Reliability of bioreactance and pulse power analysis in measuring cardiac index during cytoreductive abdominal surgery with hyperthermic intraperitoneal chemotherapy (HIPEC).

PURPOSE: Various malignancies with peritoneal carcinomatosis are treated with cytoreductive surgery and hyperthermic intraperitoneal chemotherapy (HIPEC). The hemodynamic instability resulting from fluid balance alterations during the procedure necessitates reliable hemodynamic monitoring. The aim of the study was to compare the accuracy, precision and trending ability of two less invasive hemodynamic monitors, bioreactance-based Starling SV and pulse power device LiDCOrapid with bolus thermodilution technique with pulmonary artery catheter in the setting of cytoreductive surgery with HIPEC. METHODS: Thirty-one patients scheduled for cytoreductive surgery were recruited. Twenty-three of them proceeded to HIPEC and were included to the study. Altogether 439 and 430 intraoperative bolus thermodilution injections were compared to simultaneous cardiac index readings obtained with Starling SV and LiDCOrapid, respectively. Bland-Altman method, four-quadrant plots and error grids were used to assess the agreement of the devices. RESULTS: Comparing Starling SV with bolus thermodilution, the bias was acceptable (0.13 l min- 1 m- 2, 95% CI 0.05 to 0.20), but the limits of agreement were wide (- 1.55 to 1.71 l min- 1 m- 2) and the percentage error was high (60.0%). Comparing LiDCOrapid with bolus thermodilution, the bias was acceptable (- 0.26 l min- 1 m- 2, 95% CI - 0.34 to - 0.18), but the limits of agreement were wide (- 1.99 to 1.39 l min- 1 m- 2) and the percentage error was high (57.1%). Trending ability was inadequate with both devices. CONCLUSION: Starling SV and LiDCOrapid were not interchangeable with bolus thermodilution technique limiting their usefulness in the setting of cytoreductive surgery with HIPEC.

Conserved histidine and tyrosine determine spectral responses through the water network in Deinococcus radiodurans phytochrome.

Phytochromes are red light-sensing photoreceptor proteins that bind a bilin chromophore. Here, we investigate the role of a conserved histidine (H260) and tyrosine (Y263) in the chromophore-binding domain (CBD) of Deinococcus radiodurans phytochrome (DrBphP). Using crystallography, we show that in the H260A variant, the missing imidazole side chain leads to increased water content in the binding pocket. On the other hand, Y263F mutation reduces the water occupancy around the chromophore. Together, these changes in water coordination alter the protonation and spectroscopic properties of the biliverdin. These results pinpoint the importance of this conserved histidine and tyrosine, and the related water network, for the function and applications of phytochromes.

The structural effect between the output module and chromophore-binding domain is a two-way street via the hairpin extension.

Signal transduction typically starts with either ligand binding or cofactor activation, eventually affecting biological activities in the cell. In red light-sensing phytochromes, isomerization of the bilin chromophore results in regulation of the activity of diverse output modules. During this process, several structural elements and chemical events influence signal propagation. In our study, we have studied the full-length bacteriophytochrome from Deinococcus radiodurans as well as a previously generated optogenetic tool where the native histidine kinase output module has been replaced with an adenylate cyclase. We show that the composition of the output module influences the stability of the hairpin extension. The hairpin, often referred as the PHY tongue, is one of the central structural elements for signal transduction. It extends from a distinct domain establishing close contacts with the chromophore binding site. If the coupling between these interactions is disrupted, the dynamic range of the enzymatic regulation is reduced. Our study highlights the complex conformational properties of the hairpin extension as a bidirectional link between the chromophore-binding site and the output module, as well as functional properties of diverse output modules.

The hairpin extension controls solvent access to the chromophore binding pocket in a bacterial phytochrome: a UV-vis absorption spectroscopy study.

Solvent access to the protein interior plays an important role in the function of many proteins. Phytochromes contain a specific structural feature, a hairpin extension that appears to relay structural information from the chromophore to the rest of the protein. The extension interacts with amino acids near the chromophore, and hence shields the chromophore from the surrounding solvent. We envision that the detachment of the extension from the protein surface allows solvent exchange reactions in the vicinity of the chromophore. This can facilitate for example, proton transfer processes between solvent and the protein interior. To test this hypothesis, the kinetics of the protonation state of the biliverdin chromophore from Deinococcus radiodurans bacteriophytchrome, and thus, the pH of the surrounding solution, is determined. The observed absorbance changes are related to the solvent access of the chromophore binding pocket, gated by the hairpin extension. We therefore propose a model with an "open" (solvent-exposed, deprotonation-active on a (sub)second time-scale) state and a "closed" (solvent-gated, deprotonation inactive) state, where the hairpin fluctuates slowly between these conformations thereby controlling the deprotonation process of the chromophore on a minute time scale. When the connection between the hairpin and the biliverdin surroundings is destabilized by a point mutation, the amplitude of the deprotonation phase increases considerably. In the absence of the extension, the chromophore deprotonates essentially without any "gating". Hence, we introduce a straightforward method to study the stability and fluctuation of the phytochrome hairpin in its photostationary state. This approach can be extended to other chromophore-protein systems where absorption changes reflect dynamic processes of the protein.

Site-by-site tracking of signal transduction in an azidophenylalanine-labeled bacteriophytochrome with step-scan FTIR spectroscopy.

Signal propagation in photosensory proteins is a complex and multidimensional event. Unraveling such mechanisms site-specifically in real time is an eligible but a challenging goal. Here, we elucidate the site-specific events in a red-light sensing phytochrome using the unnatural amino acid azidophenylalanine, vibrationally distinguishable from all other protein signals. In canonical phytochromes, signal transduction starts with isomerization of an excited bilin chromophore, initiating a multitude of processes in the photosensory unit of the protein, which eventually control the biochemical activity of the output domain, nanometers away from the chromophore. By implementing the label in prime protein locations and running two-color step-scan FTIR spectroscopy on the Deinococcus radiodurans bacteriophytochrome, we track the signal propagation at three specific sites in the photosensory unit. We show that a structurally switchable hairpin extension, a so-called tongue region, responds to the photoconversion already in microseconds and finalizes its structural changes concomitant with the chromophore, in milliseconds. In contrast, kinetics from the other two label positions indicate that the site-specific changes deviate from the chromophore actions, even though the labels locate in the chromophore vicinity. Several other sites for labeling resulted in impaired photoswitching, low structural stability, or no changes in the difference spectrum, which provides additional information on the inner dynamics of the photosensory unit. Our work enlightens the multidimensionality of the structural changes of proteins under action. The study also shows that the signaling mechanism of phytochromes is accessible in a time-resolved and site-specific approach by azido probes and demonstrates challenges in using these labels.

Tips and turns of bacteriophytochrome photoactivation.

Phytochromes are ubiquitous photosensor proteins, which control the growth, reproduction and movement in plants, fungi and bacteria. Phytochromes switch between two photophysical states depending on the light conditions. In analogy to molecular machines, light absorption induces a series of structural changes that are transduced from the bilin chromophore, through the protein, and to the output domains. Recent progress towards understanding this structural mechanism of signal transduction has been manifold. We describe this progress with a focus on bacteriophytochromes. We describe the mechanism along three structural tiers, which are the chromophore-binding pocket, the photosensory module, and the output domains. We discuss possible interconnections between the tiers and conclude by presenting future directions and open questions. We hope that this review may serve as a compendium to guide future structural and spectroscopic studies designed to understand structural signaling in phytochromes.

Signal amplification and transduction in phytochrome photosensors.

Sensory proteins must relay structural signals from the sensory site over large distances to regulatory output domains. Phytochromes are a major family of red-light-sensing kinases that control diverse cellular functions in plants, bacteria and fungi. Bacterial phytochromes consist of a photosensory core and a carboxy-terminal regulatory domain. Structures of photosensory cores are reported in the resting state and conformational responses to light activation have been proposed in the vicinity of the chromophore. However, the structure of the signalling state and the mechanism of downstream signal relay through the photosensory core remain elusive. Here we report crystal and solution structures of the resting and activated states of the photosensory core of the bacteriophytochrome from Deinococcus radiodurans. The structures show an open and closed form of the dimeric protein for the activated and resting states, respectively. This nanometre-scale rearrangement is controlled by refolding of an evolutionarily conserved 'tongue', which is in contact with the chromophore. The findings reveal an unusual mechanism in which atomic-scale conformational changes around the chromophore are first amplified into an ångstrom-scale distance change in the tongue, and further grow into a nanometre-scale conformational signal. The structural mechanism is a blueprint for understanding how phytochromes connect to the cellular signalling network.

Hyperthermic intraperitoneal chemotherapy enhances blood coagulation perioperatively evaluated by thromboelastography: a pilot study.

Background and Objectives: Postoperative thromboembolism is a significant cause of prolonged recovery in patients undergoing cytoreductive surgery (CRS) and hyperthermic intraperitoneal chemotherapy (HIPEC). Thromboelastography (TEG) can detect hypercoagulable states and predict thromboembolic complications after surgery. This study assessed the impact of CRS and HIPEC on TEG values.Methods: TEG parameters reaction time (R), kinetics time (K), angle (α), maximum amplitude (MA), and lysis percent at 60 min (LY60) were determined preoperatively, and at the end of CRS, during HIPEC, and at the end of the operation using blood samples from 15 HIPEC patients. Platelets, P-TT, and aPTT were also determined before and after CRS.Results: A total of 75 samples were analyzed. During CRS, there was a significant reduction in the mean MA (3.06 mm, p = 0.001). The mean P-TT declined by 32% (p < 0.001) and mean platelets by 55 × 109/L (p < 0.001). During HIPEC, the mean R and K shortened by 1.04 min (p = 0.015) and 0.18 min (p = 0.018), respectively, whereas α increased by 2.48° (p = 0.005).Conclusions: During CRS, both TEG and conventional laboratory tests indicated hypocoagulation. During HIPEC, however, the initiation of coagulation and the kinetics of thrombin formation were accelerated.

On the (un)coupling of the chromophore, tongue interactions, and overall conformation in a bacterial phytochrome.

Phytochromes are photoreceptors in plants, fungi, and various microorganisms and cycle between metastable red light-absorbing (Pr) and far-red light-absorbing (Pfr) states. Their light responses are thought to follow a conserved structural mechanism that is triggered by isomerization of the chromophore. Downstream structural changes involve refolding of the so-called tongue extension of the phytochrome-specific GAF-related (PHY) domain of the photoreceptor. The tongue is connected to the chromophore by conserved DIP and PRXSF motifs and a conserved tyrosine, but the role of these residues in signal transduction is not clear. Here, we examine the tongue interactions and their interplay with the chromophore by substituting the conserved tyrosine (Tyr263) in the phytochrome from the extremophile bacterium Deinococcus radiodurans with phenylalanine. Using optical and FTIR spectroscopy, X-ray solution scattering, and crystallography of chromophore-binding domain (CBD) and CBD-PHY fragments, we show that the absence of the Tyr263 hydroxyl destabilizes the ß-sheet conformation of the tongue. This allowed the phytochrome to adopt an α-helical tongue conformation regardless of the chromophore state, hence distorting the activity state of the protein. Our crystal structures further revealed that water interactions are missing in the Y263F mutant, correlating with a decrease of the photoconversion yield and underpinning the functional role of Tyr263 in phytochrome conformational changes. We propose a model in which isomerization of the chromophore, refolding of the tongue, and globular conformational changes are represented as weakly coupled equilibria. The results also suggest that the phytochromes have several redundant signaling routes.

Coordination of the biliverdin D-ring in bacteriophytochromes.

Phytochrome proteins translate light into biochemical signals in plants, fungi and microorganisms. Light cues are absorbed by a bilin chromophore, leading to an isomerization and a rotation of the D-ring. This relays the signal to the protein matrix. A set of amino acids, which is conserved across the phytochrome superfamily, holds the chromophore in the binding pocket. However, the functional role of many of these amino acids is not yet understood. Here, we investigate the hydrogen bonding network which surrounds the D-ring of the chromophore in the resting (Pr) state. We use UV/vis spectroscopy, infrared absorption spectroscopy and X-ray crystallography to compare the photosensory domains from Deinococcus radiodurans, the phytochrome 1 from Stigmatella aurantiaca, and a D. radiodurans H290T mutant. In the latter two, an otherwise conserved histidine next to the D-ring is replaced by a threonine. Our infrared absorption data indicate that the carbonyl of the D-ring is more strongly coordinated by hydrogen bonds when the histidine is missing. This is in apparent contrast with the crystal structure of the PAS-GAF domain of phytochrome 1 from S. aurantiaca (pdb code 4RPW), which did not resolve any obvious binding partners for the D-ring carbonyl. We present a new crystal structure of the H290T mutant of the PAS-GAF from D. radiodurans phytochrome. The 1.4 Å-resolution structure reveals additional water molecules, which fill the void created by the mutation. Two of the waters are significantly disordered, suggesting that flexibility might be important for the photoconversion. Finally, we report a spectral analysis which quantitatively explains why the histidine-less phytochromes do not reach equal Pfr-type absorption in the photoequilibrium compared to the Deinococcus radiodurans wild-type protein. The study highlights the importance of water molecules and the hydrogen bonding network around the chromophore for controlling the isomerization reaction and spectral properties of phytochromes.

Registries on peritoneal surface malignancies throughout the world, their use and their options.

AIM: The treatment of peritoneal surface malignancies ranges from palliative care to full cytoreductive surgery (CRS) and heated intraperitoneal chemotherapy, HIPEC. Ongoing monitoring of patient recruitment and volume is usually carried out through dedicated registries. With multiple registries available worldwide, we sought to investigate the nature, extent and value of existing worldwide CRS and HIPEC registries. METHODS: A questionnaire was sent out to all known major treatment centres. The questionnaire covers: general purpose of the registry; inclusion criteria in the registry; the date the registry was first established; volume of patients in the registry and description of the data fields in the registries. Finally, the population size of the catchment area of the registry was collected. RESULTS: Twenty-seven questionnaires where returned. National databases are established in northwest European countries. There are five international general databases. Most database collect data on patients who have undergone an attempt to CRS and HIPEC. Two registries collect data on all patients with peritoneal carcinomatosis regardless the treatment. Most registries are primarily used for tracking outcomes and complications. When correlating the number of cases of CRS and HIPEC that are performed to the catchment area of the various registry, a large variation in the number of performed procedures related to the overall population was noted, ranging from 1.3 to 57 patients/million year with an average of 15 patients/1 million year. CONCLUSIONS: CRS and HIPEC is a well-established treatment for peritoneal surface malignancies worldwide. However, the coverage as well as the registration of treatment procedures differs widely. The most striking difference is the proportion of HIPEC procedures per capita which ranges from 1.3 to 57 patients per million. This suggests either a difference in patient selection, lack of access to HIPEC centres or lack of appropriate data collection.

Light-induced Changes in the Dimerization Interface of Bacteriophytochromes.

Phytochromes are dimeric photoreceptor proteins that sense red light levels in plants, fungi, and bacteria. The proteins are structurally divided into a light-sensing photosensory module consisting of PAS, GAF, and PHY domains and a signaling output module, which in bacteriophytochromes typically is a histidine kinase (HK) domain. Existing structural data suggest that two dimerization interfaces exist between the GAF and HK domains, but their functional roles remain unclear. Using mutational, biochemical, and computational analyses of the Deinococcus radiodurans phytochrome, we demonstrate that two dimerization interfaces between sister GAF and HK domains stabilize the dimer with approximately equal contributions. The existence of both dimerization interfaces is critical for thermal reversion back to the resting state. We also find that a mutant in which the interactions between the GAF domains were removed monomerizes under red light. This implies that the interactions between the HK domains are significantly altered by photoconversion. The results suggest functional importance of the dimerization interfaces in bacteriophytochromes.

The role of C-reactive protein in prediction of the severity of acute diverticulitis in an emergency unit.

OBJECTIVE: Computed tomography (CT) is the most appropriate initial imaging modality for the assessment of acute diverticulitis. The aim here was to determine the usefulness of C-reactive protein (CRP) in predicting the severity of the diverticulitis process and the need for a CT examination. METHODS: The CRP values of 350 patients who presented first time with symptoms of acute diverticulitis and underwent CT imaging on admission to Oulu University Hospital were compared with the CT findings and clinical parameters by means of both univariate and multivariate analyses. RESULTS: The receiver operating characteristic curve showed that a CRP cut-off value of 149.5 mg/l significantly discriminated acute uncomplicated diverticulitis from complicated diverticulitis (specificity 65%, sensitivity 85%, area under the curve 0.811, p = 0.0001). In multivariate analysis, a CRP value over 150 mg/l and old age were independent risk factors for acute complicated diverticulitis. The mean CRP value was significantly higher in the patients who died, 207 (84 SD), than in those who survived, 139 (SD 83). In addition, a CRP value over 150 mg/l and free abdominal fluid in CT were independent variables predicting postoperative mortality. CONCLUSIONS: CRP is useful for the predicting the severity of acute diverticulitis on admission. Patients with a CRP value higher than 150 mg/l have an in increased risk of complicated diverticulitis and a CT examination should always be carried out.

Connection between absorption properties and conformational changes in Deinococcus radiodurans phytochrome.

Phytochromes consist of several protein domains and a linear tetrapyrrole molecule, which interact as a red-light-sensing system. In this study, size-exclusion chromatography and light-scattering techniques are combined with UV-vis spectroscopy to investigate light-induced changes in dimeric Deinococcus radiodurans bacterial phytochrome (DrBphP) and its subdomains. The photosensory unit (DrCBD-PHY) shows an unusually stable Pfr state with minimal dark reversion, whereas the histidine kinase (HK) domain facilitates dark reversion to the resting state. Size-exclusion chromatography reveals that all phytochrome fragments remain as dimers in the illuminated state and dark state. Still, the elution profiles of all phytochrome fragments differ between the illuminated and dark states. The differences are observed reliably only when the whole UV-vis spectrum is characterized along the elution profile and show more Pfr-state characteristics at later elution volumes in DrBphP and DrCBD-PHY fragments. This implies that the PHY domain has an important role in amplifying and relaying light-induced conformational changes to the HK domain. In the illuminated state, the HK domain appears partially unfolded and prone to form oligomers. The oligomerization of DrBphP can be diminished by converting the molecule back to the resting Pr state by using far-red light.

Multimodal Control of Bacterial Gene Expression by Red and Blue Light.

By applying sensory photoreceptors, optogenetics realizes the light-dependent control of cellular events and state. Given reversibility, noninvasiveness, and exquisite spatiotemporal precision, optogenetic approaches enable innovative use cases in cell biology, synthetic biology, and biotechnology. In this chapter, we detail the implementation of the pREDusk, pREDawn, pCrepusculo, and pAurora optogenetic circuits for controlling bacterial gene expression by red and blue light, respectively. The protocols provided here guide the practical use and multiplexing of these circuits, thereby enabling graded protein production in bacteria at analytical and semi-preparative scales.

Leveraging the histidine kinase-phosphatase duality to sculpt two-component signaling.

Bacteria must constantly probe their environment for rapid adaptation, a crucial need most frequently served by two-component systems (TCS). As one component, sensor histidine kinases (SHK) control the phosphorylation of the second component, the response regulator (RR). Downstream responses hinge on RR phosphorylation and can be highly stringent, acute, and sensitive because SHKs commonly exert both kinase and phosphatase activity. With a bacteriophytochrome TCS as a paradigm, we here interrogate how this catalytic duality underlies signal responses. Derivative systems exhibit tenfold higher red-light sensitivity, owing to an altered kinase-phosphatase balance. Modifications of the linker intervening the SHK sensor and catalytic entities likewise tilt this balance and provide TCSs with inverted output that increases under red light. These TCSs expand synthetic biology and showcase how deliberate perturbations of the kinase-phosphatase duality unlock altered signal-response regimes. Arguably, these aspects equally pertain to the engineering and the natural evolution of TCSs.

[Fulminant Clostridium difficile colitis]. / Fulminantin Clostridium difficilen aiheuttama koliitti--milloin kirurginen hoito on aiheellinen?

Clostridium difficile is a common cause of nosocomial diarrhea and it's most difficult Fulminant form is increasing in prevalence and severity in hospitalized patients. The early diagnostics and intensive conservative therapy are essential in the treatment of this ominous disease. The physicians must be aware of the possibility of this disease, when treating patients, who have received preceding antibiotic therapy. These patients have to be followed up in the surgical units. If the general condition of these patients is deteriorating, they must be transferred to emergency units. Surgery becomes mandatory before the development of multi organ failure.

The interconnecting hairpin extension "arm": An essential allosteric element of phytochrome activity.

In red-light sensing phytochromes, isomerization of the bilin chromophore triggers structural and dynamic changes across multiple domains, ultimately leading to control of the output module (OPM) activity. In between, a hairpin structure, "arm", extends from an interconnecting domain to the chromophore region. Here, by removing this protein segment in a bacteriophytochrome from Deinococcus radiodurans (DrBphP), we show that the arm is crucial for signal transduction. Crystallographic, spectroscopic, and biochemical data indicate that this variant maintains the properties of DrBphP in the resting state. Spectroscopic data also reveal that the armless systems maintain the ability to respond to light. However, there is no subsequent regulation of OPM activity without the arms. Thermal denaturation reveals that the arms stabilize the DrBphP structure. Our results underline the importance of the structurally flexible interconnecting hairpin extensions and describe their central role in the allosteric coupling of phytochromes.

Assembly of a filamin four-domain fragment and the influence of splicing variant-1 on the structure.

Filamins are scaffold proteins that bind to various proteins, including the actin cytoskeleton, integrin adhesion receptors, and adaptor proteins such as migfilin. Alternative splicing of filamin, largely constructed from 24 Ig-like domains, is thought to have a role in regulating its interactions with other proteins. The filamin A splice variant-1 (FLNa var-1) lacks 41 amino acids, including the last ß-strand of domain 19, FLNa(19), and the first ß-strand of FLNa(20) that was previously shown to mask a key binding site on FLNa(21). Here, we present a structural characterization of domains 18-21, FLNa(18-21), in the FLNa var-1 as well as its nonspliced counterpart. A model of nonspliced FLNa(18-21), obtained from small angle x-ray scattering data, shows that these four domains form an L-shaped structure, with one arm composed of a pair of domains. NMR spectroscopy reveals that in the splice variant, FLNa(19) is unstructured whereas the other domains retain the same fold as in their canonical counterparts. The maximum dimensions predicted by small angle x-ray scattering data are increased upon migfilin binding in the FLNa(18-21) but not in the splice variant, suggesting that migfilin binding is able to displace the masking ß-strand and cause a rearrangement of the structure. Possible function roles for the spliced variants are discussed.

Structural mechanism of signal transduction in a phytochrome histidine kinase.

Phytochrome proteins detect red/far-red light to guide the growth, motion, development and reproduction in plants, fungi, and bacteria. Bacterial phytochromes commonly function as an entrance signal in two-component sensory systems. Despite the availability of three-dimensional structures of phytochromes and other two-component proteins, the conformational changes, which lead to activation of the protein, are not understood. We reveal cryo electron microscopy structures of the complete phytochrome from Deinoccocus radiodurans in its resting and photoactivated states at 3.6 Å and 3.5 Å resolution, respectively. Upon photoactivation, the photosensory core module hardly changes its tertiary domain arrangement, but the connector helices between the photosensory and the histidine kinase modules open up like a zipper, causing asymmetry and disorder in the effector domains. The structures provide a framework for atom-scale understanding of signaling in phytochromes, visualize allosteric communication over several nanometers, and suggest that disorder in the dimeric arrangement of the effector domains is important for phosphatase activity in a two-component system. The results have implications for the development of optogenetic applications.