Indian Institute of Science Bangalore
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A cavity electromechanical device for superconducting vortex charge sensing
No full textThe penetration of magnetic fields in the form of quantized vortices in type-II superconductors is well known. However, it is not well known that such vortices can be electrically charged. The effect is quite subtle and originates from the particle-hole symmetry in a superconductor. The high Tc superconductors (HTS) are predicted to be better candidates for vortex-charge detection due to their large superconducting gap. Thus far, a direct measurement of charged cores has remained challenging due to their small value and electrostatic screening by the surrounding opposite charges.
In recent years, cavity-optomechanical techniques have emerged as an attractive method to improve the sensitivity of various measurements. Such methods have shown exquisite force sensitivities down to the standard quantum limit and control over the quantum states of the motion. Recently, such techniques have also drawn attention to probe the thermodynamic properties of atomically thin two-dimensional (2D) materials. The 2D crystals are particularly attractive for developing mechanical resonators and their integration in optomechanical device due to their low mass, and hence larger coupling with light field. Motivated from these aspects, we develop a device to directly detect the charges in the flux-vortices by measuring the electromechanical response. Here the electrostatic effect of vortex-charge is transduced to the mechanical response.
To study the vortex charge, a few UC thick crystals of high-transition temperature superconductor Bi2Sr2CaCu2O8+δ (BSCCO) is used for the mechanical resonator. One important parameter of the mechanical resonator is its resonant frequency. However, estimating the resonant frequency requires elastic modulus like Young's modulus and pre-tension in the flake. While the elastic coefficients of the bulk crystals of BSCCO have been observed with large variations, there is no investigation into the elastic properties of a few UC thick nanoscale samples. Further, the mechanical properties of a few unit cells (UC) thick exfoliated crystals could be significantly different from their bulk counterpart.
To begin with, we present systematic measurements of the mechanical properties of a few unit cells (UC) thick exfoliated crystals of a high-Tc cuprate superconductor BSCCO. We determine the elastic properties of these crystals by deformation using an atomic force microscope (AFM) at room temperature. With the spatial measurements of local compliance and their detailed modelling, we determine Young's modulus of rigidity and the pre-stress. Young's modulus of rigidity is found to be in the range of 22 GPa to 30 GPa for flakes with thickness from 5 UC to 18 UC. The pre-stress spreads over the range of 5 MPa - 46 MPa, indicating a run-to-run variation during the exfoliation process. The determination of Young's modulus of rigidity for thin flakes is further verified from the recently reported buckling technique [1].
In the next chapter, we present nanoelectromechanical resonators fabricated with thin exfoliated crystals of BSCCO. The mechanical r= eadout is performed by capacitively coupling their motion to a coplanar waveguide microwave cavity fabricated with a superconducting alloy of molybdenum-rhenium (MoRe). We demonstrate mechanical frequency tunability with external dc-bias voltage and quality factors up to ~36600. Our spectroscopic and time-domain measurements show that mechanical dissipation in these systems is limited by the contact resistance arising from resistive outer layers. The temperature dependence of dissipation indicates the presence of tunnelling states, further suggesting that their intrinsic performance could be as good as other two-dimensional atomic crystals such as grap= hene [2].
Learning from these two experiments, we improve the performance of the device and carry out the mechanical exfoliation in inert atmosphere. We integrate a mechanical resonator made of a thin flake of HTS BSCCO into a microwave circuit to realize a cavity-electromechanical device. In the final chapter, we studied the electromechanical response of the mechanical resonator when a magnetic field perpendicular to the CuO2 plane is applied. As the magnetic field penetrates the surface of a superconductor, it results in the formation of flux-vortices. These flux-vortices will have charged vortex core and create a dipolelike electric field. Due to the exquisite sensitivity of cavity-based devices to the external forces, we directly detect the charges in the flux vortices by measuring the electromechanical response of the mechanical resonator [3]. Our measurements reveal the strength of surface electric dipole moment due to a single vortex core to be approximately 30 |e|aB, where aB is the Bohr radius and e is the electron charge. Further, using the value of surface dipole moment, we have estimated the vortex line charge to be +4.9 × 10-2|e|/nm, which is equivalent to a charge per CuO_2 layer to be +3.7 × 10-2|e|
Deformation characteristics of soil under sequential dynamic torsional and cyclic axial loading
No full textEarthquake-induced structural damages caused due to site effects, soil liquefaction failure, and associated excessive settlement are well-known worldwide. The primary controlling factor responsible for the superstructure’s response under such dynamic (earthquake) loading conditions depends on the behaviour of local soil properties underneath it. These dynamic properties are usually measured in the laboratory by performing the shear deformation characteristics test employing a resonant column and cyclic triaxial test. The small strain (10-05 to 0.01 %) test is mainly performed using a resonant column, and the medium to large strain (> 0.01 % to 1 %) test is performed using a cyclic triaxial test. Thus, measurement of these dynamic properties often requires a minimum of two or multiple specimens, which involves immense effort and expense in facilitating the low and high strain loading separately.
In the field, however, the characteristics of seismic waves propagating from a wave source to a site are a complex combination of different types of waves, including compressional and shear waves. Therefore, in the laboratory, it is crucial to test soils under the combined loadings of dynamic torsional and cyclic axial loading (using a single specimen) to mimic the field condition as practically as possible. The main objective of this research is to study the deformation characteristics of soils at low to high strain levels while a single specimen is subjected to combined dynamic torsional and cyclic axial (sequential) loading. For this purpose, an operational resonant column and cyclic triaxial apparatus were modified with advanced instrumentations, and dynamic tests were performed on one hundred twenty-one test specimens. Reconstituted clean sands, including a United Kingdom’s sand and in-situ soil samples (Indo-Gangetic plain region, West and South India Region, North-Eastern Region soils, and liquified soil deposits) were selected for the study. The influence of different parameters on the dynamic properties such as the confining pressure, relative density (void ratio), particle shape, number of loading cycles, soil type, and previous loading history (or pre-straining effect) was studied explicitly. After extensive studies of dynamic properties under dry conditions, dynamic behaviour under full saturated conditions, i.e., liquefaction and re-liquefication of silty sand, riverbed sand, and in-situ soils, were explored.
The study brings out the following significant contributions as a part of the research work:
1) A low-cost novel Foldscope approach was introduced for quantifying the particle shape and imaging of the soil particles
2) Established appropriate methods and number of loading waveforms required for the reliable damping estimation using Resonant Column tests (at low strain levels)
3) A modified method was proposed for estimating the damping for non-symmetric hysteresis loop undergoing cyclic loading (at high strain levels)
4) Established the effects of confining pressure, relative density (void ratio), particle shape, number of loading cycles, soil type, and previous loading history (or pre-straining effect) on dynamic properties of Sand and Silty Sand
5) Shear modulus and damping model was developed for sands and natural soils with fines
6) Shear modulus and damping values were obtained for strain greater than 1 %, and a mechanism for unique behaviour of damping was established
7) Investigation of liquefaction and re-liquefaction potential of the liquefied site was made through field and laboratory experiments. Finally, a liquefaction strength curve was developed
One of the key aspects of this study is that the design curves developed in this study can be incorporated into practice, such as in carrying out any dynamic analysis
Inclusive and Eye-Gaze Controlled Human-Robot Interaction for Persons with Severe Speech and Motor Impairment
No full textMost individuals perform various activities of daily living without having to con sciously think and plan about them. People talk, cook their food, move around the house, drive to their workplace and schools, play sports, and so on. However, a sig nificant proportion of the world population is not so fortunate enough. About 15% of the world’s population lives with some form of disability caused by Neurodevel opmental disorders, accidents, or injury to the brain or spinal cord. India had around 27 million people living with disabilities [138]. There are special provisions and laws
enforced by national governments and worldwide organizations for supporting ed ucation, healthcare, and employment of persons with disabilities [161]. However, persons with disabilities are less likely to ever attend school, tend to leave education mid-way, and are less likely to possess basic literacy skills. A similar trend is observed in their employment and social inclusion in general. The persons with disabilities majorly belong to a spectrum of functional impair ments given their medical and health conditions. The most isolated section of the disability spectrum are persons with severe speech and motor impairment. Such in dividuals face challenges in having natural interactions with their environments. Per sons with SSMI are unable to use their limbs for grabbing and movement, or speech and other bodily gestures for communication. They are confined to a wheelchair and heavily depend on a caregiver or a family member to carry out even the activities of daily living (ADL). The caregivers and family members must rely on manual analysis of the eye-gaze of persons with SSMI in order to communicate and understand their needs. Advances in robotics and technology, in general, have opened new doors for inno vative solutions for supporting persons with disabilities. Researchers have come up with numerous solutions in the fields of assistive, rehabilitation, and social robotics. There are smart motorized wheelchairs, with intelligent robotic arms mounted on them, and social companion robots to talk and play with as pets.
This dissertation work proposes an eye-gaze controlled, safe, and affordable human robot interaction system for persons with severe speech and motor impairment. This research takes a user-centered design approach to design the assistive robotics sys tem based on initial interactions and pilot studies with the end users. The proposed human-robot interaction systems are evaluated with end users in various user trials over the last four years
Mechanistic insights into the role of miR‐6741‐3p in oral squamous cell carcinoma pathogenesis
No full textMany functional studies and clinical analysis have linked miRNA dysregulation as a causative factor for tumor initiation and progression. Overexpression of oncogenic miRNAs and downregulation of tumor suppressor miRNAs are widely reported to drive the process of carcinogenesis. Epigenetic silencing of miRNAs with tumor suppressor features by DNA hypermethylation is a common hallmark of many cancers including OSCC. We, therefore, used the drug 5-Azacytidine, a DNMT inhibitor, for reactivation of tumor suppressor miRNAs silenced due to DNA hypermethylation. Treatment of cells from an OSCC cell line SCC131 with control and 5-Azacytidine, followed by the miRNA microarray analysis revealed upregulation and downregulation of 50 and 28 miRNAs respectively. miR-6741-3p was one of the miRNAs found to be upregulated following the 5-Azacytidine treatment.
We validated the upregulation of miR-6741-3p following the 5-Azacytidine treatment using qRT-PCR and demonstrated its anti-proliferative activity in OSCC cells. miRNAs are epigenetic regulators which exert their effect through binding to one or more cognate target mRNAs and regulating their expression. Using bioinformatics and molecular approaches, we showed that miR-6741-3p post-transcriptionally regulates the expression of the oncogene SRSF3 at the protein level in OSCC by interacting with its 3’UTR in a sequence-specific manner. We analyzed the expression of miR-6741-3p transcript and SRSF3 protein in different cell lines and OSCC patient samples, and the results showed that the interaction between miR-6741-3p and SRSF3 is of physiological and clinical relevance. We demonstrated that miR-6741-3p reduced cell proliferation and anchorage-independent growth of OSCC cells, in part, by targeting the 3’UTR of SRSF3 and promoted apoptosis of OSCC cells independent of SRSF3. OSCC xenografts in nude mice using a synthetic miR-6741-3p mimic and a synthetic miR-6741-3p inhibitor indicated that miR-6741-3p suppresses tumor formation in OSCC, in part, by targeting SRSF3. We also demonstrated that miR-6741-3p decreases signaling through both the PI3K-AKT-MTOR and the ERK/MAPK pathways, in part, via the miR-6741-3p-SRSF3-ERK1/2-S6K1 axis.
We also demonstrated that the intronic miR-6741-3p is involved in the autoregulation of its host gene PYCR2 and inhibits its expression at both the transcript and protein levels in OSCC cells by interacting with a target site present in the 3’UTR in a sequence-specific manner. Expression analysis of miR-6741-3p and PYCR2 across different cell lines and OSCC patient samples suggested that the interaction between miR-6741-3p and PYCR2 is of physiological and clinical relevance.
We observed that MIR6741 lacks an independent promoter and seems to be co-transcribed with the PYCR2 transcript, using its host gene PYCR2 promoter. We identified and validated the host gene promoter activity and identified PAX6 as a positive regulator of the PYCR2 promoter activity. Using a combination of in silico analysis, ChIP analysis, and dual-luciferase reporter assays, we propose that PAX6 interacts with the PYCR2 promoter via an indirect mechanism. We further demonstrated that promoter methylation of the tumor suppressor gene PAX6 is responsible for the downregulation of miR-6741-3p in OSCC cells and speculate the involvement of the same mechanism in other cancers.CSIR, DB
Boolean Functional Synthesis using Gated Continuous Logic Networks
No full textBoolean Functional Synthesis (BFS) is a well-known challenging problem in the domain of automated program synthesis from logical specifications. This problem aims to synthesize a Boolean function that is correct-by-construction with respect to the declared specification; this specification symbolically relates the inputs and outputs of the function to be synthesized. Since Boolean functions are the basic building blocks of modern digital systems, BFS has applications in a wide range of areas, including QBF-SAT solving, circuit repair and debugging. This has motivated the community to develop practically efficient algorithms for synthesizing compact Boolean functions, which is a non-trivial endeavor. However, to the best of our knowledge, current techniques are unable to specify a bound on the Boolean function size during synthesis. Specifying a bound on the size of the formula offers flexibility in synthesizing minimal-sized Boolean functions.
Learning Boolean functions from logical specifications using neural networks is a difficult problem as it requires the network to represent Boolean functions. Boolean functions are discrete functions and consequently, non-differentiable. Thus, learning a Boolean function directly using traditional neural networks is not possible. Recently Ryan et al proposed the Gated Continuous Logic Network (GCLN) model that builds on Fuzzy Logic to represent Boolean and linear integer operator, in the context of learning invariants for programs. In this work, we investigate the use of the GCLN model to synthesize solutions to the BFS problem. Our model lets us bound the number of clauses used in the synthesized Boolean function.
We implement this approach in our tool BNSynth (for Bounded Neural Synthesis) which also uses sampling and counter-example-guided techniques to synthesize Boolean functions. We validate our hypothesis that this system can learn smaller functions as compared to a state-of-the-art tool over custom benchmarks and LUT-based benchmarks from the ISCAS85 benchmark suite. We observe a 15.8X average improvement in formula size by the number of clauses for these benchmarks. This empirically shows that our system can synthesize smaller Boolean functions compared to the state-of-the-art
Study of gas-fine flow in a packed bed with an application to the ironmaking blast furnace
No full textThe iron and steelmaking industry uses coal as the primary reducing agent. The carbon that is the major component of coal is finally released into the environment as carbon dioxide. One of the effective ways to reduce the coke consumption and thus the reduction in greenhouse gases is to introduce coal or other carbonaceous materials through the tuyere. However, this can be done only up to some extent as injection of these materials reduces the bed permeability which in turn affects the operation and productivity of the blast furnace (BF). Previous studies have shown that at a higher pulverised coal injection (PCI) rate and depending on the operating conditions of the furnace, some amount of coal remains unburnt and consequently the ashes and coal particles, in the form of powders, may be entrained in the gas stream or be deposited into the lower zones of the BF. However, the physics of the pulverised coal within the furnace is still not well understood, especially in presence of raceway and tuyere protrusion. Raceway is very important in iron-making BF as its size and shape determines the aerodynamics of BF and thus the heat and mass transfer. Therefore, the study of fine flow in a packed bed is a necessary precursor to understand the above-mentioned processes/phenomena.
For the validation purpose, two-dimensional experimental studies were performed at room temperature on the various packed bed such as a rectangular bed with and without a cohesive zone and a cylindrical bed without a cohesive zone. Both the packing and fine particles were made of glass material. Steady state of experiment with fines was achieved based on the recently developed accurate mass balance method which dictates that fines into the system must be equal to fines out of the system.
Most research neglect vital characteristics of BF, such as lateral inflow, presence of tuyere protrusion, the raceway’s shape and size, and cohesive zone leading to a significant incongruence with the BF being modelled. The present work incorporates these features into a 2D numerical study of a gas–fines–solid system. The mathematical modelling considers the gas and fines as a Eulerian–Eulerian system with the constant voidage model for the solid phase representing the packing particles. In presence of a cohesive zone, the cohesive blocks were assigned zero porosity. Well-established theoretical relations and correlations are used to determine the inter-phase forces and fines accumulation regions. Particular emphasis is placed on the accurate representation of the raceway formed at the tuyere exit, and three approaches are considered, viz., its absence, a correlation-based prediction, and an iso-stress-based model. The sensitivity analysis is done with a marked interest in the raceway shape and size and accumulation profiles of the fines which are important parameters in the overall flow characterization. Additionally, structural parameters such as cohesive zone configuration, cohesive block porosity, and tuyere protrusion are also varied, and their effect on the raceway and static holdup profiles are characterised. The sensitivity analysis shows that the raceway shape and size play a vital role in the flow of gas-fines and influence the accumulation of fines. While most of the parameters varied have a noticeable effect, certain factors such as fines feed rate and size exhibit negligible consequences.
The model predicts the static holdup profile through a correlation based on experimental data. Developed model is able to predict the pressure loss reasonably well against experimental and published data for both clean gas and gas–fine flow in a packed bed with a lateral and bottom injection of gas and fine. Similarly, the model is able to predict correctly the raceway shape & size, pressure profiles in both vertical and horizontal directions as well as fines static holdup both in the presence and absence of cohesive blocks. All the results indicate an excellent agreement with the experimental data and lend credibility to the model and solver.CSIR, DS
Auditory Timbre and Spatialisation: Signal Analysis and Perception of Source Widening
No full textIn this work, auditory perception of source widening is examined in the context of different source signal timbre. Perception of widening of source or auditory source width (ASW) arises in three cases: (i) In the presence of reverberation, which has been referred to as reverberant source width (RSW); (ii) Distributed sources such as an ensemble, where multiple sources are physically placed widely, referred as ensemble source width (ESW); and (iii) In hearing disabilities, where localisation is poor in the presence of interfering sources and hence a widened or diffused source width (DSW) is perceived. Though the physics of the problem is different in each of the above cases, we observe that the perception of source widening occurs in all the three cases. We also show analytically that in the case of localised, reverb and ensemble sources located about a particular direction, binaural cross-correlation has
interesting properties: (i) for localised source, cross-correlation is energy compact, followed by (ii) reverb source and (iii) ESW has highly dispersed cross-correlation compared to localised and reverb sources with the same angle of arrival and degree of decorrelation as reverb sources. Traditionally, (1-IACC) has been used as a measure for RSW and in the literature this measure is used for ESW also. We propose a combination of timbre-independent phase-based angular measure for the physical extent of the sources, localising all or many individual sources using HRIR correlation functions and timbre dependent mean time-bandwidth energy (MTBE) measure for relative perceptual weighting to compare ensemble of different timbres. This analysis gives rise to possible applications in ensemble rendering and insights
into improving hearing aids for hearing impaired listeners. Frequency sensitivity to change in IACC, and hence ASW, has been studied using binaural presentation of modulated sinusoids. In this work, we observe a
similar sensitivity to ESW by presenting listeners with spatially wide sources using narrow band noise signals. We observe that frequency sensitivity of ASW and ESW are similar. We also study bandwidth sensitivity and observe that with increase in bandwidth, the perceived width of the ensemble increases. We simulate ensemble-like music signals of different spectro-temporal distribution to probe the timbre dependency of human perception. The listeners are asked
to rate the ESW of the simulated distributed sources. Broadly, music signals can be classified as sustained instruments, partially sustained, partially transient and predominantly transient signals. Low frequency sustained instruments give rise to a wider percept than semi-sustained and transient signals. We also explore the difference between discrete and continuous spectral sources for spatialisation. We observe that continuous spectra do give rise to stable mono-
tonic width perception with change in physical width. On the other hand, in the case of discrete spectra, we do not perceive a stable monotonic perception. We developed a MUSHRA like (Multiple stimulus hidden reference and anchor)
listening methodology for estimating the accuracy of direction perception of the target signals with and without interference by normal listeners. We observe that the accuracy of direction perception of the target is high without interference. In the presence of interference, we observe that the perceived target direction is away
from that of interference, thus increasing the perceived angular separation. This perceptual effect may be used in the design of binaural hearing-aids to enhance binaural perception of localised sources in the presence of interference.
Overall, in this work, we study the perceptual interaction of signal timbre and spatialisation in the perception of ensemble source width. We study the sensitivity of several parameters like frequency, bandwidth, spectro-temporal energy distribution and role of fine AM-FM parameters
Scalable Asynchrony-Tolerant PDE Solver for Multi-GPU Systems
No full textPartial differential equations (PDEs) are used to model various natural phenomena and engineered
systems. At conditions of practical interest, these equations are highly non-linear and demand
massive computations. Current state-of-the-art simulations are routinely being performed on
supercomputers with hundreds of thousands of processing elements. With an increase in compute
intensity per node and an increase in node count as the world moves towards exa-scale machines,
communication and synchronization costs pose a major bottleneck on the performance of PDE
solvers. A standard approach to mitigate these bottlenecks is to enhance the overlap between
communication and computation in an algorithmic implementation. Another approach also looks at
relaxing communication and synchronization requirements, but at the base mathematical or
numerical method level. Asynchrony-tolerant (AT) numerical schemes follow this second approach
where larger stencils are used to relax these boundary value communications while maintaining the
required order of accuracy.
In the first part of this work, the performance of previously derived finite difference AT schemes
was investigated on GPUs. GPUs are designed to deliver a high throughput, but suffer from high
latency for data movement. Therefore, AT schemes can be used to hide the latency and achieve
scalable performance. Two algorithms to apply such AT schemes, namely the communication
avoiding which is deterministic and the synchronization avoiding which is probabilistic, have been
implemented to develop a solver for turbulence problems based on the compressible Navier-Stokes
equations. The solver was developed for multi-GPU multi-node systems using the MPI+CUDA
model. The code was profiled and optimised with techniques such as tiling to increase coalesced
memory access and manage register usage. Scaling studies were then performed and up to 50%
improvement in performance has been obtained over the baseline synchronous implementation in
benchmarks running up til 1024 nodes on OLCF Summit supercomputer.
In the second part of the work, new asynchrony-tolerant schemes for the multi-stage Runge-Kutta
methods have been developed, particularly in the context of low storage explicit Runge-Kutta
(LSERK) schemes. The performance of the LSERK-AT schemes was demonstrated using a mini-
app that solves the non-linear Burgers’ equations and parallelised with MPI. Benchmarks performed
on SahasraT showed a peak speedup of 6x at an extreme scale of 27,000 cores
Reconstruction of temperature for Cenozoic and Proterozoic Ocean water using clumped isotope thermometry
No full textClumped isotope thermometry is a novel tool which is used for the determination of formation temperature for carbonates of diverse origin and understanding different marine and terrestrial processes. The primary interest of this study is to develop use clumped as well as stable isotope as the tool to deduce temperature of ocean water from Cenozoic and Proterozoic time window. Stable and clumped isotope studies have previously shown that both of these isotopes experience different degrees of fractionation for different mineralogy of carbonates (e.g., calcite, dolomite, aragonite etc.) during the sample preparation procedures conducted at different temperature conditions. Major highlights of my research work include development of analytical procedure and revisiting the numerical equation for thermometry using aragonite and dolomite minerals. Exclusive, experiments are conducted at different temperatures to understand the isotopic fractionation during sample preparation and analysis.
As a part of the present study, the natural aragonite specimens with well characterized temperature information are analysed in order to study the fractionation during acid digestion and clumped isotope thermometry calibration using sealed vessel or Break-seal method developed at IISc. Calcite and aragonite react or fractionate in a similar manner during phosphoric acid digestion which is evident from the previous theoretical and experimental studies. However, the protocol specific variation of temperature sensitivity and intercept values of the clumped isotope thermometric calibrations demarcates the existence of inconsistencies associated with the acid digestion protocols. This study proposes revised calibration for aragonite clumped isotope thermometry using the otoliths. As an application of the calibration study, palaeo-otoliths and travertine are analysed for temperature estimates infer about the freshwater availability near coastal United States covering the time from early Cretaceous, Eocene, Oligocene and Pleistocene time. Further, we validated the temperature of travertine formation analysing modern day tufa with known temperature record extending the thermometry to 40°C. A second analytical accomplishment includes designing and executing a new isotope dilution method for the clumped isotope analysis for small carbonate powder (2-3 mg).
We have also established acid fractionation for dolomite during reaction with the phosphoric acid digestion at 25℃ using seal vessel method. We proposed acid fractionation correction factor of 0.069 for the break seal method, suitable for our experimentation. Using this approach, we determined clumped isotope ratio in the pristine relic carbonate mud from the Vempalle Formation of Proterozoic Ocean. Our observation revealed lowest temperature value of 21.7℃ and δ18OVSMOW value of water as -9.96‰. This is the first evidence documenting lighter oxygen isotopic composition of Proterozoic Ocean The overlying stromatolites with dolomites registered consistently higher temperatures (72.8-106.2℃) that might suggests precipitation from hydrothermal fluid or early phase burial diagenesis promoting water-rock interaction. Our observations resolved long-standing dispute about existence of mild and lighter composition of water in the Proterozoic Ocean
Homeostasis of cyclic-di-AMP in Mycobacterium smegmatis: Functional and structural contributions of c-di-AMP synthase (MsDisA) and hydrolase (MsPDE)
No full textIn bacteria, cyclic-di-nucleotide based second messengers regulate various physiological processes including the stress response. For the past decades, cyclic diadenosine monophosphate (c-di-AMP) has emerged as a crucial second messenger in the bacterial world. It is an essential molecule implicated in fatty acid metabolism, antibiotic resistance, biofilm formation, virulence, DNA repair, potassium and osmotic homeostasis, sporulation etc. The level of c-di-AMP is maintained within the cell by the action of two opposing enzymes, namely diadenylate cyclases and phosphodiesterases. In mycobacteria, this molecule is essential for its regulatory role in bacterial physiology and host-pathogen interactions. However, such modulation of c-di-AMP remains to be explored in Mycobacterium smegmatis. Here, we systematically investigated the c-di-AMP synthase (MsDisA) and a hydrolase (MsPDE) from M. smegmatis at different pH and osmolytic conditions in vitro. Our biochemical assays show that the MsDisA activity is enhanced during the alkaline stress and c-di-AMP is readily produced without any intermediates. At pH 9.4, the MsDisA promoter activity in vivo increases significantly, strengthening this observation. However, under physiological conditions, the activity of MsDisA was moderate with the formation of intermediates. We also observed that the size of MsDisA is significantly increased upon incubation with the substrate. To get further insights into the structural characteristics, we solved a 3.02 Å cryo-EM structure of the MsDisA, revealing some of interesting properties. Analysis of individual domains shows that the N-terminal minimal region alone can form a functional octamer. Altogether, our results reveal the biochemical and structural regulation of mycobacterial c-di-AMP in response to various environmental stresses.
Chapter 1 reviews the available literature in the field of second messengers and provide rational behind this study. The discovery of c-di-AMP was accidental, which later had known to regulate plethora of functions. This chapter stresses upon the need to investigate the significance of c-di-AMP homeostasis in Mycobacteria and the scope of the current study.
Chapter 2 reports the homeostasis of c-di-AMP by DisA and PDE in M. smegmatis and the substrate-induced inhibitory mechanism of MsDisA. Promoter study suggested MsDisA is expressed in all the growth phases and the changes in the extracellular pH during mycobacterial growth give valuable hints about the trans-membrane pH regulation by the c-di-AMP molecule.
Chapter 3 further elaborates the enzyme-substrate reaction and domain movement. The substrate-induced change of the MsDisA structure, which is demonstrated using biophysical characterization and Transmission Electron Microscopy (TEM) image analysis. The details on the structural characteristics of the MsDisA were obtained by cryo-EM. We reconstructed a 3.02 Å structure of MsDisA by Electron Cryomicroscopy (Cryo-EM). We observed an open-complex formation of this protein, which gives insight into the enzyme's active site.
Chapter 4 describes the importance of individual domain of MsDisA in c-di-AMP synthesis and critical residues at catalytic core. This study provides a few interesting observations about the oligomerization and activity of the mutant proteins. Further, this chapter also talks about the second activity of DisA protein which binds to 4-way junctions DNA, resulting in an allosteric inhibition of c-di-AMP synthesis activity in Bacillus subtilis. Interestingly, the cryo-EM open structure of MsDisA shows a unique feature where two monomers stay apart to break D4 symmetry, preventing the protein from directly interacting with branched DNA which helps mycobacterial cell for the continuous synthesis of c-di-AMP under stress.
Chapter 5 summarizes the results of the study and points out the future directions for the work.
Appendix Chapters includes the work which I have carried out in my first three years.
In appendix chapter 1, we worked on bacterial RNA polymerase and its smallest subunit ω. Here the emphasis of the work was to understand the mechanistic details of lethality by silent mutant of ω. Wild type ω shows a predominantly unstructured circular dichroic profile and becomes α-helical in the enzyme complex. This structural transition is perhaps the reason for the lack of function. We generated several silent mutants of ω to investigate the role of codon bias and the effect of rare codons with respect to their position in rpoZ. Not all silent mutations affect the structure. RNA polymerase when reconstituted with structurally altered silent mutants of ω is transcriptionally inactive. The Codon Plus strain, which has surplus tRNA, was used to assess for the rescue of the phenotype in lethal silent mutants.
In appendix chapter 2, we have utilized 7-Aza-tryptophan to investigate whether there is binding coupled folding for omega. The photo physical properties of free 7-Aza-tryptophan give a strong red-shifted fluorescence (403 nm), which would allow us to trap the conformational changes that occur during the ω-β' interaction and sigma attachment to the core RNAP. The incorporation of non-natural analogue 7-Aza-Trp in ω was achieved by way of using an E. coli Trp auxotrophic strain (RF12) where we have overcharged the system with Tryptophanyl-tRNA synthetase (trpS) gene for proper incorporation. The interaction of 7-Aza-Trp incorporated ω with the core1 (α2ββ') RNAP is being studied here