Gene Silencing via RNA Interference in Cryptococcus.

RNA interference (RNAi) is a molecular biology technique for silencing specific eukaryotic genes without altering the DNA sequence in the genome. The silencing effect occurs because of decreased levels of mRNA that then result in decreased protein levels for the gene. The specificity of the silencing is dependent upon the presence of sequence-specific double-stranded RNA (dsRNA) that activates the cellular RNAi machinery. This chapter describes the process of silencing a specific target gene in Cryptococcus using a dual promoter vector. The plasmid, pIBB103, was designed with two convergent GAL7 promoters flanking a ura5 fragment that acts as a reporter for efficient RNAi. The target gene fragment is inserted between the promoters to be transcribed from both directions leading to the production of dsRNA in cells that activate the RNAi pathway.

Molecular Pathogenesis of Nipah Virus.

Viral diseases are causing mayhem throughout the world. One of the zoonotic viruses that have emerged as a potent threat to community health in the past few decades is Nipah virus. Nipah viral sickness is a zoonotic disease whose main carrier is bat. This disease is caused by Nipah virus (NiV). It belongs to the henipavirous group and of the family paramyxoviridae. Predominantly Pteropus spp. is the carrier of this virus. It was first reported from the Kampung Sungai Nipah town of Malaysia in 1998. Human-to-human transmission can also occur. Several repeated outbreaks were reported from South and Southeast Asia in the recent past. In humans, the disease is responsible for rapid development of acute illness, which can result in severe respiratory illness and serious encephalitis. Therefore, this calls for an urgent need for health authorities to conduct clinical trials to establish possible treatment regimens to prevent any further outbreaks.

Emergent phenomena in living systems:A statistical mechanical perspective.

A natural phenomenon occurring in a living system is an outcome of the dynamics of the specific biological network underlying the phenomenon. The collective dynamics have both deterministic and stochastic components. The stochastic nature of the key processes like gene expression and cell differentiation give rise to fluctuations (noise) at the levels of the biomolecules, and this combined with nonlinear interactions gives rise to a number of emergent phenomena. In this review, we describe and discuss some of these phenomena which have the character of phase transitions in physical systems. We specifically focus on noise-induced transitions in a stochastic model of gene expression and in a population genetics model which have no analogs when the dynamics are solely deterministic in nature. Some of these transitions exhibit critical-point phenomena belonging to the mean-field Ising universality class of equilibrium phase transitions. A number of other examples, ranging from biofilms to homeostasis in adult tissues, are also discussed, which exhibit behaviour similar to critical phenomena in equilibrium and nonequilbrium phase transitions. The examples illustrate how the subject of statistical mechanics provides a bridge between theoretical models and experimental observations.

Student Attitudes Contribute to the Effectiveness of a Genomics CURE.

The Genomics Education Partnership (GEP) engages students in a course-based undergraduate research experience (CURE). To better understand the student attributes that support success in this CURE, we asked students about their attitudes using previously published scales that measure epistemic beliefs about work and science, interest in science, and grit. We found, in general, that the attitudes students bring with them into the classroom contribute to two outcome measures, namely, learning as assessed by a pre- and postquiz and perceived self-reported benefits. While the GEP CURE produces positive outcomes overall, the students with more positive attitudes toward science, particularly with respect to epistemic beliefs, showed greater gains. The findings indicate the importance of a student's epistemic beliefs to achieving positive learning outcomes.

Tipping the Balance: A Criticality Perspective.

Cell populations are often characterised by phenotypic heterogeneity in the form of two distinct subpopulations. We consider a model of tumour cells consisting of two subpopulations: non-cancer promoting (NCP) and cancer-promoting (CP). Under steady state conditions, the model has similarities with a well-known model of population genetics which exhibits a purely noise-induced transition from unimodality to bimodality at a critical value of the noise intensity σ2. The noise is associated with the parameter λ representing the system-environment coupling. In the case of the tumour model, λ has a natural interpretation in terms of the tissue microenvironment which has considerable influence on the phenotypic composition of the tumour. Oncogenic transformations give rise to considerable fluctuations in the parameter. We compute the λ-σ2 phase diagram in a stochastic setting, drawing analogies between bifurcations and phase transitions. In the region of bimodality, a transition from a state of balance to a state of dominance, in terms of the competing subpopulations, occurs at λ = 0. Away from this point, the NCP (CP) subpopulation becomes dominant as λ changes towards positive (negative) values. The variance of the steady state probability density function as well as two entropic measures provide characteristic signatures at the transition point.

Facilitating Growth through Frustration: Using Genomics Research in a Course-Based Undergraduate Research Experience.

A hallmark of the research experience is encountering difficulty and working through those challenges to achieve success. This ability is essential to being a successful scientist, but replicating such challenges in a teaching setting can be difficult. The Genomics Education Partnership (GEP) is a consortium of faculty who engage their students in a genomics Course-Based Undergraduate Research Experience (CURE). Students participate in genome annotation, generating gene models using multiple lines of experimental evidence. Our observations suggested that the students' learning experience is continuous and recursive, frequently beginning with frustration but eventually leading to success as they come up with defendable gene models. In order to explore our "formative frustration" hypothesis, we gathered data from faculty via a survey, and from students via both a general survey and a set of student focus groups. Upon analyzing these data, we found that all three datasets mentioned frustration and struggle, as well as learning and better understanding of the scientific process. Bioinformatics projects are particularly well suited to the process of iteration and refinement because iterations can be performed quickly and are inexpensive in both time and money. Based on these findings, we suggest that a dynamic of "formative frustration" is an important aspect for a successful CURE.

Osmolytes ameliorate the effects of stress in the absence of the heat shock protein Hsp104 in Saccharomyces cerevisiae.

Aggregation of the prion protein has strong implications in the human prion disease. Sup35p is a yeast prion, and has been used as a model protein to study the disease mechanism. We have studied the pattern of Sup35p aggregation inside live yeast cells under stress, by using confocal microscopy, fluorescence activated cell sorting and western blotting. Heat shock proteins are a family of proteins that are produced by yeast cells in response to exposure to stressful conditions. Many of the proteins behave as chaperones to combat stress-induced protein misfolding and aggregation. In spite of this, yeast also produce small molecules called osmolytes during stress. In our work, we tried to find the reason as to why yeast produce osmolytes and showed that the osmolytes are paramount to ameliorate the long-term effects of lethal stress in Saccharomyces cerevisiae, either in the presence or absence of Hsp104p.

Criticality in cell differentiation.

Cell differentiation is an important process in living organisms. Differentiation is mostly based on binary decisions with the progenitor cells choosing between two specific lineages. The differentiation dynamics have both deterministic and stochastic components. Several theoretical studies suggest that cell differentiation is a bifurcation phenomenon, well-known in dynamical systems theory. The bifurcation point has the character of a critical point with the system dynamics exhibiting specific features in its vicinity. These include the critical slowing down, rising variance and lag-1 autocorrelation function, strong correlations between the fluctuations of key variables and non-Gaussianity in the distribution of fluctuations. Recent experimental studies provide considerable support to the idea of criticality in cell differentiation and in other biological processes like the development of the fruit fly embryo. In this review, an elementary introduction is given to the concept of criticality in cell differentiation. The correspondence between the signatures of criticality and experimental observations on blood cell differentiation in mice is further highlighted.

Biolistic transformation of a fluorescent tagged gene into the opportunistic fungal pathogen Cryptococcus neoformans.

The basidiomycete Cryptococcus neoformans, an invasive opportunistic pathogen of the central nervous system, is the most frequent cause of fungal meningitis worldwide resulting in more than 625,000 deaths per year worldwide. Although electroporation has been developed for the transformation of plasmids in Cryptococcus, only biolistic delivery provides an effective means to transform linear DNA that can be integrated into the genome by homologous recombination.  Acetate has been shown to be a major fermentation product during cryptococcal infection, but the significance of this is not yet known. A bacterial pathway composed of the enzymes xylulose-5-phosphate/fructose-6-phosphate phosphoketolase (Xfp) and acetate kinase (Ack) is one of three potential pathways for acetate production in C. neoformans. Here, we demonstrate the biolistic transformation of a construct, which has the gene encoding Ack fused to the fluorescent tag mCherry, into C. neoformans. We then confirm integration of the ACK-mCherry fusion into the ACK locus.

Non-genetic heterogeneity, criticality and cell differentiation.

The different cell types in a living organism acquire their identity through the process of cell differentiation in which multipotent progenitor cells differentiate into distinct cell types. Experimental evidence and analysis of large-scale microarray data establish the key role played by a two-gene motif in cell differentiation in a number of cell systems. The two genes express transcription factors which repress each other's expression and autoactivate their own production. A number of theoretical models have recently been proposed based on the two-gene motif to provide a physical understanding of how cell differentiation occurs. In this paper, we study a simple model of cell differentiation which assumes no cooperativity in the regulation of gene expression by the transcription factors. The latter repress each other's activity directly through DNA binding and indirectly through the formation of heterodimers. We specifically investigate how deterministic processes combined with stochasticity contribute in bringing about cell differentiation. The deterministic dynamics of our model give rise to a supercritical pitchfork bifurcation from an undifferentiated stable steady state to two differentiated stable steady states. The stochastic dynamics of our model are studied using the approaches based on the Langevin equations and the linear noise approximation. The simulation results provide a new physical understanding of recent experimental observations. We further propose experimental measurements of quantities like the variance and the lag-1 autocorrelation function in protein fluctuations as the early signatures of an approaching bifurcation point in the cell differentiation process.

Ordered kinetochore assembly in the human-pathogenic basidiomycetous yeast Cryptococcus neoformans.

UNLABELLED: Kinetochores facilitate interaction between chromosomes and the spindle apparatus. The formation of a metazoan trilayered kinetochore is an ordered event in which inner, middle, and outer layers assemble during disassembly of the nuclear envelope during mitosis. The existence of a similar strong correlation between kinetochore assembly and nuclear envelope breakdown in unicellular eukaryotes is unclear. Studies in the hemiascomycetous budding yeasts Saccharomyces cerevisiae and Candida albicans suggest that an ordered kinetochore assembly may not be evolutionarily conserved. Here, we utilized high-resolution time-lapse microscopy to analyze the localization patterns of a series of putative kinetochore proteins in the basidiomycetous budding yeast Cryptococcus neoformans, a human pathogen. Strikingly, similar to most metazoa but atypical of yeasts, the centromeres are not clustered but positioned adjacent to the nuclear envelope in premitotic C. neoformans cells. The centromeres gradually coalesce to a single cluster as cells progress toward mitosis. The mitotic clustering of centromeres seems to be dependent on the integrity of the mitotic spindle. To study the dynamics of the nuclear envelope, we followed the localization of two marker proteins, Ndc1 and Nup107. Fluorescence microscopy of the nuclear envelope and components of the kinetochore, along with ultrastructure analysis by transmission electron microscopy, reveal that in C. neoformans, the kinetochore assembles in an ordered manner prior to mitosis in concert with a partial opening of the nuclear envelope. Taken together, the results of this study demonstrate that kinetochore dynamics in C. neoformans is reminiscent of that of metazoans and shed new light on the evolution of mitosis in eukaryotes. IMPORTANCE: Successful propagation of genetic material in progeny is essential for the survival of any organism. A proper kinetochore-microtubule interaction is crucial for high-fidelity chromosome segregation. An error in this process can lead to loss or gain of chromosomes, a common feature of most solid cancers. Several proteins assemble on centromere DNA to form a kinetochore. However, significant differences in the process of kinetochore assembly exist between unicellular yeasts and multicellular metaozoa. Here, we examined the key events that lead to formation of a proper kinetochore in a basidiomycetous budding yeast, Cryptococcus neoformans. We found that, during the progression of the cell cycle, nonclustered centromeres gradually clustered and kinetochores assembled in an ordered manner concomitant with partial opening of the nuclear envelope in this organism. These events have higher similarity to mitotic events of metazoans than to those previously described in other yeasts.

Noise-induced regime shifts: A quantitative characterization.

Diverse complex dynamical systems are known to exhibit abrupt regime shifts at bifurcation points of the saddle-node type. The dynamics of most of these systems, however, have a stochastic component resulting in noise-driven regime shifts even if the system is away from the bifurcation points. In this paper, we propose a new quantitative measure, namely, the propensity transition point as an indicator of stochastic regime shifts. The concepts and the methodology are illustrated for the one-variable May model, a well-known model in ecology and the genetic toggle, a two-variable model of a simple genetic circuit. The general applicability and usefulness of the method for the analysis of regime shifts is further demonstrated in the case of the mycobacterial switch to persistence for which experimental data are available.

Early signatures of regime shifts in gene expression dynamics.

Recently, a large number of studies have been carried out on the early signatures of sudden regime shifts in systems as diverse as ecosystems, financial markets, population biology and complex diseases. The signatures of regime shifts in gene expression dynamics are less systematically investigated. In this paper, we consider sudden regime shifts in the gene expression dynamics described by a fold-bifurcation model involving bistability and hysteresis. We consider two alternative models, models 1 and 2, of competence development in the bacterial population B. subtilis and determine some early signatures of the regime shifts between competence and noncompetence. We use both deterministic and stochastic formalisms for the purpose of our study. The early signatures studied include the critical slowing down as a transition point is approached, rising variance and the lag-1 autocorrelation function, skewness and a ratio of two mean first passage times. Some of the signatures could provide the experimental basis for distinguishing between bistability and excitability as the correct mechanism for the development of competence.

Efficient implementation of RNA interference in the pathogenic yeast Cryptococcus neoformans.

An improved method has been developed for RNA interference in Cryptococcus neoformans, using opposing promoters to facilitate cloning and RNA interference targeting URA5 to allow selection of cells in which silencing is most effective. These advances significantly reduce the variability of silencing and the effort required for interference plasmid construction.

Phenotypic heterogeneity in mycobacterial stringent response.

BACKGROUND: A common survival strategy of microorganisms subjected to stress involves the generation of phenotypic heterogeneity in the isogenic microbial population enabling a subset of the population to survive under stress. In a recent study, a mycobacterial population of M. smegmatis was shown to develop phenotypic heterogeneity under nutrient depletion. The observed heterogeneity is in the form of a bimodal distribution of the expression levels of the Green Fluorescent Protein (GFP) as reporter with the gfp fused to the promoter of the rel gene. The stringent response pathway is initiated in the subpopulation with high rel activity. RESULTS: In the present study, we characterise quantitatively the single cell promoter activity of the three key genes, namely, mprA, sigE and rel, in the stringent response pathway with gfp as the reporter. The origin of bimodality in the GFP distribution lies in two stable expression states, i.e., bistability. We develop a theoretical model to study the dynamics of the stringent response pathway. The model incorporates a recently proposed mechanism of bistability based on positive feedback and cell growth retardation due to protein synthesis. Based on flow cytometry data, we establish that the distribution of GFP levels in the mycobacterial population at any point of time is a linear superposition of two invariant distributions, one Gaussian and the other lognormal, with only the coefficients in the linear combination depending on time. This allows us to use a binning algorithm and determine the time variation of the mean protein level, the fraction of cells in a subpopulation and also the coefficient of variation, a measure of gene expression noise. CONCLUSIONS: The results of the theoretical model along with a comprehensive analysis of the flow cytometry data provide definitive evidence for the coexistence of two subpopulations with overlapping protein distributions.

Transient pulse formation in jasmonate signaling pathway.

The jasmonate (JA) signaling pathway in plants is activated as defense response to a number of stresses like attacks by pests or pathogens and wounding by animals. Some recent experiments provide significant new knowledge on the molecular detail and connectivity of the pathway. The pathway has two major components in the form of feedback loops, one negative and the other positive. We construct a minimal mathematical model, incorporating the feedback loops, to study the dynamics of the JA signaling pathway. The model exhibits transient gene expression activity in the form of JA pulses in agreement with experimental observations. The dependence of the pulse amplitude, duration and peak time on the key parameters of the model is determined computationally. The deterministic and stochastic aspects of the pathway dynamics are investigated using both the full mathematical model and a reduced version of it. We also compare the mechanism of pulse formation with the known mechanisms of pulse generation in some bacterial and viral systems.

Entanglement in a molecular three-qubit system.

We study the entanglement properties of a molecular three-qubit system described by the Heisenberg spin Hamiltonian with anisotropic exchange interactions and including an external magnetic field. The system exhibits first-order quantum phase transitions by tuning two parameters, x and y, of the Hamiltonian to specific values. The three-qubit chain is open-ended so that there are two types of pairwise entanglement: nearest-neighbour (nn) and next-nearest-neighbour (nnn). We calculate the ground and thermal state concurrences, quantifying pairwise entanglement, as a function of the parameters x, y and the temperature T. The entanglement threshold and gap temperatures are also determined as a function of the anisotropy parameter x. The results obtained are of relevance in understanding the entanglement features of the recently engineered molecular Cr(7)Ni-Cu(2+)-Cr(7)Ni complex which serves as a three-qubit system at sufficiently low temperatures.

Singularity in polarization: rewiring yeast cells to make two buds.

For budding yeast to ensure formation of only one bud, cells must polarize toward one, and only one, site. Polarity establishment involves the Rho family GTPase Cdc42, which concentrates at polarization sites via a positive feedback loop. To assess whether singularity is linked to the specific Cdc42 feedback loop, we disabled the yeast cell's endogenous amplification mechanism and synthetically rewired the cells to employ a different positive feedback loop. Rewired cells violated singularity, occasionally making two buds. Even cells that made only one bud sometimes initiated two clusters of Cdc42, but then one cluster became dominant. Mathematical modeling indicated that, given sufficient time, competition between clusters would promote singularity. In rewired cells, competition occurred slowly and sometimes failed to develop a single "winning" cluster before budding. Slowing competition in normal cells also allowed occasional formation of two buds, suggesting that singularity is enforced by rapid competition between Cdc42 clusters.

Functional characteristics of a double positive feedback loop coupled with autorepression.

We study the functional characteristics of a two-gene motif consisting of a double positive feedback loop and an autoregulatory negative feedback loop. The motif appears in the gene regulatory network controlling the functional activity of pancreatic beta-cells. The model exhibits bistability and hysteresis in appropriate parameter regions. The two stable steady states correspond to low (OFF state) and high (ON state) protein levels, respectively. Using a deterministic approach, we show that the region of bistability increases in extent when the copy number of one of the genes is reduced from 2 to 1. The negative feedback loop has the effect of reducing the size of the bistable region. Loss of a gene copy, brought about by mutations, hampers the normal functioning of the beta-cells giving rise to the genetic disorder, maturity-onset diabetes of the young (MODY). The diabetic phenotype makes its appearance when a sizable fraction of the beta-cells is in the OFF state. Using stochastic simulation techniques we show that, on reduction of the gene copy number, there is a transition from the monostable ON to the ON state in the bistable region of the parameter space. Fluctuations in the protein levels, arising due to the stochastic nature of gene expression, can give rise to transitions between the ON and OFF states. We show that as the strength of autorepression increases, the ON --> OFF state transitions become less probable whereas the reverse transitions are more probable. The implications of the results in the context of the occurrence of MODY are pointed out.

Positive feedback and noise activate the stringent response regulator rel in mycobacteria.

Phenotypic heterogeneity in an isogenic, microbial population enables a subset of the population to persist under stress. In mycobacteria, stresses like nutrient and oxygen deprivation activate the stress response pathway involving the two-component system MprAB and the sigma factor, SigE. SigE in turn activates the expression of the stringent response regulator, rel. The enzyme polyphosphate kinase 1 (PPK1) regulates this pathway by synthesizing polyphosphate required for the activation of MprB. The precise manner in which only a subpopulation of bacterial cells develops persistence, remains unknown. Rel is required for mycobacterial persistence. Here we show that the distribution of rel expression levels in a growing population of mycobacteria is bimodal with two distinct peaks corresponding to low (L) and high (H) expression states, and further establish that a positive feedback loop involving the mprAB operon along with stochastic gene expression are responsible for the phenotypic heterogeneity. Combining single cell analysis by flow cytometry with theoretical modeling, we observe that during growth, noise-driven transitions take a subpopulation of cells from the L to the H state within a "window of opportunity" in time preceding the stationary phase. It is these cells which adapt to nutrient depletion in the stationary phase via the stringent response. We find evidence of hysteresis in the expression of rel in response to changing concentrations of PPK1. Hysteresis promotes robustness in the maintenance of the induced state. Our results provide, for the first time, evidence that bistability and stochastic gene expression could be important for the development of "heterogeneity with an advantage" in mycobacteria and suggest strategies for tackling tuberculosis like targeting transitions from the low to the high rel expression state.