Quantification of early biofilm growth in microtiter plates through a novel image analysis software.

Given the significant impact of biofilms on human health and material corrosion, research in this field urgently needs more accessible techniques to facilitate the testing of new control agents and general understanding of biofilm biology. Microtiter plates offer a convenient format for standardized evaluations, including high-throughput assays of alternative treatments and molecular modulators. This study introduces a novel Biofilm Analysis Software (BAS) for quantifying biofilms from microtiter plate images. We focused on early biofilm growth stages and compared BAS quantification to common techniques: direct turbidity measurement, intrinsic fluorescence detection linked to pyoverdine production, and standard crystal violet staining which enables image analysis and optical density measurement. We also assessed their sensitivity for detecting subtle growth effects caused by cyclic AMP and gentamicin. Our results show that BAS image analysis is at least as sensitive as the standard method of spectrophotometrically quantifying the crystal violet retained by biofilms. Furthermore, we demonstrated that bacteria adhered after short incubations (from 10 min to 4 h), isolated from planktonic populations by a simple rinse, can be monitored until their growth is detectable by intrinsic fluorescence, BAS analysis, or resolubilized crystal violet. These procedures are widely accessible for many laboratories, including those with limited resources, as they do not require a spectrophotometer or other specialized equipment.

Highly Sensitive Whole-Cell Mercury Biosensors for Environmental Monitoring.

Whole-cell biosensors could serve as eco-friendly and cost-effective alternatives for detecting potentially toxic bioavailable heavy metals in aquatic environments. However, they often fail to meet practical requirements due to an insufficient limit of detection (LOD) and high background noise. In this study, we designed a synthetic genetic circuit specifically tailored for detecting ionic mercury, which we applied to environmental samples collected from artisanal gold mining sites in Peru. We developed two distinct versions of the biosensor, each utilizing a different reporter protein: a fluorescent biosensor (Mer-RFP) and a colorimetric biosensor (Mer-Blue). Mer-RFP enabled real-time monitoring of the culture's response to mercury samples using a plate reader, whereas Mer-Blue was analysed for colour accumulation at the endpoint using a specially designed, low-cost camera setup for harvested cell pellets. Both biosensors exhibited negligible baseline expression of their respective reporter proteins and responded specifically to HgBr2 in pure water. Mer-RFP demonstrated a linear detection range from 1 nM to 1 µM, whereas Mer-Blue showed a linear range from 2 nM to 125 nM. Our biosensors successfully detected a high concentration of ionic mercury in the reaction bucket where artisanal miners produce a mercury-gold amalgam. However, they did not detect ionic mercury in the water from active mining ponds, indicating a concentration lower than 3.2 nM Hg2+-a result consistent with chemical analysis quantitation. Furthermore, we discuss the potential of Mer-Blue as a practical and affordable monitoring tool, highlighting its stability, reliance on simple visual colorimetry, and the possibility of sensitivity expansion to organic mercury.

A review of shared decision-making: A call to arms for health care professionals.

OBJECTIVE: To illustrate the use of shared decision-making (SDM) and SDM tools and aids as the essential components in the care of asthma. DATA SOURCES: We reviewed individual randomized controlled studies conducted between 1998 and 2020 to compare SDM interventions and the use of SDM tools and aids for the care of asthma. All studies were published or translated in English. STUDY SELECTIONS: We excluded studies of interventions that involved multiple components other than the SDM intervention unless the control group also received these interventions. We evaluated the existing literature on both SDM tools and aids and the process of SDM to summarize in this review. RESULTS: Shared decision-making tools and aids most commonly clarify the diagnostics and options for a treatment. The 6 elements of SDM were clearly supported. We found no considerable association between the presence of these elements of SDM and asthma outcomes. CONCLUSION: We found that SDM for asthma and SDM tools and aids were often made to transfer information about asthma treatment options and their harms and benefits. The correlation between their support of SDM key elements and their impact on asthma outcomes is often difficult to ascertain but when present, there was positive correlation to improving risk communication, adherence, patient satisfaction, and possibly decreasing liability.

Author Correction: The energy cost of polypeptide knot formation and its folding consequences.

The original version of this article contained an error in the spelling of the author Christian A.M. Wilson, which was incorrectly given as Christian M.A. Wilson. This has now been corrected in both the PDF and HTML versions of the article.

The energy cost of polypeptide knot formation and its folding consequences.

Knots are natural topologies of chains. Yet, little is known about spontaneous knot formation in a polypeptide chain-an event that can potentially impair its folding-and about the effect of a knot on the stability and folding kinetics of a protein. Here we used optical tweezers to show that the free energy cost to form a trefoil knot in the denatured state of a polypeptide chain of 120 residues is 5.8 ± 1 kcal mol-1. Monte Carlo dynamics of random chains predict this value, indicating that the free energy cost of knot formation is of entropic origin. This cost is predicted to remain above 3 kcal mol-1 for denatured proteins as large as 900 residues. Therefore, we conclude that naturally knotted proteins cannot attain their knot randomly in the unfolded state but must pay the cost of knotting through contacts along their folding landscape.

Production and characterization of a highly pure RNA polymerase holoenzyme from Mycobacterium tuberculosis.

Recent publications have shown that active RNA polymerase (RNAP) from Mycobacterium tuberculosis (MtbRNAP) can be produced by expressing all four subunits in a single recombinant Escherichia coli strain [1-3]. By reducing the number of plasmids and changing the codon usage of the Mtb genes in the co-expression system published by Banerjee et al. [1], we present a simplified, detailed and reproducible protocol for the purification of recombinant MtbRNAP containing the ω subunit. Moreover, we describe the formation of ternary elongation complexes (TECs) with a short fluorescence-labeled RNA primer and DNA oligonucleotides, suitable for transcription elongation studies. The purification of milligram quantities of the pure and highly active holoenzyme omits ammonium sulfate or polyethylene imine precipitation steps [4] and requires only 5 g of wet cells. Our results indicate that subunit assemblies other than α2ßß'ω·σA can be separated by ion-exchange chromatography on Mono Q column and that assemblies with the wrong RNAP subunit stoichiometry lack transcriptional activity. We show that MtbRNAP TECs can be stalled by NTP substrate deprivation and chased upon the addition of missing NTP(s) without the need of any accessory proteins. Finally, we demonstrate the ability of the purified MtbRNAP to initiate transcription from a promoter and establish that its open promoter complexes are stabilized by the M. tuberculosis protein CarD.

New insights into the regulatory mechanisms of ppGpp and DksA on Escherichia coli RNA polymerase-promoter complex.

The stringent response modulators, guanosine tetraphosphate (ppGpp) and protein DksA, bind RNA polymerase (RNAP) and regulate gene expression to adapt bacteria to different environmental conditions. Here, we use Atomic Force Microscopy and in vitro transcription assays to study the effects of these modulators on the conformation and stability of the open promoter complex (RPo) formed at the rrnA P1, rrnB P1, its discriminator (dis) variant and λ pR promoters. In the absence of modulators, RPo formed at these promoters show different extents of DNA wrapping which correlate with the position of UP elements. Addition of the modulators affects both DNA wrapping and RPo stability in a promoter-dependent manner. Overall, the results obtained under different conditions of ppGpp, DksA and initiating nucleotides (iNTPs) indicate that ppGpp allosterically prevents the conformational changes associated with an extended DNA wrapping that leads to RPo stabilization, while DksA interferes directly with nucleotide positioning into the RNAP active site. At the iNTPs-sensitive rRNA promoters ppGpp and DksA display an independent inhibitory effect, while at the iNTPs-insensitive pR promoter DksA reduces the effect of ppGpp in accordance with their antagonistic role.

The mitochondrial FAD-dependent glycerol-3-phosphate dehydrogenase of Trypanosomatidae and the glycosomal redox balance of insect stages of Trypanosoma brucei and Leishmania spp.

The genes for the mitochondrial FAD-dependent glycerol-3-phosphate dehydrogenase were identified in Trypanosoma brucei and Leishmania major genomes. We have expressed the L. major gene in Saccharomyces cerevisiae and confirmed the subcellular localization and activity of the produced enzyme. Using cultured T. brucei procyclic and Leishmania mexicana promastigote cells with a permeabilized plasma membrane and containing intact glycosomes, it was shown that dihydroxyacetone phosphate is converted into pyruvate, and stimulates oxygen consumption, indicating that all components of the glycerol 3-phosphate/dihydoxyacetone phosphate shuttle between glycosomes and mitochondrion are present in these insect stages of both organisms. A computer model has been prepared for the energy and carbohydrate metabolism of these cells. It was used in an elementary mode analysis to get insight into the metabolic role of the shuttle in these insect-stage parasites. Our analysis suggests that the shuttle fulfils important roles for these organisms, albeit different from its well-known function in the T. brucei bloodstream form. It allows (1) a high yield of further metabolizable glycolytic products by decreasing the need to produce a secreted end product of glycosomal metabolism, succinate; (2) the consumption of glycerol and glycerol 3-phosphate derived from lipids; and (3) to keep the redox balance of the glycosome finely tuned due to a highly flexible and redundant system.

Characterization of the cofactor-independent phosphoglycerate mutase from Leishmania mexicana mexicana. Histidines that coordinate the two metal ions in the active site show different susceptibilities to irreversible chemical modification.

Phosphoglycerate mutase (PGAM) activity in promastigotes of the protozoan parasite Leishmania mexicana is found only in the cytosol. It corresponds to a cofactor-independent PGAM as it is not stimulated by 2,3-bisphosphoglycerate and is susceptible to EDTA and resistant to vanadate. We have cloned and sequenced the gene and developed a convenient bacterial expression system and a high-yield purification protocol. Kinetic properties of the bacterially produced protein have been determined (3-phosphoglycerate: K(m) = 0.27 +/- 0.02 mm, k(cat) = 434 +/- 54 s(-1); 2-phosphoglycerate: K(m) = 0.11 +/- 0.03 mm, k(cat) = 199 +/- 24 s(-1)). The activity is inhibited by phosphate but is resistant to Cl(-) and SO(4) (2-). Inactivation by EDTA is almost fully reversed by incubation with CoCl(2) but not with MnCl(2), FeSO(4), CuSO(4), NiCl(2) or ZnCl(2). Alkylation by diethyl pyrocarbonate resulted in irreversible inhibition, but saturating concentrations of substrate provided full protection. Kinetics of the inhibitory reaction showed the modification of a new group of essential residues only after removal of metal ions by EDTA. The modified residues were identified by MS analysis of peptides generated by trypsin digestion. Two substrate-protected histidines in the proximity of the active site were identified (His136, His467) and, unexpectedly, also a distant one (His160), suggesting a conformational change in its environment. Partial protection of His467 was observed by the addition of 25 micro m CoCl(2) to the EDTA treated enzyme but not of 125 micro m MnCl(2), suggesting that the latter metal ion cannot be accommodated in the active site of Leishmania PGAM.

Expression, purification, crystallization and preliminary crystallographic analysis of Leishmania mexicana phosphoglycerate mutase.

Bacterially expressed 2,3-bisphosphoglycerate-independent phosphoglycerate mutase (iPGAM) from Leishmania mexicana with a six-His tag fused at its C-terminus was expressed from plasmid pET28a after IPTG induction in Escherichia coli cells and gave a yield of 20 mg of highly purified iPGAM per litre of cell culture. Crystals of the protein complexed with 3-phosphoglycerate were obtained by the hanging-drop method of vapour diffusion with PEG 4000 as the precipitating agent in the presence of cobalt chloride and diffracted synchrotron radiation to beyond 1.90 A. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 62.46, b = 72.27, c = 129.68 A. A model of Bacillus stearothermophilus iPGAM (33% identity) was used to provide an initial molecular-replacement solution. X-ray data to 2.05 A for the structure of L. mexicana iPGAM complexed with 2-phosphoglycerate have also been collected.