High-throughput system for the thermostability analysis of proteins.

Thermal stability of proteins is a primary metric for evaluating their physical properties. Although researchers attempted to predict it using machine learning frameworks, their performance has been dependent on the quality and quantity of published data. This is due to the technical limitation that thermodynamic characterization of protein denaturation by fluorescence or calorimetry in a high-throughput manner has been challenging. Obtaining a melting curve that derives solely from the target protein requires laborious purification, making it far from practical to prepare a hundred or more samples in a single workflow. Here, we aimed to overcome this throughput limitation by leveraging the high protein secretion efficacy of Brevibacillus and consecutive treatment with plate-scale purification methodologies. By handling the entire process of expression, purification, and analysis on a per-plate basis, we enabled the direct observation of protein denaturation in 384 samples within 4 days. To demonstrate a practical application of the system, we conducted a comprehensive analysis of 186 single mutants of a single-chain variable fragment of nivolumab, harvesting the melting temperature (Tm) ranging from -9.3 up to +10.8°C compared to the wild-type sequence. Our findings will allow for data-driven stabilization in protein design and streamlining the rational approaches.

Structural Organization of Brevilaterin Biosynthesis in Brevibacillus laterosporus S62-9: A Novel MbtH-Independent Cationic Antimicrobial Peptide Synthetase System.

Cationic antimicrobial peptides, produced by nonribosomal peptide synthetases (NRPSs), have received great attention in different applications, including as biocontrol and antimicrobial agents against foodborne pathogenic bacteria. Also, Brevibacillus spp. is a competent microorganism to produce cationic antimicrobial peptides yet has received little attention. Herein, Brevibacillus laterosporus S62-9 genome mining revealed an integrated cationic antimicrobial peptide synthetase system that synthesized brevilaterin. Combining biochemical analysis with bioinformatics elucidated that the A domain from this system was the MbtH-independent enzyme and showed activity against the same amino acid in the structure of brevilaterin. Moreover, the creations of the first three and position 12 residues in the sequence were targeted to bre261, bre270, bre2691A, and bre2662, respectively. Further analysis of the specificity-conferring code of the A domain suggested that a tiny difference would make the activity of the A domain very diverse and the range of substrate selection would be enlarged or narrowed by changing some residues in the code. The dissection of this biosynthesis mechanism would contribute to the successful realization of reasonable artificial design and the modification of bioactive peptides, and this capable organism also would be more fully utilized.

Oligomerisation of tryptocidine C, a Trp-rich cyclodecapeptide from the antimicrobial tyrothricin complex.

Tryptocidine C (TpcC, cyclo[D-Phe1-Pro2-Trp3-D-Trp4-Asn5-Gln6-Trp7-Val8-Orn9-Leu10]) is a broad-spectrum antimicrobial peptide in the tyrothricin complex produced by a soil bacterium, Brevibacillus parabrevis. Electrospray mass spectrometric studies reveal the oligomerisation of TpcC into dimers and higher oligomers, analogous to tyrocidine C (TrcC, Trp7 replaced by Tyr7). Ion mobility mass spectrometry (IMMS) further confirms the formation of stable peptide dimers and tetramers with diameters of 2.7 nm and 3.3 nm, respectively, calculated from collisional cross section (CCS). Molecular dynamic simulations and docking studies support the formation of amphipathic dimers, with a diameter of 2.5 ± 0.07 nm calculated from low energy model CCS. Circular dichroism and IMMS studies point towards dynamic hydrogen-bonded conformational changes up to 28-33 µM after which the structures become more static (or in equilibrium). Fluorescence studies indicate aromatic stacking of Trp residues with a CMC of 18 µM in aqueous solutions. The concentration and time dependent interaction of Trp in oligomers indicate cooperativity in the TpcC oligomerisation that leads to the formation of higher order microscopic structures. Scanning electron microscopy studies unequivocally shows that TpcC forms nanospheres with a mean diameter of 25 nm. Repeated smaller oligomeric units, possibly dimers and tetramers, self-assemble to form these nanospheres.

Atomistic Characterization of Gramicidin Channel Formation.

We investigated gramicidin A (gA) subunit dimerization in lipid bilayers using microsecond-long replica-exchange umbrella sampling simulations, millisecond-long unbiased molecular dynamics simulations, and machine learning. Our simulations led to a dimer structure that is indistinguishable from the experimentally determined gA channel structures, with the two gA subunits joined by six hydrogen bonds (6HB). The simulations also uncovered two additional dimer structures, with different gA-gA stacking orientations that were stabilized by four or two hydrogen bonds (4HB or 2HB). When examining the temporal evolution of the dimerization, we found that two bilayer-inserted gA subunits can form the 6HB dimer directly, with no discernible intermediate states, as well as through paths that involve the 2HB and 4HB dimers.

Insecticidal potential of Brevibacillus laterosporus against dipteran pest species in a wide ecological range.

In order to increase our understanding of the insecticidal potential of the entomopathogenic bacterium Brevibacillus laterosporus strain UNISS 18 against insect pests, investigations were conducted on a selection of dipteran species including fruit flies, house flies, blow flies, and mosquitoes, characterized by adaptations to very diverse habitats. According to lethal concentration (LC50) values, the common house mosquito Culex pipiens (LC50 = 0.10 × 106 spores/mL) and the yellow fever mosquito Aedes aegypti (LC50 = 0.18 × 106 spores/mL) were significantly more susceptible than the flies. The blow flies were the second taxon in term of susceptibility to B. laterosporus spores, with a higher mortality in Calliphora vomitoria (LC50 = 78.84 × 106 spores/mL) than Lucilia caesar (LC50 = 148.30 × 106 spores/mL). The effectiveness of B. laterosporus spores was reduced by half in the house fly Musca domestica (LC50 = 82.41 × 106 spores/mL). The lowest susceptibility was observed in the fruit flies, among which the spotted wing drosophila (SWD), Drosophila suzukii, was the most susceptible (LC50 = 217.51 × 106 spores/mL) in comparison with the medfly Ceratitis capitata and the olive fly Bactrocera oleae (LC50 = 2567.32 and 2567.36 × 106 spores/mL, respectively). The present study demonstrated that significantly different degrees of susceptibility are associated with diverse dipteran species including plant and animal parasites, and we suggest that B. laterosporus established different relationships with dipteran species in different ecosystems.

Discovery of a Novel Antimicrobial Lipopeptide, Brevibacillin V, from Brevibacillus laterosporus fmb70 and Its Application on the Preservation of Skim Milk.

Increasing cases of infections by foodborne pathogenic bacteria resulted in a great demand to find safe and novel antimicrobial compounds that can be used in the food industry. The isolation and application of antimicrobial peptides including lipopeptides has been increasing tremendously in the past years. In this study, a new bacterial strain called Brevibacillus laterosporus fmb70 (fmb70) was isolated and exhibited strong antimicrobial activities against Gram-positive, Gram-negative bacteria, and fungi. Two major antimicrobial components produced by fmb70 were respectively identified as lipopeptide: brevibacillin V (MW: 1570.12 Da) and brevibacillin (MW: 1583.75 Da), of which brevibacillin V was a new compound. Both of them consisted of 13 amino acids and C6 fatty acyl (FA) chain. Brevibacillin V and brevibacillin showed significant antimicrobial activities against most foodborne pathogenic bacteria and phytopathogenic fungi. They stayed activity at 100 °C and remained 50% of their antimicrobial activities at pH 3 for 22 h. Hemolytic activities of them were lower than 8%. They effectively eliminated the S. aureus GIM 1.142 and L. monocytogenes ATCC 21633 in skim milk. In conclusion, the Brevibacillus laterosporus fmb70 and its major antimicrobial components has remarkable potentials in the food industry.

Tyrocidine A interactions with saccharides investigated by CD and NMR spectroscopies.

Tyrocidines are a family of cyclic decapeptides produced by the soil bacterium, Brevibacillus parabrevis. These antibiotic peptides can be used to prevent infections in agriculture and food industry but also to prepare antimicrobial lozenges, creams, and dressings for medical applications. It has been observed that the tyrocidines interact with saccharides such as cellulose from their soil environment, as well as sugars in culture media and glycans in fungal cell walls. Here, we investigated the interactions of tyrocidines with glucose, sucrose, and cellotetraose (as cellulose model) in a quantitative fashion utilising CD and NMR spectroscopy. The CD and NMR spectra of tyrocidine A (TrcA) were analysed as a function of solvent composition, and the spectral properties agree with the formation of oligomeric structures that are governed by ß-sheet secondary structures once the acetonitrile content of the solvent is increased. Saccharides seem to also induce TrcA spectral changes reverting those induced by organic solvents. The CD spectral changes of TrcA in the presence of glucose agree with new ordered H-bonding, possibly ß-sheet structures. The amides involved in intramolecular H-bonding remained largely unaffected by the environmental changes. In contrast, amides exposed to the exterior and/or involved in TrcA intermolecular association show the largest 1 H chemical shift changes. CD and NMR spectroscopic investigations correlated well with TrcA-glucose interactions characterized by a dissociation constant around 200 µM. Interestingly, the association of cellotetraose corresponds closely to the additive effect from four glucose moieties, while a much higher dissociation constant was observed for sucrose. Similar trends to TrcA for binding to the three saccharides were observed for the analogous tyrocidines, tyrocidine B, and tyrocidine C. These results therefore indicate that the tyrocidine interactions with the glucose monosaccharide unit are fairly specific and reversible.

X-ray crystallographic analysis of the catalytic domain of α-1,3-glucanase FH1 from Paenibacillus glycanilyticus overexpressed in Brevibacillus choshinensis.

α-1,3-Glucanase hydrolyzes α-1,3-glucan, an insoluble linear α-1,3-linked homopolymer of glucose that is found in the extracellular polysaccharides produced by oral streptococci in dental plaque and in fungal cell walls. This enzyme could be of application in dental care and the development of fungal cell-wall lytic enzymes, but its three-dimensional structure has not been available to date. In this study, the recombinant catalytic domain of α-1,3-glucanase FH1 from Paenibacillus glycanilyticus FH11, which is classified into glycoside hydrolase family 87, was prepared using a Brevibacillus choshinensis expression system and purified in a soluble form. Crystals of the purified protein were produced by the sitting-drop vapor-diffusion method. Diffraction data were collected to a resolution of 1.6 Šusing synchrotron radiation. The crystals obtained belonged to the tetragonal space group P41212 or P43212, with unit-cell parameters a = b = 132.6, c = 76.1 Å. The space group and unit-cell parameters suggest that there is one molecule in the asymmetric unit.

A novel design to screen chlorogenic acid-producing microbial strains from the environment.

The present study aimed to develop a plate-screening method, based on the specific color development of complexes formed between chlorogenic acid, a valuable plant-derived compound, and aluminum (III), to detect chlorogenic acid-producing microbial strains. Modified media with 0.75 mM aluminum chloride were developed to identify CGA-producing bacteria (based on beef extract agar medium) or fungi (based on the potato dextrose agar medium). Compared with conventional screening, the modified media let to 3.3 times more CGA producers from plants, at 90.9% selective accuracy. Novel chlorogenic acid-biosynthesizing strains included Brevibacillus borstelensis B14, Bacillus amyloliquefaciens B17, Bacillus badius B19, Sphingomonas yabuuchiae N21, Enterobacter tabaci N22, and Lodderomyces elongisporus S216 and P212. Strain S216 produced the highest chlorogenic acid yield (23.39 mg L-1). This study provides a highly efficient and low-cost tool for quick detection and subsequent identification of several newly isolated strains with chlorogenic acid-producing potential.

Biologically Inspired Total Synthesis of Ulbactin F, an Iron-Binding Natural Product.

Natural products from environmental microbiomes provide exquisite templates for elucidating biological activity in the search for new drugs. A recently discovered marine Brevibacillus sp. metabolite, ulbactin F, was found to inhibit tumor cell migration and invasion at IC50 < 3 µM. Herein, we disclose the first total synthesis of ulbactin F and epi-ulbactin F, which was modeled after the biosynthetic pathway. The scaffold bears structural similarity to siderophores of human pathogens but contains a novel tricyclic ring system derived from cysteine. We have found that ulbactin F forms low-affinity metal complexes, with a preference for Fe3+ and Cu2+, which may hint both at its environmental role and its antimetastatic mechanism of action.

Subsurface Endospore-Forming Bacteria Possess Bio-Sealant Properties.

Concrete is a strong and fairly inexpensive building substance, but has several disadvantages like cracking that allows corrosion, thus reducing its lifespan. To mitigate these complications, long-lasting microbial self-healing cement is an alternative that is eco-friendly and also actively repairs cracks. The present paper describes the detailed experimental investigation on compressive strength of cement mortars, mixed with six alkaliphilic bacteria, isolated from subsurface mica mines of high alkalinity. The experiments showed that the addition of alkaliphilic isolates at different cell concentrations (104 and 106 cells/ml) enhanced the compressive strength of cement mortar, because the rapid growth of bacteria at high alkalinity precipitates calcite crystals that lead to filling of pores and densifying the concrete mix. Thus, Bacillus subtilis (SVUNM4) showed the highest compressive strength (28.61%) of cement mortar at 104 cells/ml compared to those of other five alkaliphilic isolates (Brevibacillus sp., SVUNM15-22.1%; P. dendritiformis, SVUNM11-19.9%; B. methylotrophicus, SVUNM9-16%; B. licheniformis, SVUNM14-12.7% and S. maltophilia, SVUNM13-9.6%) and controlled cement mortar as well. This method resulted in the filling of cracks in concrete with calcite (CaCO3), which was observed by scanning electron microscopy (SEM). Our results showed that the alkaliphilic bacterial isolates used in the study are effective in self-healing and repair of concrete cracks.

Brevibacillus laterosporus isolated from the digestive tract of honeybees has high antimicrobial activity and promotes growth and productivity of honeybee's colonies.

The development of novel antimicrobial drugs, as well as the discovery of novel compounds able to promote honeybee's growth, represents major challenges for modern entomology. The main aim of this study was to investigate whether Brevibacillus laterosporus isolated from the digestive tract of Saudi honeybees, Apis mellifera, was able to stimulate colony strength parameters of honeybees and to evaluate its ability to produce antimicrobial agents. Honeybees were collected in Dirab, Riyadh Region, Saudi Arabia, and microorganisms were isolated and identified by 16S ribosomal RNA analysis. Microscopic identification of the microorganism in its native state was facilitated by atomic force microscopy at high-resolution imaging. Active biological compounds were produced by submerged fermentation with B. laterosporus. The fermented broth was subjected to extraction and purification, and then semi-pure compounds were analyzed by gas chromatography-mass spectrometry. The effectiveness of the crude extract and semi-pure compounds as antimicrobial agents was evaluated by susceptibility assays. More than 22% of the microorganisms isolated from the digestive tract of healthy honeybees have been identified as B. laterosporus, this kind of species has a unique shape and morphological structure. The cyclic dipeptide cyclo(Leu-Pro) produced by B. laterosporus showed biological activity against several pathogenic microorganisms. Furthermore, the total counts of workers, closed brood, and open brood, as well as the production of bee pollen and honey, were better in honeybees treated with a B. laterosporus suspension. The data indicated that the B. laterosporus strain isolated from a healthy honeybee might be a novel probiotic and a producer of important biological compounds.

Biosurfactants and their role in the inhibition of the biofilmforming pathogens.

Resistance of pathogenic bacteria is currently one of the major medical problems. Most microbial infections are based on the formation of biofilms, which are a significant reservoir of pathogens. The aim of this study is to determine the antibiofilm and antimicrobial activity of biosurfactants isolated from intestinal lactobacilli and marine bacteria. Biosurfactants (BS) isolated from the strains L. fermentum 2I3, L. fermentum B2/6, L. reuteri SL16, L. reuteri B6/1, S. luteola 3/22, Brevibacillus sp. 4/9, Brevibacillus sp. 2/30 and B. amyloliquefaciens 1/6K significantly (p < 0.001) inhibited the biofilm formation of S. aureus CCM 3953 and P. mirabilis CCM 7188, with higher inhibition detected in BS of marine bacteria when compared to BS isolated from lactobacilli. The results suggest that the mechanism of the antibiofilm effect of BS isolated from lactobacilli against both the reference strains is the same and it is not the result of their antimicrobial action. In contrast, the mechanism of the antibiotic effect of BS isolated from marine bacteria probably depends on the properties of the indicator strain. Key words: biosurfactants biofilm pathogens inhibition.

Bacterial Inoculant Treatment of Bermudagrass Alters Ovipositional Behavior, Larval and Pupal Weights of the Fall Armyworm (Lepidoptera: Noctuidae).

Nonpathogenic soil bacteria can colonize the rhizosphere and induce unique plant phenotypes that may influence plant-insect interactions. However, few studies have considered the influences of bacteria-plant interactions on insect feeding and oviposition. The objective of this study was to determine how rhizobacterial inoculation of bermudagrass affects larval development and ovipositional behaviors of the fall armyworm (Spodoptera frugiperda J.E. Smith). Eight blends of rhizobacteria known to induce root or shoot growth in grasses were applied weekly to hybrid bermudagrass for 5 wk. Oviposition was evaluated in two no-choice trials with bacteria-treated, fertilized, or nontreated grass. Grass blades from these treatments were extracted in polar and nonpolar solvents and assayed for oviposition responses. Another experiment compared the development of fall armyworm larvae on bermudagrass treated with each of the eight rhizobacterial blends for 5 wk to larvae fed nontreated bermudagrass. Females deposited more eggs on nontreated and fertilized grass and ≤34% of eggs on grass treated with rhizobacterial blends. Moths exposed to polar and nonpolar extracts were unable to reproduce these results. Larval and pupal weights at days 10 and 12 and the number of adults to eclose were lower for larvae fed some, but not all, bacteria-treated bermudagrass relative to controls. This is one of the few studies to investigate plant-microbe-insect interactions in an economically important system. Although the effects noted with fall armyworm are limited, induced changes in roots also reported for these bacteria may have greater utility than foliar changes for mediating interactions with biotic or abiotic stresses.

Antibacterial and antitumor activity of Bogorol B-JX isolated from Brevibacillus laterosporus JX-5.

Antimicrobial peptides are promising anti-infective agent candidates because they have a broad antimicrobial spectrum and bioactivity and are unlikely to elicit antibiotic resistance. The bogorols represent a new cationic antibiotic peptide and possess great therapeutic potential because of their bioactivity and precise mode of action. Here, we report that Bogorol B-JX (BBJX), a peptide previously isolated from Brevibacillus laterosporus JX-5 by us, has significant antibacterial and antitumor activities in vitro. BBJX was found to inhibit methicillin-resistant Staphylococcus aureus (MRSA) at 2.5 µg/mL with distinct mechanisms of action from those against Bacillus bombyseptieus and Escherichia coli. It penetrates MRSA membrane with little visible destruction and binds to genomic DNA. BBJX could inhibit the proliferation of human histiocytic lymphoma cell line U-937 and ConA-activated spleen cells at 5 µg/mL, but was not cytotoxic to the Jurkat cells, resting spleen cells or differentiated macrophage-like U-937 immunocytes. Moreover, BBJX caused apoptosis of U-937 cells by opening the mitochondrial permeability transition pore and stimulating the production of reactive oxygen species. Taken together, these studies provided basis for future medical application of the bogorols.

Paclitaxel Biosynthesis: Adenylation and Thiolation Domains of an NRPS TycA PheAT Module Produce Various Arylisoserine CoA Thioesters.

Structure-activity relationship studies show that the phenylisoserinyl moiety of paclitaxel (Taxol) is largely necessary for the effective anticancer activity. Several paclitaxel analogues with a variant isoserinyl side chain have improved pharmaceutical properties versus those of the parent drug. To produce the isoserinyl CoAs as intermediates needed for enzyme catalysis on a semibiosynthetic pathway to paclitaxel analogues, we repurposed the adenylation and thiolation domains (Phe-AT) of a nonribosomal peptide synthetase (TycA) so that they would function as a CoA ligase. Twenty-eight isoserine analogue racemates were synthesized by an established procedure based on the Staudinger [2+2] cycloaddition reaction. Phe-AT converted 16 substituted phenylisoserines, one ß-(heteroaryl)isoserine, and one ß-(cyclohexyl)isoserine to their corresponding isoserinyl CoAs. We imagine that these CoA thioesters can likely serve as linchpin biosynthetic acyl donors transferred by a 13-O-acyltransferase to a paclitaxel precursor baccatin III to make drug analogues with better efficacy. It was also interesting to find that an active site mutant [Phe-AT (W227S)] turned over 2-pyridylisoserine and the sterically demanding p-methoxyphenylisoserine substrates to their CoA thioesters, while Phe-AT did not. This mutant is promising for further development to make 3-fluoro-2-pyridylisoserinyl CoA, a biosynthetic precursor of the oral pharmaceutical tesetaxel used for gastric cancers.

Brevibacillin, a cationic lipopeptide that binds to lipoteichoic acid and subsequently disrupts cytoplasmic membrane of Staphylococcus aureus.

Brevibacillin is a newly-discovered antimicrobial lipopeptide produced by Brevibacillus laterosporus OSY-I1. It is active against Gram-positive bacteria, including antibiotic resistant strains. This research was initiated to investigate the mechanism of action of brevibacillin against an indicator strain, Staphylococcus aureus ATCC 6538. Results of the study proved that brevibacillin binds to lipoteichoic acid (LTA) on cell wall before interacting with cell membrane. Additionally, brevibacillin disrupts S. aureus cytoplasmic membrane by increasing its permeability, depolarization and potassium leakage. Therefore, cytoplasmic membrane serves as a major target for brevibacillin. Despite the presence of multiple sites on S. aureus cell envelope, scanning electron microscope observation didn't reveal evidence of cell lysis or any morphological defects in cells treated with brevibacillin. Based on the results of this study, we propose that the electrostatic interaction between the cationic brevibacillin and the anionic LTA helped the accumulation of the antimicrobial agent at cell surface; this was followed by translocation of the lipopeptide to the cytoplasmic membrane and disrupting its vital functions.

Manipulation of an existing crystal form unexpectedly results in interwoven packing networks with pseudo-translational symmetry.

Nonribosomal peptide synthetases (NRPSs) are multimodular enzymes that synthesize a myriad of diverse molecules. Tailoring domains have been co-opted into NRPSs to introduce further variety into nonribosomal peptide products. Linear gramicidin synthetase contains a unique formylation-tailoring domain in its initiation module (F-A-PCP). The structure of the F-A di-domain has previously been determined in a crystal form which had large solvent channels and no density for the minor Asub subdomain. An attempt was made to take advantage of this packing by removing the Asub subdomain from the construct (F-AΔsub) in order to produce a crystal that could accommodate the PCP domain. In the resulting crystal the original packing network was still present, but a second network with the same packing and almost no contact with the original network took the place of the solvent channels and changed the space group of the crystal.

Classification of Bacillus and Brevibacillus species using rapid analysis of lipids by mass spectrometry.

Bacillus are aerobic spore-forming bacteria that are known to lead to specific diseases, such as anthrax and food poisoning. This study focuses on the characterization of these bacteria by the detection of lipids extracted from 33 well-characterized strains from the Bacillus and Brevibacillus genera, with the aim to discriminate between the different species. For the purpose of analysing the lipids extracted from these bacterial samples, two rapid physicochemical techniques were used: matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF-MS) and liquid chromatography in conjunction with mass spectrometry (LC-MS). The findings of this investigation confirmed that MALDI-TOF-MS could be used to identify different bacterial lipids and, in combination with appropriate chemometrics, allowed for the discrimination between these different bacterial species, which was supported by LC-MS. The average correct classification rates for the seven species of bacteria were 62.23 and 77.03 % based on MALDI-TOF-MS and LC-MS data, respectively. The Procrustes distance for the two datasets was 0.0699, indicating that the results from the two techniques were very similar. In addition, we also compared these bacterial lipid MALDI-TOF-MS profiles to protein profiles also collected by MALDI-TOF-MS on the same bacteria (Procrustes distance, 0.1006). The level of discrimination between lipids and proteins was equivalent, and this further indicated the potential of MALDI-TOF-MS analysis as a rapid, robust and reliable method for the classification of bacteria based on different bacterial chemical components. Graphical abstract MALDI-MS has been successfully developed for the characterization of bacteria at the subspecies level using lipids and benchmarked against HPLC.

Ulbactins F and G, Polycyclic Thiazoline Derivatives with Tumor Cell Migration Inhibitory Activity from Brevibacillus sp.

Two new structurally unique compounds bearing a nitrogen- and sulfur-containing tricyclic ring system, ulbactin F (1) and its diastereomeric isomer ulbactin G (2), were isolated from the culture extract of a sponge-derived Brevibacillus sp. The structures and absolute configurations of 1 and 2 were determined by NMR analysis and X-ray crystallographic analysis. These compounds inhibit the migration of tumor cells in the submicromolar to micromolar range.