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Background: Aphthous stomatitis or mouth ulcers is the most common condition that we encounter. Clinically the lesions are single or multiple superficial and deep sealed and are associated with microbial invasions. Aim: This study was conducted with the aim of evaluating the effectiveness of herbal drugs for treatment of Aphthous stomatitis. Materials and Methods: In the research work, mouth ulcer gels were formulated incorporating the ethanolic extracts of as Aloe barbedensis, Ocimum tenuiflorum and Azadirachta indica using carbopol 934 as the gelling agent. Seven batches were formulated by varying the concentration of the herbal ingredients (F1 to F7)The prepared formulations were evaluated for various parameters like physical appearance, pH, Spreadability, Homogeneity and antimicrobial activity against fungi and bacteria. The antimicrobial activity was also compared with a marketed gel formulation. Results and Discussion: All the prepared formulation using different concentration of plant extract showed the pH values in between 6.10.2 to 7.00.1. The spreadability values ranged between the 5.0 to 8.0 cm. Out of all the formulations, formulation F7 containing all the three herbal extracts showed a good spreadability and very promising antimicrobial activity comparable with a marketed gel. Conclusion: Thus stable, effective gels containing herbal ingredients for management of mouth ulcers can be developed. Keywords: Aphthous stomatitis, Aloe barbedensis, Ocimum tenuiflorum, Azadirachta indica, Carbopol 940.

Cells use signaling pathways to receive and process information about their environment. These nonlinear systems rely on feedback and feedforward regulation to respond appropriately to changing environmental conditions. Mathematical models describing signaling pathways often lack predictive power because they are not trained on data that encompass the diverse time scales on which these regulatory mechanisms operate. We addressed this limitation by measuring transcriptional changes induced by the mating response in Saccharomyces cerevisiae exposed to different dynamic patterns of pheromone. We found that pheromone-induced transcription persisted after pheromone removal and showed long-term adaptation upon sustained pheromone exposure. We developed a model of the regulatory network that captured both characteristics of the mating response. We fit this model to experimental data with an evolutionary algorithm and used the parameterized model to predict scenarios for which it was not trained, including different temporal stimulus profiles and genetic perturbations to pathway components. Our model allowed us to establish the role of four architectural elements of the network in regulating gene expression. These network motifs are incoherent feedforward, positive feedback, negative feedback, and repressor binding. Experimental and computational perturbations to these network motifs established a specific role for each in coordinating the mating response to persistent and dynamic stimulation.

Cells use signaling pathways to receive and process information about their environment. These systems are nonlinear, relying on feedback and feedforward regulation to respond appropriately to changing environmental conditions. Mathematical models developed to describe signaling pathways often fail to show predictive power, because the models are not trained on data that probe the diverse time scales on which feedforward and feedback regulation operate. We addressed this limitation using microfluidics to expose cells to a broad range of dynamic environmental conditions. In particular, we focus on the well-characterized mating response pathway of S. cerevisiae (yeast). This pathway is activated by mating pheromone and initiates the transcriptional changes required for mating. Although much is known about the molecular components of the mating response pathway, less is known about how these components function as a dynamical system. Our experimental data revealed that pheromone-induced transcription persists following removal of pheromone and that long-term adaptation of the transcriptional response occurs when pheromone exposure is sustained. We developed a model of the regulatory network that captured both persistence and long-term adaptation of the mating response. We fit this model to experimental data using an evolutionary algorithm and used the parameterized model to predict scenarios for which it was not trained, including different temporal stimulus profiles and genetic perturbations to pathway components. Our model allowed us to establish the role of four regulatory motifs in coordinating pathway response to persistent and dynamic stimulation.Competing Interest StatementThe authors have declared no competing interest.View Full Text

The risk stratification of prostate cancer and breast cancer tumours from patients relies on histopathology, selective genomic testing, or on other methods employing fixed formalin tissue samples. However, static biomarker measurements from bulk fixed-tissue samples provide limited accuracy and actionability. Here, we report the development of a live-primary-cell phenotypic-biomarker assay with single-cell resolution, and its validation with prostate cancer and breast cancer tissue samples for the prediction of post-surgical adverse pathology. The assay includes a collagen-I/fibronectin extracellular-matrix formulation, dynamic live-cell biomarkers, a microfluidic device, machine-vision analysis and machine-learning algorithms, and generates predictive scores of adverse pathology at the time of surgery. Predictive scores for the risk stratification of 59 prostate cancer patients and 47 breast cancer patients, with values for area under the curve in receiver-operating-characteristic curves surpassing 80%, support the validation of the assay and its potential clinical applicability for the risk stratification of cancer patients.

MAPKs are activated by dual phosphorylation. Much of the MAPK Fus3 is monophosphorylated and acts to inhibit signaling in vivo. Computational models reveal how a kinase scaffold and phosphatase act together to dynamically regulate dual-phosphorylated and monophosphorylated MAPKs and the downstream signal., Protein kinases regulate a broad array of cellular processes and do so through the phosphorylation of one or more sites within a given substrate. Many protein kinases are themselves regulated through multisite phosphorylation, and the addition or removal of phosphates can occur in a sequential (processive) or a stepwise (distributive) manner. Here we measured the relative abundance of the monophosphorylated and dual-phosphorylated forms of Fus3, a member of the mitogen-activated protein kinase (MAPK) family in yeast. We found that upon activation with pheromone, a substantial proportion of Fus3 accumulates in the monophosphorylated state. Introduction of an additional copy of Fus3 lacking either phosphorylation site leads to dampened signaling. Conversely, cells lacking the dual-specificity phosphatase (msg5Δ) or that are deficient in docking to the MAPK-scaffold (Ste5ND) accumulate a greater proportion of dual-phosphorylated Fus3. The double mutant exhibits a synergistic, or “synthetic,” supersensitivity to pheromone. Finally, we present a predictive computational model that combines MAPK scaffold and phosphatase activities and is sufficient to account for the observed MAPK profiles. These results indicate that the monophosphorylated and dual-phosphorylated forms of the MAPK act in opposition to one another. Moreover, they reveal a new mechanism by which the MAPK scaffold acts dynamically to regulate signaling.

G proteins and their associated receptors process information from a variety of environmental stimuli to induce appropriate cellular responses. Generally speaking, each cell in a population responds within defined limits, despite large variation in the expression of protein signaling components. Therefore, we postulated that noise suppression is encoded within the signaling system. Using the yeast mating pathway as a model, we evaluated the ability of a regulator of G protein signaling (RGS) protein to suppress noise. We found that the RGS protein Sst2 limits variability in transcription and morphogenesis in response to pheromone stimulation. While signal suppression is a result of both the GAP (GTPase accelerating) and receptor binding functions of Sst2, noise suppression requires only the GAP activity. Taken together, our findings reveal a hitherto overlooked role of RGS proteins as noise suppressors and demonstrate an ability to uncouple signal and noise in a prototypical stimulus-response pathway.

Heterotrimeric G proteins are well known to transmit signals from cell surface receptors to intracellular effector proteins. There is growing appreciation that G proteins are also present at endomembrane compartments, where they can potentially interact with a distinct set of signaling proteins. Here, we examine the cellular trafficking function of the G protein α subunit in yeast, Gpa1. Gpa1 contains a unique 109-amino acid insert within the α-helical domain that undergoes a variety of posttranslational modifications. Among these is monoubiquitination, catalyzed by the NEDD4 family ubiquitin ligase Rsp5. Using a newly optimized method for G protein purification together with biophysical measures of structure and function, we show that the ubiquitination domain does not influence enzyme activity. By screening a panel of 39 gene deletion mutants, each lacking a different ubiquitin binding domain protein, we identify seven that are necessary to deliver Gpa1 to the vacuole compartment including four proteins (Ede1, Bul1, Ddi1, and Rup1) previously not known to be involved in this process. Finally, we show that proper endocytosis of the G protein is needed for sustained cellular morphogenesis and mating in response to pheromone stimulation. We conclude that a cascade of ubiquitin-binding proteins serves to deliver the G protein to its final destination within the cell. In this instance and in contrast to the previously characterized visual system, endocytosis from the plasma membrane is needed for proper signal transduction rather than for signal desensitization.

This paper reports the simulative analyses to investigate the impact of different pulse shapes on DP-DQPSK modulation for high-spectral efficient DWDM transmission at 112 Gbps per channel. In the simulation model we have considered three different pulse shapes viz. NRZ, 50% duty cycle RZ (RZ50) and 67% duty cycle RZ (RZ67) in symbol-aligned and symbol-interleaved format. The analysis has been carried out for various linear and nonlinear system impairments where the pulse is subjected to degradation under the influence of amplified spontaneous emission (ASE) noise, group velocity dispersion (GVD), cross-phase modulation (XPM) and polarization mode dispersion (PMD) and thus, limiting the system performance. Results show that the RZ50 pulse shape for DP-DQPSK with symbol interleaving shows the maximum tolerance for the various system degradations in long haul DWDM transmission. Even the system based on the NRZ pulse shape can gain significant improvement from the symbol-interleaving.

Background: Probiotics are live microorganisms present in food and dietary supplements that beneficially affect the individual by improving the intestinal microbial balance properties. Their market value and biological potential is enormous because of their health-promoting properties. Therefore, comparative studies on probiotic potential of three selected strains of Lactobacillus acidophilus were carried out during this work. Method: The selected strains of Lactobacillus acidophilus (NCIM 2660, NCIM 2903 and NCIM 2285) were assessed for tolerance to pH (pH 2.5) and oxgall (0.3%), adhesion to Caco-2 cells, antimicrobial properties against test microorganisms and susceptibility to antibiotics. Results: The distinguished characteristics of strain NCIM 2903 were high acid tolerance, antagonistic activity against all test micro-organisms and adhesion to Caco-2 cells (225±33 cells adhering to 100 Caco-2 cells). Strain NCIM 2285 exhibited bile tolerance and inability to grow in presence of five of the antibiotics used. Strain NCIM 2285 indicated moderate adhesion to Caco-2 cells. The strain NCIM 2660 was unable to grow in presence of any of the twelve antibiotics. In contrast, control plates without discs of antibiotics exhibited confluent growth. It was a desirable feature. However, NCIM 2660 exhibited poor adhesive property. The optimum temperature of growth for all three strains was found to be 37oC. Conclusions: Significant differences in the probiotic characteristics of the three strains were recorded. In the cumulative assessment, strain NCIM 2903 could be regarded as the potential probiotic contender. These studies will help in selecting the probiotic micro organisms in a more rational manner for further applications.

G protein–coupled receptor signaling is negatively regulated by both receptor internalization and regulator of G protein signaling (RGS) protein–stimulated inactivation of the G protein. The RGS protein can also positively regulate receptor signaling by binding to the receptor, thereby reducing receptor internalization., G protein–coupled receptor (GPCR) signaling is fundamental to physiological processes such as vision, the immune response, and wound healing. In the budding yeast Saccharomyces cerevisiae, GPCRs detect and respond to gradients of pheromone during mating. After pheromone stimulation, the GPCR Ste2 is removed from the cell membrane, and new receptors are delivered to the growing edge. The regulator of G protein signaling (RGS) protein Sst2 acts by accelerating GTP hydrolysis and facilitating pathway desensitization. Sst2 is also known to interact with the receptor Ste2. Here we show that Sst2 is required for proper receptor recovery at the growing edge of pheromone-stimulated cells. Mathematical modeling suggested pheromone-induced synthesis of Sst2 together with its interaction with the receptor function to reestablish a receptor pool at the site of polarized growth. To validate the model, we used targeted genetic perturbations to selectively disrupt key properties of Sst2 and its induction by pheromone. Together our results reveal that a regulator of G protein signaling can also regulate the G protein–coupled receptor. Whereas Sst2 negatively regulates G protein signaling, it acts in a positive manner to promote receptor retention at the growing edge.

Summary Background Septins are well known to form a boundary between mother and daughter cells in mitosis, but their role in other morphogenic states is poorly understood. Results Using microfluidics and live-cell microscopy, coupled with new computational methods for image analysis, we investigated septin function during pheromone-dependent chemotropic growth in yeast. We show that septins colocalize with the regulator of G protein signaling (RGS) Sst2, a GTPase-activating protein that dampens pheromone receptor signaling. We show further that the septin structure surrounds the polar cap, ensuring that cell growth is directed toward the source of pheromone. When RGS activity is abrogated, septins are partially disorganized. Under these circumstances, the polar cap travels toward septin structures and away from sites of exocytosis, resulting in a loss of gradient tracking. Conclusions Septin organization is dependent on RGS protein activity. When assembled correctly, septins promote turning of the polar cap and proper tracking of a pheromone gradient.

Extracellular signals, such as nutrients and hormones, cue intracellular pathways to produce adaptive responses. Often, cells must coordinate their responses to multiple signals to produce an appropriate outcome. We showed that components of a glucose-sensing pathway acted on components of a heterotrimeric guanine nucleotide–binding protein (G protein)–mediated pheromone signaling pathway in the yeast Saccharomyces cerevisiae. We demonstrated that the G protein α subunit Gpa1 was phosphorylated in response to conditions of reduced glucose availability and that this phosphorylation event contributed to reduced pheromone-dependent stimulation of mitogen-activated protein kinases, gene transcription, cell morphogenesis, and mating efficiency. We found that Elm1, Sak1, and Tos3, the kinases that phosphorylate Snf1, the yeast homolog of adenosine monophosphate–activated protein kinase (AMPK), in response to limited glucose availability, also phosphorylated Gpa1 and contributed to the diminished mating response. Reg1, the regulatory subunit of the phosphatase PP1 that acts on Snf1, was likewise required to reverse the phosphorylation of Gpa1 and to maintain the mating response. Thus, the same kinases and phosphatase that regulate Snf1 also regulate Gpa1. More broadly, these results indicate that the pheromone signaling and glucose-sensing pathways communicate directly to coordinate cell behavior.

e16567Background: A novel tissue based biomarker panel is developed to objectively assess disease aggressiveness and invasive potential of Prostate Cancer (CaP). The biomarker diagnostic platform i...

338 Background: A novel tissue based biomarker panel is introduced to objectively assess disease aggressiveness and invasive potential of Prostate Cancer (CaP). The biomarker diagnostic platform incorporates both molecular and phenotypic data that may allow an improved understanding of local growth and metastatic potential. The tissue based diagnostic incorporates matrix biology, phenotypic biomarkers, microfluidics, and machine vision. This technology presents the opportunity to culture samples, and both determine and automate biomarker measurements from machine vision algorithm analysis. Data are presented towards clinical validation, the ability to risk stratify, and prediction of local aggressiveness and metastasis. Methods: Conditions were optimized for reliably culturing primary cancer cells in vitro by simulating in vivo conditions on an extracellular matrix formulation. A mcirofluidics device was used to culture live tumor samples ex vivo enabling automated imaging of the label free and label-based biomarkers. Results: The validation study was IRB approved and performed in 200 consecutive CaP radical prostatectomy derived specimens collected between 03/2014 and 09/2015. Data was analyzed with receiver operating characteristics (ROC) generated Area-under-the-Curve (AUC) and specifically included capsular penetration, seminal vesicle invasion, as well as margin-positive disease. AUC Graphs are presented. The study further demonstrated that a normal set of phenotypic biomarkers can produce secondary metrics termed oncogenic potential (OP) and metastatic potential (MP). Concordance analysis supports that OP and MP are integral for distinguishing between benign histology and malignancy, predicting both stage and adverse pathology such as extra-prostatic extension (EPE) and lympho-vascular invasion (LVI). The study results demonstrate AUCs greater than 0.90 in predicting EPE and LVI. Conclusions: Results support the clinical validation of a novel live- cell phenotypic in vitro tumor diagnostic test. This test has the potential to predict adverse pathologies for CaP and may have extended clinical applications to optimize staging and risk stratification.