Measuring Β-galactosidase Activity Research Articles

Bioinformatics analysis of type II toxin-antitoxin systems and regulatory functional assessment of HigBA and SS-ATA in Streptococcus suis1

Toxin-antitoxin (TA) systems, which are prevalent in bacteria and archaea, play diverse roles in bacterial physiology and have been proposed to be significant in stress adaptation. Despite the extensive characterization of numerous TA systems in various bacteria, the investigation of these systems within Streptococcus suis (S. suis) is still limited. Here, we systematically analyzed the type II TA systems of 95 S. suis genomes available in the GenBank database using TAfinder. A total of 612 putative type II TA systems were retrieved and classified into 10 categories by phylogenetic analysis. Notably, an elevated occurrence of these TA systems was observed among the important prevalent serotypes 2, 4, 5, 9, 14, Chz, NCL1, and NCL3 strains. The following study identified the activities of TA systems using two strategies and confirmed the regulatory effect of HigBA on the type VII secretion system in S. suis by measuring β-galactosidase activity and transcriptional changes. Moreover, we unveiled a hitherto uncharacterized, highly prevalent novel TA system, with the composition of antitoxin-toxin-antitoxin (SS-ATA), which regulates the downstream two-component signaling system. Altogether, this study systematically analyzed the type II TA systems within S. suis, highlighting the widespread distribution of HigBA and SS-ATA as important regulatory elements in S. suis.

Measuring β-galactosidase activity in opaque dairy solutions under optimum conditions for galactooligosaccharide synthesis by isothermal titration calorimetry

The dairy industry uses enzymes to make cheese, alter product flavor, and eliminate lactose. The activities of these enzymes have been measured in clear buffered solutions, but because of the limitations of spectrophotometric methods, enzyme activities have not been measured in opaque or colored dairy products where they are used. Isothermal titration calorimetry (ITC) can be used to determine reaction kinetics in opaque and colored solutions by measuring the heat rate (thermal power) from enzyme-catalyzed reactions as a function of time. This study used ITC to measure β-galactosidase activity in opaque solutions of milk, sweet whey, sweet whey permeate, acid whey, and acid whey permeate with 2 β-galactosidase (Enzyme Commission number 3.2.1.23) isozymes derived from Aspergillus oryzae and Kluyveromyces lactis. The components of the dairy fluids alter the enzyme kinetics and reaction thermodynamics, and the reactions catalyzed by the 2 homologues differ as shown by differing thermodynamic profiles. The study demonstrates that ITC can be used to measure enzyme activity in opaque and colored dairy fluids and identify reactions by their thermodynamic properties.

A Simple Quantitative Assay for Measuring β‐Galactosidase Activity Using X‐Gal in Yeast‐Based Interaction Analyses

Yeast-based interaction assays to determine protein-protein and protein-nucleic acid interactions commonly rely on the reconstitution of chimeric transcription factors that activate the expression of target reporter genes. The enzyme β-galactosidase (β-gal), coded by the LacZ gene of Escherichia coli, is a widely used reporter in yeast systems, and its expression is commonly assessed by evaluating its activity. X-gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside) is an inexpensive and sensitive substrate of β-gal, whose hydrolysis results in an intensely blue colored and easily detectable end product, 5,5'-dibromo-4,4'-dichloro-indigo. The insoluble nature of this end product, however, makes X-gal-based assays unsuitable for direct spectrophotometric absorbance quantification. As such, the use of X-gal is mostly restricted to solid-support approaches, such as colony lift or agar plate assays, which often only provide a qualitative readout. In this article, we describe a quantitative solid-phase X-gal assay to measure protein-protein interaction strength in yeast cells using a simple and low-cost experimental setup. We have optimized multiple aspects of the assay, namely sample preparation, reaction time, and quantification method, for speed and consistency. By integrating the use of a freely available ImageJ-based plugin, we have further standardized the assay for reliability and reproducibility. This improved quantitative X-gal assay can be performed in a standard molecular biology lab without the need for any specialized equipment other than an inexpensive and widely accessible smartphone camera. To exemplify the protocol, we provide detailed step-by-step instructions to perform a quantitative X-gal assay to assess the interaction between two Arabidopsis thaliana proteins, SUPPRESSOR OF PHYA-105 1 (SPA1) and PRODUCTION OF ANTHOCYANIN PIGMENT 2 (PAP2). To demonstrate the sensitivity of our assay in detecting weaker interactions, we also compare the results with a liquid-phase assay that uses ONPG (ortho-nitrophenyl-β-galactopyranoside) as a substrate for β-gal. The quantitative X-gal assay described here can easily be adapted for high-throughout interaction studies and protein domain mapping, even in yeast strains with low levels of LacZ expression. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Preparation of competent yeast cells and transformation Alternate Protocol 1: In-house preparation of yeast competent cells for use in lithium acetate (LiAc)-mediated yeast transformation Support Protocol: Long-term storage and revival of frozen yeast strain stocks Basic Protocol 2: Measuring β-galactosidase activity via the quantitative X-gal assay Alternate Protocol 2: Quantification of interaction strength using liquid ONPG assay.

Metformin alleviates oxidative stress-induced senescence of human lens epithelial cells via AMPK activation and autophagic flux restoration.

Cataracts are the leading cause of blindness worldwide owing to the increasing proportion of elderly individuals in the population. The purpose of this study was to investigate whether metformin could alleviate the occurrence and development of age‐related cataract (ARC) and the underlying mechanism. In the present study, we established a senescence model induced by oxidative stress, which was confirmed by measuring β‐galactosidase activity, qRT‐PCR and Western blotting. In addition, we showed that metformin alleviated the oxidative stress‐induced senescence of HLE‐B3 cells via the activation of AMPK. Next, we provided evidence that oxidative stress impaired autophagic flux and induced lysosomal dysfunction. Subsequently, we found that metformin restored autophagic flux that had been impaired by oxidative stress by activating AMPK. Additionally, we found that metformin suppressed HLE‐B3 cell senescence by improving lysosomal function and inactivating mTOR. Furthermore, the inactivation of AMPK, impairment of autophagic flux and lysosomal dysfunction were observed in the human lens epithelium of ARC. In summary, our data suggest that the activation of AMPK may be a potential strategy for preventing ARC, and metformin may be an emerging candidate to alleviate the formation and development of ARC.

Bactericidal activity of copper-ascorbic acid mixture against Staphylococcus aureus spp.

The purpose of this study was to investigate the bactericidal activity of copper (Cu, 10 ppm) and ascorbic acid (AA, 0.3% v/v) mixture against Staphylococcus aureus (S. aureus) ATCC 25923 (methicillin-susceptible) and ATCC 700698 (methicillin-resistant) strains. Approximately 5.5 log10 CFU/ml of each strain was individually inoculated into brain heart infusion (BHI) broth containing Cu, AA, and a Cu-AA mixture, and a time-kill assay was conducted to assess the bactericidal activity. In order to determine the efficacy of Cu-AA mixture as a surface decontaminating solution, the cells were first attached to glass slides (~6.5–7.5 log10 CFU per slide) and treated with a Cu-AA mixture for 1 min. The inner membrane permeability of the S. aureus strains was determined by measuring β-Galactosidase activity. Similarly, changes in the protein profile of treated bacterial cells were analyzed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The time-kill study revealed >3.0 log10 CFU/ml reduction in both strains within 24 h of incubation at 37 °C. Attached cells on glass slides were also reduced by > 3.0 log10 CFU per slide after 1 min of exposure to the Cu-AA mixture. The results indicate that the Cu-AA mixture was bactericidal against S. aureus strains and the damage to the inner cell membrane could be attributed to this effect.

In Vitro Antibacterial Activity and Mechanism of Monocaprylin against Escherichia coli and Staphylococcus aureus

In the present study, the antibacterial activity of monocaprylin in comparison with sodium benzoate and potassium sorbate against Staphylococcus aureus and Escherichia coli was assessed by measuring MIC, MBC, effect of pH on MIC, and incubation temperature on bactericidal efficacy. Results showed that monocaprylin exhibited an excellent antibacterial activity against both strains, with the lowest MIC and MBC of 1.28 mg/mL. A MIC of monocaprylin remained unchanged despite the pH values of culture medium, ranging from 5 to 9, unlike that of potassium sorbate or sodium benzoate. Furthermore, monocaprylin at MBC effectively reduced the population of E. coli and S. aureus by >5.5 log CFU/mL at 25°C within 6 h and decreased E. coli by approximately 5.0 log CFU/mL and S. aureus by 2.9 log CFU/mL at 12 h. The underlying mechanism of monocaprylin was then investigated by measuring β-galactosidase activity, membrane potential, release of cellular contents, scanning electron microscopy, and transmission electron microscopy observations. Results indicated that monocaprylin killed E. coli by the rapid change in permeability and integrity of cell membrane, leading to decline of membrane potential, leakage of nucleic acids and proteins, and ultimately cell membrane disintegration and lysis. On the other hand, monocaprylin might exert its antibacterial activity against S. aureus mainly by diffusing across the cell wall, collapsing the cell membrane, and disturbing the order of intracellular contents. These findings indicated that monocaprylin had better antibacterial ability compared with traditional synthetic preservatives and might be a potential antibacterial additive independent of pH.

Root exudates from citrus plants subjected to abiotic stress conditions have a positive effect on rhizobacteria

Plants are constantly releasing root exudates to the rhizosphere. These compounds are responsible for different (positive or negative) interactions with other organisms, including plants, fungi or bacteria. In this work, the effect of root exudates obtained from in vitro cultured citrus plants on two rhizobacteria (Pseudomonas putida KT2440 and Novosphingobium sp. HR1a) was evaluated. Root exudates were obtained from two citrus genotypes differing in their sensitivity to salt and heat stress and differentially affected the growth of both rhizobacteria. Root exudates from salt-stressed plants of C. macrophylla (salt tolerant) induced an increase in bacterial growth higher than that obtained from Carrizo citrange exudates (salt sensitive). Root exudates from heat-stressed plants also had a positive effect on bacterial growth, which was more evident in the heat-sensitive C. macrophylla. These results reveal that the growth of these rhizobacteria can be modulated through citrus root exudates and can change depending on both the stress conditions as well as the genotype. Biosensors P. putida KT2442 (pMIS5) and Novosphingobium sp. HR1a (pPAH) were used to test the presence of proline and salicylates in root exudates by measuring β-galactosidase activity. This activity increased in the presence of root exudates obtained from stressed plants to a higher extent in the case of exudates obtained from the genotype resistant to each particular stress, indicating that those root exudates contain larger quantities of proline and salicylates, as it has been described previously. Our data reveals that both P. putida KT2442 (pMIS5) and Novosphingobium sp. HR1a (pPAH), could be used as biosensors of plant stress.

Abstract 136: Phospho-S6 levels correlate with response to Copanlisib (BAY 80-6946) in multiple myeloma

Abstract Background: Copanlisib (BAY 80-6946) is a reversible, pan-class I PI3K inhibitor with preferential activity for the alpha isoform, which may be of particular importance in multiple myeloma. Here we demonstrated the in vitro efficacy of copanlisib in a panel of 20 multiple myeloma cell lines. Baseline levels of phospho-S6 (P-S6) correlated with sensitivity to copanlisib, resulting in a potential biomarker of response. In addition, the change of P-S6 post-treatment could be used as a pharmacodynamic biomarker for copanlisib treatment. Methods: We screened a panel of 20 multiple myeloma cell lines and selected 3 sensitive: NCI-H929, MM.1S, L-363, and 3 resistant: AMO-1, JJN3, COLO-677 for further analysis. We performed apoptosis and cell senescence assays following 72 hours of 50nM and 100nM copanlisib exposure. Cell cycle analysis and induction of apoptosis were performed by FACS after propidium iodide or PI/ANX-V FITC staining, respectively. Cellular senescence was determined by measuring β-galactosidase activity in cells treated for 96 hours. Reverse phase protein array (RPPA) was performed at baseline and post treatment for proteomic analysis with confirmatory western blots. Flow cytometry was also performed to monitor the post-treatment P-S6 level changes. Results: Copanlisib treatment induced apoptosis in the sensitive cell lines (50-80% AN-V+ cells) but not in resistant cell lines (1-5% AN-V+ cells). An increased cell cycle arrest in G1 was also observed in the sensitive cell lines but not in the resistant lines. The cell senescence assays confirmed apoptosis rather than cell senescence as the mechanism of inhibition of proliferation. RPPA analysis demonstrated lower baseline p-S6 (S235/236, S240/244) protein levels in sensitive compared to resistant cell lines and this was confirmed with western blot analysis. Treatment with copanlisib resulted in a greater decrease in p-S6 in the sensitive cell lines NCI-H929 and L363 (53-83%, 73-93% respectively) than in the resistant cell lines COLO-677 and JJN3 (5-27%, and 38-67%, respectively), which was validated by western blot and phospho-flow. We also showed by RPPA and WB that copanlisib down-regulates pro-survival and proliferation molecules including p-S6K1, p-S6 and p-4EBP1, and upregulates pro-apoptotic PDCD4 in all cell lines, but to a greater extent in sensitive cell lines. Finally, pharmacodynamic p-S6 response remained at different post-treatment time points. Discussion: A differential response to copanlisib is seen in the myeloma cell line panel. A subgroup of multiple myeloma cell lines demonstrated median IC50 values in the low nanomolar range (5-100nM), and responses correlated with low baseline P-S6. This p-S6 stratified response was only observed with PI3K-alpha inhibitors, but not with inhibitors targeting other PI3K isoforms or pan-PI3K inhibitors. Further studies may allow development of a new patient screening method or companion diagnostic. Citation Format: Sarah Larson, Mao Yu Peng, Monica Mead, Andrae Vandross, Dylan Conklin, Erika Von Euw, Dennis J. Slamon. Phospho-S6 levels correlate with response to Copanlisib (BAY 80-6946) in multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 136. doi:10.1158/1538-7445.AM2017-136

KINESIN-1, AMYLOID PRECURSOR PROTEIN (APP)-VESICLE TRANSPORT MOTOR INTERACTS WITH FUN14 DOMAIN CONTAINING 1 (FUNDC1) VIA THE TETRATRICOPEPTIDE REPEAT DOMAIN OF KLC1

Mutations of the amyloid precursor protein (APP) cause for the formation of amyloid-β peptides. These peptides play a key role in Alzheimer's diseases. The tetratricopeptide repeat (TPR) domain of kinesin light chain 1 (KLC1) may be responsible for binding APP either directly or via interaction with C-jun N-terminal kinase-interacting protein 1 (JIP1). However, the binding partners of the TPR domain of KLCs have not yet been fully identified. We were used the yeast two-hybrid system to identify the binding proteins that interact with the TPR domain of KLC1. The binding affinity was quantified by measuring β-galactosidase activity in liquid cultures of yeast transformed cells. Direct interaction between binding proteins and KLC1 in mammalian cells as well as in vitro was assayed using the co-immunoprecipitation with the antibodies. The cellular co-localization in cells was used the immunocytochemistry. We revealed an interaction between the TPR domain of KLC1 and FUN14 domain-containing protein 1 (FUNDC1), which is a mitochondrial outer membrane protein mediating hypoxia-induced mitophagy. FUNDC1 bound to the six TPR motif-containing regions of KLC1 and did not interact with KIF5B (a motor subunit of kinesin-1) and KIF3A (a motor subunit of kinesin-2). The cytoplasmic amino N-terminal domain of FUNDC1 is essential for interaction with KLC1. When co-expressed in HEK-293T cells, FUNDC1 co-localized with KLC1 and co-immunoprecipitated with KLC1, but not KIF5B. These results suggest that KLC1 may modulate mitophagy through the interaction with FUNDC1.

Development and characterization of a fusion mutant with the truncated lacZ to screen regulatory genes for phenazine biosynthesis in Pseudomonas chlororaphis G05

Pseudomonas chlororaphis G05 can produce biologically active agent phenazine-1-carbosylic acid and its derivatives, which contribute to suppression of mycelial growth of some pathogenic fungi and protection of crops in modern agriculture. A seven-gene operon phz (phzABCDEFG) in its genome was identified to be responsible for expression of enzymes involving phenazine biosynthesis. Although a few regulators mediating the phz expression have been identified, it is not enough for us to generate clear regulatory pathways or networks governing phenazine biosynthesis in detail. To identify novel genes involved in regulation of the phz expression, we successfully constructed a translational phzC-lacZ fusion mutant ΔG05Z, in which most of the coding region of the phzCDEFG operon was deleted and the phzC was in frame fused with the truncated lacZ in chromosome. The bacterial colony of this mutant turned blue on LB medium plates supplemented with 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-Gal). This is a convenient and simple method to check the level of the phz expression by measuring β-galactosidase activity because dark blue colony indicates that the phz operon promoter is up-regulated and overexpressed. On the contrary, light blue or white colony means that the phz promoter is down-regulated and expressed weakly. Therefore, employing this fusion mutant as a recipient makes it easy and possible for us to screen new regulatory genes involved in the phz expression by random insertional mutagenesis mediated with transposon mini-Tn5Kan later.

Fluorometric cell-based assay for β-galactosidase activity in probiotic gram-positive bacterial cells — Lactobacillus helveticus

Although methods for measuring β-galactosidase activity in intact gram-negative bacterial cells have been reported, the methods may not be applicable to measuring β-galactosidase activity in gram-positive bacterial cells. This report focuses on the development of a fluorometric cell-based assay for measuring β-galactosidase activity in gram-positive cells.

Gene therapy for GM1 gangliosidosis: challenges of translational medicine.

G M1 gangliosidosis is an autosomal recessive lysosomal storage disorder with an estimated incidence of between 1 in 100,000 and 1 in 200,000 (1). Clinical features are predominantly those of a neurodegenerative disorder due to G M1 ganglioside deposition in the central nervous system (CNS). Other clinical features include hepatosplenomegaly, coarse facial features and skeletal dysostosis. The underlying biochemical abnormality is a deficiency of β-galactosidase whose level of activity is inversely proportional to disease severity. While the disease is relatively heterogeneous three clinical subtypes are recognised. In the severe infantile form enzyme activity may be 0.07-1.3% of normal and clinical signs emerge between birth and 6 months of age. The clinical signs are a combination of a neurolipidosis (i.e., neurodegeneration and macular cherry-red spots) and a mucopolysaccharidosis (i.e., organomegaly, dysostosis and coarse facial features). The infantile form is characterised by a rapidly progressive course with severe CNS degeneration and death by 1-2 years of age commonly due to aspiration pneumonia or cardiomyopathy. The juvenile form is associated with enzyme activity in the order of 0.3-4.8% of normal and presents between 7 months and 3 years of age. It is characterised by psychomotor retardation with the other features of the infantile form being present in variable degrees. Disease progression is slower than the infantile form and death typically occurs before the second decade of life. The adult form occurs in those with an enzyme activity in the region of 5-10% of normal and is characterised by normal early neurological development with symptom onset between 3-30 years of age. Characteristic features are of a slowly progressive dementia, parkinsonism and dystonia. There is marked phenotypic variability in the adult form and the age of death varies widely. The diagnosis can be made by measuring β-galactosidase activity in peripheral blood leucocytes or by β-galactosidase gene ( GLB1 ) molecular testing. Over 100 mutations have been reported in GLB1 (1) and there is no clear correlation between genotype and phenotype.

0080: Dietary acrylamide induces accelerated endothelial aging in murine and human cell model

Replicative senescence is characterized by a limited ability of cells to divide in-vitro . Senescence induces an endothelial dysfunction. Many compounds in food can induce an earlier vascular senescence. Acrylamide (AAM) is a recent discovery in food, as a Maillard reaction product. After ingestion, AAM is in part metabolized to glycidamide (GA) in the liver. AAM and GA have been described as “probably carcinogenic to human”, reprotoxic and neurotoxic in animals. However, endothelial toxicity of AAM has never been studied. To assess the effects of AAM on endothelium. To evaluate endothelial dysfunction, mice have been fed with AAM-enriched diets (25, 50 or 100μg/kgBW/day) for 3, 6 or 9 months. Endothelium-dependent relaxation (EDR) of aortic rings was measured. AAM (1-50mM) and GA (1-5mM) toxicity was assessed by measuring Human Umbilical Vein Endothelial Cells (HUVEC) proliferation by MTS test and cell death induction by flow cytometry. Senescence was evaluated in HUVEC cultured over 3 months with AAM or GA (10 or 100μm) by measuring β-galactosidase activity (flow cytometry) and telomere length (qPCR). After 6 months of diet, AAM (100[μg/kgBW/day) decreased aortic EDR (p<0.01). In-vitro , AAM (≥5 mm) inhibited in dose- and time-dependent manners HUVEC proliferation (p<0.001) and induced apoptosis (p<0.05). GA exhibited higher toxicity since lower doses were needed to induce same results. The extended culture with AAM or GA demonstrated a significant cell division slowdown and increased β-galactosidase activity.AAM or GA (100μm) accelerated telomere shortening (p<0.05). The toxicity of AAM and GA on endothelial cells in vitro is characterized by decreased proliferation and induction of cell death at high doses. Chronic exposure at lower concentrations induce an accelerated senescence, potentially the cause of endothelial dysfunction in-vivo .

Upstream regulatory regions controlling the expression of the Candida utilis maltase gene

Candida utilis represents a promising expression host, generating relatively high levels of recombinant proteins. The current study presents preliminary characterization of the upstream regulatory regions controlling the carbon source-dependent expression of the C. utilis maltase gene. Cellobiose and soluble starch were recognised as inducers of maltase promoter, besides maltose. We successfully applied the Cre-loxP system to acquire a null mutant strain with disrupted maltase gene and promoter in polyploid yeast C. utilis. Furthermore, the strength and minimal functional region of the promoter was evaluated by measuring β-galactosidase activity. Our results suggest, the qPCR was shown itself a very smart method for relatively easy characterization of the transformants and correlation of the expression levels with gene dosage.

The use of nano-sized acicular material, sliding friction, and antisense DNA oligonucleotides to silence bacterial genes.

Viable bacterial cells impaled with a single particle of a nano-sized acicular material formed when a mixture containing the cells and the material was exposed to a sliding friction field between polystyrene and agar gel; hereafter, we refer to these impaled cells as penetrons. We have used nano-sized acicular material to establish a novel method for bacterial transformation. Here, we generated penetrons that carried antisense DNA adsorbed on nano-sized acicular material (α-sepiolite) by providing sliding friction onto the surface of agar gel; we then investigated whether penetron formation was applicable to gene silencing techniques. Antisense DNA was artificially synthesized as 15 or 90mer DNA oligonucleotides based on the sequences around the translation start codon of target mRNAs. Mixtures of bacterial cells with antisense DNA adsorbed on α-sepiolite were stimulated by sliding friction on the surface of agar gel for 60 s. Upon formation of Escherichia coli penetrons, β-lactamase and β-galactosidase expression was evaluated by counting the numbers of colonies formed on LB agar containing ampicillin and by measuring β-galactosidase activity respectively. The numbers of ampicillin resistant colonies and the β-galactosidase activity derived from penetrons bearing antisense DNA (90mer) was repressed to 15% and 25%, respectively, of that of control penetrons which lacked antisense DNA. Biphenyl metabolite, ring cleavage yellow compound produced by Pseudomonas pseudoalcaligenes penetron treated with antisense oligonucleotide DNA targeted to bphD increased higher than that lacking antisense DNA. This result indicated that expression of bphD in P. pseudoalcaligenes penetrons was repressed by antisense DNA that targeted bphD mRNA. Sporulation rates of Bacillus subtilis penetrons treated with antisense DNA (15mer) targeted to spo0A decreased to 24.4% relative to penetrons lacking antisense DNA. This novel method of gene silencing has substantial promise for elucidation of gene function in bacterial species that have been refractory to experimental introduction of exogenous DNA.

Two mechanisms for putrescine-dependent transcriptional expression of the putrescine aminotransferase gene, ygjG, in Escherichia coli.

In this study, on evaluating the physiological function and mechanism of putrescine, we found that putrescine supplementation (1mM) increases transcription of the putrescine aminotransferase gene, ygjG. Putrescine-dependent expression was confirmed by measuring β-galactosidase activity and with reverse transcription-polymerase chain reaction. To understand the role of putrescine in ygjG expression, we genetically characterized and found that a knockout mutation in an alternative sigma factor, rpoS, abolished putrescine-dependent ygjG-lacZ expression. In the rpoS mutant, RpoS overexpression complemented the mutant phenotype. However, RpoS overexpression induced ygjG-lacZ expression with putrescine supplementation but not without supplementation. We also found that the loss of putrescine-dependent ygjG-lacZ expression induced by rpoS was completely restored under nitrogen-starvation conditions. The putrescine-dependent expression of ygjG-lacZ under this condition was clearly dependent on another alternative sigma factor, rpoN, and its cognate activator ntrC. These results show that rpoS is required for putrescine-dependent ygjG-lacZ expression, but the effect of putrescine on this expression is not caused by simple modulation of RpoS synthesis. Putrescine-dependent expression of ygjG-lacZ was controlled by at least two sigma factors: rpoS under excess nitrogen conditions and rpoN under nitrogen-starvation conditions. These results suggest that putrescine plays an important role in the nitrogen regulation system.

Enhanced Gene Transfer to Pancreatic Islets Using Glucagon-Like Peptide-1

ObjectiveThe efficient transfer of genes into intact islets is difficult since islets exist as clusters of differentiated cells with little replication potential. Cell proliferation in response to growth factors is known to be accompanied by loosening of cell-to-cell contacts and increasing paracellular permeability. In this study, we investigated whether gene delivery into intact islet cells was facilitated by modulating β-cell proliferation. MethodsIsolated rat islets were pretreated with glucagon-like peptide (GLP)-1 or human growth hormone for 24 hours, or with 300 mg/dL of glucose for 48 hours before transduction with a suboptimal dose of recombinant adenoviral vector expressing green fluorescent protein (GFP) and β-galactosidase (multiplicity of infection of 25). Transduction efficiency was assessed by measuring β-galactosidase activity and GFP expression using enzyme-linked immunosorbent assay, flow cytometry, and fluorescence microscopy. The numbers of 7-aminoactinomycin D-positive dead cells and 5-ethynyl-2-deoxyuridine (EdU)-positive proliferating cells were also monitored using flow cytometry and fluorescence microscopy. ResultsThe transduction efficiency of rat islet cells by a suboptimal dose of viral vector was significantly improved by GLP-1 pretreatment, accompanied by enhanced cell viability and cell proliferation. An increased GFP expression in islet cells after GLP-1 pretreatment was observed among the increased numbers of EdU-positive proliferating cells. ConclusionPretreatment of rat islets with GLP-1 enhanced the transduction efficiency of an adenoviral vector, reducing viral dose burden while improving islet cell viability. From a therapeutic standpoint, genetic modification of pancreatic islets combined with GLP-1 pretreatment may be a promising option for ex vivo gene therapy prior to islet transplantation.

Effect of Preservatives on Shiga Toxigenic Phages and Shiga Toxin of Escherichia coli O157:H7

Toxin synthesis by Shiga toxin-producing Escherichia coli (STEC) appears to be coregulated through the induction of the integrated bacteriophages that encode the toxin genes. These phages might be the principal means for the dissemination and release of Shiga toxins. We evaluated the effect of three common food preservatives, potassium sorbate, sodium benzoate, and sodium propionate, on the propagation of the phages and Shiga toxins. We tested each preservative at four concentrations, 1, 1.25, 2.5, and 5 mg/ml, both on free phages and on lysogenic phages in bacteria. We also evaluated the expression of a lambdoid phage, which was exposed to increasing concentrations of preservatives, by measuring β-galactosidase activity from SPC105, a transductant strain. Furthermore, we tested the effect of the preservatives on cytotoxigenic activity of Shiga toxin on Vero cells. We detected an increase of the inhibitory effect of the phage lytic activity, both in lysogenic and free phages, as the preservative concentration increased. However, the inhibition was higher on the lysogenic phages release than on free phages. Sodium benzoate and potassium sorbate were about equal at inhibiting phages; they were more effective than sodium propionate. A significant decrease of lacZ expression, encoded in a lambda phage, was observed. We also found a reduction in Shiga toxin titer caused by exposure of E. coli O157:H7 to 5 mg/ml sodium benzoate or potassium sorbate. These results imply that these three preservatives, used to inhibit microbial spoilage of foods, also act to inhibit lytic activity and dispersion of a phage carrying the gene encoding powerful Shiga cytotoxins. Also notable was the inactivation of Shiga toxin activity, although this effect was detected using concentrations of preservatives greater than those allowed by the Argentine Food Code.

Mechanisms of Host Receptor Adaptation by Severe Acute Respiratory Syndrome Coronavirus

The severe acute respiratory syndrome coronavirus (SARS-CoV) from palm civets has twice evolved the capacity to infect humans by gaining binding affinity for human receptor angiotensin-converting enzyme 2 (ACE2). Numerous mutations have been identified in the receptor-binding domain (RBD) of different SARS-CoV strains isolated from humans or civets. Why these mutations were naturally selected or how SARS-CoV evolved to adapt to different host receptors has been poorly understood, presenting evolutionary and epidemic conundrums. In this study, we investigated the impact of these mutations on receptor recognition, an important determinant of SARS-CoV infection and pathogenesis. Using a combination of biochemical, functional, and crystallographic approaches, we elucidated the molecular and structural mechanisms of each of these naturally selected RBD mutations. These mutations either strengthen favorable interactions or reduce unfavorable interactions with two virus-binding hot spots on ACE2, and by doing so, they enhance viral interactions with either human (hACE2) or civet (cACE2) ACE2. Therefore, these mutations were viral adaptations to either hACE2 or cACE2. To corroborate the above analysis, we designed and characterized two optimized RBDs. The human-optimized RBD contains all of the hACE2-adapted residues (Phe-442, Phe-472, Asn-479, Asp-480, and Thr-487) and possesses exceptionally high affinity for hACE2 but relative low affinity for cACE2. The civet-optimized RBD contains all of the cACE2-adapted residues (Tyr-442, Pro-472, Arg-479, Gly-480, and Thr-487) and possesses exceptionally high affinity for cACE2 and also substantial affinity for hACE2. These results not only illustrate the detailed mechanisms of host receptor adaptation by SARS-CoV but also provide a molecular and structural basis for tracking future SARS-CoV evolution in animals.

Amperometric detection of Enterobacteriaceae in river water by measuring β-galactosidase activity at interdigitated microelectrode arrays

Two simple methodologies are compared for the detection of faecal contamination in water using amperometry at gold interdigitated microelectrodes. They rely on the detection of β-galactosidase (β-gal) by redox cycling amperometry of the p-aminophenol (PAP) produced by the enzyme from the 4-aminophenyl β- d-galactopyranoside (PAPG) substrate. The use of phages as specific agents for the release of the bacteria-enclosed enzyme allowed the detection of 6 × 10 5 CFU mL −1 Escherichia coli in 2 h without any pre-enrichment or preconcentration steps. Better limits of detection were achieved for the second strategy in the absence of phages. In this case, bacteria were enriched in the presence of both β- d-1-thiogalactopyranoside (IPTG) and substrate but in the absence of phages. Under such experimental conditions, 5 × 10 4 CFU mL −1 E. coli could be detected after 2 h of incubation, while 7 h of incubation were enough to detect down to 10 CFU mL −1 in river water samples. This represents a straightforward one-step method for the detection of faecal contamination that can be conducted in a single working day with minimal sample manipulation by the user.