Growth Inhibitory Compounds Research Articles

8801 Towards Safe and Efficacious Medical Therapy for Patients with Clinically Non-Functioning Pituitary Tumors

Abstract Disclosure: D. Zhang: None. M. Bergsneider: None. W. Kim: None. M.B. Wang: None. H. Vinters: None. A.P. Heaney: None. Clinically non-functioning pituitary tumors (CNFTs) account for one-third of all pituitary adenomas. They do not cause clinical and/or biochemical evidence of tumor-related hormone hypersecretion, grow insidiously and typically present with mass effect symptoms such as headache and visual field deficits. Surgical resection is the current standard of care for CNFTs but complete resection is infrequently achieved due to their size and invariable invasion of locally adjacent structures. CNFT remnant tumor regrowth rates approach 50% and these patients may need repeat surgery and/or radiotherapy (RT). The latter is quite effective in achieving tumor control, but carries a risk of hypopituitarism of ∼ 40% at 10 years. Although dopamine agonists and somatostatin receptor ligands have been explored as medical therapy, there are currently no consistently effective medical treatments for CNFTs. There is a clear unmet need for novel safe and effective medical therapies for these comparatively common tumors. In a highly innovative approach we have used patient-derived ex vivo 3D tumoroid human CNFTs in an automated high throughput compound screen (HTS) to identify novel tumoricidal and growth inhibitory compounds for CNFTs. To date, we have screened ∼4,000 compounds and have identified 13 potential hits that inhibited tumor cell proliferation (a reduction of 76±15%) and induced apoptosis (induction rate of 8.2 ± 6.6) in the CNFT tumoroids respectively. The hit compounds to date fall into 3 categories based on their targets, namely Na+/K+-ATPase inhibitors (n=6), reactive oxygen species (ROS) regulators (n=4), and cell cycle inhibitors (n=3). Representative “hit” compounds from each group identified in our primary screen were rescreened in triplicate in 20 concentrations from 25µM to 50pM at 2-fold dilutions. Dose-response curves characterized compound doses that inhibited cell viability by 50% (IC50) and ranged from 13nM to 103nM. Two of these 3 compounds also induced 2-3-fold apoptosis at 0.1uM with the final compound displaying a potent 5-10-fold pro-apoptotic effect at 0.15uM concentration. In parallel, we used single cell RNA sequencing to validate targets that emerged from our HTS and to characterize the mechanism of action (MOA) of these hit compounds. As proof of concept, several of the known drug targets were noted to be amongst the highest differentially expressed genes in the CNFT tumor cell population. This approach validating a candidate target in conjunction with demonstrable activity of a drug for that same target in CNFTs may simultaneously provide us with a back-bone molecule to further develop with structural activity relationship analysis and/or allow us to individualize drug choice in patients based on immunocytochemical tumor target expression. Presentation: 6/1/2024

Integrated processes for olive mill wastewater treatment and its revalorization for microalgae culture.

The olive oil industry generates 30 million cubic meters of olive mill wastewaters (OMWWs) annually. OMWWs are a major environmental concern in the Mediterranean region due to their high organic matter content, suspended solids, unpleasant odor, and dark color. The application of primary treatments such as coagulation-flocculation, adsorption, and hybrid systems combining coagulation-flocculation with adsorption has enabled to remove part of the organic matter, color, turbidity, and growth-inhibiting compounds from OMWWs. Among these methods, the hybrid system combining activated carbon and chitosan has proven to be the best removal efficiency. Subsequently, secondary treatment involving the cultivation of Chlorella sp. on OMWWs pretreated with chitosan achieved the highest maximal specific growth rate (0.513 ± 0.022day⁻1) and biomass productivity (0.621 ± 0.021g/L/day). Notably, the fatty acids (FA) profile produced by Chlorella sp. cells grown under these conditions differed, underscoring the potential of OMWWs as a microalgal growth medium. This innovative approach not only addresses environmental issues but also opens new avenues for sustainable bioproducts.

Bioprospecting of inhibitors of EPEC virulence from metabolites of marine actinobacteria from the Arctic Sea.

A considerable number of antibacterial agents are derived from bacterial metabolites. Similarly, numerous known compounds that impede bacterial virulence stem from bacterial metabolites. Enteropathogenic Escherichia coli (EPEC) is a notable human pathogen causing intestinal infections, particularly affecting infant mortality in developing regions. These infections are characterized by microvilli effacement and intestinal epithelial lesions linked with aberrant actin polymerization. This study aimed to identify potential antivirulence compounds for EPEC infections among bacterial metabolites harvested from marine actinobacteria (Kocuria sp. and Rhodococcus spp.) from the Arctic Sea by the application of virulence-based screening assays. Moreover, we demonstrate the suitability of these antivirulence assays to screen actinobacteria extract fractions for the bioassay-guided identification of metabolites. We discovered a compound in the fifth fraction of a Kocuria strain that interferes with EPEC-induced actin polymerization without affecting growth. Furthermore, a growth-inhibiting compound was identified in the fifth fraction of a Rhodococcus strain. Our findings include the bioassay-guided identification, HPLC-MS-based dereplication, and isolation of a large phospholipid and a likely antimicrobial peptide, demonstrating the usefulness of this approach in screening for compounds capable of inhibiting EPEC virulence.

PSXII-29 Development of an in Vitro Rumen Fermentation Model to Assess Fusobacterium Necrophorum Growth Inhibition

Abstract This experiment aimed to determine the impact of growth-inhibitory compounds on F. necrophorum subsp. necrophorum (F.n.n.), a primary etiologic agent of liver abscesses in feedlot cattle, in an in vitro rumen fermentation system. The assessment of inhibition was by quantitative PCR. We hypothesized that the addition of growth inhibitory compounds would reduce F.n.n. concentrations. A 2 x 2 x 6 factorial arrangement of treatments was used. Factors included: 1) sodium lactate (0.0 or 100 mM); 2) bacteria (0 or 1x108 CFU/ml of F.n.n.); and 3) 1 mL of test compound [Control (solvent with no additive); Tylosin: 5 μg/mL; Compound A; Compound B; Compound C; and Compound D]. Prior to morning feeding, rumen contents were collected from a single ruminally canulated steer fed a grain-based finishing diet. Rumen fluid was strained through 4 layers of cheesecloth, mixed with McDougall’s buffer (1:1 ratio; RFB), and 30 mL of the RFB was added into 100 mL-serum bottles containing the appropriate treatments. Serum bottles (n = 3 replicates per treatment) were flushed with CO2 closed with rubber stoppers, crimped with aluminum seals, and incubated at 39ºC for 0, 6, 12, and 24 h. Following each incubation time point, a 2-mL aliquot was removed from each serum bottle under a stream of CO2 to maintain anaerobic conditions. Immediately after sample collection, pH was determined, and DNA was extracted by boiling the sample, centrifugation, and purifying with a GeneClean Turbo DNA extraction kit. The concentration of F.n.n was determined by a quantitative PCR assay targeting the promoter region of the leukotoxin genes. Concentrations of F.n.n. from the RFB were used to adjust F.n.n. concentrations for all treatments at all time points. There was a lactate x test compound x incubation time interaction (P < 0.05) for F.n.n. concentration. In the absence of lactate, serum bottles containing tylosin or test compound treatments had decreased (P < 0.05) F.n.n. concentrations by 24 h post incubation. However, when sodium lactate was added, no treatments had F.n.n. concentrations less than the control treatment at 24 h post incubation (P > 0.05). These data suggest that compounds used in this experiment reduced the concentrations of F.n.n by 24 h post incubation in the absence of additional lactate. However, when lactate, a substrate for F.n.n, was added to the in vitro system, the reduction in F.n.n. growth was not observed.

Xylose consumption and ethanol production by Pichia guilliermondii and Candida oleophila in the presence of furans, phenolic compounds, and organic acids commonly produced during the pre-treatment of plant biomass.

For 2G ethanol production, pentose fermentation and yeast tolerance to lignocellulosic hydrolyzate components are essential to improve biorefinery yields. Generally, physicochemical pre-treatment methodologies are used to facilitate access to cellulose and hemicellulose in plant material, which consequently can generate microbial growth inhibitory compounds, such as furans, weak acids, and phenolic compounds. Because of the unsatisfactory yield of wild-type Saccharomyces cerevisiae during pentose fermentation, the search for xylose-fermenting yeasts tolerant to microbial growth inhibitors has gained attention. In this study, we investigated the ability of the yeasts Pichia guilliermondii G1.2 and Candida oleophila G10.1 to produce ethanol from xylose and tolerate the inhibitors furfural, 5-hydroxymethylfurfural (HMF), acetic acid, formic acid, ferulic acid, and vanillin. We demonstrated that both yeasts were able to grow and consume xylose in the presence of all single inhibitors, with greater growth limitation in media containing furfural, acetic acid, and vanillin. In saline medium containing a mixture of these inhibitors (2.5-3.5 mM furfural and HMF, 1 mM ferulic acid, 1-1.5 mM vanillin, 10-13 mM acetic acid, and 5-7 mM formic acid), both yeasts were able to produce ethanol from xylose, similar to that detected in the control medium (without inhibitors). In future studies, the proteins involved in the transport of pentose and tolerance to these inhibitors need to be investigated.

High-Throughput Cellular Heterogeneity Analysis in Cell Migration at the Single-Cell Level.

Cancer cell migration represents an essential step toward metastasis and cancer deaths. However, conventional drug discovery focuses on cytotoxic and growth-inhibiting compounds rather than inhibitors of migration. Drug screening assays generally measure the average response of many cells, masking distinct cell populations that drive metastasis and resist treatments. Here, this work presents a high-throughput microfluidic cell migration platform that coordinates robotic liquid handling and computer vision for rapidly quantifying individual cellular motility. Using this innovative technology, 172 compoundsweretested and a surprisingly low correlation between migration and growth inhibitionwasfound. Notably, many compoundswerefound to inhibit migration of most cells while leaving fast-moving subpopulations unaffected. This work further pinpoints synergistic drug combinations, including Bortezomib and Danirixin, to stop fast-moving cells. To explain the observed cell behaviors, single-cell morphological and molecular analysiswereperformed. These studies establish a novel technology to identify promising migration inhibitors for cancer treatment and relevant applications.

Assessment of the Phytotoxic Potential of Dregea volubilis (L.f.) Benth. ex Hook.f. and Identification of its Phytotoxic Substances for Weed Control

The phytotoxic potential of plants and the effect of their active components on another plant species is being explored as a potential alternative to synthetic herbicides for weed control. In the current study, we investigated the phytotoxic potential of the leaves of Dregea volubilis (L.f.) Benth. ex Hook.f. against four test plants [timothy (Phleum pratense L.), barnyard grass (Echinochloa crus-galli (L.) P. Beauv), lettuce (Lactuca sativa L.), and alfalfa (Medicago sativa L.)] and observed significant growth inhibition on those plants at concentrations >3 mg D.W. equivalent extract mL−1. A bioassay-governed purification of the D. volubilis extracts using different chromatography phases produced two growth inhibitory compounds, 3-hydroxy-α-ionone (compound 1) and 5-hydroxy-3,4-dimethyl-5-pentylfuran-2(5H)-one (compound 2). The compounds retarded the growth of barnyard grass and cress (Lepidium sativum L.) with I50 (concentration required for 50% growth suppression) values ranging from 0.098 to 0.450 mM for 3-hydroxy-α-ionone and 0.029 to 0.420 mM for 5-hydroxy-3,4-dimethyl-5-pentylfuran-2(5H)-one. Thus, the extracts and identified compounds may have the possibility to be utilized as bioagents for weed control.

Characterization of Conyza bonariensis Allelochemicals against Broomrape Weeds.

The study of allelopathic activity of plants and the isolation and characterization of the responsible allelochemicals can lead to the development of environment friendly alternative approaches to weed control. Conyza species are invasive weeds that use allelopathic activity as part of a successful strategy to outcompete neighboring plants. Broomrape weeds are parasitic plants that use host-induced germination and the formation of a haustorium as strategies to infect host plants. The control of broomrape infection in most affected crops is limited or non-existing. In the current study, we investigated the allelopathic activity of Conyza bonariensis organic extracts in suicidal germination and radicle growth of four broomrape species (Orobanche crenata, Orobanche cumana, Orobanche minor and Phelipanche ramosa). A bioactivity-driven fractionation of Conyza bonariensis extracts led to the identification of two germination-inducing molecules and two growth-inhibitory compounds. The germination-inducing metabolites had species-specific activity being hispidulin active on seeds of O. cumana and methyl 4-hydroxybenzoate active in P. ramosa. The growth-inhibitory metabolites (4Z)-lachnophyllum lactone and (4Z,8Z)-matricaria lactone strongly inhibited the radicle growth of all parasitic weed species studied. Some structure-activity relationships were found as result of the study herein presented.

Forward Chemical Genetic Screen for Oxygen-Dependent Cytotoxins Uncovers New Covalent Fragments that Target GPX4.

The identification of growth inhibitory compounds with the ability to selectively target the cellular oxygenation state may be of therapeutic interest. Here, a phenotypic screen of a covalent fragment library revealed diverse compounds containing propiolamide warheads with selective toxicity for liver cancer cells in normoxic conditions. Target identification and validation through CETSA and direct pulldown experiments demonstrated that several compounds target glutathione peroxidase 4 (GPX4) and induce ferroptotic cell death. Although being an oxidative cell death mechanism, ferroptosis can be induced also under hypoxic conditions. Prompted by the selective toxicity discovered in the screen, we mapped the oxygen-dependence of several ferroptosis-inducing compounds across three different cell lines. These studies revealed combinations with notable reductions in sensitivity under hypoxic conditions. These observations are mechanistically interesting and may be relevant for the use of ferroptosis-inducers as anti-cancer agents.

Growth of wood-inhabiting yeasts of the Faroe Islands in the presence of spent sulphite liquor

In the microbial community of decaying wood, yeasts are important for the recycling of nutrients. Nevertheless, information on their biodiversity in this niche in the Northern hemisphere is limited. Wood-colonising yeasts encounter identical and similar growth-inhibitory compounds as those in spent sulphite liquor (SSL), an energy-rich, acid hydrolysate and waste product from the paper industry, which may render them well-suited for cultivation in SSL. In the present study, yeasts were isolated from decaying wood on the Faroe Islands and identified based on sequence homology of the ITS and D1/D2 regions. Among the yeasts isolated, Candida argentea, Cystofilobasidium infirmominiatum, Naganishia albidosimilis, Naganishia onofrii, Holtermanniella takashimae and Goffeauzyma gastrica were new to decaying wood in cold and temperate climates. C. argentea and Rhodotorula are rarely-isolated species, with no previous documentation from cold and maritime climates. The isolates were further tested for growth in a medium with increasing concentrations of softwood SSL. Most grew in the presence of 10% SSL. Isolates of Debaryomyces sp., C. argentea and Rhodotorula sp. were the most tolerant. Representatives of Debaryomyces and Rhodotorula have previously been found in decaying wood. In contrast, the least tolerant isolates belonged to species that are rarely reported from decaying wood. The relative importance of individual inhibitors to yeast growth is discussed. To our knowledge, none of the present yeast species have previously been cultivated in SSL medium. Decaying wood can be a useful future source of yeasts for valorisation of various hydrolysates to industrial chemicals and biofuels.

Isolation and identification of three potential phytotoxic compounds from Chrysopogon aciculatus (Retz.) Trin

We conducted an investigation to search for the possibility of phytotoxic compounds and then identify them in C. aciculatus. The aqueous methanol extracts of C. aciculatus imposed a strong suppressive effect on the shoots and roots growth of Medicago sativa and Echinochloa crus-galli. The C. aciculatus extracts were subsequently purified through chromatography and three growth-inhibitory compounds were identified by spectral data as methyl cis-p-coumarate, methyl trans-p-coumarate, and trans-ferulaldehyde. The seedlings growth of Lepidium sativum was suppressed by methyl cis-p-coumarate, methyl trans-p-coumarate, and trans-ferulaldehyde. The I50 values (concentration required for 50% growth inhibition) for the seedling growth of L. sativum ranged from 1.25 to 1.73 mM for methyl cis-p-coumarate, 0.74 to 1.23 mM for methyl trans-p-coumarate, and 1.05 to 1.84 mM for trans-ferulaldehyde. Our finding indicates that methyl cis-p-coumarate, methyl trans-p-coumarate, and trans-ferulaldehyde may contribute to the phytotoxic effects of C. aciculatus.

Induction of antibiotic specialized metabolism by co-culturing in a collection of phyllosphere bacteria.

A diverse set of bacteria live on the above-ground parts of plants, composing the phyllosphere, and play important roles for plant health. Phyllosphere microbial communities assemble in a predictable manner and diverge from communities colonizing other plant organs or the soil. However, how these communities differ functionally remains obscure. We assembled a collection of 258 bacterial isolates representative of the most abundant taxa of the phyllosphere of Arabidopsis and a shared soil inoculum. We screened the collection for the production of metabolites that inhibit the growth of Gram-positive and Gram-negative bacteria either in isolation or in co-culture. We found that isolates capable of constitutive antibiotic production in monoculture were significantly enriched in the soil fraction. In contrast, the proportion of binary cultures resulting in the production of growth inhibitory compounds differed only marginally between the phyllosphere and soil fractions. This shows that the phyllosphere may be a rich resource for potentially novel molecules with antibiotic activity, but that production or activity is dependent upon induction by external signals or cues. Finally, we describe the isolation of antimicrobial acyloin metabolites from a binary culture of Arabidopsis phyllosphere isolates, which inhibit the growth of clinically relevant Acinetobacter baumannii.

Isolation, identification and evaluation of Lactic acid synthesis of bacteria in traditional fermented products in Vietnam

Nowadays, many food products are promoted as being particularly healthy due to the characteristics of certain strains of lactic acid bacteria. Lactic acid bacteria (LAB) play a vital role in different fields, including food, pharmacy due to their benefit for young children, pregnant women and the elderly. LAB is one of the most significant groups of microorganisms used in food fermentations. They improve the flavour and texture of fermented foods. Moreover, LAB kills food spoilage bacteria by developing growth-inhibiting compounds. In this study, seven strains of LAB were isolated from traditional fermented products in VietNamm including fermented rice, kimchi, and yoghurt. Moreover, LAB isolated was identified by amplification and sequencing of 16s rDNA gene. The isolated strains were Lactobacillus plantarum (M2 and M7), Lactobacillus paracasei (M4), Lactobacillus sakei (M10), Leuconostoc mesenteroides (M15), Lactobacillus curvatus (M17), Lactobacillus delbruekii sub sp. (M19). Cultural characteristics of M1 and M7 surface colony are 2mm wide, raised, smooth and white on MRS agar. The morphology of the colonies M4 and M10 is rounded, smooth, light yellow. Nevertheless, M4 and M10 are quite different in colour, white and light yellow, respectively. Moreover, M15 colonies are convex, with flat edges, smooth, semitransparent. M17 and M19 are small, normally rough and non-pigment. Acid lactic content produced by the isolated strains determined by spectral absorbance at 390 nm with FeCl3 (0.2%), was highest in M2 strain (37.814g/l), followed by M17 (32.357g/l), M19 (27.336g/l), M4 (20.853g/l), M10 (9.408g/l), M7 (8.864g/l), and M15 (6.645g/l). In this study, the highest lactic acid content produced was M2 stain (Lactobacillus plantarum) and the lowest was M15 (Leuconostoc mesenteroides).

Multiple Drug-Induced Stress Responses Inhibit Formation of Escherichia coli Biofilms.

In most ecosystems, bacteria exist primarily as structured surface-associated biofilms that can be highly tolerant to antibiotics and thus represent an important health issue. Here, we explored drug repurposing as a strategy to identify new antibiofilm compounds, screening over 1,000 compounds from the Prestwick Chemical Library of approved drugs for specific activities that prevent biofilm formation by Escherichia coli Most growth-inhibiting compounds, which include known antibacterial but also antiviral and other drugs, also reduced biofilm formation. However, we also identified several drugs that were biofilm inhibitory at doses where only a weak effect or no effect on planktonic growth could be observed. The activities of the most specific antibiofilm compounds were further characterized using gene expression analysis, proteomics, and microscopy. We observed that most of these drugs acted by repressing genes responsible for the production of curli, a major component of the E. coli biofilm matrix. This repression apparently occurred through the induction of several different stress responses, including DNA and cell wall damage, and homeostasis of divalent cations, demonstrating that biofilm formation can be inhibited through a variety of molecular mechanisms. One tested drug, tyloxapol, did not affect curli expression or cell growth but instead inhibited biofilm formation by suppressing bacterial attachment to the surface.IMPORTANCE The prevention of bacterial biofilm formation is one of the major current challenges in microbiology. Here, by systematically screening a large number of approved drugs for their ability to suppress biofilm formation by Escherichia coli, we identified a number of prospective antibiofilm compounds. We further demonstrated different mechanisms of action for individual compounds, from induction of replicative stress to disbalance of cation homeostasis to inhibition of bacterial attachment to the surface. Our work demonstrates the potential of drug repurposing for the prevention of bacterial biofilm formation and suggests that also for other bacteria, the activity spectrum of antibiofilm compounds is likely to be broad.

Valorization of xylose-rich hydrolysate from rice straw, an agroresidue, through biosurfactant production by the soil bacterium Serratia nematodiphila

Biosurfactants, amphiphilic compounds that reduce interfacial tension in oil-aqueous mixtures, are used in the petroleum, pharmaceutical, food, and agriculture industries. Fermentative production of biosurfactants requires expensive sugar or lipid substrates. Lignocellulosic biomass is a relatively cheap and abundant agricultural residue that can be used as an alternative substrate. Currently, several million tonnes of rice and wheat straw are generated globally as agricultural residues, most of which is disposed by open-field burning thereby leading to severe environmental pollution. This study aimed to produce biosurfactants in xylose-rich hydrolysates generated from rice straw. The hydrolysate is also a byproduct of 2G biofuel processes that often goes underutilized. A soil bacterium capable of growing and producing biosurfactants in rice straw hydrolysates, which typically contain growth-inhibitory compounds such as furfural and hydroxymethyl furfural, was isolated. Interestingly, the organism, identified as Serratia nematodiphila, exhibited higher glycolipid formation (4.5 ± 0.6 gL−1) in xylose-rich hydrolysate than in glucose-rich enzymatic hydrolysate (3.1 ± 0.2 gL−1) despite the higher bacterial cell density observed with the latter. The biosurfactants were thermostable and possessed promising emulsifying property and anti-microbial activity against bacteria and yeast. Further optimization of C:N resulted in a 2.8-fold increase in glycolipid production from xylose-rich hydrolysates. This study demonstrates the production of glycolipid biosurfactants from lignocellulosic biomass, a low-cost substrate and offers a plausible strategy for the management of these residues. Further, it also provides insights into the generation of additional high-value compounds in a bioethanol biorefinery to improve its commercial feasibility.

Tequila Agave Bagasse Hydrolysate for the Production of Polyhydroxybutyrate by Burkholderia sacchari.

Tequila agave bagasse (TAB) is the fibrous waste from the Tequila production process. It is generated in large amounts and its disposal is an environmental problem. Its use as a source of fermentable sugars for biotechnological processes is of interest; thus, it was investigated for the production of polyhydroxybutyrate (PHB) by the xylose-assimilating bacteria Burkholderia sacchari. First, it was chemically hydrolyzed, yielding 20.6 g·L−1 of reducing sugars, with xylose and glucose as the main components (7:3 ratio). Next, the effect of hydrolysis by-products on B. sacchari growth was evaluated. Phenolic compounds showed the highest toxicity (> 60% of growth inhibition). Then, detoxification methods (resins, activated charcoal, laccases) were tested to remove the growth inhibitory compounds from the TAB hydrolysate (TABH). The highest removal percentage (92%) was achieved using activated charcoal (50 g·L−1, pH 2, 4 h). Finally, detoxified TABH was used as the carbon source for the production of PHB in a two-step batch culture, reaching a biomass production of 11.3 g·L−1 and a PHB accumulation of 24 g PHB g−1 dry cell (after 122 h of culture). The polymer structure resulted in a homopolymer of 3-hydroxybutyric acid. It is concluded that the TAB could be hydrolyzed and valorized as a carbon source for producing PHB.

Transcriptomic analysis of the oleaginous yeast Lipomyces starkeyi during lipid accumulation on enzymatically treated corn stover hydrolysate

BackgroundEfficient and economically viable production of biofuels from lignocellulosic biomass is dependent on mechanical and chemical pretreatment and enzymatic hydrolysis of plant material. These processing steps yield simple sugars as well as plant-derived and process-added organic acids, sugar-derived dehydration products, aldehydes, phenolics and other compounds that inhibit the growth of many microorganisms. Lipomyces starkeyi is an oleaginous yeast capable of robust growth on a variety of sugars and lipid accumulation on pretreated lignocellulosic substrates making it attractive as an industrial producer of biofuels. Here, we examined gene expression during batch growth and lipid accumulation in a 20-L bioreactor with either a blend of pure glucose and xylose or pretreated corn stover (PCS) that had been enzymatically hydrolyzed as the carbon sources.ResultsWe monitored sugar and ammonium utilization as well as biomass accumulation and found that growth of L. starkeyi is inhibited with PCS hydrolysate as the carbon source. Both acetic acid and furfural are present at concentrations toxic to L. starkeyi in PCS hydrolysate. We quantified gene expression at seven time-points for each carbon source during batch growth and found that gene expression is similar at physiologically equivalent points. Analysis of promoter regions revealed that gene expression during the transition to lipid accumulation is regulated by carbon and nitrogen catabolite repression, regardless of carbon source and is associated with decreased expression of the translation machinery and suppression of the cell cycle. We identified 73 differentially expressed genes during growth phase in the bioreactor that may be involved in detoxification of corn stover hydrolysate.ConclusionsGrowth of L. starkeyi is inhibited by compounds present in PCS hydrolysate. Here, we monitored key metabolites to establish physiologically equivalent comparisons during a batch bioreactor run comparing PCS hydrolysate and purified sugars. L. starkeyi’s response to PCS hydrolysate is primarily at the beginning of the run during growth phase when inhibitory compounds are presumably at their highest concentration and inducing the general detoxification response by L. starkeyi. Differentially expressed genes identified herein during growth phase will aid in the improvement of industrial strains capable of robust growth on substrates containing various growth inhibitory compounds.

Transcriptional Regulation Contributes to Prioritized Detoxification of Hydrogen Peroxide over Nitric Oxide.

Hydrogen peroxide (H2O2) and nitric oxide (NO·) are toxic metabolites that immune cells use to attack pathogens. These antimicrobials can be present at the same time in phagosomes, and it remains unclear how bacteria deal with these insults when simultaneously present. Here, using Escherichia coli, we observed that simultaneous exposure to H2O2 and NO· leads to prioritized detoxification, where enzymatic removal of NO· is impeded until H2O2 has been eliminated. This phenomenon is reminiscent of carbon catabolite repression (CCR), where preferred carbon sources are catabolized prior to less desirable substrates; however, H2O2 and NO· are toxic, growth-inhibitory compounds rather than growth-promoting nutrients. To understand how NO· detoxification is delayed by H2O2 whereas H2O2 detoxification proceeds unimpeded, we confirmed that the effect depended on Hmp, which is the main NO· detoxification enzyme, and used an approach that integrated computational modeling and experimentation to delineate and test potential mechanisms. Plausible interactions included H2O2-dependent inhibition of hmp transcription and translation, direct inhibition of Hmp catalysis, and competition for reducing equivalents between Hmp and H2O2-degrading enzymes. Experiments illustrated that Hmp catalysis and NAD(P)H supply were not impaired by H2O2, whereas hmp transcription and translation were diminished. A dependence of this phenomenon on transcriptional regulation parallels CCR, and we found it to involve the transcriptional repressor NsrR. Collectively, these data suggest that bacterial regulation of growth inhibitor detoxification has similarities to the regulation of growth substrate consumption, which could have ramifications for infectious disease, bioremediation, and biocatalysis from inhibitor-containing feedstocks.IMPORTANCE Bacteria can be exposed to H2O2 and NO· concurrently within phagosomes. In such multistress situations, bacteria could have evolved to simultaneously degrade both toxic metabolites or preferentially detoxify one over the other. Here, we found that simultaneous exposure to H2O2 and NO· leads to prioritized detoxification, where detoxification of NO· is hampered until H2O2 has been eliminated. This phenomenon resembles CCR, where bacteria consume one substrate over others in carbon source mixtures. Further experimentation revealed a central role for transcriptional regulation in the prioritization of H2O2 over NO·, which is also important to CCR. This study suggests that regulatory scenarios observed in bacterial consumption of growth-promoting compound mixtures can be conserved in bacterial detoxification of toxic metabolite mixtures.

Facile processing of Microchloropsis salina biomass for phosphate recycle

Algal biomass is a proposed feedstock for sustainable production of petroleum displacing commodities. However, production of 10% of US demand for liquid transportation fuel from algae would require a 60–150% increase over current agricultural demand for phosphorus fertilizers. Without efforts to recycle major nutrients, algal biomass production can be expected to catalyze a food versus fuel crisis. We have developed a novel and simple process for efficient liberation of phosphate from algal biomass and have demonstrated recycling at both laboratory and pilot scale, of up to 70% of total cellular phosphate from osmotically-shocked but non-denatured Microchloropsis salina biomass using a range of mild incubation conditions. The phosphate released in this process is bioavailable, can support the same level of algal growth as standard nutrients, and does not contain any growth inhibitory compounds as evidenced by its ability to support multiple sequential cycles of growth and remineralization.

HAOCHEN BAI, The Linsly School, Wheeling, WV, 26003. Evaluation of allelopathic effects of suspensions of black walnut (Juglans nigra), Japanese knotweed (Fallopia japonica), and slender speedwell (Veronica filiformis) on seedlings of five species of garden plants (carrot, dandelion, lettuce, collard green and kale).

Plants use different strategies to compete with each other including the allelopathic effect, which helps one plant gain advantage over others by chemically inhibiting their growth. While allelopathic effects of Black Walnut are well-known, the purpose of this study is to also evaluate possible allelopathic effects of two common invasive species, Japanese knotweed and slender speedwell. To determine whether their foliage has allelopathic effects, filtered homogenates of dried leaves of black walnut, Japanese knotweed and slender speedwell were prepared and used as the water source in germination experiments. Seeds of five common garden plants (carrot, dandelion, lettuce, collard green, and kale) were placed in standard germination chambers (layers of paper towels in plastic petri dishes) and measured for germination rate and shoot and root elongation at regular intervals. Black walnut homogenate significantly reduced shoot and root growth (t-tests) of all five species of garden seeds. Japanese knotweed and slender speedwell homogenates did not show a negative effect on any of the garden seeds. In fact, these homogenates significantly enhanced shoot growth (t-tests) when compared to the distilled water control. Thus, the experiment supports the hypothesis that Japanese knotweed and slender speedwell foliage do not contain allelopathic chemicals that inhibit growth of other plant’s seedlings. It is uncertain if the homogenates provided growth-enhancing nutrients not available in the distilled water. The experiment confirms previously published research that determined the allelopathic effects of black walnut extracts. Recent research shows juglone, the growth-inhibiting compound involved, inhibits RNA polyermase II and genetic transcription.