Photocatalytic Activation of Aryl(trifluoromethyl) Diazos to Carbenes for High-Resolution Protein Labeling with Red Light.

State-of-the-art methods in photoproximity labeling center on the targeted generation and capture of short-lived reactive intermediates to provide a snapshot of local protein environments. Diazirines are the current gold standard for high-resolution proximity labeling, generating short-lived aryl(trifluoromethyl) carbenes. Here, we present a method to access aryl(trifluoromethyl) carbenes from a stable diazo source via tissue-penetrable, deep red to near-infrared light (600-800 nm). The operative mechanism of this activation involves Dexter energy transfer from photoexcited osmium(II) photocatalysts to the diazo, thus revealing an aryl(trifluoromethyl) carbene. The labeling preferences of the diazo probe with amino acids are studied, showing high reactivity toward heteroatom-H bonds. Upon the synthesis of a biotinylated diazo probe, labeling studies are conducted on native proteins as well as proteins conjugated to the Os photocatalyst. Finally, we demonstrate that the conjugation of a protein inhibitor to the photocatalyst also enables selective protein labeling in the presence of spectator proteins and achieves specific labeling of a membrane protein on the surface of mammalian cells via a two-antibody photocatalytic system.

A field study to evaluate PMI estimation methods for advanced decomposition stages.

Estimating the postmortem interval (PMI) is one of the major tasks and a continuous challenge in forensic pathology. It is often an exclusion process of available methods, which ultimately can lead to an unsatisfactory outcome due to poor reliability. This problem is most acute in the late PMI, when decomposition proceeds and some methods (such as rigor, livor, and algor mortis) are no longer applicable. Several methods, such as forensic entomology, skeletal muscle protein degradation, and the study of body decomposition by application of a morphological scoring, are expected to provide further information; however, all have certain limitations and weaknesses. Availability of a tool-box of methods allows a case-specific selection of the most appropriate one(s), or eventually provides improvements in the overall accuracy and precision of the PMI estimation by merging and combining methods. To investigate practical (field) application, eventual interferences, and/or synergetic effects, as well as the robustness of these methods towards specific influencing factors, a field study was conducted, using eight pig cadavers of different body weights and physical coverage, left to decompose under natural conditions for 16 days. Morphological changes during decomposition were assessed using the total body score (TBS), muscle samples were collected to analyze protein degradation, and insect colonization was evaluated. The results reveal strengths and current limitations of all tested methods, as well as promising synergistic effects, and thus, provide a baseline for targeted future research.

Concurrent presence of on- and off-pathway folding intermediates of apoflavodoxin at physiological ionic strength.

Flavodoxins have a protein topology that can be traced back to the universal ancestor of the three kingdoms of life. Proteins with this type of architecture tend to temporarily misfold during unassisted folding to their native state and form intermediates. Several of these intermediate species are molten globules (MGs), which are characterized by a substantial amount of secondary structure, yet without the tertiary side-chain packing of natively folded proteins. An off-pathway MG is formed at physiological ionic strength in the case of the F44Y variant of Azotobacter vinelandii apoflavodoxin (i.e., flavodoxin without flavin mononucleotide (FMN)). Here, we show that at this condition actually two folding species of this apoprotein co-exist at equilibrium. These species were detected by using a combination of FMN fluorescence quenching upon cofactor binding to the apoprotein and of polarized time-resolved tryptophan fluorescence spectroscopy. Besides the off-pathway MG, we observe the simultaneous presence of an on-pathway folding intermediate, which is native-like. Presence of concurrent intermediates at physiological ionic strength enables future exploration of how aspects of the cellular environment, like for example involvement of chaperones, affect these species.

The Ribosome Restrains Molten Globule Formation in Stalled Nascent Flavodoxin.

Folding of proteins usually involves intermediates, of which an important type is the molten globule (MG). MGs are ensembles of interconverting conformers that contain (non-)native secondary structure and lack the tightly packed tertiary structure of natively folded globular proteins. Whereas MGs of various purified proteins have been probed to date, no data are available on their presence and/or effect during protein synthesis. To study whether MGs arise during translation, we use ribosome-nascent chain (RNC) complexes of the electron transfer protein flavodoxin. Full-length isolated flavodoxin, which contains a non-covalently bound flavin mononucleotide (FMN) as cofactor, acquires its native α/ß parallel topology via a folding mechanism that contains an off-pathway intermediate with molten globular characteristics. Extensive population of this MG state occurs at physiological ionic strength for apoflavodoxin variant F44Y, in which a phenylalanine at position 44 is changed to a tyrosine. Here, we show for the first time that ascertaining the binding rate of FMN as a function of ionic strength can be used as a tool to determine the presence of the off-pathway MG on the ribosome. Application of this methodology to F44Y apoflavodoxin RNCs shows that at physiological ionic strength the ribosome influences formation of the off-pathway MG and forces the nascent chain toward the native state.

Spirohexene-Tetrazine Ligation Enables Bioorthogonal Labeling of Class B G Protein-Coupled Receptors in Live Cells.

A new bioorthogonal reactant pair, spiro[2.3]hex-1-ene (Sph) and 3,6-di(2-pyridyl)-s-tetrazine (DpTz), for the strain-promoted inverse electron-demand Diels-Alder cycloaddition, that is, tetrazine ligation, is reported. As compared to the previously reported strained alkenes such as trans-cyclooctene (TCO) and 1,3-disubstituted cyclopropene, Sph exhibits balanced reactivity and stability in tetrazine ligation with the protein substrates. A lysine derivative of Sph, SphK, was site-selectively incorporated into the extracellular loop regions (ECLs) of GCGR and GLP-1R, two members of class B G protein-coupled receptors (GPCRs) in mammalian cells with the incorporation efficiency dependent on the location. Subsequent bioorthogonal reactions with the fluorophore-conjugated DpTz reagents afforded the fluorescently labeled GCGR and GLP-1R ECL mutants with labeling yield as high as 68%. A multitude of functional assays were performed with these GPCR mutants, including ligand binding, ligand-induced receptor internalization, and ligand-stimulated intracellular cAMP accumulation. Several positions in the ECL3s of GCGR and GLP-1R were identified that tolerate SphK mutagenesis and subsequent bioorthogonal labeling. The generation of functional, fluorescently labeled ECL3 mutants of GCGR and GLP-1R should allow biophysical studies of conformation dynamics of this important class of GPCRs in their native environment in live cells.

Diatoms in drowning cases in forensic veterinary context: a preliminary study.

In this preliminary study, a digestive method used in forensic context to extract diatoms has been applied in organs taken from ten wildlife animals belonging three species of mammals (a deer, a roe and five otters) and one species of birds (two magpies). Only four carcasses were recovered from aquatic environments (bath trough for animals, bathtub irrigation, river) and only in three cases out of ten that the cause of death was ruled out as drowning. In seven cases, the death was due to other causes: gunshot injuries for one otter, blunt trauma for a magpie, and traumatic injuries followed by motor vehicle collision in other four otters and a roe. Post-mortem examination was performed in all carcasses. The diatom test protocol was performed according to the Italian guidelines for analysis of benthic diatoms for ecological status assessment of inland waters. Five grams of lung, liver, and kidney was taken from all the animal carcasses. In some cases, additional tissue samples were also available among which brain, heart, spleen, and bone marrow. In all four cases found in water, the drowning medium was also available. Hydrogen peroxide (H2O2) digestion was performed, and identification of 21 genera of diatoms was obtained. The method proved to be suitable for the identification of diatoms in the organs of the animals drowned supporting the final diagnosis of drowning. Only in otters, all died for causes other than drowning, diatoms did not prove to be suitable for the diagnosis of drowning since their presence in the internal organs was mainly related to their main diet based on fishmeal. The authors believe that this first trial is very promising, and the results suggest that diatom test can be easily applied in forensic veterinary context.

Supratrochlear Neuralgia: A Prospective Case Series of 15 Patients.

OBJECTIVE: The aim of this study was to describe clinical features unique to supratrochlear neuralgia. BACKGROUND: The supratrochlear nerve supplies the medial aspect of the forehead. Due to the intricate relationship between supraorbital and supratrochlear nerves, neuralgic pain in this region has been traditionally attributed to supraorbital neuralgia. No cases of supratrochlear neuralgia have been reported so far. METHODS: From 2009 through 2016, we prospectively recruited patients with pain confined to the territory of the supratrochlear nerve. RESULTS: Fifteen patients (13 women, 2 men; mean age 51.4 years, standard deviation 14.9) presented with pain in the lower paramedian forehead, extending to the eyebrow in two patients and to the internal angle of the orbit in another. Pain was unilateral in 11 patients (six on the right, five on the left), and bilateral in four. Six patients had continuous pain and nine described intermittent pain. Palpation of the supratrochlear nerve at the medial third of the supraorbital rim resulted in hypersensitivity in all cases. All but one patient exhibited sensory disturbances within the painful area. Fourteen patients underwent anesthetic blockades of the supratrochlear nerve, with immediate relief in all cases and long-term remission in three. Six of them had received unsuccessful anesthetic blocks of the supraorbital nerve. Five patients were treated successfully with oral drugs and one patient was treated with radiofrequency. CONCLUSIONS: Supratrochlear neuralgia is an uncommon disorder causing pain in the medial region of the forehead. It may be differentiated from supraorbital neuralgia and other similar headaches and neuralgias based on the topography of the pain and the response to anesthetic blockade.

Primary cutaneous CD4-positive small/medium-sized pleomorphic T-cell lymphoproliferative disorder: Report of a case and review of the literature.

Primary cutaneous small/medium-sized T-cell lymphoma (PCSM-TCL), which was included in the World Health Organization - European Organization for Research and Treatment of Cancer (WHO-EORTC) classification for cutaneous lymphomas as a provisional entity in 2008, has recently been reclassified as primary cutaneous small/medium-sized T-cell lymphoproliferative disorder (PCSM-TCLPD) because of its indolent behavior and uncertain malignant potential. Treatment with local therapies is usually curative, although there have been reports of aggressive, systemic disease. This spectrum of disease behavior evokes the consideration that this entity may actually be multiple diseases with a shared clinicopathologic features rather than a singular disease process with a variety of behaviors. PCSM-TCLPD retained its designation as a provisional entity under the updated WHO-EORTC guidelines; however, additional cases of PCSM-TCLPD are needed to shed more light on this rare disorder.

Stalled flavodoxin binds its cofactor while fully exposed outside the ribosome.

Correct folding of proteins is crucial for cellular homeostasis. More than thirty percent of proteins contain one or more cofactors, but the impact of these cofactors on co-translational folding remains largely unknown. Here, we address the binding of flavin mononucleotide (FMN) to nascent flavodoxin, by generating ribosome-arrested nascent chains that expose either the entire protein or C-terminally truncated segments thereof. The native α/ß parallel fold of flavodoxin is among the most ancestral and widely distributed folds in nature and exploring its co-translational folding is thus highly relevant. In Escherichia coli (strain BL21(DE3) Δtig::kan) FMN turns out to be limiting for saturation of this flavoprotein on time-scales vastly exceeding those of flavodoxin synthesis. Because the ribosome affects protein folding, apoflavodoxin cannot bind FMN during its translation. As a result, binding of cofactor to released protein is the last step in production of this flavoprotein in the cell. We show that once apoflavodoxin is entirely synthesized and exposed outside the ribosome to which it is stalled by an artificial linker containing the SecM sequence, the protein is natively folded and capable of binding FMN.

Sequence-Specific 2-Cyanobenzothiazole Ligation.

The use of small, natural chemical reporters in conjunction with catalyst-free bioorthogonal reactions will greatly streamline protein labeling in a cellular environment with minimum perturbation to their function. Here we report the discovery of a 2-cyanobenzothiazole (CBT)-reactive peptide tag, CX10R7, from a cysteine-encoded peptide phage library using the phage-assisted interrogation of reactivity method. Fusion of CX10R7 with a protein of interest allows site-specific labeling of the protein with CBT both in vitro and on the surface of E. coli cells. Mutagenesis studies indicated that the reactivity and specificity of CX10R7 are attributed to the sequence environment, in which the residues surrounding cysteine help to stabilize the ligation product.

Combined effect of genetic variants in the GluN2B coding gene (GRIN2B) on prefrontal function during working memory performance.

BACKGROUND: The GluN2B subunit of N-methyl-d-aspartate receptors is crucially involved in the physiology of the prefrontal cortex during working memory (WM). Consistently, genetic variants in the GluN2B coding gene (GRIN2B) have been associated with cognitive phenotypes. However, it is unclear how GRIN2B genetic variation affects gene expression and prefrontal cognitive processing. Using a composite score, we tested the combined effect of GRIN2B variants on prefrontal activity during WM performance in healthy subjects. METHOD: We computed a composite score to combine the effects of single nucleotide polymorphisms on post-mortem prefrontal GRIN2B mRNA expression. We then computed the composite score in independent samples of healthy participants in a peripheral blood expression study (n = 46), in a WM behavioural study (n = 116) and in a WM functional magnetic resonance imaging study (n = 122). RESULTS: Five polymorphisms were associated with GRIN2B expression: rs2160517, rs219931, rs11055792, rs17833967 and rs12814951 (all corrected p < 0.05). The score computed to account for their combined effect reliably indexed gene expression. GRIN2B composite score correlated negatively with intelligence quotient, WM behavioural efficiency and dorsolateral prefrontal cortex activity. Moreover, there was a non-linear association between GRIN2B genetic score and prefrontal activity, i.e. both high and low putative genetic score levels were associated with high blood oxygen level-dependent signals in the prefrontal cortex. CONCLUSIONS: Multiple genetic variants in GRIN2B are jointly associated with gene expression, prefrontal function and behaviour during WM. These results support the role of GRIN2B genetic variants in WM prefrontal activity in human adults.

Fourth case of louse-borne relapsing fever in Young Migrant, Sicily, Italy, December 2015. Mini Review Article.

OBJECTIVES: Currently louse-borne relapsing fever (LBRF) is primarily found in limited endemic foci in Ethiopia, Somalia and Sudan; no case of imported LBRF has been reported in Europe in the 9 years prior to 2015. The aim of our paper is to describe a new case of imported LBRF detected in Sicily, Italy, and to review all cases reported in migrants arrived in Europe in the last 10 years. STUDY DESIGN: Mini review of all published cases of louse-borne relapsing fever in Europe in the last 10 years. METHODS: A computerized search without language restriction was conducted using PubMed combining the terms '(louse-borne relapsing fever or LBRF or recurrentis) and (refugee or Europe or migrant)' without limits. Furthermore, the 'Ahead-of-Print Articles' of the top 10 journals (ranked by Impact factor - Web of Science) of Infectious diseases and of Epidemiology were checked. RESULTS: Our search identified 26 cases of LBRF between July and October 2015 in migrants recently arrived in Europe: 8 had been described in Italy; 1 in Switzerland; 2 in the Netherlands; 15 in Germany. We describe data regarding the clinical characteristics, diagnostic methods, therapy and outcome of these patients and of the new case. CONCLUSIONS: LBRF by Borrelia recurrentis should be considered among the clinical hypotheses in migrants presenting with fever, headache, chills, sweating, arthralgia, myalgia, dizziness, nausea and vomiting.

Reversible Temperature-Switching of Hydrogel Stiffness of Coassembled, Silk-Collagen-Like Hydrogels.

Recombinant protein polymers, which can combine different bioinspired self-assembly motifs in a well-defined block sequence, have large potential as building blocks for making complex, hierarchically structured materials. In this paper we demonstrate the stepwise formation of thermosensitive hydrogels by combination of two distinct, orthogonal self-assembly mechanisms. In the first step, fibers are coassembled from two recombinant protein polymers: (a) a symmetric silk-like block copolymer consisting of a central silk-like block flanked by two soluble random coil blocks and (b) an asymmetric silk-collagen-like block copolymer consisting of a central random-coil block flanked on one side by a silk-like block and on the other side a collagen-like block. In the second step, induced by cooling, the collagen-like blocks form triple helices and thereby cross-link the fibers, leading to hydrogels with a thermo-reversibly switchable stiffness. Our work demonstrates how complex self-assembled materials can be formed through careful control of the self-assembly pathway.

Skin Biopsy, the Allergists' Tool: How to Interpret a Report.

Inflammatory dermatoses are frequently encountered by the allergist, and histologic evaluation achieved through skin biopsy can be of tremendous value clinically. There is no substitute for a thorough history and physical exam; however, the skin biopsy is a simple, in-office procedure with little risk of complication that can provide invaluable information when a diagnosis is uncertain. Histopathologically, many inflammatory eruptions can look similar or overlap, but information provided by the dermatopathologist can help the clinician render or refine the clinical diagnosis and guide management. This review will discuss descriptive elements contained in the pathology report to provide a framework that can be used by the allergist to comfortably and confidently diagnose inflammatory dermatologic conditions.

The effect of tooth loss on gait stability of community-dwelling older adults.

OBJECTIVE: To investigate the effects of tooth loss on gait stability in a healthy elderly population. METHODS: A case-control study was conducted among healthy and prosthetically well-restored seniors over the age of 65 years. The test group comprised 24 edentulous participants who were restored with complete dentures in the upper jaw and an overdenture fixed on two implants in the lower jaw. The control group comprised 25 dentate participants who either still had their natural teeth or were restored with conventional fixed partial dentures. Gait stability was evaluated by measuring the parameters 'gait velocity' and 'cycle-time variability' during self-selected normal walking speed and under dual-task performance conditions. Measurements were conducted using the GAITRite(®) electronic walkway system. RESULTS: Dentated and fixed restored participants (the control group) had a significantly higher gait velocity compared with denture wearers (the test group) under both normal walking (p = 0.03) and dual-task performance conditions (p = 0.01). In each test condition, among edentulous participants, gait velocity did not significantly differ according to whether the participant wore their dentures. CONCLUSION: The present results suggest that tooth loss in healthy seniors is associated with lower gait velocity and therefore may have a negative impact on gait stability.

Calcineurin upregulates local Ca(2+) signaling through ryanodine receptor-1 in airway smooth muscle cells.

Local Ca(2+) signals (Ca(2+) sparks) play an important role in multiple cellular functions in airway smooth muscle cells (ASMCs). Protein kinase Cϵ is known to downregulate ASMC Ca(2+) sparks and contraction; however, no complementary phosphatase has been shown to produce opposite effects. Here, we for the first time report that treatment with a specific calcineurin (CaN) autoinhibitory peptide (CAIP) to block CaN activity decreases, whereas application of nickel to activate CaN increases, Ca(2+) sparks in both the presence and absence of extracellular Ca(2+). Treatment with xestospogin-C to eliminate functional inositol 1,4,5-trisphosphate receptors does not prevent CAIP from inhibiting local Ca(2+) signaling. However, high ryanodine treatment almost completely blocks spark formation and prevents the nickel-mediated increase in sparks. Unlike CAIP, the protein phosphatase 2A inhibitor endothall has no effect. Local Ca(2+) signaling is lower in CaN catalytic subunit Aα gene knockout (CaN-Aα(-/-)) mouse ASMCs. The effects of CAIP and nickel are completely lost in CaN-Aα(-/-) ASMCs. Neither CAIP nor nickel produces an effect on Ca(2+) sparks in type 1 ryanodine receptor heterozygous knockout (RyR1(-/+)) mouse ASMCs. However, their effects are not altered in RyR2(-/+) or RyR3(-/-) mouse ASMCs. CaN inhibition decreases methacholine-induced contraction in isolated RyR1(+/+) but not RyR1(-/+) mouse tracheal rings. Supportively, muscarinic contractile responses are also reduced in CaN-Aα(-/+) mouse tracheal rings. Taken together, these results provide novel evidence that CaN regulates ASMC Ca(2+) sparks specifically through RyR1, which plays an important role in the control of Ca(2+) signaling and contraction in ASMCs.

Synthesis of cell-permeable stapled BH3 peptide-based Mcl-1 inhibitors containing simple aryl and vinylaryl cross-linkers.

We report the synthesis of a series of distance-matching aryl and vinylaryl cross-linkers for constructing stapled peptides containing cysteines at i,i+7 positions. Langevin dynamics simulation studies helped to classify these cross-linkers into two categories: the rigid cross-linkers with narrower S-S distance distribution and the flexible cross-linkers with wider S-S distance distribution. The stapled Noxa BH3 peptides with the flexible distance-matching cross-linkers gave the highest degree of helicity as well as the most potent inhibitory activity against Mcl-1. However, the stapled peptides with the highest hydrophobicity showed the most efficient cellular uptake. Together, this work illustrates the divergent nature of binding affinity and cellular uptake, and the vital importance of choosing appropriate cross-linkers in constructing stapled peptides with the drug-like properties.

Instrumental monitoring of the birth and development of truffles in a Tuber melanosporum orchard.

Mycorrhizal symbiotic plants, soil suitability, temperature, and humidity are, by general consensus, considered decisive factors in truffle production. However, experimental approaches to define the environmental conditions that stimulate formation of truffle primordia and promote their growth to maturity have been lacking. By analysis of data of many atmospheric and soil parameters collected since 2009 within a Tuber melanosporum orchard, the trends of metabolic activity, detected as CO2 production in the soil, have been identified as the most reliable parameter to indicate the 'birth' of the truffle primordia. They seem to be produced when mycelial activity is intense and undergoes water stress, after which it resumes. About 6-18 days after recovery of metabolic activity, we could collect primordia of T. melanosporum. Many die or develop too early and consequently rot or are eaten by insect larvae. These events occur several times during summer and autumn, those that 'sprout' in late summer or later grow steadily and reach maturity. Using a particular ground-penetrating radar (GPR) setup to discriminate truffles, we could identify individual truffles in the soil after they have enlarged to at least 6 mm in diameter and follow their growth in volume and diameter over time. These two instrumental methods (CO2 sensor and GPR), although yet to be improved, open new important perspectives to better understand truffle biology and manage truffle orchards to support the newly acquired demonstration of the fundamental role of host plants for the nutrient transfer to the ectomycorrhiza-mycelium-fruiting body complex of T. melanosporum.

Rise-time of FRET-acceptor fluorescence tracks protein folding.

Uniform labeling of proteins with fluorescent donor and acceptor dyes with an equimolar ratio is paramount for accurate determination of Förster resonance energy transfer (FRET) efficiencies. In practice, however, the labeled protein population contains donor-labeled molecules that have no corresponding acceptor. These FRET-inactive donors contaminate the donor fluorescence signal, which leads to underestimation of FRET efficiencies in conventional fluorescence intensity and lifetime-based FRET experiments. Such contamination is avoided if FRET efficiencies are extracted from the rise time of acceptor fluorescence upon donor excitation. The reciprocal value of the rise time of acceptor fluorescence is equal to the decay rate of the FRET-active donor fluorescence. Here, we have determined rise times of sensitized acceptor fluorescence to study the folding of double-labeled apoflavodoxin molecules and show that this approach tracks the characteristics of apoflavodoxin's complex folding pathway.

Evaluating the efficacy of current treatments for reducing postoperative ileus: a randomized clinical trial in a single center.

AIM: Postoperative ileus has been considered an inevitable consequence of abdominal surgery. The aim of the study was to investigate the efficacy of same treatments in resolving postoperative ileus in various surgical approaches. METHODS: A total of 360 patients underwent abdominal surgery, and was divided into four groups: videolaparoscopic cholecystectomy, laparotomic colo-rectal surgery, laparotomic Hartmann procedure, laparotomic gastric surgery. In each group, patients received different postoperative treatments: chewing gum, olive oil, both, and water. Each group was compared with a control group. RESULTS: In patients who underwent videolaparoscopic cholecystectomy, median postoperative first passage of flatus and stool in the water group was 10 and 34 hours, respectively (P=0.006, P=0.021) and significantly earlier than in the control group (median postoperative 24 and 72 hours). Postoperative stay for the water group was lower (median day 1, 3rd interquartile 2.5) compared with control (median day 3; 3rd interquartile 7.0, P=0.01). In patients who underwent gastric surgery, median postoperative first passage of stool in the olive oil and chewing gum group was 48 hours, significantly earlier than in the control (median postoperative hour 120, P=0.04). Median time to first passage of flatus and stool was also earlier in the other groups compared with the control group, though this difference was not significant. CONCLUSION: Chewing gum, olive oil or both do not induce a relevant reduction of ileus after surgery. Water may be a safe and inexpensive option in reducing ileus. (United States National Institutes of Health, www.clinicaltrial.gov, number NCT01869231).