Mycobacterium tuberculosis Response Regulators, DevR and NarL, Interact in Vivo and Co-regulate Gene Expression during Aerobic Nitrate Metabolism

Mycobacterium tuberculosis genes Rv0844c/Rv0845 encoding the NarL response regulator and NarS histidine kinase are hypothesized to constitute a two-component system involved in the regulation of nitrate metabolism. However, there is no experimental evidence to support this. In this study, we established M. tuberculosis NarL/NarS as a functional two-component system and identified His(241) and Asp(61) as conserved phosphorylation sites in NarS and NarL, respectively. Transcriptional profiling between M. tuberculosis H37Rv and Delta narL mutant strain during exponential growth in broth cultures with or without nitrate defined an similar to 30-gene NarL regulon that exhibited significant overlap with DevR-regulated genes, thereby implicating a role for the DevR response regulator in the regulation of nitrate metabolism. Notably, expression analysis of a subset of genes common to NarL and DevR regulons in M. tuberculosis Delta devR, Delta devS Delta dosT, and Delta narL mutant strains revealed that in response to nitrite produced during aerobic nitrate metabolism, the DevRS/DosT regulatory system plays a primary role that is augmented by NarL. Specifically, NarL itself was unable to bind to the narK2, acg, and Rv3130c promoters in phosphorylated or unphosphorylated form; however, its interaction with DevR similar to P resulted in cooperative binding, thereby enabling co-regulation of these genes. These findings support the role of physiologically derived nitrite as a metabolic signal in mycobacteria. We propose NarL-DevR binding, possibly as a heterodimer, as a novel mechanism for co-regulation of gene expression by the DevRS/DosT and NarL/NarS regulatory systems.

LDPC Code Designs Based on root I Matrices

LDPC codes can be constructed by tiling permutation matrices that belong to the square root of identity type and similar algebraic structures. We investigate into the properties of such codes. We also present code structures that are amenable for efficient encoding.

Coding of relative size in monkey inferotemporal cortex

We seldom mistake a closer object as being larger, even though its retinal image is bigger. One underlying mechanism could be to calculate the size of the retinal image relative to that of another nearby object. Here we set out to investigate whether single neurons in the monkey inferotemporal cortex (IT) are sensitive to the relative size of parts in a display. Each neuron was tested on shapes containing two parts that could be conjoined or spatially separated. Each shape was presented in four versions created by combining the two parts at each of two possible sizes. In this design, neurons sensitive to the absolute size of parts would show the greatest response modulation when both parts are scaled up, whereas neurons encoding relative size would show similar responses. Our main findings are that 1) IT neurons responded similarly to all four versions of a shape, but tuning tended to be more consistent between versions with proportionately scaled parts; 2) in a subpopulation of cells, we observed interactions that resulted in similar responses to proportionately scaled parts; 3) these interactions developed together with sensitivity to absolute size for objects with conjoined parts but developed slightly later for objects with spatially separate parts. Taken together, our results demonstrate for the first time that there is a subpopulation of neurons in IT that encodes the relative size of parts in a display, forming a potential neural substrate for size constancy.

A Multiantigenic DNA Vaccine That Induces Broad Hepatitis C Virus-Specific T-Cell Responses in Mice

There are 3 to 4 million new hepatitis C virus (HCV) infections annually around the world, but no vaccine is available. Robust T-cell mediated responses are necessary for effective clearance of the virus, and DNA vaccines result in a cell-mediated bias. Adjuvants are often required for effective vaccination, but during natural lytic viral infections damage-associated molecular patterns (DAMPs) are released, which act as natural adjuvants. Hence, a vaccine that induces cell necrosis and releases DAMPs will result in cell-mediated immunity (CMI), similar to that resulting from natural lytic viral infection. We have generated a DNA vaccine with the ability to elicit strong CMI against the HCV nonstructural (NS) proteins (3, 4A, 4B, and 5B) by encoding a cytolytic protein, perforin (PRF), and the antigens on a single plasmid. We examined the efficacy of the vaccines in C57BL/6 mice, as determined by gamma interferon enzyme-linked immunosorbent spot assay, cell proliferation studies, and intracellular cytokine production. Initially, we showed that encoding the NS4A protein in a vaccine which encoded only NS3 reduced the immunogenicity of NS3, whereas including PRF increased NS3 immunogenicity. In contrast, the inclusion of NS4A increased the immunogenicity of the NS3, NS4B, andNS5B proteins, when encoded in a DNA vaccine that also encoded PRF. Finally, vaccines that also encoded PRF elicited similar levels of CMI against each protein after vaccination with DNA encoding NS3, NS4A, NS4B, and NS5B compared to mice vaccinated with DNA encoding only NS3 or NS4B/5B. Thus, we have developed a promising ``multiantigen'' vaccine that elicits robust CMI. IMPORTANCE Since their development, vaccines have reduced the global burden of disease. One strategy for vaccine development is to use commercially viable DNA technology, which has the potential to generate robust immune responses. Hepatitis C virus causes chronic liver infection and is a leading cause of liver cancer. To date, no vaccine is currently available, and treatment is costly and often results in side effects, limiting the number of patients who are treated. Despite recent advances in treatment, prevention remains the key to efficient control and elimination of this virus. Here, we describe a novel DNA vaccine against hepatitis C virus that is capable of inducing robust cell-mediated immune responses in mice and is a promising vaccine candidate for humans.

State Transition Based Embedding in Cepstrum Domain for Audio Copyright Protection

In this paper, we propose a new state transition based embedding (STBE) technique for audio watermarking with high fidelity. Furthermore, we propose a new correlation based encoding (CBE) scheme for binary logo image in order to enhance the payload capacity. The result of CBE is also compared with standard run-length encoding (RLE) compression and Huffman schemes. Most of the watermarking algorithms are based on modulating selected transform domain feature of an audio segment in order to embed given watermark bit. In the proposed STBE method instead of modulating feature of each and every segment to embed data, our aim is to retain the default value of this feature for most of the segments. Thus, a high quality of watermarked audio is maintained. Here, the difference between the mean values (Mdiff) of insignificant complex cepstrum transform (CCT) coefficients of down-sampled subsets is selected as a robust feature for embedding. Mdiff values of the frames are changed only when certain conditions are met. Hence, almost 50% of the times, segments are not changed and still STBE can convey watermark information at receiver side. STBE also exhibits a partial restoration feature by which the watermarked audio can be restored partially after extraction of the watermark at detector side. The psychoacoustic model analysis showed that the noise-masking ratio (NMR) of our system is less than -10dB. As amplitude scaling in time domain does not affect selected insignificant CCT coefficients, strong invariance towards amplitude scaling attacks is also proved theoretically. Experimental results reveal that the proposed watermarking scheme maintains high audio quality and are simultaneously robust to general attacks like MP3 compression, amplitude scaling, additive noise, re-quantization, etc.

Design of symmetric TIM barrel proteins from first principles

Background: Computational protein design is a rapidly maturing field within structural biology, with the goal of designing proteins with custom structures and functions. Such proteins could find widespread medical and industrial applications. Here, we have adapted algorithms from the Rosetta software suite to design much larger proteins, based on ideal geometric and topological criteria. Furthermore, we have developed techniques to incorporate symmetry into designed structures. For our first design attempt, we targeted the (alpha/beta)(8) TIM barrel scaffold. We gained novel insights into TIM barrel folding mechanisms from studying natural TIM barrel structures, and from analyzing previous TIM barrel design attempts. Methods: Computational protein design and analysis was performed using the Rosetta software suite and custom scripts. Genes encoding all designed proteins were synthesized and cloned on the pET20-b vector. Standard circular dichroism and gel chromatographic experiments were performed to determine protein biophysical characteristics. 1D NMR and 2D HSQC experiments were performed to determine protein structural characteristics. Results: Extensive protein design simulations coupled with ab initio modeling yielded several all-atom models of ideal, 4-fold symmetric TIM barrels. Four such models were experimentally characterized. The best designed structure (Symmetrin-1) contained a polar, histidine-rich pore, forming an extensive hydrogen bonding network. Symmetrin-1 was easily expressed and readily soluble. It showed circular dichroism spectra characteristic of well-folded alpha/beta proteins. Temperature melting experiments revealed cooperative and reversible unfolding, with a T-m of 44 degrees C and a Gibbs free energy of unfolding (Delta G degrees) of 8.0 kJ/mol. Urea denaturing experiments confirmed these observations, revealing a C-m of 1.6 M and a Delta G degrees of 8.3 kJ/mol. Symmetrin-1 adopted a monomeric conformation, with an apparent molecular weight of 32.12 kDa, and displayed well resolved 1D-NMR spectra. However, the HSQC spectrum revealed somewhat molten characteristics. Conclusions: Despite the detection of molten characteristics, the creation of a soluble, cooperatively folding protein represents an advancement over previous attempts at TIM barrel design. Strategies to further improve Symmetrin-1 are elaborated. Our techniques may be used to create other large, internally symmetric proteins.

Simultaneous presence of fhs and purT genes is disadvantageous for the fitness of Escherichia coli growth

In bacteria, alternate mechanisms are known to synthesize N-10-formyltetrahydrofolate (N10-formyl-THF) and formyl glycinamide ribotide (fGAR), which are important in purine biosynthesis. In one of the mechanisms, a direct transfer of one carbon unit from formate allows Fhs to convert tetrahydrofolate to N-10-formyl-THF, and PurT to convert glycinamide ribotide (GAR) to fGAR. Our bioinformatics analysis of fhs and purT genes (encoding Fhs and PurT) showed that in a majority of bacteria (similar to 94%), their presence was mutually exclusive. A large number of organisms possessing fhs lacked purT and vice versa. The phenomenon is so penetrating that even within a genus (Bacillus) if a species possessed fhs it lacked purT and vice versa. To investigate physiological importance of this phenomenon, we used Escherichia coli, which naturally lacks fhs (and possesses purT) as model. We generated strains, which possessed fhs and purT genes in singles or together. Deletion of purT from E. coli in the presence or absence of fhs did not confer a detectable growth disadvantage in pure cultures. However, growth competition assays revealed that the strains possessing either of the single genes outcompeted those possessing both the genes suggesting that mutual exclusion of purT and fhs in organisms confers fitness advantage in mixed cultures.

A Cause and Effect Analysis of Motion Trajectories for Modeling Actions

An action is typically composed of different parts of the object moving in particular sequences. The presence of different motions (represented as a 1D histogram) has been used in the traditional bag-of-words (BoW) approach for recognizing actions. However the interactions among the motions also form a crucial part of an action. Different object-parts have varying degrees of interactions with the other parts during an action cycle. It is these interactions we want to quantify in order to bring in additional information about the actions. In this paper we propose a causality based approach for quantifying the interactions to aid action classification. Granger causality is used to compute the cause and effect relationships for pairs of motion trajectories of a video. A 2D histogram descriptor for the video is constructed using these pairwise measures. Our proposed method of obtaining pairwise measures for videos is also applicable for large datasets. We have conducted experiments on challenging action recognition databases such as HMDB51 and UCF50 and shown that our causality descriptor helps in encoding additional information regarding the actions and performs on par with the state-of-the art approaches. Due to the complementary nature, a further increase in performance can be observed by combining our approach with state-of-the-art approaches.

Asparagine requirement in Plasmodium berghei as a target to prevent malaria transmission and liver infections

The proteins of Plasmodium, the malaria parasite, are strikingly rich in asparagine. Plasmodium depends primarily on host haemoglobin degradation for amino acids and has a rudimentary pathway for amino acid biosynthesis, but retains a gene encoding asparagine synthetase (AS). Here we show that deletion of AS in Plasmodium berghei (Pb) delays the asexual-and liver-stage development with substantial reduction in the formation of ookinetes, oocysts and sporozoites in mosquitoes. In the absence of asparagine synthesis, extracellular asparagine supports suboptimal survival of PbAS knockout (KO) parasites. Depletion of blood asparagine levels by treating PbASKO-infected mice with asparaginase completely prevents the development of liver stages, exflagellation of male gametocytes and the subsequent formation of sexual stages. In vivo supplementation of asparagine in mice restores the exflagellation of PbASKO parasites. Thus, the parasite life cycle has an absolute requirement for asparagine, which we propose could be targeted to prevent malaria transmission and liver infections.

Functional expression and purification of Anabaena PCC 7120 XisA protein

Anabaena PCC 7120 xisA gene product mediates the site-specific excision of 11,278 bp nifD element in heterocysts formed under nitrogen starvation conditions. Although XisA protein possesses both site-specific recombinase and endonuclease activities, till date neither xisA transcript nor XisA protein has been detected. Gene encoding XisA protein was isolated from plasmid pMX25 and overexpressed in Escherichia coli BL21 DE3 yielding 7.7 mg enzyme per L of growth culture in soluble fraction. His-tagged XisA was purified using Ni-NTA affinity chromatography with 95% recovery. The purified XisA showed a single band on SDS-PAGE with molecular mass of 52 kDa. Identity of XisA was confirmed by MALDI-TOF analysis and functionality of enzyme was confirmed using restriction digestion. A PCR based method was developed to monitor excision by XisA, which displayed near 100% activity in E. coli within 1 h at 37 degrees C on LB under static condition. (C) 2015 Elsevier Inc. All rights reserved.

An Endophytic Fungus, Talaromyces radicus, Isolated from Catharanthus roseus, Produces Vincristine and Vinblastine, Which Induce Apoptotic Cell Death

Endophytic fungi isolated from Catharanthus roseus were screened for the production of vincristine and vinblastine. Twenty-two endophytic fungi isolated from various tissues of C. roseus were characterized taxonomically by sequence analysis of the internal transcribed spacer (ITS) region of rDNA and grouped into 10 genera: Alternaria, Aspergillus, Chaetomium, Colletotrichum, Dothideomycetes, Eutypella, Eutypa, Flavodon, Fusarium and Talaromyces. The antiproliferative activity of these fungi was assayed in HeLa cells using the MTT assay. The fungal isolates Eutypella sp-CrP14, obtained from stem tissues, and Talaromyces radicus-CrP20, obtained from leaf tissues, showed the strongest antiproliferative activity, with IC50 values of 13.5 mu g/ml and 20 mu g/ml, respectively. All 22 endophytic fungi were screened for the presence of the gene encoding tryptophan decarboxylase (TDC), the key enzyme in the terpenoid indole alkaloid biosynthetic pathway, though this gene could only be amplified from T. radicus-CrP20 (NCBI GenBank accession number KC920846). The production of vincristine and vinblastine by T. radicus-CrP20 was confirmed and optimized in nine different liquid media. Good yields of vincristine (670 mu g/l) in modified M2 medium and of vinblastine (70 mu g/l) in potato dextrose broth medium were obtained. The cytotoxic activity of partially purified fungal vincristine was evaluated in different human cancer cell lines, with HeLa cells showing maximum susceptibility. The apoptosis-inducing activity of vincristine derived from this fungus was established through cell cycle analysis, loss of mitochondrial membrane potential and DNA fragmentation patterns.

The mycobacterial iron-dependent regulator IdeR induces ferritin (bfrB) by alleviating Lsr2 repression

Emerging evidence indicates that precise regulation of iron (Fe) metabolism and maintenance of Fe homeostasis in Mycobacterium tuberculosis (Mtb) are essential for its survival and proliferation in the host. IdeR is a central transcriptional regulator of Mtb genes involved in Fe metabolism. While it is well understood how IdeR functions as a repressor, how it induces transcription of a subset of its targets is still unclear. We investigated the molecular mechanism of IdeR-mediated positive regulation of bfrB, the gene encoding the major Fe-storage protein of Mtb. We found that bfrB induction by Fe required direct interaction of IdeR with a DNA sequence containing four tandem IdeR-binding boxes located upstream of the bfrB promoter. Results of in vivo and in vitro transcription assays identified a direct repressor of bfrB, the histone-like protein Lsr2. IdeR counteracted Lsr2-mediated repression in vitro, suggesting that IdeR induces bfrB transcription by antagonizing the repressor activity of Lsr2. Together, these results elucidate the main mechanism of bfrB positive regulation by IdeR and identify Lsr2 as a new factor contributing to Fe homeostasis in mycobacteria.

The Feedback Capacity of the Binary Erasure Channel With a No-Consecutive-Ones Input Constraint

The input-constrained erasure channel with feedback is considered, where the binary input sequence contains no consecutive ones, i.e., it satisfies the (1, infinity)-RLL constraint. We derive the capacity for this setting, which can be expressed as C-is an element of = max(0 <= p <= 0.5) (1-is an element of) H-b (p)/1+(1-is an element of) p, where is an element of is the erasure probability and Hb(.) is the binary entropy function. Moreover, we prove that a priori knowledge of the erasure at the encoder does not increase the feedback capacity. The feedback capacity was calculated using an equivalent dynamic programming (DP) formulation with an optimal average-reward that is equal to the capacity. Furthermore, we obtained an optimal encoding procedure from the solution of the DP, leading to a capacity-achieving, zero-error coding scheme for our setting. DP is, thus, shown to be a tool not only for solving optimization problems, such as capacity calculation, but also for constructing optimal coding schemes. The derived capacity expression also serves as the only non-trivial upper bound known on the capacity of the input-constrained erasure channel without feedback, a problem that is still open.

SYNGAP1: Mind the Gap

A cardinal feature of early stages of human brain development centers on the sensory, cognitive, and emotional experiences that shape neuronal-circuit formation and refinement. Consequently, alterations in these processes account for many psychiatric and neurodevelopmental disorders. Neurodevelopment disorders affect 3-4% of the world population. The impact of these disorders presents a major challenge to clinicians, geneticists, and neuroscientists. Mutations that cause neurodevelopmental disorders are commonly found in genes encoding proteins that regulate synaptic function. Investigation of the underlying mechanisms using gain or loss of function approaches has revealed alterations in dendritic spine structure, function, and plasticity, consequently modulating the neuronal circuit formation and thereby raising the possibility of neurodevelopmental disorders resulting from synaptopathies. One such gene, SYNGAP1 (Synaptic Ras-GTPase-activating protein) has been shown to cause Intellectual Disability (ID) with comorbid Autism Spectrum Disorder (ASD) and epilepsy in children. SYNGAP1 is a negative regulator of Ras, Rap and of AMPA receptor trafficking to the postsynaptic membrane, thereby regulating not only synaptic plasticity, but also neuronal homeostasis. Recent studies on the neurophysiology of SYNGAP1, using Syngapl mouse models, have provided deeper insights into how downstream signaling proteins and synaptic plasticity are regulated by SYNGAP1. This knowledge has led to a better understanding of the function of SYNGAP1 and suggests a potential target during critical period of development when the brain is more susceptible to therapeutic intervention.

SYNGAP1: Mind the Gap

A cardinal feature of early stages of human brain development centers on the sensory, cognitive, and emotional experiences that shape neuronal-circuit formation and refinement. Consequently, alterations in these processes account for many psychiatric and neurodevelopmental disorders. Neurodevelopment disorders affect 3-4% of the world population. The impact of these disorders presents a major challenge to clinicians, geneticists, and neuroscientists. Mutations that cause neurodevelopmental disorders are commonly found in genes encoding proteins that regulate synaptic function. Investigation of the underlying mechanisms using gain or loss of function approaches has revealed alterations in dendritic spine structure, function, and plasticity, consequently modulating the neuronal circuit formation and thereby raising the possibility of neurodevelopmental disorders resulting from synaptopathies. One such gene, SYNGAP1 (Synaptic Ras-GTPase-activating protein) has been shown to cause Intellectual Disability (ID) with comorbid Autism Spectrum Disorder (ASD) and epilepsy in children. SYNGAP1 is a negative regulator of Ras, Rap and of AMPA receptor trafficking to the postsynaptic membrane, thereby regulating not only synaptic plasticity, but also neuronal homeostasis. Recent studies on the neurophysiology of SYNGAP1, using Syngapl mouse models, have provided deeper insights into how downstream signaling proteins and synaptic plasticity are regulated by SYNGAP1. This knowledge has led to a better understanding of the function of SYNGAP1 and suggests a potential target during critical period of development when the brain is more susceptible to therapeutic intervention.