What constitutes a community? A co-occurrence exploration of the Costa Rican avifauna.

The concept of a "community" as a form of organization for natural biological systems is both widespread and widely accepted within the ecological and biological sciences. Communities have been defined as groups of organisms that interact in ways that denote interdependence between individuals and taxa (e.g. as defined by "food webs") but they have also been defined as groups of co-occurring organisms that are assumed to interact by virtue of their shared spatiotemporal existence. The latter definition has been debated and challenged in the literature, with mounting evidence for co-occurrence being more indicative of coincident ecological niches in space and time rather than being evidence of ecological interaction or dependency. Using a dataset of 460 Costa Rican bird species divided into breeding and non-breeding season datasets, we empirically demonstrate the ways in which co-occurrence can create illusory communities based on similar occupied ecological niches and similar patterns of co-occurrence at different times of year. We discuss the importance of discerning coincidental co-occurrence from true ecological interactions that would manifest a true community, and further address the importance of differentiating communities of co-occurrence from communities of demonstrable ecological interaction. While co-occurrence is a necessary aspect of interspecific interactions, we discuss and demonstrate here that such co-occurrence does not make a community, nor should explicit patterns of co-occurrence be seen as evidence for evolutionarily important ecological interactions.

Step-by-Step Regulation of Productive and Abortive Transcription Initiation by Pyrophosphorolysis.

An understanding of the kinetics and mechanism of bacterial transcription initiation is needed to understand regulation of gene expression and advance fields from antibiotic discovery to promoter design. The step-by-step forward kinetics and mechanism of initiation and RNA-DNA hybrid growth, made irreversible by omitting pyrophosphate (PPi) byproduct, were determined recently for E. coli RNA polymerase (RNAP)-λPR promoter complexes. Strong position-dependences of overall rate constants (kcat/Km analogs) for each nucleotide-addition step were observed because of coupling of hybrid growth to disruption of promoter contacts, bubble closing, and RNAP escape. Here we investigate reversal of these steps (pyrophosphorolysis) at PPi concentrations ([PPi]) found in exponentially-growing cells. We quantify [PPi] effects on the amount and rate of synthesis of long (>10-mer, post-escape) and short (stalled, abortive) RNA to determine how PPi regulates initiation. Physiological [PPi] makes uridine incorporation and some other initiation steps significantly reversible. Physiological [PPi] reduces the fraction of RNAP-promoter complexes that productively initiate and the rate of RNA synthesis per productive complex, while increasing the fraction of complexes that abortively initiate, affecting abortive rates, and shifting the abortive-product distribution to shorter RNAs. Pyrophosphorolysis rates for some initiation complexes are orders of magnitude larger than for removal of the same nucleotide from elongation complexes because of the strong bias toward the pre-translocated state in initiation, and exhibit even stronger dependences on nucleotide identity (pyrimidine ≫ purine). Because cytoplasmic [PPi] is much higher in exponential-phase than stationary-phase cells, these [PPi] effects on initiation rates and amounts of RNA synthesis must be physiologically-relevant.

Temperature effects on RNA polymerase initiation kinetics reveal which open complex initiates and that bubble collapse is stepwise.

Transcription initiation is highly regulated by promoter sequence, transcription factors, and ligands. All known transcription inhibitors, an important class of antibiotics, act in initiation. To understand regulation and inhibition, the biophysical mechanisms of formation and stabilization of the "open" promoter complex (OC), of synthesis of a short RNA-DNA hybrid upon nucleotide addition, and of escape of RNA polymerase (RNAP) from the promoter must be understood. We previously found that RNAP forms three different OC with λPR promoter DNA. The 37 °C RNAP-λPR OC (RPO) is very stable. At lower temperatures, RPO is less stable and in equilibrium with an intermediate OC (I3). Here, we report step-by-step rapid quench-flow kinetic data for initiation and growth of the RNA-DNA hybrid at 25 and 37 °C that yield rate constants for each step of productive nucleotide addition. Analyzed together, with previously published data at 19 °C, our results reveal that I3 and not RPO is the productive initiation complex at all temperatures. From the strong variations of rate constants and activation energies and entropies for individual steps of hybrid extension, we deduce that contacts of RNAP with the bubble strands are disrupted stepwise as the hybrid grows and translocates. Stepwise disruption of RNAP-strand contacts is accompanied by stepwise bubble collapse, base stacking, and duplex formation, as the hybrid extends to a 9-mer prior to disruption of upstream DNA-RNAP contacts and escape of RNAP from the promoter.

Mobile monitoring of air and noise pollution in Philadelphia neighborhoods during 2017.

Mobile monitoring is a useful approach for measuring intra-urban variation of air pollution in urban environments. In this study, we used a mobile monitoring approach to study the spatial-temporal variability of air and noise pollution in urban neighborhoods of Philadelphia. During summer 2017, we used portable instruments to measure PM2.5, black carbon (BC), and noise levels along 5 km paths in four residential neighborhoods (Tioga, Mill Creek, Chestnut Hill, and Northern Liberties) and one commercial district (Center City) in Philadelphia, Pennsylvania, USA. A total of 62 sets of measurements were made at three different times of day (during morning rush hour, mid-afternoon, and during afternoon rush hour) from June 5 to July 7, 2017. Spatially, there was a significant difference in PM2.5 concentrations among the four residential neighborhoods. Overall, the Chestnut Hill neighborhood had the highest PM2.5 concentrations (13.25 ±â€¯6.89 µg/m3), followed by Tioga (9.58 ±â€¯4.83 µg/m3), Northern Liberties (7.02 ±â€¯4.17 µg/m3), and Mill Creek (3.9 ±â€¯4.5 µg/m3). There was temporal variability of pollutants depending on the neighborhood; Northern Liberties demonstrated the highest temporal variability in these data. The highest PM2.5 (18.86 ±â€¯3.17 mg/m3) was measured in the Chestnut Hill neighborhood during mid-afternoon. Mean PM2.5, BC, and noise levels based on mobile measurements at Philadelphia during summer 2017 were 8.41 ±â€¯4.31 µg/m3, 0.99 ±â€¯0.44 µg C/m3, and 62.01 ±â€¯3.20 dBA, respectively. Environmental noise showed the highest temporal variation of the monitored components for 3 time periods. In general, tree cover showed a weak and inconclusive association with particulate pollution levels.

RNA Polymerase: Step-by-Step Kinetics and Mechanism of Transcription Initiation.

To determine the step-by-step kinetics and mechanism of transcription initiation and escape by E. coli RNA polymerase from the λPR promoter, we quantify the accumulation and decay of transient short RNA intermediates on the pathway to promoter escape and full-length (FL) RNA synthesis over a wide range of NTP concentrations by rapid-quench mixing and phosphorimager analysis of gel separations. Experiments are performed at 19 °C, where almost all short RNAs detected are intermediates in FL-RNA synthesis by productive complexes or end-products in nonproductive (stalled) initiation complexes and not from abortive initiation. Analysis of productive-initiation kinetic data yields composite second-order rate constants for all steps of NTP binding and hybrid extension up to the escape point (11-mer). The largest of these rate constants is for incorporation of UTP into the dinucleotide pppApU in a step which does not involve DNA opening or translocation. Subsequent steps, each of which begins with reversible translocation and DNA opening, are slower with rate constants that vary more than 10-fold, interpreted as effects of translocation stress on the translocation equilibrium constant. Rate constants for synthesis of 4- and 5-mer, 7-mer to 9-mer, and 11-mer are particularly small, indicating that RNAP-promoter interactions are disrupted in these steps. These reductions in rate constants are consistent with the previously determined ∼9 kcal cost of escape from λPR. Structural modeling and previous results indicate that the three groups of small rate constants correspond to sequential disruption of in-cleft, -10, and -35 interactions. Parallels to escape by T7 RNAP are discussed.

Phenotype of CM-AVM2 caused by variants in EPHB4: how much overlap with hereditary hemorrhagic telangiectasia (HHT)?

PURPOSE: EPHB4 variants were recently reported to cause capillary malformation-arteriovenous malformation 2 (CM-AVM2). CM-AVM2 mimics RASA1-related CM-AVM1 and hereditary hemorrhagic telangiectasia (HHT), as clinical features include capillary malformations (CMs), telangiectasia, and arteriovenous malformations (AVMs). Epistaxis, another clinical feature that overlaps with HHT, was reported in several cases. Based on the clinical overlap of CM-AVM2 and HHT, we hypothesized that patients considered clinically suspicious for HHT with no variant detected in an HHT gene (ENG, ACVRL1, or SMAD4) may have an EPHB4 variant. METHODS: Exome sequencing or a next-generation sequencing panel including EPHB4 was performed on individuals with previously negative molecular genetic testing for the HHT genes and/or RASA1. RESULTS: An EPHB4 variant was identified in ten unrelated cases. Seven cases had a pathogenic EPHB4 variant, including one with mosaicism. Three cases had an EPHB4 variant of uncertain significance. The majority had epistaxis (6/10 cases) and telangiectasia (8/10 cases), as well as CMs. Two of ten cases had a central nervous system AVM. CONCLUSIONS: Our results emphasize the importance of considering CM-AVM2 as part of the clinical differential for HHT and other vascular malformation syndromes. Yet, these cases highlight significant differences in the cutaneous presentations of CM-AVM2 versus HHT.

Additional weekend allied health services reduce length of stay in subacute rehabilitation wards but their effectiveness and cost-effectiveness are unclear in acute general medical and surgical hospital wards: a systematic review.

QUESTION: Are additional weekend allied health services effective and cost-effective for acute general medical and surgical wards, and subacute rehabilitation hospital wards? DESIGN: Systematic review and meta-analysis of studies published between January 2000 and May 2017. Two reviewers independently screened studies for inclusion, extracted data, and assessed methodological quality. Meta-analyses were conducted for relative measures of effect estimates. PARTICIPANTS: Patients admitted to acute general medical and surgical wards, and subacute rehabilitation wards. INTERVENTION: All services delivered by allied health professionals during weekends (Saturday and/or Sunday). This study limited allied health professions to: occupational therapy, physiotherapy, social work, speech pathology, dietetics, art therapy, chiropractic, exercise physiology, music therapy, oral health (not dentistry), osteopathy, podiatry, psychology, and allied health assistants. OUTCOME MEASURES: Hospital length of stay, hospital re-admission, adverse events, discharge destination, functional independence, health-related quality of life, and cost of hospital care. RESULTS: Nineteen articles (20 studies) were identified, comprising 10 randomised and 10 non-randomised trials. Physiotherapy was the most commonly investigated profession. A meta-analysis of randomised, controlled trials showed that providing additional weekend allied health services in subacute rehabilitation wards reduced hospital length of stay by 2.35days (95% CI 0.45 to 4.24, I2=0%), and may be a cost-effective way to improve function (SMD 0.09, 95% CI -0.01 to 0.19, I2=0%), and health-related quality of life (SMD 0.10, 95% CI -0.01 to 0.20, I2=0%). For acute general medical and surgical hospital wards, it was unclear whether the weekend allied health service model provided in the two identified randomised trials led to significant changes in measured outcomes. CONCLUSION: The benefit of providing additional allied health services is clearer in subacute rehabilitation settings than for acute general medical and surgical wards in hospitals. REGISTRATION: PROSPERO CRD76771. [Sarkies MN, White J, Henderson K, Haas R, Bowles J, Evidence Translation in Allied Health (EviTAH) Group (2018) Additional weekend allied health services reduce length of stay in subacute rehabilitation wards but their effectiveness and cost-effectiveness are unclear in acute general medical and surgical hospital wards: a systematic review. Journal of Physiotherapy 64: 142-158].

The Irving-Williams series and the 2-His-1-carboxylate facial triad: a thermodynamic study of Mn2+, Fe2+, and Co2+ binding to taurine/α-ketoglutarate dioxygenase (TauD).

Taurine/α-ketoglutarate (αKG) dioxygenase (TauD) is an E. coli nonheme Fe2+- and αKG-dependent metalloenzyme that catalyzes the hydroxylation of taurine, leading to the production of sulfite. The metal-dependent active site in TauD is formed by two histidine and one aspartate that coordinating to one face of an octahedral coordination geometry, known as the 2-His-1-carboxylate facial triad. This motif is found in many nonheme Fe2+ proteins, but there is limited information on the thermodynamic parameters that govern metal-ion binding to this site. Here, we report data from calorimetry and related biophysical techniques to generate complete thermodynamic profiles of Mn2+ and Co2+ binding to TauD, and these values are compared to the Fe2+ data reported earlier Henderson et al. (Inorg Chem 54: 2278-2283, 2015). The buffer-independent binding constants (K) were measured to be 1.6 × 106, 2.4 × 107, and 1.7 × 109, for Mn2+, Fe2+, and Co2+, respectively. The corresponding ΔG° values were calculated to be - 8.4, - 10.1, and - 12.5 kcal/mol, respectively. The metal-binding enthalpy changes (ΔH) for these binding events are - 11.1 (± 0.1), - 12.2 (± 0.1), and - 16.0 (± 0.6) kcal/mol, respectively. These data are fully consistent with the Irving-Williams series, which show an increasing affinity for transition metal ions across the periodic table. It appears that the periodic increase in affinity, however, is a result of a complicated summation of enthalpy terms (including favorable metal-ion coordination processes and unfavorable ionization events) and related entropy terms.

Reducing challenging behaviour of adults with intellectual disabilities in supported accommodation: A cluster randomized controlled trial of setting-wide positive behaviour support.

BACKGROUND: Improving the quality of social care through the implementation of setting-wide positive behaviour support (SWPBS) may reduce and prevent challenging behaviour. METHOD: Twenty-four supported accommodation settings were randomized to experimental or control conditions. Settings in both groups had access to individualized PBS either via the organisation's Behaviour Support Team or from external professionals. Additionally, within the experimental group, social care practice was reviewed and improvement programmes set going. Progress was supported through coaching managers and staff to enhance their performance and draw more effectively on existing resources, and through monthly monitoring over 8-11 months. Quality of support, quality of life and challenging behaviour were measured at baseline and after intervention with challenging behaviour being additionally measured at long-term follow-up 12-18 months later. RESULTS: Following intervention there were significant changes to social care practice and quality of support in the experimental group. Ratings of challenging behaviour declined significantly more in the experimental group and the difference between groups was maintained at follow-up. There was no significant difference between the groups in measurement of quality of life. Staff, family members and professionals evaluated the intervention and its outcomes positively. CONCLUSIONS: Some challenging behaviour in social care settings may be prevented by SWPBS that improves the quality of support provided to individuals.

Active physiotherapy interventions following total knee arthroplasty in the hospital and inpatient rehabilitation settings: a systematic review and meta-analysis.

BACKGROUND: Physiotherapy is a routine component of postoperative management following total knee arthroplasty (TKA). As the demand for surgery increases it is vital that postoperative physiotherapy interventions are effective and efficient. OBJECTIVES: Determine the most beneficial active physiotherapy interventions in acute hospital and inpatient rehabilitation for improving pain, activity, range of motion and reducing length of stay for adults who have undergone TKA. DATA SOURCES: Electronic databases MEDLINE, CINAHL, PUBMED and EMBASE. STUDY ELIGIBILITY CRITERIA: Randomised controlled trials investigating the effect of active physiotherapy interventions in the acute hospital or inpatient rehabilitation setting for adults who have undergone TKA. STUDY APPRAISAL AND SYNTHESIS METHODS: Risk of bias for individual studies was assessed using the Physiotherapy Evidence Database (PEDro) scale. Standardised Mean Differences (SMD) or Mean Differences (MD) and 95% confidence intervals were calculated and combined in meta-analyses. Quality of meta-analyses was assessed using the Grades of Research, Assessment, Development and Evaluation approach. RESULTS: Accelerated physiotherapy regimens were effective for reducing acute hospital length of stay (MD -3.50 days, 95% CI -5.70 to -1.30). Technology-assisted physiotherapy did not show any difference for activity (SMD -0.34, 95% CI -0.82 to 0.13). From high quality individual studies pain, activity and range of motion improved with accelerated physiotherapy regimens and activity improved with hydrotherapy. LIMITATIONS: Lack of blinding and small sample sizes across the included trials. CONCLUSION: After TKA, there is low level evidence that accelerated physiotherapy regimens can reduce acute hospital length of stay. Systematic review registration number PROSPERO (Registration number CRD42014013414) http://www.crd.york.ac.uk/PROSPERO.

Cortical Thickness in Adolescents with a Family History of Alcohol Use Disorder.

BACKGROUND: Individuals with a family history (FH+) of alcohol use disorder (AUD) have a higher risk for developing an AUD than those with no family history (FH-) of AUD. In addition, FH+ individuals tend to perform worse on neuropsychological measures and show heightened impulsivity, which may be due to underlying differences in brain structure such as cortical thickness. The primary aim of this study was to investigate differences in cortical thickness in FH+ compared to FH- adolescents. Secondary aims were to (i) investigate differences in executive functioning and impulsivity, and (ii) examine associations between brain structure and behavior. METHODS: Brain scans of 95 FH- and 93 FH+ subjects aged 13 to 18 were obtained using magnetic resonance imaging. FH+ subjects were required to have at least 1 biological parent with a history of an AUD. FH+ and FH- individuals had limited or no past alcohol use, thereby minimizing potential effects of alcohol. Subjects were evaluated on impulsivity and executive functioning tasks. Thicknesses of cortical lobes and subregions were analyzed using FreeSurfer. Regions showing group differences were examined for group-by-age interactions and correlations with neuropsychological and personality measures. RESULTS: FH+ adolescents had thinner cortices in frontal and parietal lobes, notably in the medial orbitofrontal, lateral orbitofrontal, and superior parietal cortices. The difference in cortical thickness between family history groups was strongest among the youngest subjects. FH+ subjects were also more impulsive and had poorer performance on a spatial memory task. CONCLUSIONS: These findings demonstrate frontal and parietal structural differences in FH+ adolescents that might underlie cognitive and behavioral characteristics associated with AUD risk.

Mechanism of transcription initiation and promoter escape by E. coli RNA polymerase.

To investigate roles of the discriminator and open complex (OC) lifetime in transcription initiation by Escherichia coli RNA polymerase (RNAP; α2ßß'ωσ70), we compare productive and abortive initiation rates, short RNA distributions, and OC lifetime for the λPR and T7A1 promoters and variants with exchanged discriminators, all with the same transcribed region. The discriminator determines the OC lifetime of these promoters. Permanganate reactivity of thymines reveals that strand backbones in open regions of long-lived λPR-discriminator OCs are much more tightly held than for shorter-lived T7A1-discriminator OCs. Initiation from these OCs exhibits two kinetic phases and at least two subpopulations of ternary complexes. Long RNA synthesis (constrained to be single round) occurs only in the initial phase (<10 s), at similar rates for all promoters. Less than half of OCs synthesize a full-length RNA; the majority stall after synthesizing a short RNA. Most abortive cycling occurs in the slower phase (>10 s), when stalled complexes release their short RNA and make another without escaping. In both kinetic phases, significant amounts of 8-nt and 10-nt transcripts are produced by longer-lived, λPR-discriminator OCs, whereas no RNA longer than 7 nt is produced by shorter-lived T7A1-discriminator OCs. These observations and the lack of abortive RNA in initiation from short-lived ribosomal promoter OCs are well described by a quantitative model in which ∼1.0 kcal/mol of scrunching free energy is generated per translocation step of RNA synthesis to overcome OC stability and drive escape. The different length-distributions of abortive RNAs released from OCs with different lifetimes likely play regulatory roles.

Global stability of an α-ketoglutarate-dependent dioxygenase (TauD) and its related complexes.

BACKGROUND: TauD is a nonheme iron(II) and α-ketoglutarate (αKG) dependent dioxygenase, and a member of a broader family of enzymes that oxidatively decarboxylate αKG to succinate and carbon dioxide thereby activating O2 to perform a range of oxidation reactions. However before O2 activation can occur, these enzymes bind both substrate and cofactor in an effective manner. Here the thermodynamics associated with substrate and cofactor binding to FeTauD are explored. METHODS: Thermal denaturation of TauD and its enzyme-taurine, enzyme-αKG, and enzyme-taurine-αKG complexes are explored using circular dichroism (CD) spectroscopy and differential scanning calorimetry (DSC). RESULTS: Taurine binding is endothermic (+26kcal/mol) and entropically driven that includes burial of hydrophobic surfaces to close the lid domain. Binding of αKG is enthalpically favorable and shows cooperativity with taurine binding, where the change in enthalpy associated with αKG binding (δΔHcal) increases from -30.1kcal/mol when binding to FeTauD to -65.2kcal/mol when binding to the FeTauD-taurine complex. CONCLUSIONS: The intermolecular interactions that govern taurine and αKG binding impact the global stability of TauD and its complexes, with clear and dramatic cooperativity between substrate and cofactor. GENERAL SIGNIFICANCE: Thermal denaturation of TauD and its enzyme-taurine, enzyme-αKG, and enzyme-taurine-αKG complexes each exhibited increased temperature stability over the free enzyme. Through deconvolution of the energetic profiles for all species studied, a thermodynamic cycle was generated that shows significant cooperativity between substrate and cofactor binding which continues to clarity the events leading up O2 activation.

ITC Methods for Assessing Buffer/Protein Interactions from the Perturbation of Steady-State Kinetics: A Reactivity Study of Homoprotocatechuate 2,3-Dioxygenase.

Isothermal titration calorimetry (ITC) can be used to study the thermodynamics of enzyme substrate binding or the kinetics of substrate turnover (or both). Substrate-binding interactions are observed in a typical ITC titration experiment in which the heat change for the addition of an aliquot of substrate to a solution containing the enzyme is determined for a number of titrant (i.e., substrate) injections and the data fit for the thermodynamic parameters (ΔG, ΔH, and -TΔS) for substrate binding. Of course, these measurements must be made under conditions where the substrate binds but does not turnover. In the ITC "kinetics" experiment, the power change observed after injection of an excess of substrate into a solution of the enzyme is a direct measure of the rate at which substrate is converted to product, and the ITC data can be analyzed for the kinetic parameters (Vmax, kcat, KM, and kcat/KM). The ITC technique is particularly versatile in that it can be applied to systems where there might not be a change in a spectroscopic signal for either substrate binding or the reaction of the substrate to form product. A complication is that if there are competing reactions, for example, buffer protonation, or product binding, to name just two, the enthalpy change measured for either substrate binding or for substrate turnover will be a summation of all of the reaction heats. Enzyme studies are typically done in buffered solutions at constant pH. The general, and often incorrect, assumption is that the buffer components are simply spectators and not participants in either substrate binding or substrate turnover. This chapter describes how we have used ITC measurements to identify problem buffers that impact the kinetics for an enzyme catalyzed reaction. Herein, we show the effects of several buffers on the steady-state kinetics for the conversion of the substrate, 3,4-dihydroxyphenyl acetate (homoprotocatechuate), to the ring-opened product, 5-carboxymethyl-2-hydroxymuconic semialdehyde by the nonheme iron(II) metalloenzyme, homoprotocatechuate 2,3-dioxygenase. Several buffers were observed to engage in buffer/enzyme interactions within the active site pocket. These enzyme-buffer interactions were shown to inhibit substrate turnover and to contribute additional enthalpy terms to the overall heat of reaction observed for substrate turnover (and for substrate binding).

Calorimetric and spectroscopic investigations of the binding of metallated porphyrins to G-quadruplex DNA.

BACKGROUND: The human telomere contains tandem repeat of (TTAGG) capable of forming a higher order DNA structure known as G-quadruplex. Porphyrin molecules such as TMPyP4 bind and stabilize G-quadruplex structure. METHODS: Isothermal titration calorimetry (ITC), circular dichroism (CD), and mass spectroscopy (ESI/MS), were used to investigate the interactions between TMPyP4 and the Co(III), Ni(II), Cu(II), and Zn(II) complexes of TMPyP4 (e.g. Co(III)-TMPyP4) and a model human telomere G-quadruplex (hTel22) at or near physiologic ionic strength ([Na(+)] or [K(+)]≈0.15M). RESULTS: The apo-TMPyP4, Ni(II)-TMPyP4, and Cu(II)-TMPyP4 all formed complexes having a saturation stoichiometry of 4:1, moles of ligand per mole of DNA. Binding of apo-TMPyP4, Ni(II)-TMPyP4, and Cu(II)-TMPyP4 is described by a "four-independent-sites model". The two highest-affinity sites exhibit a K in the range of 10(8) to 10(10)M(-1) with the two lower-affinity sites exhibiting a K in the range of 10(4) to 10(5)M(-1). Binding of Co(III)-TMPyP4, and Zn(II)-TMPyP4, is best described by a "two-independent-sites model" in which only the end-stacking binding mode is observed with a K in the range of 10(4) to 10(5)M(-1). CONCLUSIONS: In the case of apo-TMPyP4, Ni(II)-TMPyP4, and Cu(II)-TMPyP4, the thermodynamic signatures for the two binding modes are consistent with an "end stacking" mechanism for the higher affinity binding mode and an "intercalation" mechanism for the lower affinity binding mode. In the case of Co(III)-TMPyP4 and Zn(II)-TMPyP4, both the lower affinity for the "end-stacking" mode and the loss of the intercalative mode for forming the 2:1 complexes with hTel22 are attributed to the preferred metal coordination geometry and the presence of axial ligands. GENERAL SIGNIFICANCE: The preferred coordination geometry around the metal center strongly influences the energetics of the interactions between the metallated-TMPyP4 and the model human telomeric G-quadruplex.

Thermodynamics of substrate binding to the metal site in homoprotocatechuate 2,3-dioxygenase: Using ITC under anaerobic conditions to study enzyme-substrate interactions.

BACKGROUND: Extradiol dioxygenases are a family of nonheme iron (and sometimes manganese) enzymes that catalyze an O2-dependent ring-opening reaction in a biodegradation pathway of aromatic compounds. Here we characterize the thermodynamics of two substrates binding in homoprotocatechuate 2,3-dioxygenase (HPCD) prior to the O2 activation step. METHODS: This study uses microcalorimetry under an inert atmosphere to measure thermodynamic parameters associated with catechol binding to nonheme metal centers in HPCD. Several stopped-flow rapid mixing experiments were used to support the calorimetry experiments. RESULTS: The equilibria constant for 4-nitrocatechol and homoprotocatechuate binding to the iron(II) and manganese(II) forms of HPCD range from 2×10(4) to 1×10(6), suggesting there are distinctive differences in how the enzyme-substrate complexes are stabilized. Further experiments in multiple buffers allowed us to correct the experimental ΔH for substrate ionization and to fully derive the pH and buffer independent thermodynamic parameters for substrate binding to HPCD. Fewer protons are released from the iron(II) dependent processes than their manganese(II) counterparts. CONCLUSIONS: Condition independent thermodynamic parameters for 4-nitrocatechol and homoprotocatechuate binding to HPCD are highly consistent with each other, suggesting these enzyme-substrate complexes are more similar than once thought, and the ionization state of metal coordinated waters may be playing a role in tuning redox potential and in governing reactivity. GENERAL SIGNIFICANCE: Substrate binding to HPCD is a complex set of equilibria that includes ionization of substrate and water release, yet it is also the key step in O2 activation.

Arachidonoyl-ethanolamide activates endoplasmic reticulum stress-apoptosis in tumorigenic keratinocytes: Role of cyclooxygenase-2 and novel J-series prostamides.

Non-melanoma skin cancer and other epithelial tumors overexpress cyclooxygenase-2 (COX-2), differentiating them from normal cells. COX-2 metabolizes arachidonic acid to prostaglandins including, the J-series prostaglandins, which induce apoptosis by mechanisms including endoplasmic reticulum (ER) stress. Arachidonoyl-ethanolamide (AEA) is a cannabinoid that causes apoptosis in diverse tumor types. Previous studies from our group demonstrated that AEA was metabolized by COX-2 to J-series prostaglandins. Thus, the current study examines the role of COX-2, J-series prostaglandins, and ER stress in AEA-induced apoptosis. In tumorigenic keratinocytes that overexpress COX-2, AEA activated the PKR-like ER kinase (PERK), inositol requiring kinase-1 (IRE1), and activating transcription factor-6 (ATF6) ER stress pathways and the ER stress apoptosis-associated proteins, C/EBP homologous protein-10 (CHOP10), caspase-12, and caspase-3. Using an ER stress inhibitor, it was determined that ER stress was required for AEA-induced apoptosis. To evaluate the role of COX-2 in ER stress-apoptosis, HaCaT keratinocytes with low endogenous COX-2 expression were transfected with COX-2 cDNA or an empty vector and AEA-induced ER stress-apoptosis occurred only in the presence of COX-2. Moreover, LC-MS analysis showed that the novel prostaglandins, 15-deoxyΔ(12,14) PGJ2 -EA and Δ(12) PGJ2 /PGJ2-EA, were synthesized from AEA. These findings suggest that AEA will be selectively toxic in tumor cells that overexpress COX-2 due to the metabolism of AEA by COX-2 to J-series prostaglandin-ethanolamides (prostamides). Hence, AEA may be an ideal topical agent for the elimination of malignancies that overexpress COX-2.

Calorimetric assessment of Fe(2+) binding to α-ketoglutarate/taurine dioxygenase: ironing out the energetics of metal coordination by the 2-His-1-carboxylate facial triad.

The thermodynamic properties of Fe(2+) binding to the 2-His-1-carboxylate facial triad in α-ketoglutarate/taurine dioxygenase (TauD) were explored using isothermal titration calorimetry. Direct titrations of Fe(2+) into TauD and chelation experiments involving the titration of ethylenediaminetetraacetic acid into Fe(2+)-TauD were performed under an anaerobic environment to yield a binding equilibrium of 2.4 (±0.1) × 10(7) (Kd = 43 nM) and a ΔG° value of -10.1 (±0.03) kcal/mol. Further analysis of the enthalpy/entropy contributions indicates a highly enthalpic binding event, where ΔH = -11.6 (±0.3) kcal/mol. Investigations into the unfavorable entropy term led to the observation of water molecules becoming organized within the Fe(2+)-TauD structure.

Productivity of Premodern Agriculture in the Cucuteni-Trypillia Area.

We present paleoeconomy reconstructions for premodern agriculture, selecting, wherever required, features and parameter values specific for the Cucuteni-Trypillia cultural unity (CTU; 5,400-2,700 BC, mostly the territory of modern Ukraine, Moldova, and Romania). We verify the self-consistency and viability of the archaeological evidence related to all major elements of the agricultural production cycle within the constraints provided by environmental and technological considerations. The starting point of our analysis is the paleodiet structure suggested by archaeological data, stable isotope analyses of human remains, and palynology studies in the CTU area. We allow for the archeologically attested contributions of domesticated and wild animal products to the diet, develop plausible estimates of the yield of ancient cereal varieties cultivated with ancient techniques, and quantify the yield dependence on the time after initial planting and on rainfall (as a climate proxy). Our conclusions involve analysis of the labor costs of various seasonal parts of the agricultural cycle of both an individual and a family with a majority of members that do not engage in productive activities that require physical fitness, such as tillage. Finally, we put our results into the context of the exploitation territory and catchment analysis, to project various subsistence strategies into the exploitation territory of a farming settlement. The simplest economic complex based on cereals and domestic and wild animal products, with fallow cropping, appears to be capable of supporting an isolated, relatively small farming settlement of 50-300 people (2-10 ha in area) even without recourse to technological improvements such as the use of manure fertilizer. Our results strongly suggest that dairy products played a significant role in the dietary and labor balance. The smaller settlements are typical of the earliest Trypillia A stage but remain predominant at the later stages. A larger settlement of several hundred people could function in isolation, perhaps with a larger fraction of cereals in the diet, only with technological innovations, such as manure fertilizer and, most important, ard tillage. The ard radically relieves the extreme time pressure associated with soil preparation for sowing. It appears that very large settlements of a few hundred hectares in area, found in the CTU region, could function only if supported by satellite farming villages and stable exchange networks. In turn, this implies social division of labor and occupation, sufficiently complex social relations, stable exchange channels, and so on: altogether, a proto-urban character of such settlements. A model is proposed for the lifetime of a farming settlement, assuming that it is limited by the soil fertility (the depleted resources model), that provides a lifetime estimate consistent with the archaeological evidence available (100-150 years). The model shows that the lifetime strongly depends on the fraction of the arable land area kept fallow. We also discuss, quantify, and assess some strategies to mitigate the risks of arable agriculture associated with strong temporal fluctuations in the cereal yield, such as manure fertilization, and increased fraction of cereals in the diet combined with producing grain surplus for emergency storage.

Biased T cell receptor usage directed against human leukocyte antigen DQ8-restricted gliadin peptides is associated with celiac disease.

Celiac disease is a human leukocyte antigen (HLA)-DQ2- and/or DQ8-associated T cell-mediated disorder that is induced by dietary gluten. Although it is established how gluten peptides bind HLA-DQ8 and HLA-DQ2, it is unclear how such peptide-HLA complexes are engaged by the T cell receptor (TCR), a recognition event that triggers disease pathology. We show that biased TCR usage (TRBV9(∗)01) underpins the recognition of HLA-DQ8-α-I-gliadin. The structure of a prototypical TRBV9(∗)01-TCR-HLA-DQ8-α-I-gliadin complex shows that the TCR docks centrally above HLA-DQ8-α-I-gliadin, in which all complementarity-determining region-ß (CDRß) loops interact with the gliadin peptide. Mutagenesis at the TRBV9(∗)01-TCR-HLA-DQ8-α-I-gliadin interface provides an energetic basis for the Vß bias. Moreover, CDR3 diversity accounts for TRBV9(∗)01(+) TCRs exhibiting differing reactivities toward the gliadin epitopes at various deamidation states. Accordingly, biased TCR usage is an important factor in the pathogenesis of DQ8-mediated celiac disease.