusion and ligand binding in enzyme crystals using high-repetition-rate mix-and-inject serial crystallography.

Here, we illustrate what happens inside the catalytic cleft of an enzyme when substrate or ligand binds on single-millisecond timescales. The initial phase of the enzymatic cycle is observed with near-atomic resolution using the most advanced X-ray source currently available: the European XFEL (EuXFEL). The high repetition rate of the EuXFEL combined with our mix-and-inject technology enables the initial phase of ceftriaxone binding to the Mycobacterium tuberculosis ß-lactamase to be followed using time-resolved crystallography in real time. It is shown how a diffusion coefficient in enzyme crystals can be derived directly from the X-ray data, enabling the determination of ligand and enzyme-ligand concentrations at any position in the crystal volume as a function of time. In addition, the structure of the irreversible inhibitor sulbactam bound to the enzyme at a 66 ms time delay after mixing is described. This demonstrates that the EuXFEL can be used as an important tool for biomedically relevant research.

The primary structural photoresponse of phytochrome proteins captured by a femtosecond X-ray laser.

Phytochrome proteins control the growth, reproduction, and photosynthesis of plants, fungi, and bacteria. Light is detected by a bilin cofactor, but it remains elusive how this leads to activation of the protein through structural changes. We present serial femtosecond X-ray crystallographic data of the chromophore-binding domains of a bacterial phytochrome at delay times of 1 ps and 10 ps after photoexcitation. The data reveal a twist of the D-ring, which leads to partial detachment of the chromophore from the protein. Unexpectedly, the conserved so-called pyrrole water is photodissociated from the chromophore, concomitant with movement of the A-ring and a key signaling aspartate. The changes are wired together by ultrafast backbone and water movements around the chromophore, channeling them into signal transduction towards the output domains. We suggest that the observed collective changes are important for the phytochrome photoresponse, explaining the earliest steps of how plants, fungi and bacteria sense red light.

Plants adapt to the availability of light throughout their lives because it regulates so many aspects of their growth and reproduction. To detect the level of light, plant cells use proteins called phytochromes, which are also found in some bacteria and fungi. Phytochrome proteins change shape when they are exposed to red light, and this change alters the behaviour of the cell. The red light is absorbed by a molecule known as chromophore, which is connected to a region of the phytochrome called the PHY-tongue. This region undergoes one of the key structural changes that occur when the phytochrome protein absorbs light, turning from a flat sheet into a helix. Claesson, Wahlgren, Takala et al. studied the structure of a bacterial phytochrome protein almost immediately after shining a very brief flash of red light using a laser. The experiments revealed that the structure of the protein begins to change within a trillionth of a second: specifically, the chromophore twists, which disrupts its attachment to the protein, freeing the protein to change shape. Claesson, Wahlgren, Takala et al. note that this structure is likely a very short-lived intermediate state, which however triggers more changes in the overall shape change of the protein. One feature of the rearrangement is the disappearance of a particular water molecule. This molecule can be found at the core of many different phytochrome structures and interacts with several parts of the chromophore and the phytochrome protein. It is unclear why the water molecule is lost, but given how quickly this happens after the red light is applied it is likely that this disappearance is an integral part of the reshaping process. Together these events disrupt the interactions between the chromophore and the PHY-tongue, enabling the PHY-tongue to change shape and alter the structure of the phytochrome protein. Understanding and controlling this process could allow scientists to alter growth patterns in plants, such as crops or weeds.

High-resolution crystal structures of a myxobacterial phytochrome at cryo and room temperatures.

Phytochromes (PHYs) are photoreceptor proteins first discovered in plants, where they control a variety of photomorphogenesis events. PHYs as photochromic proteins can reversibly switch between two distinct states: a red light (Pr) and a far-red light (Pfr) absorbing form. The discovery of Bacteriophytochromes (BphPs) in nonphotosynthetic bacteria has opened new frontiers in our understanding of the mechanisms by which these natural photoswitches can control single cell development, although the role of BphPs in vivo remains largely unknown. BphPs are dimeric proteins that consist of a photosensory core module (PCM) and an enzymatic domain, often a histidine kinase. The PCM is composed of three domains (PAS, GAF, and PHY). It holds a covalently bound open-chain tetrapyrrole (biliverdin, BV) chromophore. Upon absorption of light, the double bond between BV rings C and D isomerizes and reversibly switches the protein between Pr and Pfr states. We report crystal structures of the wild-type and mutant (His275Thr) forms of the canonical BphP from the nonphotosynthetic myxobacterium Stigmatella aurantiaca (SaBphP2) in the Pr state. Structures were determined at 1.65 Å and 2.2 Å (respectively), the highest resolution of any PCM construct to date. We also report the room temperature wild-type structure of the same protein determined at 2.1 Å at the SPring-8 Angstrom Compact free electron LAser (SACLA), Japan. Our results not only highlight and confirm important amino acids near the chromophore that play a role in Pr-Pfr photoconversion but also describe the signal transduction into the PHY domain which moves across tens of angstroms after the light stimulus.

Structural basis for light control of cell development revealed by crystal structures of a myxobacterial phytochrome.

Phytochromes are red-light photoreceptors that were first characterized in plants, with homologs in photosynthetic and non-photosynthetic bacteria known as bacteriophytochromes (BphPs). Upon absorption of light, BphPs interconvert between two states denoted Pr and Pfr with distinct absorption spectra in the red and far-red. They have recently been engineered as enzymatic photoswitches for fluorescent-marker applications in non-invasive tissue imaging of mammals. This article presents cryo- and room-temperature crystal structures of the unusual phytochrome from the non-photosynthetic myxo-bacterium Stigmatella aurantiaca (SaBphP1) and reveals its role in the fruiting-body formation of this photomorphogenic bacterium. SaBphP1 lacks a conserved histidine (His) in the chromophore-binding domain that stabilizes the Pr state in the classical BphPs. Instead it contains a threonine (Thr), a feature that is restricted to several myxobacterial phytochromes and is not evolutionarily understood. SaBphP1 structures of the chromophore binding domain (CBD) and the complete photosensory core module (PCM) in wild-type and Thr-to-His mutant forms reveal details of the molecular mechanism of the Pr/Pfr transition associated with the physiological response of this myxobacterium to red light. Specifically, key structural differences in the CBD and PCM between the wild-type and the Thr-to-His mutant involve essential chromophore contacts with proximal amino acids, and point to how the photosignal is transduced through the rest of the protein, impacting the essential enzymatic activity in the photomorphogenic response of this myxobacterium.

Sub-pixel Area Calculation Methods for Estimating Irrigated Areas.

The goal of this paper was to develop and demonstrate practical methods forcomputing sub-pixel areas (SPAs) from coarse-resolution satellite sensor data. Themethods were tested and verified using: (a) global irrigated area map (GIAM) at 10-kmresolution based, primarily, on AVHRR data, and (b) irrigated area map for India at 500-mbased, primarily, on MODIS data. The sub-pixel irrigated areas (SPIAs) from coarse-resolution satellite sensor data were estimated by multiplying the full pixel irrigated areas(FPIAs) with irrigated area fractions (IAFs). Three methods were presented for IAFcomputation: (a) Google Earth Estimate (IAF-GEE); (b) High resolution imagery (IAF-HRI); and (c) Sub-pixel de-composition technique (IAF-SPDT). The IAF-GEE involvedthe use of "zoom-in-views" of sub-meter to 4-meter very high resolution imagery (VHRI)from Google Earth and helped determine total area available for irrigation (TAAI) or netirrigated areas that does not consider intensity or seasonality of irrigation. The IAF-HRI isa well known method that uses finer-resolution data to determine SPAs of the coarser-resolution imagery. The IAF-SPDT is a unique and innovative method wherein SPAs aredetermined based on the precise location of every pixel of a class in 2-dimensionalbrightness-greenness-wetness (BGW) feature-space plot of red band versus near-infraredband spectral reflectivity. The SPIAs computed using IAF-SPDT for the GIAM was within2 % of the SPIA computed using well known IAF-HRI. Further the fractions from the 2 methods were significantly correlated. The IAF-HRI and IAF-SPDT help to determine annualized or gross irrigated areas (AIA) that does consider intensity or seasonality (e.g., sum of areas from season 1, season 2, and continuous year-round crops). The national census based irrigated areas for the top 40 irrigated nations (which covers about 90% of global irrigation) was significantly better related (and had lesser uncertainties and errors) when compared to SPIAs than FPIAs derived using 10-km and 500-m data. The SPIAs were closer to actual areas whereas FPIAs grossly over-estimate areas. The research clearly demonstrated the value and the importance of sub-pixel areas as opposed to full pixel areas and presented 3 innovative methods for computing the same.

Structural enzymology using X-ray free electron lasers.

Mix-and-inject serial crystallography (MISC) is a technique designed to image enzyme catalyzed reactions in which small protein crystals are mixed with a substrate just prior to being probed by an X-ray pulse. This approach offers several advantages over flow cell studies. It provides (i) room temperature structures at near atomic resolution, (ii) time resolution ranging from microseconds to seconds, and (iii) convenient reaction initiation. It outruns radiation damage by using femtosecond X-ray pulses allowing damage and chemistry to be separated. Here, we demonstrate that MISC is feasible at an X-ray free electron laser by studying the reaction of M. tuberculosis ß-lactamase microcrystals with ceftriaxone antibiotic solution. Electron density maps of the apo-ß-lactamase and of the ceftriaxone bound form were obtained at 2.8 Å and 2.4 Å resolution, respectively. These results pave the way to study cyclic and non-cyclic reactions and represent a new field of time-resolved structural dynamics for numerous substrate-triggered biological reactions.

Periodontium bestows vision!!

The role of periodontium in supporting the tooth structures is well-known. However, less is known about its contribution to the field of ophthalmology. Corneal diseases are among major causes of blindness affecting millions of people worldwide, for which synthetic keratoprosthesis was considered the last resort to restore vision. Yet, these synthetic keratoprosthesis suffered from serious limitations, especially the foreign body reactions invoked by them resulting in extrusion of the whole prosthesis from the eye. To overcome these shortcomings, an autologous osteo-odonto keratoprosthesis utilizing intraoral entities was introduced that could positively restore vision even in cases of severely damaged eyes. The successful functioning of this prosthesis, however, predominantly depended on the presence of a healthy periodontium for grafting. Therefore, the following short communication aims to acknowledge this lesser-known role of the periodontium and other oral structures in bestowing vision to the blind patients.

The room temperature crystal structure of a bacterial phytochrome determined by serial femtosecond crystallography.

Phytochromes are a family of photoreceptors that control light responses of plants, fungi and bacteria. A sequence of structural changes, which is not yet fully understood, leads to activation of an output domain. Time-resolved serial femtosecond crystallography (SFX) can potentially shine light on these conformational changes. Here we report the room temperature crystal structure of the chromophore-binding domains of the Deinococcus radiodurans phytochrome at 2.1 Å resolution. The structure was obtained by serial femtosecond X-ray crystallography from microcrystals at an X-ray free electron laser. We find overall good agreement compared to a crystal structure at 1.35 Å resolution derived from conventional crystallography at cryogenic temperatures, which we also report here. The thioether linkage between chromophore and protein is subject to positional ambiguity at the synchrotron, but is fully resolved with SFX. The study paves the way for time-resolved structural investigations of the phytochrome photocycle with time-resolved SFX.

A rare case report of grade III gingival enlargement associated with chronic periodontitis: Comparison of two treatment techniques.

Idiopathic gingival enlargement (GE) is a rare entity characterized by massive enlargement of the gingiva. It may be associated with other diseases/conditions characterizing a syndrome, but rarely documented in literature occurring along with chronic periodontitis. This case report describes a rare case of long standing massive GE in a systemically healthy, nonsyndromic young female involving both the arches, thereby posing a diagnostic dilemma. Furthermore, in this case, we compared two surgical approaches, that is, scalpel and electrosurgery for the convenience as well as the postoperative comfort of the patient. Quadrants 1 and 3 were treated by ledge and wedge technique using scalpel and blade while quadrants 2 and 4 were treated by electrosurgery. The patient was followed postoperatively up to 1-year. The massive GE subsided without recurrence and patient was completely satisfied with the treatment, though better compliance was observed at the site treated by conventional scalpel and blade technique.

Glucometric assessment of gingival crevicular blood in diabetic and non-diabetic patients: A randomized clinical trial.

BACKGROUND: Diabetes mellitus is one of the most incessant metabolic afflictions with high prevalence rate in Indians. Diagnosis of diabetics in the initial stage helps to prevent its long term complications that are responsible for high morbidity and mortality. The aim of the present study was to assess whether glucometric analysis using Gingival Crevicular Blood (GCB) can be used for screening of diabetic patients in dental chair. MATERIALS AND METHODS: Present study was a double blinded randomized controlled trial. A total of 50 patients, 25 diabetic and 25 non-diabetic with chronic periodontitis were enrolled in the study. Blood oozing from the gingival crevices after periodontal pocket probing of anterior teeth and Finger Prick Blood (FPB) was taken and analysed by glucometer. RESULT: Mean ± S.D was 195.84 ± 27.19 and 138.78 ± 29.95 for GCB and FPB respectively in diabetic group. For non-diabetic group Mean ± S.D was 103.84 ± 12.56 and 84.36 ± 10.36 respectively for GCB and FPB. A Karl Pearson correlation coefficient value of r = +0.735 for diabetic and r = +0.802 for non-diabetic group comparing GCB and FPB. CONCLUSION: GCB cannot be used for screening blood glucose during periodontal examination.

Treatment of localized gingival recession using the free rotated papilla autograft combined with coronally advanced flap by conventional (macrosurgery) and surgery under magnification (microsurgical) technique: A comparative clinical study.

BACKGROUND: The aim of the present study was to evaluate and compare the conventional (macro-surgical) and microsurgical approach in performing the free rotated papilla autograft combined with coronally advanced flap surgery in treatment of localized gingival recession. MATERIALS AND METHODS: A total of 20 sites from 10 systemically healthy patients were selected for the study. The selected sites were randomly divided into experimental site A and experimental site B by using the spilt mouth design. Conventional (macro-surgical) approach for site A and micro-surgery for site B was applied in performing the free rotated papilla autograft combined with coronally advanced flap. Recession depth (RD), recession width (RW) clinical attachment level (CAL.) and width of keratinized tissue (WKT.) were recorded at baseline, 3 months and 6 months post-operatively. RESULTS: Both (macro- and microsurgery) groups showed significant clinical improvement in all the parameters (RD, RW, CAL and WKT). However, on comparing both the groups, these parameters did not reach statistical significance. CONCLUSION: Both the surgical procedures were equally effective in treatment of localized gingival recession by the free rotated papilla autograft technique combined with coronally advanced flap. However, surgery under magnification (microsurgery) may be clinically better than conventional surgery in terms of less post-operative pain and discomfort experienced by patients at the microsurgical site.