High Fructose Negatively Impacts Proliferation of NSC-34 Motor Neuron Cell Line

Objectives The main aim of this study is to identify the deleterious effects of indiscriminately consumed high fructose on motor neurons that are critically affected in many neurological conditions causing movement disorders including paralysis. Materials and Methods Neuroblastoma x mouse spinal cord motor neuron cell line (NSC-34) motor neuron cell lines were treated with high fructose and oxygen supplementation (18.8%) and assayed for cell proliferation/death, reactive oxygen species (ROS) generation, and oxidative stress response induction Statistical Analysis Mean and standard deviation, significance with and without high fructose (F)-5%, were estimated by t-tests using GraphPad Prism ver. 8.2.1 Results F-5% along with O2 (18.8%) annihilates the cells (∼85%) by day10 and inhibits cell division as observed by the presence of multinucleated cells. Unexpectedly, 1 to 2% of cells that survived, differentiated and displayed progressive neurite extension. Though not healthy, they were viable up to 80 days. F-5% increased ROS levels (∼34%) not accompanied by concomitant enhanced expression of oxidative stress response regulator, the transcription factor, nrf-2, or downstream effector, sod-1. Conclusion High fructose is extremely harmful to NSC-34 motor neuron cell line.

Evolution of two-component quorum sensing systems

Quorum sensing (QS) is a cell-to-cell communication system that enables bacteria to coordinate their gene expression depending on their population density, via the detection of small molecules called autoinducers. In this way bacteria can act collectively to initiate processes like bioluminescence, virulence and biofilm formation. Autoinducers are detected by receptors, some of which are part of two-component signal transduction systems (TCS), which comprise of a (usually membrane-bound) sensor histidine kinase (HK) and a cognate response regulator (RR). Different QS systems are used by different bacterial taxa, and their relative evolutionary relationships have not been extensively studied. To address this, we used the Kyoto Encyclopedia of Genes and Genomes (KEGG) database to identify all the QS HKs and RRs that are part of TCSs and examined their conservation across microbial taxa. We compared the combinations of the highly conserved domains in the different families of receptors and response regulators using the Simple Modular Architecture Research Tool (SMART) and KEGG databases, and we also carried out phylogenetic analyses for each family, and all families together. The distribution of the different QS systems across taxa, indicates flexibility in HK–RR pairing and highlights the need for further study of the most abundant systems. For both the QS receptors and the response regulators, our results indicate close evolutionary relationships between certain families, highlighting a common evolutionary history which can inform future applications, such as the design of novel inhibitors for pathogenic QS systems.

Regulation of Heterogenous LexA Expression in Staphylococcus aureus by an Antisense RNA Originating from Transcriptional Read-Through upon Natural Mispairings in the sbrB Intrinsic Terminator

Bacterial genomes are pervasively transcribed, generating a wide variety of antisense RNAs (asRNAs). Many of them originate from transcriptional read-through events (TREs) during the transcription termination process. Previous transcriptome analyses revealed that the lexA gene from Staphylococcus aureus, which encodes the main SOS response regulator, is affected by the presence of an asRNA. Here, we show that the lexA antisense RNA (lexA-asRNA) is generated by a TRE on the intrinsic terminator (TTsbrB) of the sbrB gene, which is located downstream of lexA, in the opposite strand. Transcriptional read-through occurs by a natural mutation that destabilizes the TTsbrB structure and modifies the efficiency of the intrinsic terminator. Restoring the mispairing mutation in the hairpin of TTsbrB prevented lexA-asRNA transcription. The level of lexA-asRNA directly correlated with cellular stress since the expressions of sbrB and lexA-asRNA depend on the stress transcription factor SigB. Comparative analyses revealed strain-specific nucleotide polymorphisms within TTsbrB, suggesting that this TT could be prone to accumulating natural mutations. A genome-wide analysis of TREs suggested that mispairings in TT hairpins might provide wider transcriptional connections with downstream genes and, ultimately, transcriptomic variability among S. aureus strains.

uvrY deletion and acetate reduce gut colonization of Crohn’s disease-associated adherent-invasive Escherichia coli by decreasing expression of type 1 fimbriae

Adherent-invasive Escherichia coli (AIEC) is involved in onset and/or exacerbation of Crohn’s disease. AIEC adapts to the gut environment by altering gene-expression programs, leading to successful gut-lumen colonization. However, the underlying mechanism of gut colonization is still far from clarified. Here, we show the role of UvrY, a response regulator of bacterial two-component signal transduction systems, in AIEC gut colonization. An AIEC mutant lacking the uvrY gene exhibited impairment of competitive colonization in the murine intestinal tract. UvrY contributes to functional expression of type 1 fimbriae by activating expression of small RNA CsrB, which confers adherence and invasion into epithelial cells on AIEC. In contrast, acetate suppresses the UvrY-dependent expression of type 1 fimbriae, resulting in less efficient cell invasion and attenuated gut colonization. Our findings might lead to therapeutic interventions for CD, in which inhibitions of UvrY activation and acetate supplementation reduce the colonization levels of AIEC by decreasing type-1 fimbriae expression.

The Campylobacter jejuni Response Regulator and Cyclic-Di-GMP Binding CbrR Is a Novel Regulator of Flagellar Motility

A leading cause of bacterial gastroenteritis, Campylobacter jejuni is also associated with broad sequelae, including extragastrointestinal conditions such as reactive arthritis and Guillain-Barré Syndrome (GBS). CbrR is a C. jejuni response regulator that is annotated as a diguanylate cyclase (DGC), an enzyme that catalyzes the synthesis of c-di-GMP, a universal bacterial second messenger, from GTP. In C. jejuni DRH212, we constructed an unmarked deletion mutant, cbrR−, and complemented mutant, cbrR+. Motility assays indicated a hyper-motile phenotype associated with cbrR−, whereas motility was deficient in cbrR+. The overexpression of CbrR in cbrR+ was accompanied by a reduction in expression of FlaA, the major flagellin. Biofilm assays and scanning electron microscopy demonstrated similarities between DRH212 and cbrR−; however, cbrR+ was unable to form significant biofilms. Transmission electron microscopy showed similar cell morphology between the three strains; however, cbrR+ cells lacked flagella. Differential radial capillary action of ligand assays (DRaCALA) showed that CbrR binds GTP and c-di-GMP. Liquid chromatography tandem mass spectrometry detected low levels of c-di-GMP in C. jejuni and in E. coli expressing CbrR. CbrR is therefore a negative regulator of FlaA expression and motility, a critical virulence factor in C. jejuni pathogenesis.

Comparative proteomic analysis of Fusarium oxysporum f. sp. cubense strains (Foc R1 and Foc TR4) provides better insights into mechanisms of their virulence, habitat adaptation and pathogenesis

Fusarium oxysporum f. sp. cubense (Foc), a devastative soil-borne fungal pathogen causing vascular wilt (i.e. Panama disease) which leads to severe crop losses in most of the banana-growing regions of the world. As there is no single source of effective management practices available so far, understand the pathogenicity of the organism may help in designing effective control measures through molecular approaches. The study aims to compare the proteome of the two pathogenic Foc virulent strains, Race 1 (Foc R1) and tropical race 4 (Foc TR4) that are capable of infecting the Cavendish group of bananas using 2-dimensional (2-D) gel electrophoresis, MALDI-TOF/MS and MS/MS analysis. The results of the study revealed that the proteins, peroxiredoxins, NAD-aldehyde dehydrogenase (NAD-ALDH), MAPK protein, pH-response regulator protein palA/rim-20 and isotrichodermin C15 hydroxylase have shared homology with the fungal proteins, which regulate the osmotic stress response, signal transduction, root colonization and toxin biosynthesis. These are the important functions for the pathogen survival in an unfavourable environment, and successful establishment and infection of the banana host. The present study also identified several putative pathogenicity related proteins in both Foc R1 and Foc TR4. Specifically, certain Foc TR4 specific putative pathogenicity related proteins, phytotoxins biosynthesis gene, fructose 1,6-bisphosphate aldolase class II, Synembryn-like proteins found to contribute strong virulence. Overexpression or knockout of the elective genes could help in devising better control measures for the devastative pathogens in the future. To the best of our knowledge, this is the first report on the proteomics of Foc R1 and Foc TR4 strains of Indian origin that infect Cavendish bananas.

Sequestration of histidine kinases by non-cognate response regulators establishes a threshold level of stimulation for bacterial two-component signaling

Two-component signaling systems (TCSs) in bacteria are often positively auto-regulated, where the histidine kinase (HK) and response regulator (RR) proteins comprising a TCS are expressed downstream of the signal they transduce. This auto-regulation improves the sensitivity of the TCS to stimuli and amplifies adaptive responses. The downside, however, is that the TCS may mount disproportionately large responses to weak or fleeting signals. How bacteria prevent such disproportionate responses is not known. Here, we show that sequestration of phosphorylated HKs by non-cognate RRs serves as a design to prevent such disproportionate responses. Using TCSs of M. tuberculosis as model systems, we found that with every one of the five HKs we studied, there was at least one non-cognate RR with higher affinity than that of the cognate RR for the HK. Phosphorylated HKs would thus preferentially bind the non-cognate RRs, suppressing signal transduction through the cognate pathways, which we demonstrated in vitro. Using mathematical modeling of TCS signaling in vivo, we predicted that this sequestration would introduce a threshold level of stimulation for a significant response, preventing responses to signals below this threshold. Finally, we showed in vivo using tunable expression systems in M. bovis that upregulation of a higher affinity non-cognate RR substantially suppressed the output from the cognate TCS pathway, presenting strong evidence of sequestration by non-cognate RRs as a design to regulate TCS signaling. Blocking this sequestration may be a novel intervention strategy, as it would compromise bacterial fitness by letting it respond unnecessarily to signals.

Dichotomous Feedback: A Signal Sequestration-based Feedback Mechanism for Biocontroller Design

We introduce a new design framework for implementing negative feedback regulation in Synthetic Biology, which we term "dichotomous feedback". Our approach is different from current methods, in that it sequesters existing fluxes in the process to be controlled, and in this way takes advantage of the process's architecture to design the control law. This signal sequestration mechanism appears in many natural biological systems and can potentially be easier to realise than 'molecular sequestration' and other comparison motifs that are nowadays common in biomolecular feedback control design. The loop is closed by linking the strength of signal sequestration to the process output. Our feedback regulation mechanism is motivated by two-component signalling systems, where we introduce a second response regulator competing with the natural response regulator thus sequestering kinase activity. Here, dichotomous feedback is established by increasing the concentration of the second response regulator as the level of the output of the natural process increases. Extensive analysis demonstrates how this type of feedback shapes the signal response, attenuates intrinsic noise while increasing robustness and reducing crosstalk.

GUN4 Affects the Circadian Clock and Seedlings Adaptation to Changing Light Conditions

The chloroplast is a key organelle for photosynthesis and perceiving environmental information. GENOME UNCOUPLED 4 (GUN4) has been shown to be required for the regulation of both chlorophyll synthesis, reactive oxygen species (ROS) homeostasis and plastid retrograde signaling. In this study, we found that growth of the gun4 mutant was significantly improved under medium strong light (200 μmol photons m−2s−1) compared to normal light (100 μmol photons m−2s−1), in marked contrast to wild-type (WT). Further analysis revealed that GUN4 interacts with SIGNAL RECOGNITION PARTICLE 54 KDA SUBUNIT (SRP43) and SRP54. RNA-seq analysis indicated that the expression of genes for light signaling and the circadian clock is altered in gun4 compared with (WT). qPCR analysis confirmed that the expression of the clock genes CLOCK-RELATED 1 (CCA1), LATE ELONGATION HYPOCOTYL (LHY), TIMING OF CAB EXPRESSION 1 (TOC1) and PSEUDO RESPONSE REGULATOR 7 (PRR7) is significantly changed in the gun4 and srp54 mutants under normal and medium strong light conditions. These results suggest that GUN4 may coordinate the adaptation of plants to changing light conditions by regulating the biological clock, although it is not clear whether the effect is direct or indirect.