PEP-ASSOCIATED PROTEIN 3 regulates rice tiller formation and grain yield by controlling chloroplast biogenesis.

Plastid-encoded RNA polymerase (PEP) plays a pivotal role in chloroplast development by governing the transcription of chloroplast genes, and PEP-associated proteins (PAPs) modulate PEP transcriptional activity. Therefore, PAPs provide an intriguing target for those efforts to improve yield, by enhancing chloroplast development. In this study, we identified the rice (Oryza sativa) OsPAP3 gene and characterized its function in chloroplast development. OsPAP3 expression was light-dependent and leaf-specific, similar to the PEP-dependent chloroplast gene RUBISCO LARGE SUBUNIT (OsRbcL), and OsPAP3 protein localized to chloroplast nucleoids where PEP functions. Analysis of loss-of-function and gain-of-function mutants showed that the expression of OsPAP3 is tightly linked to chloroplast gene expression and chloroplast biogenesis in rice. Homozygous knockout mutants of OsPAP3 had fewer chloroplasts than wild type, whereas plants overexpressing OsPAP3 had more chloroplasts. Also, OsPAP3 knockout suppressed the PEP-dependent expression of chloroplast genes, but OsPAP3 overexpression increased their expression. These findings indicate that OsPAP3 regulates chloroplast biogenesis in rice by controlling the PEP-dependent expression of chloroplast genes. More importantly, data from 3 seasons of field cultivation revealed that the overexpression of OsPAP3 improves rice grain yield by approximately 25%, largely due to increased tiller formation. Collectively, these observations suggest that OsPAP3 regulates rice growth and productivity by promoting chloroplast development.

A novel Escherichia coli cell-based bioreporter for quantification of salicylic acid in cosmetics.

Transcription factor-based bioreporters have been extensively studied for monitoring and detecting environmental toxicants. In Escherichia coli, the multiple antibiotic resistance regulator (MarR) induces transcription upon binding to salicylic acid (SA). We generated SA-specific E. coli cell-based bioreporters utilizing the operator region of the mar operon and MarR as components of the reporter and sensing domains, respectively. Although bioreporters based on endogenous MarR and wild-type E. coli cells responded to SA, their sensitivity and selectivity were insufficient for practical sample monitoring. To improve these parameters, we genetically engineered host strains for optimal MarR expression, which enhanced the sensitivity of the biosensor to micromolar quantities of SA with increased selectivity. Under the optimized experimental conditions, the biosensor could quantify SA in environmental samples. For validation, the SA concentration in artificially contaminated SA-containing cosmetic samples was determined using the developed biosensor. Reliability assessment by comparing the concentrations determined using LC-MS/MS revealed > 90% accuracy of the bioreporters. Although bioreporters are not considered standard tools for environmental monitoring, bacterial cell-based bioreporters may serve as alternative tools owing to their affordability and simplicity. The SA biosensor developed in this study can potentially be a valuable tool for monitoring SA in environmental systems. KEY POINTS: • SA-responsive bioreporter is generated by employing mar operon system in E. coli • SA specificity and selectivity were enhanced by genetic/biochemical engineering • The novel bioreporter would be valuable for SA monitoring in environmental systems.

SH2 Domains Serve as Lipid-Binding Modules for pTyr-Signaling Proteins.

The Src-homology 2 (SH2) domain is a protein interaction domain that directs myriad phosphotyrosine (pY)-signaling pathways. Genome-wide screening of human SH2 domains reveals that ∼90% of SH2 domains bind plasma membrane lipids and many have high phosphoinositide specificity. They bind lipids using surface cationic patches separate from pY-binding pockets, thus binding lipids and the pY motif independently. The patches form grooves for specific lipid headgroup recognition or flat surfaces for non-specific membrane binding and both types of interaction are important for cellular function and regulation of SH2 domain-containing proteins. Cellular studies with ZAP70 showed that multiple lipids bind its C-terminal SH2 domain in a spatiotemporally specific manner and thereby exert exquisite spatiotemporal control over its protein binding and signaling activities in T cells. Collectively, this study reveals how lipids control SH2 domain-mediated cellular protein-protein interaction networks and suggest a new strategy for therapeutic modulation of pY-signaling pathways.

Recent Trends in Chemical Sensors for Detecting Toxic Materials.

Industrial development has led to the widespread production of toxic materials, including carcinogenic, mutagenic, and toxic chemicals. Even with strict management and control measures, such materials still pose threats to human health. Therefore, convenient chemical sensors are required for toxic chemical monitoring, such as optical, electrochemical, nanomaterial-based, and biological-system-based sensors. Many existing and new chemical sensors have been developed, as well as new methods based on novel technologies for detecting toxic materials. The emergence of material sciences and advanced technologies for fabrication and signal-transducing processes has led to substantial improvements in the sensing elements for target recognition and signal-transducing elements for reporting interactions between targets and sensing elements. Many excellent reviews have effectively summarized the general principles and applications of different types of chemical sensors. Therefore, this review focuses on chemical sensor advancements in terms of the sensing and signal-transducing elements, as well as more recent achievements in chemical sensors for toxic material detection. We also discuss recent trends in biosensors for the detection of toxic materials.

The Biomedical Applications of Biomolecule Integrated Biosensors for Cell Monitoring.

Cell monitoring is essential for understanding the physiological conditions and cell abnormalities induced by various stimuli, such as stress factors, microbial invasion, and diseases. Currently, various techniques for detecting cell abnormalities and metabolites originating from specific cells are employed to obtain information on cells in terms of human health. Although the states of cells have traditionally been accessed using instrument-based analysis, this has been replaced by various sensor systems equipped with new materials and technologies. Various sensor systems have been developed for monitoring cells by recognizing biological markers such as proteins on cell surfaces, components on plasma membranes, secreted metabolites, and DNA sequences. Sensor systems are classified into subclasses, such as chemical sensors and biosensors, based on the components used to recognize the targets. In this review, we aim to outline the fundamental principles of sensor systems used for monitoring cells, encompassing both biosensors and chemical sensors. Specifically, we focus on biosensing systems in terms of the types of sensing and signal-transducing elements and introduce recent advancements and applications of biosensors. Finally, we address the present challenges in biosensor systems and the prospects that should be considered to enhance biosensor performance. Although this review covers the application of biosensors for monitoring cells, we believe that it can provide valuable insights for researchers and general readers interested in the advancements of biosensing and its further applications in biomedical fields.

Skeletal muscle satellite cell-derived mesenchymal stem cells ameliorate acute alcohol-induced liver injury.

Cultured human skeletal-muscle satellite cells have properties of mesenchymal stem cells (skeletal muscle satellite cell-derived mesenchymal stem cells, SkMSCs) and play anti-inflammatory roles by secreting prostaglandin E2 and hepatocyte growth factor (HGF). To evaluate the utility of SkMSCs in treating liver diseases, we determined whether SkMSCs could ameliorate acute liver and gut inflammation induced by binge ethanol administration. Binge drinking of ethanol led to weight loss in the body and spleen, liver inflammation and steatosis, and increased serum ALT and AST levels (markers of liver injury), along with increased IL-1ß, TNF-α, and iNOS expression levels in mice. However, levels of these binge-drinking-induced indicators were reduced by a single intraperitoneal treatment of SkMSCs. Furthermore, levels of bacteria-derived lipopolysaccharide decreased in the livers and sera of ethanol-exposed mice after SkMSC administration. SkMSCs decreased the extent of tissue inflammation and reduced villus and crypt lengths in the small intestine after alcohol binge drinking. SkMSCs also reduced the leakage of blood albumin, an indicator of leaky gut, in the stool of ethanol-exposed mice. Alcohol-induced damage to human colonic Caco-2/tc7 cells was also alleviated by HGF. Therefore, a single treatment with SkMSCs can attenuate alcoholic liver damage by reducing inflammatory responses in the liver and gut, suggesting that SkMSCs could be used in cell therapy to treat alcoholic liver diseases.

SH3 Domain-Containing Protein 2 Plays a Crucial Role at the Step of Membrane Tubulation during Cell Plate Formation.

During cytokinesis in plants, trans-Golgi network-derived vesicles accumulate at the center of dividing cells and undergo various structural changes to give rise to the planar cell plate. However, how this conversion occurs at the molecular level remains elusive. In this study, we report that SH3 Domain-Containing Protein 2 (SH3P2) in Arabidopsis thaliana plays a crucial role in converting vesicles to the planar cell plate. SH3P2 RNAi plants showed cytokinesis-defective phenotypes and produced aggregations of vesicles at the leading edge of the cell plate. SH3P2 localized to the leading edge of the cell plate, particularly the constricted or curved regions of the cell plate. The BAR domain of SH3P2 induced tubulation of vesicles. SH3P2 formed a complex with dynamin-related protein 1A (DRP1A) and affected DRP1A accumulation to the cell plate. Based on these results, we propose that SH3P2 functions together with DRP1A to convert the fused vesicles to tubular structures during cytokinesis.

Specific heavy metal/metalloid sensors: current state and perspectives.

Heavy metal(loid)s play pivotal roles in regulating physiological and developmental aspects in living organisms depending on their concentration. For example, a trace amount of heavy metal(loid)s is essential for living organisms, but heavy metal(loid)s in high concentrations negatively affect their physiology and development. Because of rapid industrial developments, heavy metal(loid)s have been accumulating in environmental systems, thereby becoming a threat to human health and the earth's ecosystem. Thus, the development of tools to quantify and monitor heavy metal(loid)s in environmental systems has become essential. Typically, risk has been determined through instrument-based analysis, regardless of the shortcomings regarding expense and duration. Nowadays, the need for alternative tools, besides instrumental analysis, to detect heavy metals has prompted the development of new techniques, and many different methods have been reported from various research areas, including new techniques based on electrochemistry and biological systems. Nonetheless, it seems that the gap between laboratory and fieldwork is still greater than it should be when it comes to applying these systems. In this mini-review, we discuss the current status of heavy metals/metalloid detection techniques, with an emphasis on biosensors. Moreover, we discuss the advantages and disadvantages as well as the mechanisms behind newly developed sensors and make suggestions to improve applicability and to develop new objective targeting sensors. Although many different types of metal(loid) sensors are available, we focused on metal sensors based on biological systems. Additionally, we suggest potent approaches to developing new biosensor systems based on current metal sensor mechanisms.

Antimony sensing whole-cell bioreporters derived from ArsR genetic engineering.

Despite the known hazardous effects of antimony (Sb) on human health, Sb monitoring biosensors have not been as actively investigated as arsenic (As) biosensors. Whole-cell bioreporters (WCBs) employing an arsenic-responsive operon and a regulatory protein (ArsR) are reportedly capable of monitoring arsenite, arsenate, and antimonite. However, the potential of WCBs as Sb biosensors has been largely ignored. Here, the metal-binding site of ArsR (sequenced as ELCVCDLCTA from amino acid number 30 to 39) was modified via genetic engineering to enhance Sb specificity. By relocating cysteine residues and introducing point mutations, nine ArsR mutants were generated and tested for metal(loid) ion specificity. The Sb specificity of WCBs was enhanced by the C37S/A39C and L36C/C37S mutations on the As binding site of ArsR. Additionally, WCBs with other ArsR mutants exhibited new target sensing capabilities toward Cd and Pb. Although further research is required to enhance the specificity and sensitivity of WCBs and to broaden their practical applications, our proposed strategy based on genetic engineering of regulatory proteins provides a valuable basis to generate WCBs to monitor novel targets.

Shifting the Specificity of E. coli Biosensor from Inorganic Arsenic to Phenylarsine Oxide through Genetic Engineering.

It has recently been discovered that organic and inorganic arsenics could be detrimental to human health. Although organic arsenic is less toxic than inorganic arsenic, it could form inorganic arsenic through chemical and biological processes in environmental systems. In this regard, the availability of tools for detecting organic arsenic species would be beneficial. Because As-sensing biosensors employing arsenic responsive genetic systems are regulated by ArsR which detects arsenics, the target selectivity of biosensors could be obtained by modulating the selectivity of ArsR. In this study, we demonstrated a shift in the specificity of E. coli cell-based biosensors from the detection of inorganic arsenic to that of organic arsenic, specifically phenylarsine oxide (PAO), through the genetic engineering of ArsR. By modulating the number and location of cysteines forming coordinate covalent bonds with arsenic species, an E. coli cell-based biosensor that was specific to PAO was obtained. Despite its restriction to PAO at the moment, it offers invaluable evidence of the potential to generate new biosensors for sensing organic arsenic species through the genetic engineering of ArsR.

Crosstalk with Jasmonic Acid Integrates Multiple Responses in Plant Development.

To date, extensive studies have identified many classes of hormones in plants and revealed the specific, nonredundant signaling pathways for each hormone. However, plant hormone functions largely overlap in many aspects of plant development and environmental responses, suggesting that studying the crosstalk among plant hormones is key to understanding hormonal responses in plants. The phytohormone jasmonic acid (JA) is deeply involved in the regulation of plant responses to biotic and abiotic stresses. In addition, a growing number of studies suggest that JA plays an essential role in the modulation of plant growth and development under stress conditions, and crosstalk between JA and other phytohormones involved in growth and development, such as gibberellic acid (GA), cytokinin, and auxin modulate various developmental processes. This review summarizes recent findings of JA crosstalk in the modulation of plant growth and development, focusing on JA-GA, JA-cytokinin, and JA-auxin crosstalk. The molecular mechanisms underlying this crosstalk are also discussed.

Heavy metal(loid) biosensor based on split-enhanced green fluorescent protein: development and characterization.

Heavy metal(loid)s such as Cd and Hg adversely affect human health and are therefore strictly regulated and monitored; however, their quantitation in the environment is usually performed by expensive and time-consuming instrumental analysis techniques, which necessitates the search for more practical alternatives. Herein, we prepare enhanced green fluorescent protein (eGFP)-based biomolecules for metal(loid) sensing by insertion of metal-binding loops (MBLs) into a loop region of eGFP to render this protein inactive and show that the binding of metal ions to MBLs induces a conformational change and restores the original activity. Specifically, eGFP with an MBL sequenced as CTTCGCG regains fluorescence upon exposure to Cd and Hg, which allows the above metals to be quantified in the concentration range of 0-5 µM. For practical applicability verification, the developed sensing platform is used to quantify Cd in artificially amended soil and water samples. Although the obtained results imply that sensor performance needs to be significantly improved, the presented design concept is believed to be of high value to researchers in the field of heavy metal sensing and facilitate the development of new biosensors.

A Biosensor Platform for Metal Detection Based on Enhanced Green Fluorescent Protein.

Microbial cell-based biosensors, which mostly rely on stress-responsive operons, have been widely developed to monitor environmental pollutants. Biosensors are usually more convenient and inexpensive than traditional instrumental analyses of environmental pollutants. However, the targets of biosensors are restricted by the limited number of genetic operon systems available. In this study, we demonstrated a novel strategy to overcome this limitation by engineering an enhanced green fluorescent protein (eGFP). It has been reported that combining two fragments of split-eGFP can form a native structure. Thus, we engineered new biosensors by inserting metal-binding loops (MBLs) between ß-strands 9 and 10 of the eGFP, which then undergoes conformational changes upon interaction between the MBLs and targets, thereby emitting fluorescence. The two designed MLBs based on our previous study were employed as linkers between two fragments of eGFP. As a result, an Escherichia coli biosensor exhibited a fluorescent signal only when interacting with cadmium ions, revealing the prospect of a new biosensor for cadmium detection. Although this study is a starting stage for further developing biosensors, we believe that the proposed strategy can serve as basis to develop new biosensors to target various environmental pollutants.

Modulating the sensing properties of Escherichia coli-based bioreporters for cadmium and mercury.

Despite the large number of bioreporters developed to date, the ability to detect heavy metal(loid)s with bioreporters has thus far been limited owing to the lack of appropriate genetic systems. We here present a novel approach to modulate the selectivity and sensitivity of microbial whole-cell bioreporters (WCBs) for sensing metal(loid)s via the znt-operon from Escherichia coli, which were applied to quantify the bioavailability of these contaminants in environmental samples. The WCB harboring the fusion gene zntAp::egfp was used as a microbial metal(loid) sensor, which was turned on by the interaction between ZntR and metal(loid) ions. This design makes it possible to modulate the selectivity and sensitivity to metal(loid)s simply by changing the metal-binding property of ZntR and by disrupting the metal efflux system of E. coli, respectively. In fact, the E. coli cell-based bioreporter harboring zntAp::egfp showed multi-target responses to Cd(II), Hg(II), and Zn(II). However, the WCBs showed responses toward only Cd(II) and Hg(II) when the amino acid sequence of the metal-binding loop of ZntR was changed to CNHEPGTVCPIC and CPGDDSADC, respectively. Moreover, the sensitivity toward both Cd(II) and Hg(II) was enhanced when copA, which is known to export copper and silver, was deleted. Thus, our findings provide a strong foundation for expanding the target of WCBs from the currently limited number of genetic systems available.

Enhancing the copper-sensing capability of Escherichia coli-based whole-cell bioreporters by genetic engineering.

Metals are essential to all organisms; accordingly, cells employ numerous genes to maintain metal homeostasis as high levels can be toxic. In the present study, the gene operons responsive to metal(loid)s were employed to generate bacterial cell-based biosensors to detect target metal(loid)s. The cluster of genes related to copper transport known as the cop-operon is regulated by the interaction between the copA promoter region (copAp) and CueR, turning on and off gene expression upon copper ion binding. Therefore, the detection of copper ions could be achieved by inserting a plasmid harboring the fusion of copAp and reporter genes, such as enzymes and fluorescent genes. However, copAp is not as strong a promoter as other metal-inducible promoters, such as znt-, mer-, and ars-operons; thereby, its sensitivity toward copper ions was not sufficient for quantification. To overcome this problem, we engineered Escherichia coli with a deletion of copA to interfere with copper export from cells. The engineered E. coli whole-cell bioreporter was able to detect copper ions at 0 to 10 µM in an aqueous solution. Most importantly, it was specific to copper among several tested heavy metal(loid)s. Therefore, it will likely be useful to detect copper in diverse environmental systems. Although additional improvements are still required to optimize the E. coli-based copper-sensing whole-cell bioreporters presented in this study, our results suggest that there is huge potential to generate whole-cell bioreporters for additional targets by molecular engineering.

Inhibitory potential of flavonoids on PtdIns(3,4,5)P3 binding with the phosphoinositide-dependent kinase 1 pleckstrin homology domain.

Many membrane-associated proteins are involved in various signaling pathways, including the phosphoinositide 3-kinase (PI3K) pathway, which has key roles in diverse cellular processes. Disruption of the activities of these proteins is involved in the development of disease in humans, making these proteins promising targets for drug development. In most cases, the catalytic domain is targeted; however, it is also possible to target membrane associations in order to regulate protein activity. In this study, we established a novel method to study protein-lipid interactions and screened for flavonoid-derived antagonists of PtdIns(3,4,5)P3 binding with the phosphoinositide-dependent kinase 1 (PDK1) pleckstrin homology (PH) domain. Using an enhanced green fluorescent protein (eGFP)-tagged PDK1 PH domain and 50% sucrose-loaded liposomes, the protein-lipid interaction could be efficiently evaluated using liposome pull-down assays coupled with fluorescence spectrophotometry, and a total of 32 flavonoids were screened as antagonists for PtdIns(3,4,5)P3 binding with the PDK1 PH domain. From this analysis, we found that two adjunct hydroxyl groups in the C ring were responsible for the inhibitory effects of the flavonoids. Because the flavonoids shared structural similarities, the results were then subjected to quantitative structure-activity relationship (QSAR) analysis. The results were then further confirmed by in silico docking experiments. Taken together, our strategy presented herein to screen antagonists targeting lipid-protein interactions could be an alternative method for identification and characterization of drug candidates.

Simultaneous detection of bioavailable arsenic and cadmium in contaminated soils using dual-sensing bioreporters.

Whole-cell bioreporters (WCBs) have attracted increasing attention during the last few decades because they allow fast determination of bioavailable heavy metals in contaminated sites. Various WCBs to monitor specific heavy metals such as arsenic and cadmium in diverse environmental systems are available. However, currently, no study on simultaneous analysis of arsenic and cadmium has been reported, even though soils are contaminated by diverse heavy metals and metalloids. We demonstrated herein the development of dual-sensing WCBs to simultaneously quantify bioavailable arsenic and cadmium in contaminated sites by employing the promoter regions of the ars and znt operons as separate metal-sensing domains, and egfp and mcherry as reporter genes. The dual-sensing WCBs were generated by inserting two sets of genes into E. coli DH5α. The capability of WCBs was successfully proved to simultaneously quantify bioavailable arsenic and cadmium in amended Landwirtschaftliche Untersuchungs und Forschungsanstalt (LUFA) soils, and then, it was applied to contaminated field soils collected from a smelter area in Korea. As a result, it was noticed that the bioavailable portion of cadmium was higher than that of arsenic while the absolute amount of bioavailable arsenic and cadmium level was opposite. Since both cadmium and arsenic were assessed from the same E. coli cells, the data obtained by using dual-sensing WCBs would be more efficient and convenient than that from comparative WCB assay. In spite of advantageous aspects, to our knowledge, this is the first report on a dual-sensing WCB for rapid and concurrent quantification of bioavailable arsenic and cadmium in contaminated soils.

Stochastic expression and epigenetic memory at the yeast HO promoter.

Eukaryotic gene regulation usually involves sequence-specific transcription factors and sequence-nonspecific cofactors. A large effort has been made to understand how these factors affect the average gene expression level among a population. However, little is known about how they regulate gene expression in individual cells. In this work, we address this question by mutating multiple factors in the regulatory pathway of the yeast HO promoter (HOpr) and probing the corresponding promoter activity in single cells using time-lapse fluorescence microscopy. We show that the HOpr fires in an "on/off" fashion in WT cells as well as in different genetic backgrounds. Many chromatin-related cofactors that affect the average level of HO expression do not actually affect the firing amplitude of the HOpr; instead, they affect the firing frequency among individual cell cycles. With certain mutations, the bimodal expression exhibits short-term epigenetic memory across the mitotic boundary. This memory is propagated in "cis" and reflects enhanced activator binding after a previous "on" cycle. We present evidence that the memory results from slow turnover of the histone acetylation marks.

The Rts1 regulatory subunit of PP2A phosphatase controls expression of the HO endonuclease via localization of the Ace2 transcription factor.

The RTS1 gene encodes a subunit of the PP2A phosphatase that regulates cell cycle progression. Ace2 and Swi5 are cell cycle-regulated transcription factors, and we recently showed that phosphorylation of Ace2 and Swi5 is altered in an rts1 mutant. Here we examine expression of Ace2 and Swi5 target genes and find that an rts1 mutation markedly reduces expression of the HO gene. The decreased HO expression in an rts1 mutant is significantly restored by an additional ace2 mutation, a surprising result because HO is normally activated by Swi5 but not by Ace2. Ace2 normally accumulates only in daughter cells, and only activates transcription in daughters. However, in an rts1 mutant, Ace2 is present in both mother and daughter cells. One of the genes activated by Ace2 is ASH1, a protein that normally accumulates mostly in daughter cells; Ash1 is a transcriptional repressor, and it blocks HO expression in daughters. We show that in the rts1 mutant, Ace2 accumulation in mother cells results in Ash1 expression in mothers, and the Ash1 can now repress HO expression in mothers.

Improved production of isomaltulose by a newly isolated mutant of Serratia sp. cells immobilized in calcium alginate.

Isomaltulose, also known as palatinose, is produced by sucrose isomerase and has been highlighted as a sugar substitute due to a number of advantageous properties. For the massive production of isomaltulose, high resistance to sucrose and stability of sucrose isomerase as well as sucrose conversion yields would be critical factors. We describe a series of screening procedures to isolate the mutant strain of Serratia sp. possessing enhanced isomaltulose production with improved stability. The new Serratia sp. isolated from a series of screening procedures allowed us to produce isomaltulose from 60% sucrose solution, with over 90% conversion yield. Moreover, when this strain was immobilized in calcium alginate beads and placed in a medium containing 60% sucrose, it showed over 70% sucrose conversion yields for 30 cycles of repeated-batch reactions. Thus, improved conversion activity and stability of the newly isolated Serratia sp. strain in the present study would be highly valuable for industries related to isomaltulose production.