Tomographic imaging of carbon dioxide in the exhaust plume of large commercial aero-engines.

We report here the first implementation of chemically specific imaging in the exhaust plume of a gas turbine typical of those used for propulsion in commercial aircraft. The method used is chemical species tomography (CST) and the target species is CO2, absorbing in the near-infrared at 1999.4 nm. A total of 126 beams propagate transverse to the plume axis, along 7 m paths in a coplanar geometry, to probe a central region of diameter ≈1.5m. The CO2 absorption spectrum is measured using tunable diode laser spectroscopy with wavelength modulation, using the second harmonic to first harmonic (2f/1f) ratio method. The engine is operated over the full range of thrust, while data are recorded in a quasi-simultaneous mode at frame rates of 1.25 and 0.3125 Hz. Various data inversion methodologies are considered and presented for image reconstruction. At all thrust levels a persistent ring structure of high CO2 concentration is observed in the central region of the measurement plane, with a raised region in the middle of the plume assumed to be due to the engine's boat tail. With its potential to target various exhaust species, the CST method outlined here offers a new approach to turbine combustion research, turbine engine development, and aviation fuel research and development.

Distinctive phosphoinositide- and Ca2+-binding properties of normal and cognitive performance-linked variant forms of KIBRA C2 domain.

Kidney- and brain-expressed protein (KIBRA), a multifunctional scaffold protein with around 20 known binding partners, is involved in memory and cognition, organ size control via the Hippo pathway, cell polarity, and membrane trafficking. KIBRA includes tandem N-terminal WW domains, a C2 domain, and motifs for binding atypical PKC and PDZ domains. A naturally occurring human KIBRA variant involving residue changes at positions 734 (Met-to-Ile) and 735 (Ser-to-Ala) within the C2 domain affects cognitive performance. We have elucidated 3D structures and calcium- and phosphoinositide-binding properties of human KIBRA C2 domain. Both WT and variant C2 adopt a canonical type I topology C2 domain fold. Neither Ca2+ nor any other metal ion was bound to WT or variant KIBRA C2 in crystal structures, and Ca2+ titration produced no significant reproducible changes in NMR spectra. NMR and X-ray diffraction data indicate that KIBRA C2 binds phosphoinositides via an atypical site involving ß-strands 5, 2, 1, and 8. Molecular dynamics simulations indicate that KIBRA C2 interacts with membranes via primary and secondary sites on the same domain face as the experimentally identified phosphoinositide-binding site. Our results indicate that KIBRA C2 domain association with membranes is calcium-independent and involves distinctive C2 domain-membrane relative orientations.

An Inactivation Switch Enables Rhythms in a Neurospora Clock Model.

Autonomous endogenous time-keeping is ubiquitous across many living organisms, known as the circadian clock when it has a period of about 24 h. Interestingly, the fundamental design principle with a network of interconnected negative and positive feedback loops is conserved through evolution, although the molecular components differ. Filamentous fungus Neurospora crassa is a well-established chrono-genetics model organism to investigate the underlying mechanisms. The core negative feedback loop of the clock of Neurospora is composed of the transcription activator White Collar Complex (WCC) (heterodimer of WC1 and WC2) and the inhibitory element called FFC complex, which is made of FRQ (Frequency protein), FRH (Frequency interacting RNA Helicase) and CK1a (Casein kinase 1a). While exploring their temporal dynamics, we investigate how limit cycle oscillations arise and how molecular switches support self-sustained rhythms. We develop a mathematical model of 10 variables with 26 parameters to understand the interactions and feedback among WC1 and FFC elements in nuclear and cytoplasmic compartments. We performed control and bifurcation analysis to show that our novel model produces robust oscillations with a wild-type period of 22.5 h. Our model reveals a switch between WC1-induced transcription and FFC-assisted inactivation of WC1. Using the new model, we also study the possible mechanisms of glucose compensation. A fairly simple model with just three nonlinearities helps to elucidate clock dynamics, revealing a mechanism of rhythms' production. The model can further be utilized to study entrainment and temperature compensation.

Extracellular Fibrinogen-binding Protein (Efb) from Staphylococcus aureus Inhibits the Formation of Platelet-Leukocyte Complexes.

Extracellular fibrinogen-binding protein (Efb) from Staphylococcus aureus inhibits platelet activation, although its mechanism of action has not been established. In this study, we discovered that the N-terminal region of Efb (Efb-N) promotes platelet binding of fibrinogen and that Efb-N binding to platelets proceeds via two independent mechanisms: fibrinogen-mediated and fibrinogen-independent. By proteomic analysis of Efb-interacting proteins within platelets and confirmation by pulldown assays followed by immunoblotting, we identified P-selectin and multimerin-1 as novel Efb interaction partners. The interaction of both P-selectin and multimerin-1 with Efb is independent of fibrinogen. We focused on Efb interaction with P-selectin. Excess of P-selectin extracellular domain significantly impaired Efb binding by activated platelets, suggesting that P-selectin is the main receptor for Efb on the surface of activated platelets. Efb-N interaction with P-selectin inhibited P-selectin binding to its physiological ligand, P-selectin glycoprotein ligand-1 (PSGL-1), both in cell lysates and in cell-free assays. Because of the importance of P-selectin-PSGL-1 binding in the interaction between platelets and leukocytes, we tested human whole blood and found that Efb abolishes the formation of platelet-monocyte and platelet-granulocyte complexes. In summary, we present evidence that in addition to its documented antithrombotic activity, Efb can play an immunoregulatory role via inhibition of P-selectin-PSGL-1-dependent formation of platelet-leukocyte complexes.

Absolute noninvasive measurement of CO2 mole fraction emitted by E. coli and S. aureus using calibration-free 2f WMS applied to a 2004 nm VCSEL.

We report the first demonstration, to the best of our knowledge, of accurate real-time noninvasive measurement of the absolute cumulative mole fraction of metabolic carbon dioxide emitted by Escherichia coli and Staphylococcus aureus over a period of several hours of their life cycles using a recently developed calibration-free wavelength modulation spectroscopy technique. A 1 mW vertical-cavity surface-emitting laser is used to interrogate a single rotational vibrational absorption line of carbon dioxide at 2003.5 nm. The measurements are immune to laser intensity fluctuations and variable optical coupling that is inevitable in such free-space coupled experiments that run over 10-18 h. The cumulative carbon dioxide mole fraction follows the characteristic modified Gompertz model that is typical of bacterial growth in batch cultures. The characteristic growth parameters are extracted from this curve. The technique can be readily extended to study multiple volatile organic compounds that bacteria are known to emit.

Measurement of atmospheric carbon dioxide and water vapor in built-up urban areas in the Gandhinagar-Ahmedabad region in India using a portable tunable diode laser spectroscopy system.

This paper reports open-path in situ measurements of atmospheric carbon dioxide at Gandhinagar (23.2156°N, 72.6369°E) and Ahmedabad (23.0225°N, 72.5714°E) in the heavily industrialized state of Gujarat in western India. Calibration-free second harmonic wavelength modulation spectroscopy (2f WMS) is used to carry out accurate and fully automated measurements. The mean values of the mole fraction of carbon dioxide at four locations were 438 ppm, 495 ppm, 550 ppm, and 740 ppm, respectively. These values are much higher than the current global average of 406.67 ppm. A 1 mW, 2004-nm vertical cavity surface-emitting laser is used to selectively interrogate the R16 transition of carbon dioxide at 2003.5 nm (4991.2585 cm-1). The 2f WMS signal corresponding to the gas absorption line shape is simulated using spectroscopic parameters available in the HITRAN database and relevant laser parameters that are extracted in situ from non-absorbing spectral wings of the harmonic signals. The mole fraction of carbon dioxide is extracted in real-time by a MATLAB program from least-squares fit of the simulated 2f WMS signal to the corresponding experimentally obtained signal. A 10-mW, 1392.54-nm distributed feedback laser is used at two of the locations to carry out water vapor measurements using direct absorption spectroscopy. This is the first instance of a portable tunable diode laser spectroscopy system being deployed in an urban location in India to measure atmospheric carbon dioxide and water vapor under varying traffic conditions. The measurements clearly demonstrate the need to adopt tunable diode laser spectroscopy for precise long-term monitoring of greenhouse gases in the Indian subcontinent.

Calibration-free 2f WMS with in situ real-time laser characterization and 2f RAM nulling.

This Letter demonstrates a new calibration-free 2f wavelength modulation spectroscopy (WMS) technique to measure gas concentration and pressure without the need for laser precharacterization. A 1650-nm laser diode is used for methane concentration and pressure measurements for pressures up to 4 bar and for a modulation index (m) of 2.2. All laser parameters such as the intensity, linear and nonlinear intensity modulation (IM), frequency modulation (FM) characteristics, the phase difference ψ1 between the FM and the linear IM, and the phase difference ψ2 between the FM and the nonlinear IM are accurately estimated in situ and in real time. This technique accounts for variations in these parameters that arise due to scanning of the laser's center wavelength, laser temperature variations, and aging of the laser. The laser is modulated at its phase quadrature frequency at which the linear IM and the FM are orthogonal to each other (ψ1=90°). This ensures that the two linear IM-dependent distorting Fourier components are orthogonal to the detection axis, and the undistorted 2f signal is recovered. This simplifies the simulation and gas parameter-extraction process. Finally, 2f RAM nulling is implemented to remove the significant absorption-independent 2f residual amplitude-modulation (RAM) signal that is seen to cause significant distortion of the 2f signal and detector saturation.

Post-translational modification in the archaea: structural characterization of multi-enzyme complex lipoylation.

Lipoylation, the covalent attachment of lipoic acid to 2-oxoacid dehydrogenase multi-enzyme complexes, is essential for metabolism in aerobic bacteria and eukarya. In Escherichia coli, lipoylation is catalysed by LplA (lipoate protein ligase) or by LipA (lipoic acid synthetase) and LipB [lipoyl(octanoyl) transferase] combined. Whereas bacterial and eukaryotic LplAs comprise a single two-domain protein, archaeal LplA function typically involves two proteins, LplA-N and LplA-C. In the thermophilic archaeon Thermoplasma acidophilum, LplA-N and LplA-C are encoded by overlapping genes in inverted orientation (lpla-c is upstream of lpla-n). The T. acidophilum LplA-N structure is known, but the LplA-C structure is unknown and LplA-C's role in lipoylation is unclear. In the present study, we have determined the structures of the substrate-free LplA-N-LplA-C complex and E2lipD (dihydrolipoyl acyltransferase lipoyl domain) that is lipoylated by LplA-N-LplA-C, and carried out biochemical analyses of this archaeal lipoylation system. Our data reveal the following: (i) LplA-C is disordered but folds upon association with LplA-N; (ii) LplA-C induces a conformational change in LplA-N involving substantial shortening of a loop that could repress catalytic activity of isolated LplA-N; (iii) the adenylate-binding region of LplA-N-LplA-C includes two helices rather than the purely loop structure of varying order observed in other LplA structures; (iv) LplAN-LplA-C and E2lipD do not interact in the absence of substrate; (v) LplA-N-LplA-C undergoes a conformational change (the details of which are currently undetermined) during lipoylation; and (vi) LplA-N-LplA-C can utilize octanoic acid as well as lipoic acid as substrate. The elucidated functional inter-dependence of LplA-N and LplA-C is consistent with their evolutionary co-retention in archaeal genomes.

Non-chemical weed management: Harnessing flame weeding for effective weed control.

The goal of the current study was to create and assess the effectiveness of a hand-pulled ergonomically designed flame weeder. The developed weeder was tested in the field at three operating pressures (20, 30 and 40 Psi) and forward speeds (1.00, 1.25 and 1.50 km/h) to study their effects on plant damage, survival rates, weight preservation rates, weed management effectiveness, soil temperatures, and gas and energy consumption. Thereafter, at optimized values of forward speed and operating pressure, a comparative assessment of flame weeding with traditional methods (mechanical and manual weeding) was done in terms of weed control effectiveness, operational time, energy consumption, and cost of operation. Results showed that the optimal performance of the designed flame weeder was achieved when operated at a speed of 1 km/h and an operating pressure of 40 psi. The survival rate, weight preservation rate, weed control efficiency, change in soil temperature, recovery rate, plant damage, gas consumption, and energy consumption were observed to be 27.3 %, 32.5 %, 91.1 %, 40.74 °C, 8.5 %, 2.2 %, 4.05 kg/h, and 2500.24 MJ/ha, respectively, at optimized values of forward speed (1.00 km/h) and operating pressure (40 Psi). The actual field capacity, field efficiency and operating cost of the flame weeder were 0.0755 ha/h, 94.94 %, and 3620.81 ₹/ha, respectively. Hand weeding had the best level of weed control effectiveness, but it was a laborious, time-consuming process. When compared to manual weeding, flame weeding was 50.42 % cheaper and 94.82 % faster.

Preliminary study on cytotoxicity of selegiline on different cancer cell lines: exploration of the induction of ROS-independent apoptosis in breast cancer cells.

The concept of drug repurposing is now widely utilized by biomedical scientists for drug discovery. An example of this is the use of selegiline (SEL), a monoamine oxidase inhibitor that was initially used for the management of depression but is now being considered for another purpose. This study compares the cytotoxic effects of SEL on different cancer cells. Further, the study explores the molecular mechanism of cell death, validating the possibility of its repurposing for cancer. Preliminary analysis of network pharmacological data was conducted in silico, followed by in vitro cytotoxicity tests on PC12, G361, MDA-MB231, MCF7, THP-1, and Hela cells under normoxic and hypoxic conditions, using the MTT assay. The mechanism of cell death was then confirmed by performing DAPI and FITC-conjugated Annexin V and Propidium Iodide (PI) staining assays. Additionally, ROS levels and PKC phosphorylation were also evaluated. In silico analysis has revealed that SEL is associated with ten genes linked to different cancer types. Specifically, SEL was most cytotoxic to neuronal pheochromocytoma, triple-negative human epithelial breast cancer cells, and ER+ and PR+ breast cancer cells. Furthermore, it was observed that this cell death occurred through ROS-independent apoptosis pathways. In addition, SEL was found to inhibit the phosphorylation of PKC, which may contribute to cell death. SEL induces apoptosis in breast cancer cells independently of reactive oxygen species and inhibits the phosphorylation of protein kinase C, which merits further exploration.

A novel tunable metal-clad planar waveguide with 0.62PMN-0.38PT material for detection of cancer cells.

A dynamically tunable metal clad planar waveguide having 0.62PMN-0.38PT material is simulated and optimized for detection of cancer cells. Angular interrogation of the TE0 mode of waveguide shows that critical angle increases greater than the resonance angle with increasing of cover refractive index, which limits the detection range of waveguide. To overcome this limitation, proposed waveguide applies a potential on the PMN-PT adlayer. Although a sensitivity of 105.42 degree/RIU was achieved at 70 Volts in testing the proposed waveguide, it was found that the optimal performance parameters were obtained at 60 Volts. At this voltage, the waveguide demonstrated detection range 1.3330-1.5030, a detection accuracy 2393.33, and a figure of merit 2243.59 RIU-1 , which enabled the detection of the entire range of the targeted cancer cells. Therefore, it is recommended to apply a potential of 60 Volts to achieve the best performance from the proposed waveguide.

A peptide from the staphylococcal protein Efb binds P-selectin and inhibits the interaction of platelets with leukocytes.

AIMS: P-selectin is a key surface adhesion molecule for the interaction of platelets with leukocytes. We have shown previously that the N-terminal domain of Staphylococcus aureus extracellular fibrinogen-binding protein (Efb) binds to P-selectin and interferes with platelet-leukocyte aggregate formation. Here, we aimed to identify the minimal Efb motif required for binding platelets and to characterize its ability to interfering with the formation of platelet-leukocyte aggregates. METHODS AND RESULTS: Using a library of synthetic peptides, we mapped the platelet-binding site to a continuous 20 amino acid stretch. The peptide Efb68-87 was able to bind to resting and, to a greater extent, thrombin-stimulated platelets in the absence of fibrinogen. Dot blots, pull-down assays and P-selectin glycoprotein ligand-1 (PSGL-1) competitive binding experiments identified P-selectin as the cellular docking site mediating Efb68-87 platelet binding. Accordingly, Efb68-87 did not bind to other blood cells and captured platelets from human whole blood under low shear stress conditions. Efb68-87 did not affect platelet activation as tested by aggregometry, flow cytometry and immunoblotting, but inhibited the formation of platelet-leukocyte aggregates (PLAs). Efb68-87 also interfered with the platelet-dependent stimulation of neutrophil extracellular traps (NETs) formation in vitro. CONCLUSIONS: We have identified Efb68-87 as a novel selective platelet-binding peptide. Efb68-87 binds directly to P-selectin and inhibits interactions of platelets with leukocytes that lead to PLA and NET formation. As PLAs and NETs play a key role in thromboinflammation, Efb68-87 is an exciting candidate for the development of novel selective inhibitors of the proinflammatory activity of platelets.

Heat inactivation of clinical COVID-19 samples on an industrial scale for low risk and efficient high-throughput qRT-PCR diagnostic testing.

We report the development of a large scale process for heat inactivation of clinical COVID-19 samples prior to laboratory processing for detection of SARS-CoV-2 by RT-qPCR. With more than 266 million confirmed cases, over 5.26 million deaths already recorded at the time of writing, COVID-19 continues to spread in many parts of the world. Consequently, mass testing for SARS-CoV-2 will remain at the forefront of the COVID-19 response and prevention for the near future. Due to biosafety considerations the standard testing process requires a significant amount of manual handling of patient samples within calibrated microbiological safety cabinets. This makes the process expensive, effects operator ergonomics and restricts testing to higher containment level laboratories. We have successfully modified the process by using industrial catering ovens for bulk heat inactivation of oropharyngeal/nasopharyngeal swab samples within their secondary containment packaging before processing in the lab to enable all subsequent activities to be performed in the open laboratory. As part of a validation process, we tested greater than 1200 clinical COVID-19 samples and showed less than 1 Cq loss in RT-qPCR test sensitivity. We also demonstrate the bulk heat inactivation protocol inactivates a murine surrogate of human SARS-CoV-2. Using bulk heat inactivation, the assay is no longer reliant on containment level 2 facilities and practices, which reduces cost, improves operator safety and ergonomics and makes the process scalable. In addition, heating as the sole method of virus inactivation is ideally suited to streamlined and more rapid workflows such as 'direct to PCR' assays that do not involve RNA extraction or chemical neutralisation methods.

Improving the efficiency and effectiveness of an industrial SARS-CoV-2 diagnostic facility.

On 11th March 2020, the UK government announced plans for the scaling of COVID-19 testing, and on 27th March 2020 it was announced that a new alliance of private sector and academic collaborative laboratories were being created to generate the testing capacity required. The Cambridge COVID-19 Testing Centre (CCTC) was established during April 2020 through collaboration between AstraZeneca, GlaxoSmithKline, and the University of Cambridge, with Charles River Laboratories joining the collaboration at the end of July 2020. The CCTC lab operation focussed on the optimised use of automation, introduction of novel technologies and process modelling to enable a testing capacity of 22,000 tests per day. Here we describe the optimisation of the laboratory process through the continued exploitation of internal performance metrics, while introducing new technologies including the Heat Inactivation of clinical samples upon receipt into the laboratory and a Direct to PCR protocol that removed the requirement for the RNA extraction step. We anticipate that these methods will have value in driving continued efficiency and effectiveness within all large scale viral diagnostic testing laboratories.

Structural features and ligand binding properties of tandem WW domains from YAP and TAZ, nuclear effectors of the Hippo pathway.

The paralogous multifunctional adaptor proteins YAP and TAZ are the nuclear effectors of the Hippo pathway, a central mechanism of organ size control and stem cell self-renewal. WW domains, mediators of protein-protein interactions, are essential for YAP and TAZ function, enabling interactions with PPxY motifs of numerous partner proteins. YAP has single and double WW domain isoforms (YAP1 and YAP2) whereas only a single WW domain isoform of TAZ has been described to date. Here we identify the first example of a double WW domain isoform of TAZ. Using NMR, we have characterized conformational features and peptide binding of YAP and TAZ tandem WW domains (WW1-WW2). The solution structure of YAP WW2 confirms that it has a canonical three-stranded antiparallel ß-sheet WW domain fold. While chemical shift-based analysis indicates that the WW domains in the tandem WW pairs retain the characteristic WW domain fold, 15N relaxation data show that, within the respective WW pairs, YAP WW1 and both WW1 and WW2 of TAZ undergo conformational exchange. 15N relaxation data also indicate that the linker between the WW domains is flexible in both YAP and TAZ. Within both YAP and TAZ tandem WW pairs, WW1 and WW2 bind single PPxY-containing peptide ligand concurrently and noncooperatively with sub-mM affinity. YAP and TAZ WW1-WW2 bind a dual PPxY-containing peptide with approximately 6-fold higher affinity. Our results indicate that both WW domains in YAP and TAZ are functional and capable of enhanced affinity binding to multi-PPxY partner proteins such as LATS1, ErbB4, and AMOT.

Principles underlying the complex dynamics of temperature entrainment by a circadian clock.

Autonomously oscillating circadian clocks resonate with daily environmental (zeitgeber) rhythms to organize physiology around the solar day. Although entrainment properties and mechanisms have been studied widely and in great detail for light-dark cycles, entrainment to daily temperature rhythms remains poorly understood despite that they are potent zeitgebers. Here we investigate the entrainment of the chronobiological model organism Neurospora crassa, subject to thermocycles of different periods and fractions of warm versus cold phases, mimicking seasonal variations. Depending on the properties of these thermocycles, regularly entrained rhythms, period-doubling (frequency demultiplication) but also irregular aperiodic behavior occurs. We demonstrate that the complex nonlinear phenomena of experimentally observed entrainment dynamics can be understood by molecular mathematical modeling.

Photorhabdus adhesion modification protein (Pam) binds extracellular polysaccharide and alters bacterial attachment.

BACKGROUND: Photorhabdus are Gram-negative nematode-symbiotic and insect-pathogenic bacteria. The species Photorhabdus asymbiotica is able to infect humans as well as insects. We investigated the secreted proteome of a clinical isolate of P. asymbiotica at different temperatures in order to identify proteins relevant to the infection of the two different hosts. RESULTS: A comparison of the proteins secreted by a clinical isolate of P. asymbiotica at simulated insect (28 degrees C) and human (37 degrees C) temperatures led to the identification of a small and highly abundant protein, designated Pam, that is only secreted at the lower temperature. The pam gene is present in all Photorhabdus strains tested and shows a high level of conservation across the whole genus, suggesting it is both ancestral to the genus and probably important to the biology of the bacterium. The Pam protein shows limited sequence similarity to the 13.6 kDa component of a binary toxin of Bacillus thuringiensis. Nevertheless, injection or feeding of heterologously produced Pam showed no insecticidal activity to either Galleria mellonella or Manduca sexta larvae. In bacterial colonies, Pam is associated with an extracellular polysaccharide (EPS)-like matrix, and modifies the ability of wild-type cells to attach to an artificial surface. Interestingly, Surface Plasmon Resonance (SPR) binding studies revealed that the Pam protein itself has adhesive properties. Although Pam is produced throughout insect infection, genetic knockout does not affect either insect virulence or the ability of P. luminescens to form a symbiotic association with its host nematode, Heterorhabditis bacteriophora. CONCLUSIONS: We studied a highly abundant protein, Pam, which is secreted in a temperature-dependent manner in P. asymbiotica. Our findings indicate that Pam plays an important role in enhancing surface attachment in insect blood. Its association with exopolysaccharide suggests it may exert its effect through mediation of EPS properties. Despite its abundance and conservation in the genus, we find no evidence for a role of Pam in either virulence or symbiosis.

Tradeoffs between air pollution mitigation and meteorological response in India.

To curb the staggering health burden attributed to air pollution, the sustainable solution for India would be to reduce emissions in future. Here we project ambient fine particulate matter (PM2.5) exposure in India for the year 2030 under two contrasting air pollution emission pathways for two different climate scenarios based on Representative Concentration Pathways (RCP4.5 and RCP8.5). All-India average PM2.5 is expected to increase from 41.4 ± 26.5 µg m-3 in 2010 to 61.1 ± 40.8 and 58.2 ± 37.5 µg m-3 in 2030 under RCP8.5 and RCP4.5 scenarios, respectively if India follows the current legislation (baseline) emission pathway. In contrast, ambient PM2.5 in 2030 would be 40.2 ± 27.5 (for RCP8.5) and 39.2 ± 25.4 (for RCP4.5) µg m-3 following the short-lived climate pollutant (SLCP) mitigation emission pathway. We find that the lower PM2.5 in the mitigation pathway (34.2% and 32.6%, respectively for RCP8.5 and RCP4.5 relative to the baseline emission pathway) would come at a cost of 0.3-0.5 °C additional warming due to the direct impact of aerosols. The premature mortality burden attributable to ambient PM2.5 exposure is expected to rise from 2010 to 2030, but 381,790 (5-95% confidence interval, CI 275,620-514,600) deaths can be averted following the mitigation emission pathway relative to the baseline emission pathway. Therefore, we conclude that given the expected large health benefit, the mitigation emission pathway is a reasonable tradeoff for India despite the meteorological response. However, India needs to act more aggressively as the World Health Organization (WHO) annual air quality guideline (10 µg m-3) would remain far off.

Multiple random phosphorylations in clock proteins provide long delays and switches.

Theory predicts that self-sustained oscillations require robust delays and nonlinearities (ultrasensitivity). Delayed negative feedback loops with switch-like inhibition of transcription constitute the core of eukaryotic circadian clocks. The kinetics of core clock proteins such as PER2 in mammals and FRQ in Neurospora crassa is governed by multiple phosphorylations. We investigate how multiple, slow and random phosphorylations control delay and molecular switches. We model phosphorylations of intrinsically disordered clock proteins (IDPs) using conceptual models of sequential and distributive phosphorylations. Our models help to understand the underlying mechanisms leading to delays and ultrasensitivity. The model shows temporal and steady state switches for the free kinase and the phosphoprotein. We show that random phosphorylations and sequestration mechanisms allow high Hill coefficients required for self-sustained oscillations.

The guanine-nucleotide-exchange factor BopE from Burkholderia pseudomallei adopts a compact version of the Salmonella SopE/SopE2 fold and undergoes a closed-to-open conformational change upon interaction with Cdc42.

BopE is a type III secreted protein from Burkholderia pseudomallei, the aetiological agent of melioidosis, a severe emerging infection. BopE is a GEF (guanine-nucleotide-exchange factor) for the Rho GTPases Cdc42 (cell division cycle 42) and Rac1. We have determined the structure of BopE catalytic domain (amino acids 78-261) by NMR spectroscopy and it shows that BopE(78-261) comprises two three-helix bundles (alpha1alpha4alpha5 and alpha2alpha3alpha6). This fold is similar to that adopted by the BopE homologues SopE and SopE2, which are GEFs from Salmonella. Whereas the two three-helix bundles of SopE(78-240) and SopE2(69-240) form the arms of a 'Lambda' shape, BopE(78-261) adopts a more closed conformation with substantial interactions between the two three-helix bundles. We propose that arginine and proline residues are important in the conformational differences between BopE and SopE/E2. Analysis of the molecular interface in the SopE(78-240)-Cdc42 complex crystal structure indicates that, in a BopE-Cdc42 interaction, the closed conformation of BopE(78-261) would engender steric clashes with the Cdc42 switch regions. This implies that BopE(78-261) must undergo a closed-to-open conformational change in order to catalyse guanine nucleotide exchange. In an NMR titration to investigate the BopE(78-261)-Cdc42 interaction, the appearance of additional peaks per NH for residues in hinge regions of BopE(78-261) indicates that BopE(78-261) does undergo a closed-to-open conformational change in the presence of Cdc42. The conformational change hypothesis is further supported by substantial improvement of BopE(78-261) catalytic efficiency through mutations that favour an open conformation. Requirement for closed-to-open conformational change explains the 10-40-fold lower k(cat) of BopE compared with SopE and SopE2.