Soybean isoflavones modulate gut microbiota to benefit the health weight and metabolism.

Soybean isoflavones (SIs) are widely found in food and herbal medicines. Although the pharmacological activities of SIs have been widely reported, their effects on the intestinal microecology of normal hosts have received little attention. Five-week-old Kunming (KM) mice were administered SIs (10 mg/kg/day) for 15 days. Food intake, body weight, and digestive enzyme activity were measured. Small intestine microbiota, including lumen-associated bacteria (LAB) and mucosa-associated bacteria (MAB), were analyzed using 16S ribosomal ribonucleic acid (16S rRNA) gene sequencing. Short-chain fatty acids (SCFAs) were analyzed using gas chromatography-mass spectrometry (GC-MS). The results showed that the mice that consuming SIs showed a higher food intake but a lower body weight gain rate than that of normal mice. Sucrase, cellulase, and amylase activities reduced, while protease activity increased after SIs intervention. Moreover, SIs increased the intestinal bacterial diversity in both LAB and MAB of normal mice. The composition of LAB was more sensitive to SIs than those of MAB. Lactobacillus, Adlercreutzia, Coprococcus, Ruminococcus, Butyricicoccus, and Desulfovibrio were the differential bacteria among the LAB of mice treated with SIs. In addition, acetic acid, valeric acid, isobutyric acid, isovaleric acid, and caproic acid decreased, while butyric acid and propionic acid increased in the mice treated with SIs. Taken together, SIs are beneficial for weight control, even in short-term interventions. The specific mechanism is related to regulating the gut microbiota, changing digestive enzyme activities, and further affecting carbohydrate absorption and metabolism.

The Sex Pheromone of the Pine Brown-Tail Moth, Euproctis terminalis (Lepidoptera: Erebidae).

The pine brown tail moth, Euproctis terminalis (Walker 1855), is a periodic pest in pine plantations in South Africa. The larvae feed on pine needles and can cause severe defoliation when population densities are high. Population densities fluctuate temporally and spatially, complicating the prediction of potential growth loss and tree mortality. The aim of this study was to identify the sex pheromone of the pine brown tail moth to provide stakeholders with a tool for monitoring it. Gas chromatography-electroantennogram detection and gas chromatography/mass spectrometry analyses of female pheromone gland extracts identified the major component as (Z,Z,Z,Z)-7,13,16,19-docosatetraen-1-ol isobutyrate. Traps baited with (Z,Z,Z,Z)-7,13,16,19-docosatetraen-1-ol isobutyrate caught more males than unbaited traps. A delta trap was shown to be a superior design compared to a bucket funnel trap. This pheromone can now be used for monitoring E. terminalis in pine plantations.

Identification of lysine isobutyrylation as a new histone modification mark.

Short-chain acylations of lysine residues in eukaryotic proteins are recognized as essential posttranslational chemical modifications (PTMs) that regulate cellular processes from transcription, cell cycle, metabolism, to signal transduction. Lysine butyrylation was initially discovered as a normal straight chain butyrylation (Knbu). Here we report its structural isomer, branched chain butyrylation, i.e. lysine isobutyrylation (Kibu), existing as a new PTM on nuclear histones. Uniquely, isobutyryl-CoA is derived from valine catabolism and branched chain fatty acid oxidation which is distinct from the metabolism of n-butyryl-CoA. Several histone acetyltransferases were found to possess lysine isobutyryltransferase activity in vitro, especially p300 and HAT1. Transfection and western blot experiments showed that p300 regulated histone isobutyrylation levels in the cell. We resolved the X-ray crystal structures of HAT1 in complex with isobutyryl-CoA that gleaned an atomic level insight into HAT-catalyzed isobutyrylation. RNA-Seq profiling revealed that isobutyrate greatly affected the expression of genes associated with many pivotal biological pathways. Together, our findings identify Kibu as a novel chemical modification mark in histones and suggest its extensive role in regulating epigenetics and cellular physiology.

Short chain fatty acids produced by Cutibacterium acnes inhibit biofilm formation by Staphylococcus epidermidis.

Biofilm formation by bacterial pathogens is associated with numerous human diseases and can confer resistance to both antibiotics and host defenses. Many strains of Staphylococcus epidermidis are capable of forming biofilms and are important human pathogens. Since S. epidermidis coexists with abundant Cutibacteria acnes on healthy human skin and does not typically form a biofilm in this environment, we hypothesized that C. acnes may influence biofilm formation of S. epidermidis. Culture supernatants from C. acnes and other species of Cutibacteria inhibited S. epidermidis but did not inhibit biofilms by Pseudomonas aeruginosa or Bacillus subtilis, and inhibited biofilms by S. aureus to a lesser extent. Biofilm inhibitory activity exhibited chemical properties of short chain fatty acids known to be produced from C. acnes. The addition of the pure short chain fatty acids propionic, isobutyric or isovaleric acid to S. epidermidis inhibited biofilm formation and, similarly to C. acnes supernatant, reduced polysaccharide synthesis by S. epidermidis. Both short chain fatty acids and C. acnes culture supernatant also increased sensitivity of S. epidermidis to antibiotic killing under biofilm-forming conditions. These observations suggest the presence of C. acnes in a diverse microbial community with S. epidermidis can be beneficial to the host and demonstrates that short chain fatty acids may be useful to limit formation of a biofilm by S. epidermidis.

Constituents of Xerolekia speciosissima (L.) Anderb. (Inuleae), and Anti-Inflammatory Activity of 7,10-Diisobutyryloxy-8,9-epoxythymyl Isobutyrate.

Xerolekia speciosissima (L.) Anderb., a rare plant from the north of Italy, is a member of the Inuleae-Inulinae subtribe of the Asteraceae. Despite its close taxonomic relationship with many species possessing medicinal properties, the chemical composition of the plant has remained unknown until now. A hydroalcoholic extract from the aerial parts of X. speciosissima was analyzed by HPLC-DAD-MSn, revealing the presence of caffeic acid derivatives and flavonoids. In all, 19 compounds, including commonly found chlorogenic acids and less frequently occurring butyryl and methylbutyryl conjugates of dicaffeoylquinic and tricaffeoylhexaric acids, plus two flavonoids, were tentatively identified. Chromatographic separation of a hydroalcoholic extract from the capitula of the plant led to the isolation of (+)-dehydrodiconiferyl alcohol 4-O-ß-glucopyranoside, quercimeritrin, astragalin, isoquercitrin, 6-hydroxykaempferol-7-O-ß-glucoside, quercetagitrin, methyl caffeate, caffeic acid, protocatechuic acid, chlorogenic acid and 1,5-dicaffeoylquinic acid. Composition of a nonpolar extract from the aerial parts of the plant was analyzed by chromatographic methods supported with 1H-NMR spectroscopy. The analysis revealed the presence of loliolide, reynosin, samtamarine, 2,3-dihydroaromaticin, 2-deoxy-4-epi-pulchellin and thymol derivatives as terpenoid constituents of the plant. One of the latter compounds-7,10-diisobutyryloxy-8,9-epoxythymyl isobutyrate-at concentrations 0.5, 1.0 and 2.5 µM, significantly reduced IL-8, IL-1ß and CCL2 excretion by LPS-stimulated human neutrophils.

Acetyl-CoA carboxylase 1 and 2 inhibition ameliorates steatosis and hepatic fibrosis in a MC4R knockout murine model of nonalcoholic steatohepatitis.

Acetyl-CoA carboxylase (ACC) catalyzes the rate-limiting step in de novo lipogenesis, which is increased in the livers of patients with nonalcoholic steatohepatitis. GS-0976 (firsocostat), an inhibitor of isoforms ACC1 and ACC2, reduced hepatic steatosis and serum fibrosis biomarkers such as tissue inhibitor of metalloproteinase 1 in patients with nonalcoholic steatohepatitis in a randomized controlled trial, although the impact of this improvement on fibrosis has not fully been evaluated in preclinical models. Here, we used Western diet-fed melanocortin 4 receptor-deficient mice that have similar phenotypes to nonalcoholic steatohepatitis patients including progressively developed hepatic steatosis as well as fibrosis. We evaluated the effects of ACC1/2 inhibition on hepatic fibrosis. After the confirmation of significant hepatic fibrosis with a 13-week pre-feeding, GS-0976 (4 and 16 mg/kg/day) treatment for 9 weeks lowered malonyl-CoA and triglyceride content in the liver and improved steatosis, histologically. Furthermore, GS-0976 reduced the histological area of hepatic fibrosis, hydroxyproline content, mRNA expression level of type I collagen in the liver, and plasma tissue metalloproteinase inhibitor 1, suggesting an improvement of hepatic fibrosis. The treatment with GS-0976 was also accompanied by reductions of plasma ALT and AST levels. These data demonstrate that improvement of hepatic lipid metabolism by ACC1/2 inhibition could be a new option to suppress fibrosis progression as well as to improve hepatic steatosis in nonalcoholic steatohepatitis.

GS-0976 (Firsocostat): an investigational liver-directed acetyl-CoA carboxylase (ACC) inhibitor for the treatment of non-alcoholic steatohepatitis (NASH).

Introduction: De novo lipogenesis (DNL) plays a major role in fatty acid metabolism and contributes significantly to triglyceride accumulation within the hepatocytes in patients with nonalcoholic steatohepatitis (NASH). Acetyl-CoA carboxylase (ACC) converts acetyl-CoA to malonyl CoA and is a rate-controlling step in DNL. Furthermore, malonyl-CoA is an important regulator of hepatic mitochondrial fat oxidation through its ability to inhibit carnitine palmitoyltransferase I. Therefore, inhibiting ACC pharmacologically represents an attractive approach to treating NASH.Areas covered: This article summarizes preclinical and clinical data on the efficacy and safety of the liver-targeted ACC inhibitor GS-0976 (Firsocostat) for the treatment of NASH. In a phase 2 trial that included 126 patients with NASH and fibrosis, GS-0976 20 mg daily for 12 weeks showed significant relative reduction in liver fat by 29%; however, treatment was associated with an increase in plasma triglycerides with 16 patients having levels >500 mg/dL.Expert opinion: Preclinical and preliminary clinical data support the development of GS-0976 as treatment for NASH. ACC-induced hypertriglyceridemia can be mitigated by fish oil or fibrates, but the long-term cardiovascular effects require further investigations.

Effect of dietary supplementation of sodium acetate and calcium butyrate on milk fat synthesis in lactating dairy cows.

Acetate is a major source of energy and substrate for milk fat synthesis in the dairy cow. We recently reported a linear increase in milk fat yield and greater than a 30% net apparent transfer of acetate to milk fat with ruminal infusion of neutralized acetate. Additionally, ruminal acetate infusion linearly increases plasma ß-hydroxybutyrate. The objective of the current study was to investigate the ability of acetate and butyrate fed in a diet to increase milk fat synthesis. Twelve multiparous lactating Holstein cows were randomly assigned to treatments in a 3 × 3 Latin square design with 14-d periods that included a 7-d washout followed by 7 d of treatment. Cows were fed ad libitum a basal diet with a low risk for biohydrogenation-induced milk fat depression, and treatments were mixed into the basal diet. Treatments were 3.2% NaHCO3 (control), 2.9% sodium acetate, and 2.5% calcium butyrate (carbon equivalent to acetate treatment) as a percent of diet dry matter. Feeding sodium acetate increased dry matter intake by 2.7 kg, had no effect on milk yield, and increased milk fat yield by 90 g/d and concentration by 0.2 percentage units, compared with control. Calcium butyrate decreased dry matter intake by 2.6 kg/d, milk yield by 1.65 kg/d, and milk fat yield by 60 g/d, compared with control. Sodium acetate increased concentration and yield of 16 carbon mixed source fatty acids (FA) and myristic acid, while decreasing the concentration of preformed FA, compared with control. Calcium butyrate had no effect on concentration of milk FA by source, but increased concentration of trans-10 C18:1 in milk by 18%, indicating a shift in rumen biohydrogenation pathways. Our data demonstrate that milk fat yield and concentration can be increased by feeding sodium acetate at 2.9% of diet dry matter, but not by feeding calcium butyrate at an equivalent carbon mass.

Small-chain fatty acid activates astrocytic odorant receptor Olfr920.

Odorant receptors are the largest subfamily of G protein-coupled receptors and were recently suggested to play critical roles in nonolfactory tissues. However, the expression and function of odorant receptors in astrocytes, the most abundant cells in the brain, are not well known. We demonstrate that Olfr920 is highly expressed and propose that it functions as a short-chain fatty acid sensor in primary cortical astrocytes. The short-chain fatty acid isobutyric acid (IBA) was identified via a luciferase assay as an Olfr920 ligand. We show that IBA activates the Gs protein-adenylyl cyclase-cAMP pathway via Olfr920 in primary cortical astrocytes by using cAMP and knockdown analyses. In addition, IBA reduces lipopolysaccharide-induced glial fibrillary acidic protein expression in reactive astrocytes. These results suggest that astrocytic Olfr920 is a potential novel target for increased reactive astrocytes.

GS-0976 Reduces Hepatic Steatosis and Fibrosis Markers in Patients With Nonalcoholic Fatty Liver Disease.

BACKGROUND & AIMS: De novo lipogenesis is increased in livers of patients with nonalcoholic steatohepatitis (NASH). Acetyl-coenzyme carboxylase catalyzes the rate-limiting step in this process. We evaluated the safety and efficacy of GS-0976, an inhibitor of acetyl-coenzyme A carboxylase in liver, in a phase 2 randomized placebo-controlled trial of patients with NASH. METHODS: We analyzed data from 126 patients with hepatic steatosis of at least 8%, based on the magnetic resonance imaging-estimated proton density fat fraction (MRI-PDFF), and liver stiffness of at least 2.5 kPa, based on magnetic resonance elastography measurement or historical biopsy result consistent with NASH and F1-F3 fibrosis. Patients were randomly assigned (2:2:1) to groups given GS-0976 20 mg, GS-0976 5 mg, or placebo daily for 12 weeks, from August 8, 2016 through July 18, 2017. Measures of hepatic steatosis, stiffness, serum markers of fibrosis, and plasma metabolomics were evaluated. The primary aims were to confirm previous findings and evaluate the relation between dose and efficacy. RESULTS: A relative decrease of at least 30% from baseline in MRI-PDFF (PDFF response) occurred in 48% of patients given GS-0976 20 mg (P = .004 vs placebo), 23% given GS-0976 5 mg (P = .43 vs placebo), and 15% given placebo. Median relative decreases in MRI-PDFF were greater in patients given GS-0976 20 mg (decrease of 29%) than those given placebo (decrease of 8%; P = .002). Changes in magnetic resonance elastography-measured stiffness did not differ among groups, but a dose-dependent decrease in the fibrosis marker tissue inhibitor of metalloproteinase 1 was observed in patients given GS-0976 20 mg. Plasma levels of acylcarnitine species also decreased in patients with a PDFF response given GS-0976 20 mg. GS-0976 was safe, but median relative increases of 11% and 13% in serum levels of triglycerides were observed in patients given GS-0976. CONCLUSIONS: In a randomized placebo-controlled trial of patients with NASH, we found 12-week administration of GS-0976 20 mg decreased hepatic steatosis, selected markers of fibrosis, and liver biochemistry. ClinicalTrials.gov ID NCT02856555.

Identification of a selective inhibitor of human monocarboxylate transporter 4.

The human monocarboxylate transporters (hMCTs/SLC16As) mediate the uptake of various monocarboxylates. Several isoforms of hMCTs are expressed in cancerous tissue as well as in normal tissue. In cancerous tissue, hypoxia induces the expression of hMCT4, which transports the energetic metabolite l-lactate across the plasma membrane. Since hMCT4 is involved in pH regulation and the transport of l-lactate in cancer cells, an hMCT4 inhibitor could function as an anticancer agent. Although several non specific hMCT inhibitors have been developed, a selective hMCT4 inhibitor has not yet been identified. The aim of this study was therefore to identify a selective hMCT4 inhibitor for use as a pharmacological tool for studying hMCT4. The heterologous expression system of the Xenopus oocyte was used to assess the effects of test compounds on hMCT4, whereupon isobutyrate derivatives, fibrates, and bindarit (2-[(1-benzyl-1H-indazol-3-yl)methoxy]-2-methylpropanoic acid) were demonstrated to exhibit selective inhibitory effects against this transporter. It is suggested that the structure formed from the joining of an isobutyrate moiety and two aromatic rings by appropriate linkers is important for acquiring the selective hMCT4-inhibiting activity. These findings provide novel insights into the ligand recognition of hMCT4, and contribute to the development of novel anticancer agents.

Effect of light and auxin transport inhibitors on endoreduplication in hypocotyl and cotyledon.

KEY MESSAGE: Enhancement of endoreduplication in dark-grown hypocotyl is a common feature in dicotyledonous polysomatic plants, and TIBA-mediated inhibition of the endoreduplication is partially due to abnormal actin organization. Many higher plant species use endoreduplication during cell differentiation. However, the mechanisms underlying this process have remained elusive. In this study, we examined endoreduplication in hypocotyls and cotyledons in response to light in some dicotyledonous plant species. Enhancement of endoreduplication was found in the dark-grown hypocotyls of all the polysomatic species analyzed across five different families, indicating that this process is a common feature in dicotyledonous plants having polysomatic tissues. Conversely, endoreduplication was enhanced in the light-grown cotyledons in four of the five species analyzed. We also analyzed the effect of a polar auxin transport inhibitor, 2,3,5-triiodobenzoic acid (TIBA) on endoreduplication in hypocotyl and cotyledon tissues of radish (Raphanus sativus L. var. longipinnatus Bailey). TIBA was found to inhibit and promote endoreduplication in hypocotyls and cotyledons, respectively, suggesting that the endoreduplication mechanism differs in these organs. To gain insight into the effect of TIBA, radish and spinach (Spinacia oleracea L.) seedlings were treated with a vesicle-trafficking inhibitor, brefeldin A, and an actin polymerization inhibitor, cytochalasin D. Both of the inhibitors partially inhibited endoreduplication of the dark-grown hypocotyl tissues, suggesting that the prominent inhibition of endoreduplication by TIBA might be attributed to its multifaceted role.

Characterization of an Endophytic Gloeosporium sp. and Its Novel Bioactivity with "Synergistans".

Gloeosporium sp. (OR-10) was isolated as an endophyte of Tsuga heterophylla (Western hemlock). Both ITS and 18S sequence analyses indicated that the organism best fits either Hypocrea spp. or Trichoderma spp., but neither of these organisms possess conidiophores associated with acervuli, in which case the endophytic isolate OR-10 does. Therefore, the preferred taxonomic assignment was primarily based on the morphological features of the organism as one belonging to the genus Gloeosporium sp. These taxonomic observations clearly point out that limited ITS and 18S sequence information can be misleading when solely used in making taxonomic assignments. The volatile phase of this endophyte was active against a number of plant pathogenic fungi including Phytophthora palmivora, Rhizoctonia solani, Ceratocystis ulmi, Botrytis cinerea, and Verticillium dahliae. Among several terpenes and furans, the most abundantly produced compound in the volatile phase was 6-pentyl-2H-pyran-2-one, a compound possessing antimicrobial activities. When used in conjunction with microliter amounts of any in a series of esters or isobutyric acid, an enhanced inhibitory response occurred with each test fungus that was greater than that exhibited by Gloeosporium sp. or the compounds tested individually. Compounds behaving in this manner are hereby designated "synergistans." An expression of the "median synergistic effect," under prescribed conditions, has been termed the mSE50. This value describes the amount of a potential synergistan that is required to yield an additional median 50% inhibition of a target organism. In this report, the mSE50s are reported for a series of esters and isobutyric acid. The results indicated that isoamyl acetate, allyl acetate, and isobutyric acid generally possessed the lowest mSE50 values. The value and potential importance of these microbial synergistic effects to the microbial environment are also discussed.

Effects of isobutyrate supplementation on ruminal microflora, rumen enzyme activities and methane emissions in Simmental steers.

The objective of this study was to evaluate the effects of isobutyrate supplementation on rumen microflora, enzyme activities and methane emissions in Simmental steers consuming a corn stover-based diet. Eight ruminally cannulated Simmental steers were used in a replicated 4 × 4 Latin square experiment. The treatments were control (without isobutyrate), low isobutyrate (LIB), moderate isobutyrate (MIB) and high isobutyrate (HIB) with 8.4, 16.8 and 25.2 g isobutyrate per steer per day respectively. Isobutyrate was hand-mixed into the concentrate portion. Diet consisted of 60% corn stover and 40% concentrate [dry matter (DM) basis]. Dry matter intake (averaged 9 kg/day) was restricted to a maximum of 90% of ad libitum intake. Population of total bacteria, cellulolytic bacteria and anaerobic fungi were linearly increased, whereas that of protozoa and total methanogens was linearly reduced with increasing isobutyrate supplementation. Real-time PCR quantification of population of Ruminococcus albus, Ruminococcus flavefaciens, Butyrivibrio fibrisolvens and Fibrobacter succinogenes was linearly increased with increasing isobutyrate supplementation. Activities of carboxymethyl cellulase, xylanase and ß-glucosidase were linearly increased, whereas that of protease was linearly reduced. Methane production was linearly decreased with increasing isobutyrate supplementation. Effective degradabilities of cellulose and hemicellulose of corn stover were linearly increased, whereas that of crude protein in diet was linearly decreased with increasing isobutyrate supplementation. The present results indicate that isobutyrate supplemented improved microflora, rumen enzyme activities and methane emissions in steers. It was suggested that the isobutyrate stimulated the digestive micro-organisms or enzymes in a dose-dependent manner. In the experimental conditions of this trial, the optimum isobutyrate dose was approximately 16.8 g isobutyrate per steer per day.

Filling dynamics of the Brindley's glands in the blood-sucking bug Triatoma infestans (Hemiptera: Reduviidae).

The filling dynamics of exocrine defensive glands is an important component of the defensive capacity of an insect in its natural environment. We studied the filling state and reloading rate of the Brindley's glands in the haematophagous Chagas disease vector Triatoma infestans (Hemiptera: Reduviidae). Quantitative analyses of isobutyric acid, the main secretion component, were carried out with glands dissected from adults under different scenarios of development, number of discharging events and feeding conditions. The alarm-pheromone function of the gland secretion was also assessed in bioassays with conspecific nymphs. Although pharate adults have their glands completely developed, these were not full until imaginal ecdysis. If kept undisturbed, the adults maintained a constant gland load, and discharged about 75% of the gland contents upon one disturbance event. While the glands can be discharged several times, full replenishing was not complete after one week, unless the insect had access to food. The escape behavior of nymphs in bioassays correlated with the chemical analyses, with nymphs showing significant avoidance only toward gland discharges from undisturbed or disturbed/fed adults. The results are discussed in reference to the feeding frequency and gregarious behavior of T. infestans under natural conditions, which suggest a relevant role of the filling dynamics of the Brindley's glands in the intraspecific communication of the insect.

Activation of NF-κB in basolateral amygdala is required for memory reconsolidation in auditory fear conditioning.

Posttraumatic stress disorder (PTSD) is characterized by acute and chronic changes in the stress response, manifested as conditioned fear memory. Previously formed memories that are susceptible to disruption immediately after retrieval undergo a protein synthesis-dependent process to become persistent, termed reconsolidation, a process that is regulated by many distinct molecular mechanisms that control gene expression. Increasing evidence supports the participation of the transcription factor NF-κB in the different phases of memory. Here, we demonstrate that inhibition of NF-κB in the basolateral amygdala (BLA), but not central nucleus of the amygdala, after memory reactivation impairs the retention of amygdala-dependent auditory fear conditioning (AFC). We used two independent pharmacological strategies to disrupt the reconsolidation of AFC. Bilateral intra-BLA infusion of sulfasalazine, an inhibitor of IκB kinase that activates NF-κB, and bilateral intra-BLA infusion of SN50, a direct inhibitor of the NF-κB DNA-binding complex, immediately after retrieval disrupted the reconsolidation of AFC. We also found that systemic pretreatment with sodium butyrate, a histone deacetylase inhibitor that enhances histone acetylation, in the amygdala rescued the disruption of reconsolidation induced by NF-κB inhibition in the BLA. These findings indicate that NF-κB activity in the BLA is required for memory reconsolidation in AFC, suggesting that NF-κB might be a potential pharmacotherapy target for posttraumatic stress disorder.

Peroxisomal Acyl-CoA oxidase 4 activity differs between Arabidopsis accessions.

In plants, peroxisomes are the primary site of fatty acid ß-oxidation. Following substrate activation, fatty acids are oxidized by Acyl-CoA Oxidase (ACX) enzymes. Arabidopsis has six ACX genes, although ACX6 is not expressed. Biochemical characterization has revealed that each ACX enzyme acts on specific chain-length targets, but in a partially overlapping manner, indicating a degree of functional redundancy. Genetic analysis of acx single and double mutants in the Columbia (Col-0) accession revealed only minor phenotypes, but an acx3acx4 double mutant from Wassileskija (Ws) is embryo lethal. In this study, we show that acx3acx4(Col) and acx1acx3acx4(Col) mutants are viable and that enzyme activity in these mutants is significantly reduced on a range of substrates compared to wild type. However, the triple mutant displays only minor defects in seed-storage mobilization, seedling development, and adult growth. Although the triple mutant is defective in the three most active and highly-expressed ACX proteins, increases in ACX2 expression may support partial ß-oxidation activity. Comparison of acx mutant alleles in the Col-0 and Ws accessions reveals independent phenotypes; the Ws acx4 mutant uniquely shows increased sensitivity to propionate, whereas the Col-0 acx4 allele has sucrose-dependent growth in the light. To dissect the issues between Col-0 and Ws, we generated mixed background mutants. Although alleles with the Col-0 acx4 mutant were viable, we were unable to isolate an acx3acx4 line using the Ws acx4 allele. Reducing ACX4 expression in several Arabidopsis backgrounds showed a split response, suggesting that the ACX4 gene and/or protein functions differently in Arabidopsis accessions.

An in vitro, short-term culture method for mammalian haploid round spermatids amenable for molecular manipulation.

Extensive chromatin remodeling is a characteristic feature of mammalian spermiogenesis. To date, methods for the molecular manipulation of haploid spermatids are not available as there is a lack of a well-established culture system. Biochemical experiments and knockout studies reveal only the final outcome; studying the incremental details of the intricate mechanisms involved is still a challenge. We have established an in vitro culture system for pure haploid round spermatids isolated from rat testes that can be maintained with good viability for up to 72 hr. Changes in cell morphology and flagellar growth were also studied in the cultured spermatids. Further, we have demonstrated that upon treatment of cells with specific histone deacetylase inhibitors, sodium butyrate and trichostatin A, there is an increase in the hyperacetylation status of histone H4, mimicking an important event characteristic of histone replacement process that occurs during later stages of spermiogenesis. We have also tried various methods for introducing DNA and protein into these round spermatids in culture, and report that while DNA transfection is still a challenging task, protein transfection could be achieved using Chariot™ peptide as a transfection reagent. Thus, the method described here sets a stage to study the molecular roles of spermatid-specific proteins and chromatin remodelers in the cellular context.

Histone deacetylase inhibitors sodium butyrate and valproic acid delay spontaneous cell death in purified rat retinal ganglion cells.

PURPOSE: Histone deacetylase inhibitors (HDACi) have neuroprotective effects under various neurodegenerative conditions, e.g., after optic nerve crush (ONC). HDACi-mediated protection of central neurons by increased histone acetylation has not previously been demonstrated in rat retinal ganglion cells (RGCs), although epigenetic changes were shown to be associated with cell death after ONC. We investigated whether HDACi can delay spontaneous cell death in purified rat RGCs and analyzed concomitant histone acetylation levels. METHODS: RGCs were purified from newborn (postnatal day [P] 0-P2) rat retinas by immunopanning with antibodies against Thy-1.1 and culturing in serum-free medium for 2 days. RGCs were treated with HDACi, each at several different concentrations: 0.1-10 mM sodium butyrate (SB), 0.1-2 mM valproic acid (VPA), or 0.5-10 nM trichostatin A (TSA). Negative controls were incubated in media alone, while positive controls were incubated in 0.05-0.4 IU/µl erythropoietin. Survival was quantified by counting viable cells using phase-contrast microscopy. The expression of acetylated histone proteins (AcH) 3 and 4 was analyzed in RGCs by immunohistochemistry. RESULTS: SB and VPA enhanced RGC survival in culture, with both showing a maximum effect at 0.1 mM (increase in survival to 188% and 163%, respectively). Their neuroprotective effect was comparable to that of erythropoietin at 0.05 IU/µl. TSA 0.5-1.0 nM showed no effect on RGC survival, and concentrations ≥ 5 nM increased RGC death. AcH3 and AcH4 levels were only significantly increased in RGCs treated with 0.1 mM SB. VPA 0.1 mM produced only a slight effect on histone acetylation. CONCLUSIONS: Millimolar concentrations of SB and VPA delayed spontaneous cell death in purified RGCs; however, significantly increased histone acetylation levels were only detectable in RGCs after SB treatment. As the potent HDACi TSA was not neuroprotective, mechanisms other than histone acetylation may be the basis on which SB and VPA are acting in this model. Additional studies are necessary to identify HDACi-targeted genes and pathways involved in RGC protection.

Short- and medium-chain fatty acids exhibit antimicrobial activity for oral microorganisms.

OBJECTIVES: This study assessed the antibacterial activity of short-, medium-, and long-chain fatty acids against various oral microorganisms. METHODS: The short-chain fatty acids [formic acid (C1), acetic acid (C2), propionic acid (C3), butyric acid (C4), isobutyric acid (C4), isovaleric acid (C5), hexanoic acid (C6)], medium-chain fatty acids [octanoic acid (C8), capric acid (C10), lauric acid (12)], and long-chain fatty acids [myristic acid (C14), palmitic acid (C16)], were investigated for antimicrobial activity against Streptococcus mutans, Streptococcus gordonii, Streptococcus sanguis, Candida albicans, Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum, and Porphyromonas gingivalis. RESULTS: The data demonstrated that the fatty acids exhibited patterns of inhibition against oral bacteria with some specificity that appeared related more to the bacterial species that the general structural characteristics of the microorganism. As a group the fatty acids were much less effective against C. albicans than the oral bacteria, with effectiveness limited to hexanoic, octanoic, and lauric acids. Formic acid, capric, and lauric acids were broadly inhibitory for the bacteria. Interestingly, fatty acids that are produced at metabolic end-products by a number of these bacteria, were specifically inactive against the producing species, whilst substantially inhibiting the growth of other oral microorganisms. CONCLUSIONS: The results indicate that the antimicrobial activity of short-chain fatty acids (SCFAs), medium-chain fatty acids (MCFAs), long-chain fatty acids (LCFAs) could influence the microbial ecology in the oral cavity via at least 2 potential pathways. First, the agents delivered exogenously as therapeutic adjuncts could be packaged to enhance a microbial-regulatory environment in the subgingival sulcus. Second, it would be the intrinsic nature of these fatty acid inhibitors in contributing to the characteristics of the microbial biofilms, their evolution, and emergence of species within the biofilms. Further studies on these functions are required to better understand the nature of these potential microbial interactions in the biofilms.