Transcriptional Regulatory System Research Articles

Engineering a Controllable Targeted Protein Degradation System and a Derived OR-GATE-Type Inducible Gene Expression System in Synechococcus elongatus PCC 7942.

Cyanobacteria are important model organisms for exploring the mechanisms of photosynthesis and are considered as promising microbial platforms for photosynthetic biomanufacturing. The development of efficient cyanobacteria cell factories requires efficient and convenient tools to dynamically regulate and manipulate target proteins, modules, and pathways. Targeted protein degradation is important to achieve rapid responses of cellular metabolic networks to artificial or environmental signals, and there are currently limited approaches to induce protein degradation in cyanobacteria. In this work, we developed an Escherichia coli sourced ssrA-tagging system in an important cyanobacteria strain, Synechococcus elongatus PCC 7942, to achieve inducible degradation of target proteins. A modified version of the E. coli ssrA tag (ssrADAS) proved to be immune to the native ClpXP system in Synechococcus elongatus PCC 7942, while induced expression of the E. coli sourced adaptor SspB and ClpXP resulted in effective degradation of the tagged proteins. Compared to the previously developed down-regulation approaches, the inducible ssrADAS-SspB-ClpXPEc system facilitated the smart and rapid degradation of target proteins in PCC7942 cells at different growth stages. Furthermore, when used to regulate the degradation of LacI, the repressor element of LacO-LacI transcription regulation system, an efficient and stringent inducible gene expression system was obtained based on an OR-GATE type genetic circuit design. The tools developed in this work expanded the cyanobacteria synthetic biology toolbox and will facilitate the success of future dynamic metabolic engineering.

Comparative Analysis of HSF Genes From Secale cereale and its Triticeae Relatives Reveal Ancient and Recent Gene Expansions

Plants have evolved sophisticated systems to cope with the environmental stresses, with the heat shock factor (HSF) family proteins composing an integral part of the transcriptional regulation system. Understanding the evolutionary history and functional diversity of HSFs will facilitate improving tolerance of crops to adverse environmental conditions. In this study, genome-wide analysis of Secale cereale identified 31 HSF genes. The total number of HSF genes in S. cereale is larger than that in barley and the three subgenomes of wheat, suggesting it is a valuable resource for mining functional HSFs. Chromosome analysis revealed an uneven distribution of HSF genes among the 7 S. cereale chromosomes, with no HSF gene was detected on chromosome 4. Further interspecies synteny analysis revealed that chromosome reorganization during species-speciation may lead to the escape of HSF genes from the S. cereale chromosome 4. Phylogenetic analysis revealed that S. cereale experienced more HSF gene duplications than barley and the three wheat subgenomes. Expression analysis demonstrated that S. cereale HSF genes showed diverse expression patterns across plant developmental stages and upon drought and freezing treatment, suggesting functional diversity of the gene family. Notably, we detected distinct expression patterns for a recently duplicated HSF gene pair, indicating functional divergence may have occurred between the two genes. The study presents the genome organization, evolutionary features and expression patterns of the S. cereale HSF genes. These results provide new insights into the evolution of HSF genes in Triticeae and may serve as a resource for Triticeae molecular breeding.

The regulation of DKGA2ox1 and miR171f_3 in scion dwarfing with Diospyros kaki Thunb. cv. 'Nan-tong-xiao-fang-shi' as interstocks.

High-throughput sequencing and yeast one and two-hybrid library screening reveal that DKGA2ox1 and miR171f_3 are involved in the regulation of scion dwarfing with 'Nan-tong-xiao-fang-shi' as interstocks. Diospyros kaki Thunb. cv. Nan-tong-xiao-fang-shi ('Nan-tong-xiao-fang-shi') interstocks play a critical role in the scion dwarfing. However, the understanding of the molecular signaling pathways that regulate the scion dwarfing with 'Nan-tong-xiao-fang-shi' as interstocks remain unclear. In this work, the regulatory network in the scion dwarfing with 'Nan-tong-xiao-fang-shi' as interstocks was identified. Using a yeast one-hybrid library screening, luciferase activity analysis, luciferase complementation imaging assays and GFP signal detection, a SPL transcription factor named Diospyros kaki SPL (DKSPL), potentially functioning as a transcriptional activator of the Diospyros kaki GA2ox1 (DKGA2ox1) gene, was identified as a key stimulating factor in the persimmon growth and development. The DKSPL was found in the nucleus, and might play a role in the transcriptional regulation system. A microRNA named miR171f_3 was identified, which might act as a negative regulator of Diospyros kaki SCR (DKSCR) in persimmon. The interactions between DKSCR and seven proteins were experimentally validated with a yeast two-hybrid library screening. Compared to the non-grafted wildtype persimmon, the tissue section of graft union healed well due to the increased expression of cinnamyl-alcohol dehydrogenase. These results indicate that DKGA2ox1 and miR171f_3 may co-promote the scion dwarfing by plant hormone signal transduction pathways.

Strigolactones affect the root system architecture of cherry rootstock by mediating hormone signaling pathways

Strigolactones (SLs) are new phytohormones that modulate plant root growth and development. Rootstocks affect horticulturally important traits of cultivars, including tree size, abiotic stress tolerance, and disease resistance. This study focused on understanding the effects of SLs on the transcriptional regulation and root system architecture of cherry rootstock. Here, we examined the morphology and profiled endogenous hormone levels, and transcript levels of key genes from the roots treated with synthetic SLs analog GR24 or SLs inhibitor TIS108. Cherry rootstock plantlets treated with 10 μM TIS108 exhibited fewer adventitious roots (AR). The diameter of AR at 5, 10, and 30 days after 0.1 μM GR24 treatment decreased by 23.8, 23.2, and 11.5 %, respectively, whereas AR diameter was 1.15, 1.15, and 1.16 times higher than that in the control when 10 μM TIS108 was applied, respectively. The LR number in plantlets treated with 10 μM TIS108 reached a maximum of 11.25 for each AR, much higher than those of the control and other treatments at 30 days after treatment (DAT). X-ray micro-CT scanning also showed that the treatment with 10 μM TIS108 markedly induced LR initiation but suppressed LR elongation. Furthermore, 2946, 1536, and 2146 differentially expressed genes (DEGs; p-value < 0.05, |log2Fold Change|> 1) were identified in the root samples treated with 0.1 μM GR24, 10 μM GR24, and 10 μM TIS108, respectively, compared to the control. RNA-Seq findings highlighted several DEGs, such as ARF, PP2C, ERF, LOB, and CYC, which play critical roles in hormone signaling, cell cycle, and root growth and development. UPLC-MS analysis further demonstrated that SLs markedly altered the levels of endogenous hormones in cherry rootstock. In particular, the content of endogenous indole-3-acetic acid (IAA) markedly increased by 64.6, 52.1, and 173.5 % after treatment with 0.1 μM GR24, 10 μM GR24, and 10 μM TIS108, respectively. Overall, our study indicates that SLs affect root system architecture by interacting with other plant hormones, which provides a solid theoretical basis for the effects of SLs on the root system architecture of other woody plants.

Asymmetric Lévy noise changed stability in a gene transcriptional regulatory system

• Stability analysis of gene transcriptional regulatory system under asymmetric Lévy noise is meaningful but seldom reported before, and this article's study is about it. • A recently proposed stochastic basin of attraction (SBA) is used to explore the stability of the system. The SBA is built by escape probability. The comparison of Monte Carlo simulation (MCS) shows that the numerical scheme for solving escape probability is accurate enough to observe the influence of parameters on stability. • A new concept that is constructed using the stochastic basin of attraction and the stationary probability density function was used to determine the stable steady state of the system. The change of stability degree can be observed clearly by it. Perturbation of internal molecules in gene regulation is discontinuous and asymmetrical. Therefore, it is reasonable to use asymmetric Lévy noise to simulate environmental disturbance. However, there is limited research on the stability of systems under asymmetric Lévy noise. This study, based on the stochastic basin of attraction and mean first exit time, examines the stability of the gene transcriptional regulatory system. It is found that, by changing the skewness parameter of noise, the stability of the system can change significantly. Stochastic basin stability, a new concept based on the stochastic basin of attraction and the stationary probability density function, is then used to quantify the stability of the two states of the system. We demonstrate that merely modulating the dissipation coefficient does not change the effect of the skewness parameter on the stochastic basin of attraction.

VPS38/UVRAG and ATG14, the variant regulatory subunits of the ATG6/Beclin1-PI3K complexes, are crucial for the biogenesis of the yolk organelles and are transcriptionally regulated in the oocytes of the vector Rhodnius prolixus.

In insects the reserve proteins are stored in the oocytes into endocytic-originated vesicles named yolk organelles. VPS38/UVRAG and ATG14 are the variant regulatory subunits of two class-III ATG6/Beclin1 PI3K complexes that regulate the recruitment of the endocytic (complex II) and autophagic (complex I) machineries. In a previous work from our group, we found that the silencing of ATG6/Beclin1 resulted in the formation of yolk-deficient oocytes due to defects in the endocytosis of the yolk proteins. Because ATG6/Beclin1 is present in the two above-described PI3K complexes, we could not identify the contributions of each complex to the yolk defective phenotypes. To address this, here we investigated the role of the variant subunits VPS38/UVRAG (complex II, endocytosis) and ATG14 (complex I, autophagy) in the biogenesis of the yolk organelles in the insect vector of Chagas Disease Rhodnius prolixus. Interestingly, the silencing of both genes phenocopied the silencing of ATG6/Beclin1, generating 1) accumulation of yolk proteins in the hemolymph; 2) white, smaller, and yolk-deficient oocytes; 3) abnormal yolk organelles in the oocyte cortex; and 4) unviable F1 embryos. However, we found that the similar phenotypes were the result of a specific cross-silencing effect among the PI3K subunits where the silencing of VPS38/UVRAG and ATG6/Beclin1 resulted in the specific silencing of each other, whereas the silencing of ATG14 triggered the silencing of all three PI3K components. Because the silencing of VPS38/UVRAG and ATG6/Beclin1 reproduced the yolk-deficiency phenotypes without the cross silencing of ATG14, we concluded that the VPS38/UVRAG PI3K complex II was the major contributor to the previously observed phenotypes in silenced insects. Altogether, we found that class-III ATG6/Beclin1 PI3K complex II (VPS38/UVRAG) is essential for the yolk endocytosis and that the subunits of both complexes are under an unknown transcriptional regulatory system.

Prediction of regime shifts under spatial indicators in gene transcription regulation systems

Gene transcription regulation is a noisy process that can lead to regime shifts and these shifts are difficult to predict. In this letter, we consider spatiotemporal fluctuations in gene transcription regulation, and employ a spatially extended gene transcription regulation system by using theoretical analysis and numerical simulation. We find that the system produces a regime shift from a low (high) to a high (low) protein concentration state (i.e., the transform of the switch of gene transcription regulation), and we apply spatial early warning signals (e.g., rising spatial variance, spatial skewness, spatial kurtosis, and lag-1 autocorrelation) to study the regime shifts of protein concentration. The results show that the spatial early warning signals can be used as a robust indicator of predicting gene transcription regulation switch. Once the signal is detected, we will take measures to intervene. Our results may have a positive effect on improving early treatment interventions for complex human diseases.

Streptococcus pyogenes ("Group A Streptococcus"), a Highly Adapted Human Pathogen-Potential Implications of Its Virulence Regulation for Epidemiology and Disease Management.

Streptococcus pyogenes (group A streptococci; GAS) is an exclusively human pathogen. It causes a variety of suppurative and non-suppurative diseases in people of all ages worldwide. Not all can be successfully treated with antibiotics. A licensed vaccine, in spite of its global importance, is not yet available. GAS express an arsenal of virulence factors responsible for pathological immune reactions. The transcription of all these virulence factors is under the control of three types of virulence-related regulators: (i) two-component systems (TCS), (ii) stand-alone regulators, and (iii) non-coding RNAs. This review summarizes major TCS and stand-alone transcriptional regulatory systems, which are directly associated with virulence control. It is suggested that this treasure of knowledge on the genetics of virulence regulation should be better harnessed for new therapies and prevention methods for GAS infections, thereby changing its global epidemiology for the better.

Two types of bHLH transcription factor determine the competence of the pericycle for lateral root initiation.

The molecular basis of the competence of the pericycle cell to initiate lateral root primordium formation is totally unknown. Here, we report that in Arabidopsis, two types of basic helix-loop-helix (bHLH) transcription factors, named PERICYCLE FACTOR TYPE-A (PFA) proteins and PERICYCLE FACTOR TYPE-B (PFB) proteins, govern the competence of pericycle cells to initiate lateral root primordium formation. Overexpression of PFA genes confers hallmark pericycle characteristics, including specific marker gene expression and auxin-induced cell division, and multiple loss-of-function mutations in PFA genes or the repression of PFB target genes results in the loss of this specific pericycle function. PFA and PFB proteins physically interact and are under mutual- and self-regulation, forming a positive feedback loop. This study unveils the transcriptional regulatory system that determines pericycle participation in lateral root initiation.

Comparative Phosphoproteomics of Classical Bordetellae Elucidates the Potential Role of Serine, Threonine and Tyrosine Phosphorylation in Bordetella Biology and Virulence.

The Bordetella genus is divided into two groups: classical and non-classical. Bordetella pertussis, Bordetella bronchiseptica and Bordetella parapertussis are known as classical bordetellae, a group of important human pathogens causing whooping cough or whooping cough-like disease and hypothesized to have evolved from environmental non-classical bordetellae. Bordetella infections have increased globally driving the need to better understand these pathogens for the development of new treatments and vaccines. One unexplored component in Bordetella is the role of serine, threonine and tyrosine phosphorylation. Therefore, this study characterized the phosphoproteome of classical bordetellae and examined its potential role in Bordetella biology and virulence. Applying strict identification of localization criteria, this study identified 70 unique phosphorylated proteins in the classical bordetellae group with a high degree of conservation. Phosphorylation was a key regulator of Bordetella metabolism with proteins involved in gluconeogenesis, TCA cycle, amino acid and nucleotide synthesis significantly enriched. Three key virulence pathways were also phosphorylated including type III secretion system, alcaligin synthesis and the BvgAS master transcriptional regulatory system for virulence genes in Bordetella. Seven new phosphosites were identified in BvgA with 6 located in the DNA binding domain. Of the 7, 4 were not present in non-classical bordetellae. This suggests that serine/threonine phosphorylation may play an important role in stabilizing/destabilizing BvgA binding to DNA for fine-tuning of virulence gene expression and that BvgA phosphorylation may be an important factor separating classical from non-classical bordetellae. This study provides the first insight into the phosphoproteome of classical Bordetella species and the role that Ser/Thr/Tyr phosphorylation may play in Bordetella biology and virulence.

Cellular Phosphorylation Signaling and Gene Expression in Drought Stress Responses: ABA-Dependent and ABA-Independent Regulatory Systems.

Drought is a severe and complex abiotic stress that negatively affects plant growth and crop yields. Numerous genes with various functions are induced in response to drought stress to acquire drought stress tolerance. The phytohormone abscisic acid (ABA) accumulates mainly in the leaves in response to drought stress and then activates subclass III SNF1-related protein kinases 2 (SnRK2s), which are key phosphoregulators of ABA signaling. ABA mediates a wide variety of gene expression processes through stress-responsive transcription factors, including ABA-RESPONSIVE ELEMENT BINDING PROTEINS (AREBs)/ABRE-BINDING FACTORS (ABFs) and several other transcription factors. Seed plants have another type of SnRK2s, ABA-unresponsive subclass I SnRK2s, that mediates the stability of gene expression through the mRNA decay pathway and plant growth under drought stress in an ABA-independent manner. Recent research has elucidated the upstream regulators of SnRK2s, RAF-like protein kinases, involved in early responses to drought stress. ABA-independent transcriptional regulatory systems and ABA-responsive regulation function in drought-responsive gene expression. DEHYDRATION RESPONSIVE ELEMENT (DRE) is an important cis-acting element in ABA-independent transcription, whereas ABA-RESPONSIVE ELEMENT (ABRE) cis-acting element functions in ABA-responsive transcription. In this review article, we summarize recent advances in research on cellular and molecular drought stress responses and focus on phosphorylation signaling and transcription networks in Arabidopsis and crops. We also highlight gene networks of transcriptional regulation through two major regulatory pathways, ABA-dependent and ABA-independent pathways, that ABA-responsive subclass III SnRK2s and ABA-unresponsive subclass I SnRK2s mediate, respectively. We also discuss crosstalk in these regulatory systems under drought stress.

Persistent Bacterial Coinfection of a COVID-19 Patient Caused by a Genetically Adapted Pseudomonas aeruginosa Chronic Colonizer.

Pseudomonas aeruginosa is a biofilm-forming opportunistic pathogen which causes chronic infections in immunocompromised patients and leads to high mortality rate. It is identified as a common coinfecting pathogen in COVID-19 patients causing exacerbation of illness. In our hospital, P. aeruginosa is one of the top coinfecting bacteria identified among COVID-19 patients. We collected a strong biofilm-forming P. aeruginosa strain displaying small colony variant morphology from a severe COVID-19 patient. Genomic and transcriptomic sequencing analyses were performed with phenotypic validation to investigate its adaptation in SARS-CoV-2 infected environment. Genomic characterization predicted specific genomic islands highly associated with virulence, transcriptional regulation, and DNA restriction-modification systems. Epigenetic analysis revealed a specific N6-methyl adenine (m6A) methylating pattern including methylation of alginate, flagellar and quorum sensing associated genes. Differential gene expression analysis indicated that this isolate formed excessive biofilm by reducing flagellar formation (7.4 to 1,624.1 folds) and overproducing extracellular matrix components including CdrA (4.4 folds), alginate (5.2 to 29.1 folds) and Pel (4.8–5.5 folds). In summary, we demonstrated that P. aeuginosa clinical isolates with novel epigenetic markers could form excessive biofilm, which might enhance its antibiotic resistance and in vivo colonization in COVID-19 patients.

Light-Photoreceptors and Proteins Related to Monilinia laxa Photoresponses.

Light represents a ubiquitous source of information for organisms to evaluate their environment. The influence of light on colony growth and conidiation was determined for three Monilinia laxa isolates. The highest mycelial growth rate was observed under red light for the three M. laxa isolates, followed by green light, daylight or darkness. However, reduced sporulation levels were observed in darkness and red light, but conidiation enhancement was found under daylight, black and green light with more hours of exposure to light. Putative photoreceptors for blue (white-collar and cryptochromes), green (opsins), and red light (phytochromes) were identified, and the photoresponse-related regulatory family of velvet proteins. A unique ortholog for each photoreceptor was found, and their respective domain architecture was highly conserved. Transcriptional analyses of uncovered sets of genes were performed under daylight or specific color light, and both in time course illumination, finding light-dependent triggered gene expression of MlVEL2, MlPHY2, MlOPS2, and MlCRY2, and color light as a positive inductor of MlVEL3, MlVEL4, MlPHY1, and MlCRY1 expression. M. laxa has a highly conserved set of photoreceptors with other light-responsive fungi. Our phenotypic analyses and the existence of this light-sensing machinery suggest transcriptional regulatory systems dedicated to modulating the development and dispersion of this pathogen.

DeepTFactor: A deep learning-based tool for the prediction of transcription factors

A transcription factor (TF) is a sequence-specific DNA-binding protein that modulates the transcription of a set of particular genes, and thus regulates gene expression in the cell. TFs have commonly been predicted by analyzing sequence homology with the DNA-binding domains of TFs already characterized. Thus, TFs that do not show homologies with the reported ones are difficult to predict. Here we report the development of a deep learning-based tool, DeepTFactor, that predicts whether a protein in question is a TF. DeepTFactor uses a convolutional neural network to extract features of a protein. It showed high performance in predicting TFs of both eukaryotic and prokaryotic origins, resulting in F1 scores of 0.8154 and 0.8000, respectively. Analysis of the gradients of prediction score with respect to input suggested that DeepTFactor detects DNA-binding domains and other latent features for TF prediction. DeepTFactor predicted 332 candidate TFs in Escherichia coli K-12 MG1655. Among them, 84 candidate TFs belong to the y-ome, which is a collection of genes that lack experimental evidence of function. We experimentally validated the results of DeepTFactor prediction by further characterizing genome-wide binding sites of three predicted TFs, YqhC, YiaU, and YahB. Furthermore, we made available the list of 4,674,808 TFs predicted from 73,873,012 protein sequences in 48,346 genomes. DeepTFactor will serve as a useful tool for predicting TFs, which is necessary for understanding the regulatory systems of organisms of interest. We provide DeepTFactor as a stand-alone program, available at https://bitbucket.org/kaistsystemsbiology/deeptfactor.

Comparative RNA-Seq analysis of the root revealed transcriptional regulation system for aluminum tolerance in contrasting indica rice of North East India.

Expression pattern of aluminum (Al) tolerance genes is one of the major determinants of Al avoidance/tolerance within plant cultivars. We have performed transcriptome analysis of two contrasting (Al-tolerant, Disang; Al-sensitive, Joymati) cultivars of India's North Eastern region, an indica rice diversity hotspot, on exposure to excess Al3+ treatment in acidic condition. Co-expression analysis and SNPs enrichment analysis proposed the role of both trans-acting and cis-acting polymorphisms in Al signaling in the Al-tolerant cultivar. We proposed ten major genes, including arginine decarboxylase, phytase, and beta-glucosidase aggregating factor as candidates responsible for Al tolerance based on transcriptome analysis. Al3+ stress led to changes in the alternative splicing profile of the Al-tolerant cultivar. These studies demonstrated the transcriptional variations affiliated to Al avoidance/tolerance in contrasting indica rice of North East India and provided us with several candidate genes responsible for Al tolerance.

A tunable, rapid, and precise drug control of protein expression by combining transcriptional and post-translational regulation systems

Rapid, precise, and tunable regulation of protein abundance would be significantly useful in a variety of biotechnologies and biomedical applications. Here, we describe a system that allows tunable and rapid drug control of gene expression for either gene activation or inactivation in mammalian cells. We construct the system by coupling Tet-on 3G and small molecule-assisted shutoff systems, which can respectively induce transcriptional activation and protein degradation in the presence of corresponding small molecules. This dual-input drug inducer regulation system facilitates a bidirectional control of gene expression. The gene of interest can be precisely controlled by dual small molecules in a broad dynamic range of expression from overexpression to complete silence, allowing gene function study in a comprehensive expression profile. Our results reveal that the bidirectional control system enables sensitive dosage- and time-dependent regulation for either turn-on or shutoff of gene expression. We also apply this system for inducible genome editing and gene activation mediated by clustered regularly interspaced short palindromic repeats. The system provides an integrated platform for studying multiple biological processes by manipulating gene expression in a more flexible way.

Directed evolution of the transcription factor Gal4 for development of an improved transcriptional regulation system in Saccharomyces cerevisiae

As a well-characterized eukaryotic DNA binding transcription factor, Gal4 has been extensively employed to construct controllable gene expression systems in Saccharomyces cerevisiae. The problem of insufficient inducibility arises in the constructs with multiples genes under control of GAL promoters due to the low expression level and activity of the native Gal4. In order to obtain improved transcriptional regulation systems for multi-gene pathways, Gal4 mutants with improved activity were created by directed evolution. During the preliminary screening, five positive Gal4 variants were isolated based on the lycopene-indicated high-throughput screening method. Analysis of the mutation sites revealed that the majority of positive mutations are localized in the middle homology region with unspecified function, suggesting an important role of this domain in transactivation of Gal4. Through combinatorial site-directed mutagenesis, the best variant Gal4T406A/V413A was obtained, which successfully increased the transcription of PGAL-driven lycopene pathway genes and led to 48 % higher lycopene accumulation relative to the wild-type Gal4. This study demonstrates the viability of modifying Gal4 activity by directed evolution for elevated expression of PGAL-driven genes and therefore enhanced production of the target metabolite.

Spatiotemporal Regulation of Vibrio Exotoxins by HlyU and Other Transcriptional Regulators

After invading a host, bacterial pathogens secrete diverse protein toxins to disrupt host defense systems. To ensure successful infection, however, pathogens must precisely regulate the expression of those exotoxins because uncontrolled toxin production squanders energy. Furthermore, inappropriate toxin secretion can trigger host immune responses that are detrimental to the invading pathogens. Therefore, bacterial pathogens use diverse transcriptional regulators to accurately regulate multiple exotoxin genes based on spatiotemporal conditions. This review covers three major exotoxins in pathogenic Vibrio species and their transcriptional regulation systems. When Vibrio encounters a host, genes encoding cytolysin/hemolysin, multifunctional-autoprocessing repeats-in-toxin (MARTX) toxin, and secreted phospholipases are coordinately regulated by the transcriptional regulator HlyU. At the same time, however, they are distinctly controlled by a variety of other transcriptional regulators. How this coordinated but distinct regulation of exotoxins makes Vibrio species successful pathogens? In addition, anti-virulence strategies that target the coordinating master regulator HlyU and related future research directions are discussed.

Mitochondrial dysfunction in GnRH neurons impaired GnRH production

The onset establishment and maintenance of gonadotropin-releasing hormone (GnRH) secretion is an important phenomenon regulating pubertal development and reproduction. GnRH neurons as well as other neurons in the hypothalamus have high-energy demands and require a constant energy supply from their mitochondria machinery to maintain active functioning. However, the involvement of mitochondrial function in GnRH neurons is still unclear. In this study, we examined the role of NADH Dehydrogenase (Ubiquinone) Fe–S protein 4 (Ndufs4), a member of the mitochondrial complex 1, on GnRH neurons using Ndufs4-KO mice and Ndufs4-KO GT1-7 cells. Ndufs4 was highly expressed in GnRH neurons in the medial preoptic area (MPOA) and NPY/AgRP and POMC neurons in the arcuate (ARC) nucleus in WT mice. Conversely, there was a significant decrease in GnRH expression in MPOA and median eminence of Ndufs4-KO mice, followed by impaired peripheral endocrine system. In Ndufs4-KO GT1-7 cells, Gnrh1 expression was significantly decreased with or without stimulation with either kisspeptin or NGF, whereas, stimulation significantly increased Gnrh1 expression in control cells. In contrast, there was no difference in cell signaling activity including ERK and CREB as well as the expression of GPR54, TrkA and p75NTR, suggesting that Ndufs4 is involved in the transcriptional regulation system for GnRH production. These findings may be useful in understanding the mitochondrial function in GnRH neuron.

Characterization and control of bacterial phenotypes at the single cell level

<p indent=0mm>Microbial biofilms comprise surface-associated, multicellular, highly structured matrix-enclosed, morphologically complex microbial communities. The subject of biofilms has received much attention, in part owing to scientific communities’ acknowledgements that biofilm formation of pathogen can cause many acute or chronic diseases that usually cannot be treated by antibiotic therapies, such as <italic>Pseudomonas aeruginosa</italic>, one of the highly prevalent opportunistic human pathogens, that is the leading cause of morbidity and mortality in immunocompromised patients and in patients suffering from cystic fibrosis (CF). We have learned that biofilm-forming bacteria are phenotypically distinct from those of planktonically grown cells, because they express genes in a pattern differs profoundly from that of their free-swimming, planktonic counterparts. Therefore, understanding the roles played by bacterial behaviors and phenotypes in the development of biofilms is an emerging link to disease pathogenesis. In the first half of this review, we present how to develop characteristic approaches by using high-throughput microscopical techniques together with automatic image-processing methods and customized microfluidic system to in situ visualize bacterial cells and investigate their behaviors at the single cell scale. We mainly focused on the pathogen of <italic>P. aeruginosa</italic> and provided an overview of recent related progress using those approaches in characterization of bacterial phenotypes at the single cell level in the process of biofilm formation, especially in areas of bacterial twitching motility, attachment phenotypes, social behaviors and architecture of biofilms. For instance, by monitoring single-cell motility behaviors, researchers observed single-cell twitching chemotaxis in developing biofilms and revealed that cells can modulate twitching motility behaviors to survive or thrive in slowly changing and locally heterogeneous natural environments. We then referred to optogenetics, which is a technology that allows targeted, fast control of precisely defined events in biological systems by expressing exogenous genes coding for light-sensitive proteins. In optogenetics, light as inducer can be applied more precisely in the concentration, time and space dimensions than traditional effectors such as chemical molecules. In the field of microbiology, different light responsive sensors, such as UV, blue, green, red and far-red transcriptional regulation systems, have been engineered, and application of these optogenetic systems enables little perturbations and unprecedented spatiotemporal resolution in controlling bacterial behaviors that can reveal new insights into biological function. For example, researchers have used blue light-switchable gene regulation system to optogenetic control of gene expression to spatiotemporally manipulate biofilm formation and pattern cells with high-resolution. These studies provide the ability to grow structured biofilms, with applications toward an improved understanding of natural biofilm communities, as well as the engineering of living biomaterials and bottom-up approaches to microbial consortia design. In addition, optogenetics enables characterization of bacterial gene circuit dynamics with optically programmed gene expression signals, which could advance understanding of gene circuit dynamics and cell signaling pathways. We also proposed that the development of optogenetics in bacteriology is limited by the requirement of engineered light-sensitive proteins for specific regulation networks and new methods for manipulation of living bacterial cells at the single-cell level. This review concludes with some expectations on the foreseeable future application of those single-cell analysis approaches in biofilms-related fields, and a discussion of innovative anti-biofilm strategies that are inspired from the studies of characterization and control of single-cell phenotypes.