Basic techniques associated with studying transcription elongation both in vitro and in vivo within mammalian cells.

All cellular functions and identity of every cell are directly or indirectly depend on its gene expression. Therefore, cells control their gene expression very finely at multiple layers. Cells always fine tune its gene expression profile depending on the internal and external cues to maintain best possible cellular growth condition. Regulation of mRNA production is a major step in the control of gene expression. mRNA production primarily depends on two factors. One is the level of RNA polymerase II (Pol II hereafter) recruitment at the promoter region and another is the amount of Pol II successfully elongating through the whole gene body also known as coding region. There are several proteins (individually or as part of a complex) which control elongation of Pol II both positively or negatively. It is important to understand how different transcription factors regulate this elongation step since this knowledge is important for understanding different cellular functions both under basal and stimulus-dependent contexts. Here, we have discussed both in vitro and in vivo techniques which can be used to study the effect of different factors on Pol II-mediated transcription elongation. In vitro techniques give us valuable information about the ability of a transcription factor or a complex to exert its direct effect on the overall processes. In vivo techniques give us an understanding about the effect of a transcription factor or a complex in its native condition where functions of a transcription factor can be influenced by many other factors including its associated ones.

ATM-mediated ELL phosphorylation enhances its self-association through increased EAF1 interaction and inhibits global transcription during genotoxic stress.

Mammalian cells immediately inhibit transcription upon exposure to genotoxic stress to avoid fatal collision between ongoing transcription and newly recruited DNA repair machineries to protect genomic integrity. However, mechanisms of this early transcriptional inhibition are poorly understood. In this study, we decipher a novel role of human EAF1, a positive regulator of ELL-dependent RNA Polymerase II-mediated transcription in vitro, in regulation of temporal inhibition of transcription during genotoxic stress. Our results show that, besides Super Elongation Complex (SEC) and Little Elongation Complex (LEC), human ELL (aka ELL1) also forms a complex with EAF1 alone. Interestingly, contrary to the in vitro studies, EAF1 inhibits ELL-dependent RNA polymerase II-mediated transcription of diverse target genes. Mechanistically, we show that intrinsic self-association property of ELL leads to its reduced interaction with other SEC components. EAF1 enhances ELL self-association and thus reduces its interaction with other SEC components leading to transcriptional inhibition. Physiologically, we show that upon exposure to genotoxic stress, ATM-mediated ELL phosphorylation-dependent enhanced EAF1 association results in reduced ELL interaction with other SEC components that lead to global transcriptional inhibition. Thus, we describe an important mechanism of dynamic transcriptional regulation during genotoxic stress involving post-translational modification of a key elongation factor.

Plant-Derived Phenolic Acids Limit the Pathogenesis of Salmonella Typhimurium and Protect Intestinal Epithelial Cells during Their Interactions.

The incidence of gastrointestinal illness attributable to Salmonella enterica serovar Typhimurium (ST) remains a concern for public health worldwide, as it can progress into systemic infections mediated by the type-three secretion system (T3SS), which allows for adherence and invasion to intestinal epithelial cells. The current study evaluates the ability of gallic acid (GA), protocatechuic acid (PA), and vanillic acid (VA) to impair the adhesion and invasion abilities of ST to a human epithelial (INT-407) cell monolayer while also assessing their cytotoxicity. GA, PA, and VA inhibited detectable ST growth at specific concentrations but showed cytotoxicity against INT-407 cells (>20% reduction in viability) after 3 h of treatments. Adjusting the pH of the solutions had a neutralizing effect on cytotoxicity, though it did reduce their antimicrobial potency. Adhesion of ST was reduced significantly when the cells were treated with 4.0 mg/mL of VA, whereas invasion was reduced in all treatments, with GA requiring the lowest concentration (0.5 mg/mL). Relative gene expression of virulence genes after treatment with GA showed downregulation in the T3SS regulator and effector hilA and sipA, respectively. These findings suggest further use of phenolic acids in reducing the activity of key virulence factors critical during ST infection.

DBC1, p300, HDAC3, and Siah1 coordinately regulate ELL stability and function for expression of its target genes.

Among all of the Super Elongation Complex (SEC) components, ELL1 (also known as ELL) is the only bona fide elongation factor that directly stimulates transcription elongation by RNA polymerase II. However, the mechanism(s) of functional regulation of ELL1 (referred to as ELL hereafter), through its stabilization, is completely unknown. Here, we report a function of human DBC1 in regulating ELL stability involving HDAC3, p300, and Siah1. Mechanistically, we show that p300-mediated site-specific acetylation increases, whereas HDAC3-mediated deacetylation decreases, ELL stability through polyubiquitylation by the E3 ubiquitin ligase Siah1. DBC1 competes with HDAC3 for the same binding sites on ELL and thus increases its acetylation and stability. Knockdown of DBC1 reduces ELL levels and expression of a significant number of genes, including those involved in glucose metabolism. Consistently, Type 2 diabetes patient-derived peripheral blood mononuclear cells show reduced expression of DBC1 and ELL and associated key target genes required for glucose homeostasis. Thus, we describe a pathway of regulating stability and functions of key elongation factor ELL for expression of diverse sets of genes, including ones that are linked to Type 2 diabetes pathogenesis.

Space-dependent intermittent feedback can control birhythmicity.

Birhythmicity is evident in many nonlinear systems, which include physical and biological systems. In some living systems, birhythmicity is necessary for response to the varying environment while unnecessary in some physical systems as it limits their efficiency. Therefore, its control is an important area of research. This paper proposes a space-dependent intermittent control scheme capable of controlling birhythmicity in various dynamical systems. We apply the proposed control scheme in five nonlinear systems from diverse branches of natural science and demonstrate that the scheme is efficient enough to control the birhythmic oscillations in all the systems. We derive the analytical condition for controlling birhythmicity by applying harmonic decomposition and energy balance methods in a birhythmic van der Pol oscillator. Further, the efficacy of the control scheme is investigated through numerical and bifurcation analyses in a wide parameter space. Since the proposed control scheme is general and efficient, it may be employed to control birhythmicity in several dynamical systems.

AFF1 acetylation by p300 temporally inhibits transcription during genotoxic stress response.

Soon after exposure to genotoxic reagents, mammalian cells inhibit transcription to prevent collisions with repair machinery and to mount a proper DNA damage response. However, mechanisms underlying early transcriptional inhibition are poorly understood. In this report, we show that site-specific acetylation of super elongation complex (SEC) subunit AFF1 by p300 reduces its interaction with other SEC components and impairs P-TEFb-mediated C-terminal domain phosphorylation of RNA polymerase II both in vitro and in vivo. Reexpression of wild-type AFF1, but not an acetylation mimic mutant, restores SEC component recruitment and target gene expression in AFF1 knockdown cells. Physiologically, we show that, upon genotoxic exposure, p300-mediated AFF1 acetylation is dynamic and strongly correlated with concomitant global down-regulation of transcription-and that this can be reversed by overexpression of an acetylation-defective AFF1 mutant. Therefore, we describe a mechanism of dynamic transcriptional regulation involving p300-mediated acetylation of a key elongation factor during genotoxic stress.

Impulsive feedback control of birhythmicity: Theory and experiment.

We study the dynamic control of birhythmicity under an impulsive feedback control scheme where the feedback is made ON for a certain rather small period of time and for the rest of the time, it is kept OFF. We show that, depending on the height and width of the feedback pulse, the system can be brought to any of the desired limit cycles of the original birhythmic oscillation. We derive a rigorous analytical condition of controlling birhythmicity using the harmonic decomposition and energy balance methods. The efficacy of the control scheme is investigated through numerical analysis in the parameter space. We demonstrate the robustness of the control scheme in a birhythmic electronic circuit where the presence of noise and parameter fluctuations are inevitable. Finally, we demonstrate the applicability of the control scheme in controlling birhythmicity in diverse engineering and biochemical systems and processes, such as an energy harvesting system, a glycolysis process, and a p53-mdm2 network.

Effects of propagation delay in coupled oscillators under direct-indirect coupling: Theory and experiment.

Propagation delay arises in a coupling channel due to the finite propagation speed of signals and the dispersive nature of the channel. In this paper, we study the effects of propagation delay that appears in the indirect coupling path of direct (diffusive)-indirect (environmental) coupled oscillators. In sharp contrast to the direct coupled oscillators where propagation delay induces amplitude death, we show that in the case of direct-indirect coupling, even a small propagation delay is conducive to an oscillatory behavior. It is well known that simultaneous application of direct and indirect coupling is the general mechanism for amplitude death. However, here we show that the presence of propagation delay hinders the death state and helps the revival of oscillation. We demonstrate our results by considering chaotic time-delayed oscillators and FitzHugh-Nagumo oscillators. We use linear stability analysis to derive the explicit conditions for the onset of oscillation from the death state. We also verify the robustness of our results in an electronic hardware level experiment. Our study reveals that the effect of time delay on the dynamics of coupled oscillators is coupling function dependent and, therefore, highly non-trivial.

Transient Receptor Potential Vanilloid 4 Is Required for Foreign Body Response and Giant Cell Formation.

The presence of biomaterials and devices implanted into soft tissue is associated with development of a foreign body response (FBR), a chronic inflammatory condition that can ultimately lead to implant failure, which may cause harm to or death of the patient. Development of FBR includes activation of macrophages at the tissue-implant interface, generation of destructive foreign body giant cells (FBGCs), and generation of fibrous tissue that encapsulates the implant. However, the mechanisms underlying the FBR remain poorly understood, as neither the materials composing the implants nor their chemical properties can explain triggering of the FBR. Herein, we report that genetic ablation of transient receptor potential vanilloid 4 (TRPV4), a Ca2+-permeable mechanosensitive cation channel in the transient receptor potential vanilloid family, protects TRPV4 knockout mice from FBR-related events. The mice showed diminished collagen deposition along with reduced macrophage accumulation and FBGC formation compared with wild-type mice in a s.c. implantation model. Analysis of macrophage markers in spleen tissues and peritoneal cavity showed that the TRPV4 deficiency did not impair basal macrophage maturation. Furthermore, genetic deficiency or pharmacologic antagonism of TRPV4 blocked cytokine-induced FBGC formation, which was restored by lentivirus-mediated TRPV4 reintroduction. Taken together, these results suggest an important, previously unknown, role for TRPV4 in FBR.

Streptolysin-induced endoplasmic reticulum stress promotes group A Streptococcal host-associated biofilm formation and necrotising fasciitis.

Group A Streptococcus (GAS) is a human pathogen that causes infections ranging from mild to fulminant and life-threatening. Biofilms have been implicated in acute GAS soft-tissue infections such as necrotising fasciitis (NF). However, most in vitro models used to study GAS biofilms have been designed to mimic chronic infections and insufficiently recapitulate in vivo conditions along with the host-pathogen interactions that might influence biofilm formation. Here, we establish and characterise an in vitro model of GAS biofilm development on mammalian cells that simulates microcolony formation observed in a mouse model of human NF. We show that on mammalian cells, GAS forms dense aggregates that display hallmark biofilm characteristics including a 3D architecture and enhanced tolerance to antibiotics. In contrast to abiotic-grown biofilms, host-associated biofilms require the expression of secreted GAS streptolysins O and S (SLO, SLS) that induce endoplasmic reticulum (ER) stress in the host. In an in vivo mouse model, the streptolysin null mutant is attenuated in both microcolony formation and bacterial spread, but pretreatment of soft-tissue with an ER stressor restores the ability of the mutant to form wild-type-like microcolonies that disseminate throughout the soft tissue. Taken together, we have identified a new role of streptolysin-driven ER stress in GAS biofilm formation and NF disease progression.

Oscillating synchronization in delayed oscillators with time-varying time delay coupling: Experimental observation.

The time-varying time-delayed (TVTD) systems attract the attention of research communities due to their rich complex dynamics and wide application potentiality. Particularly, coupled TVTD systems show several intriguing behaviors that cannot be observed in systems with a constant delay or no delay. In this context, a new synchronization scenario, namely, oscillating synchronization, was reported by Senthilkumar and Lakshmanan [Chaos 17, 013112 (2007)], which is exclusive to the time-varying time delay systems only. However, like most of the dynamical behavior of TVTD systems, its existence has not been established in an experiment. In this paper, we report the first experimental observation of oscillating synchronization in coupled nonlinear time-delayed oscillators induced by a time-varying time delay in the coupling path. We implement a simple yet effective electronic circuit to realize the time-varying time delay in an experiment. We show that depending upon the instantaneous variation of the time delay, the system shows a synchronization scenario oscillating among lag, complete, and anticipatory synchronization. This study may open up the feasibility of applying oscillating synchronization in engineering systems.

Effectiveness of probiotics, prebiotics, and prebiotic-like components in common functional foods.

The bioactive ingredients in commonly consumed foods include, but are not limited to, prebiotics, prebiotic-like components, probiotics, and postbiotics. The bioactive ingredients in functional foods have also been associated with beneficial effects on human health. For example, they aid in shaping of gut microflora and promotion of immunity. These functional components also contribute in preventing serious diseases such as cardiovascular malfunction and tumorigenesis. However, the specific mechanisms of these positive influences on human health are still under investigation. In this review, we aim to emphasize the major contents of probiotics, prebiotics, and prebiotic-like components commonly found in consumable functional foods, and we present an overview of direct and indirect benefits they provide on human health. The major contributors are certain families of metabolites, specifically short-chain fatty acids and polyunsaturated fatty acids produced by probiotics, and prebiotics, or prebiotic-like components such as flavonoids, polyphenols, and vitamins that are found in functional foods. These functional ingredients in foods influence the gut microbiota by stimulating the growth of beneficial microbes and the production of beneficial metabolites that, in turn, have direct benefits to the host, while also providing protection from pathogens and maintaining a balanced gut ecosystem. The complex interactions that arise among functional food ingredients, human physiology, the gut microbiota, and their respective metabolic pathways have been found to minimize several factors that contribute to the incidence of chronic disease, such as inflammation oxidative stress.

Structure of ScpC, a virulence protease from Streptococcus pyogenes, reveals the functional domains and maturation mechanism.

Group A Streptococcus (GAS; Streptococcus pyogenes) causes a wide range of infections, including pharyngitis, impetigo, and necrotizing fasciitis, and results in over half a million deaths annually. GAS ScpC (SpyCEP), a 180-kDa surface-exposed, subtilisin-like serine protease, acts as an essential virulence factor that helps S. pyogenes evade the innate immune response by cleaving and inactivating C-X-C chemokines. ScpC is thus a key candidate for the development of a vaccine against GAS and other pathogenic streptococcal species. Here, we report the crystal structures of full-length ScpC wild-type, the inactive mutant, and the ScpC-AEBSF inhibitor complex. We show ScpC to be a multi-domain, modular protein consisting of nine structural domains, of which the first five constitute the PR + A region required for catalytic activity. The four unique C-terminal domains of this protein are similar to collagen-binding and pilin proteins, suggesting an additional role for ScpC as an adhesin that might mediate the attachment of S. pyogenes to various host tissues. The Cat domain of ScpC is similar to subtilisin-like proteases with significant difference to dictate its specificity toward C-X-C chemokines. We further show that ScpC does not undergo structural rearrangement upon maturation. In the ScpC-inhibitor complex, the bound inhibitor breaks the hydrogen bond between active-site residues, which is essential for catalysis. Guided by our structure, we designed various epitopes and raised antibodies capable of neutralizing ScpC activity. Collectively, our results demonstrate the structure, maturation process, inhibition, and substrate recognition of GAS ScpC, and reveal the presence of functional domains at the C-terminal region.

Alterations of Salmonella enterica Serovar Typhimurium Antibiotic Resistance under Environmental Pressure.

Microbial horizontal gene transfer is a continuous process that shapes bacterial genomic adaptation to the environment and the composition of concurrent microbial ecology. This includes the potential impact of synthetic antibiotic utilization in farm animal production on overall antibiotic resistance issues; however, the mechanisms behind the evolution of microbial communities are not fully understood. We explored potential mechanisms by experimentally examining the relatedness of phylogenetic inference between multidrug-resistant Salmonella enterica serovar Typhimurium isolates and pathogenic Salmonella Typhimurium strains based on genome-wide single-nucleotide polymorphism (SNP) comparisons. Antibiotic-resistant S Typhimurium isolates in a simulated farm environment barely lost their resistance, whereas sensitive S Typhimurium isolates in soils gradually acquired higher tetracycline resistance under antibiotic pressure and manipulated differential expression of antibiotic-resistant genes. The expeditious development of antibiotic resistance and the ensuing genetic alterations in antimicrobial resistance genes in S Typhimurium warrant effective actions to control the dissemination of Salmonella antibiotic resistance.IMPORTANCE Antibiotic resistance is attributed to the misuse or overuse of antibiotics in agriculture, and antibiotic resistance genes can also be transferred to bacteria under environmental stress. In this study, we report a unidirectional alteration in antibiotic resistance from susceptibility to increased resistance. Highly sensitive Salmonella enterica serovar Typhimurium isolates from organic farm systems quickly acquired tetracycline resistance under antibiotic pressure in simulated farm soil environments within 2 weeks, with expression of antibiotic resistance-related genes that was significantly upregulated. Conversely, originally resistant S Typhimurium isolates from conventional farm systems lost little of their resistance when transferred to environments without antibiotic pressure. Additionally, multidrug-resistant S Typhimurium isolates genetically shared relevancy with pathogenic S Typhimurium isolates, whereas susceptible isolates clustered with nonpathogenic strains. These results provide detailed discussion and explanation about the genetic alterations and simultaneous acquisition of antibiotic resistance in S Typhimurium in agricultural environments.

Networks of coupled oscillators: From phase to amplitude chimeras.

We show that amplitude-mediated phase chimeras and amplitude chimeras can occur in the same network of nonlocally coupled identical oscillators. These are two different partial synchronization patterns, where spatially coherent domains coexist with incoherent domains and coherence/incoherence referring to both amplitude and phase or only the amplitude of the oscillators, respectively. By changing the coupling strength, the two types of chimera patterns can be induced. We find numerically that the amplitude chimeras are not short-living transients but can have a long lifetime. Also, we observe variants of the amplitude chimeras with quasiperiodic temporal oscillations. We provide a qualitative explanation of the observed phenomena in the light of symmetry breaking bifurcation scenarios. We believe that this study will shed light on the connection between two disparate chimera states having different symmetry-breaking properties.

Short chain and polyunsaturated fatty acids in host gut health and foodborne bacterial pathogen inhibition.

As a major source of microbes and their numerous beneficial effects, the gut microflora/microbiome is intimately linked to human health and disease. The exclusion of enteric pathogens by these commensal microbes partially depends upon the production of bioactive compounds such as short-chain fatty acids (SCFAs) and polyunsaturated fatty acids (PUFAs). These key intestinal microbial byproducts are crucial to the maintenance of a healthy gut microbial community. Moreover, SCFAs and PUFAs play multiple critical roles in host defense and immunity, including anti-cancer, anti-inflammation, and anti-oxidant activities, as well as out-competition of enteric bacterial pathogens. In this review article, we hereby aim to highlight the importance of SCFAs and PUFAs and the microbes involved in production of these beneficial intestinal components, and their biological functions, specifically as to their immunomodulation and interactions with enteric bacterial pathogens. Finally, we also advance potential applications of these fatty acids with regards to food safety and human gut health.

Prevalence, antimicrobial resistance, and molecular characterization of Campylobacter spp. in bulk tank milk and milk filters from US dairies.

Campylobacter spp. are frequently isolated from dairy cows as commensal organisms. Sporadic Campylobacter infections in humans in the United States are generally attributed to poultry, but outbreaks are also commonly associated with dairy products, particularly unpasteurized or raw milk. Bulk tank milk samples and milk filters from US dairy operations were collected during the National Animal Health Monitoring System Dairy 2014 study and analyzed using real-time PCR and traditional culture techniques for the presence of thermophilic Campylobacter species. The weighted prevalence of operations from which we detected Campylobacter spp. in either bulk tank milk or milk filters was 24.9%. We detected Campylobacter spp. in a higher percentage of operations with 100-499 cows (42.8%) and 500 or more cows (47.5%) than in operations with 30-99 cows (6.5%). Campylobacter spp. were also more frequently detected in operations in the west than the east (45.9 and 22.6%, respectively). We isolated Campylobacter spp. from approximately half of PCR-positive samples, representing 12.5% (weighted prevalence) of operations. The majority (91.8%) of isolates were C. jejuni, but C. lari and C. coli were also isolated. We detected resistance to tetracycline in 68.4% of C. jejuni isolates, and resistance to ciprofloxacin and nalidixic acid in 13.2% of C. jejuni isolates. Based on pulsed-field gel electrophoresis, we found that dairy-associated C. jejuni were genotypically diverse, although clonal strains were isolated from different geographic regions. These results suggest that bulk tank milk can be contaminated with pathogenic Campylobacter spp., and that the consumption of unpasteurized or raw milk presents a potential human health risk.

Control of birhythmicity: A self-feedback approach.

Birhythmicity occurs in many natural and artificial systems. In this paper, we propose a self-feedback scheme to control birhythmicity. To establish the efficacy and generality of the proposed control scheme, we apply it on three birhythmic oscillators from diverse fields of natural science, namely, an energy harvesting system, the p53-Mdm2 network for protein genesis (the OAK model), and a glycolysis model (modified Decroly-Goldbeter model). Using the harmonic decomposition technique and energy balance method, we derive the analytical conditions for the control of birhythmicity. A detailed numerical bifurcation analysis in the parameter space establishes that the control scheme is capable of eliminating birhythmicity and it can also induce transitions between different forms of bistability. As the proposed control scheme is quite general, it can be applied for control of several real systems, particularly in biochemical and engineering systems.

Prevalence and antibiotic resistance pattern of Salmonella serovars in integrated crop-livestock farms and their products sold in local markets.

Major concern in the Mixed Crop-Livestock (MCL) farms, in which livestock and vegetables grown closely in the same facility, is cross-contamination of zoonotic bacterial pathogens especially Salmonella. To investigate the distribution of Salmonella serovars in MCL and their products, a total of 1287 pre-harvest samples from various farms and 1377 post-harvest samples from retail supermarkets in Maryland and Washington D.C. areas were collected and analysed. A total of 315 Salmonella isolates were recovered, with 17.44% and 5.88%, from MCL and conventional farms samples (P < 0.001). At post-harvest level, the prevalence of Salmonella was 30.95%, 19.83%, and 8.38% in chicken meat (P < 0.001) from farmers, organic, and conventional retail markets respectively, and 16.81% and 6.06% in produce products (P < 0.001) from farmers and organic retail markets, but none from conventional retail markets. From the isolated Salmonella, 34.50% was confirmed S. Typhimurium, followed by S. Heidelberg (10.86%) and S. Enteritidis (9.90%). The overall multi-antibiotic resistance in recovered Salmonella was 23.81% versus 4.55% in conventional and MCL farms (P = 0.004) and 66.67% versus 7.76% in conventional and farmers markets (P < 0.001). Overall the data reveals higher Salmonella risks in MCL farms' environment and their products sold in farmers markets and warrants taking necessary measures to limit Salmonella transmission.

Base-pair resolution DNA methylation sequencing reveals profoundly divergent epigenetic landscapes in acute myeloid leukemia.

We have developed an enhanced form of reduced representation bisulfite sequencing with extended genomic coverage, which resulted in greater capture of DNA methylation information of regions lying outside of traditional CpG islands. Applying this method to primary human bone marrow specimens from patients with Acute Myelogeneous Leukemia (AML), we demonstrated that genetically distinct AML subtypes display diametrically opposed DNA methylation patterns. As compared to normal controls, we observed widespread hypermethylation in IDH mutant AMLs, preferentially targeting promoter regions and CpG islands neighboring the transcription start sites of genes. In contrast, AMLs harboring translocations affecting the MLL gene displayed extensive loss of methylation of an almost mutually exclusive set of CpGs, which instead affected introns and distal intergenic CpG islands and shores. When analyzed in conjunction with gene expression profiles, it became apparent that these specific patterns of DNA methylation result in differing roles in gene expression regulation. However, despite this subtype-specific DNA methylation patterning, a much smaller set of CpG sites are consistently affected in both AML subtypes. Most CpG sites in this common core of aberrantly methylated CpGs were hypermethylated in both AML subtypes. Therefore, aberrant DNA methylation patterns in AML do not occur in a stereotypical manner but rather are highly specific and associated with specific driving genetic lesions.