Lactate supports a metabolic-epigenetic link in macrophage polarization.

Lactate accumulation is a hallmark of solid cancers and is linked to the immune suppressive phenotypes of tumor-infiltrating immune cells. We report herein that interleukin-4 (IL-4)­induced M0 → M2 macrophage polarization is accompanied by interchangeable glucose- or lactate-dependent tricarboxylic acid (TCA) cycle metabolism that directly drives histone acetylation, M2 gene transcription, and functional immune suppression. Lactate-dependent M0 → M2 polarization requires both mitochondrial pyruvate uptake and adenosine triphosphate­citrate lyase (ACLY) enzymatic activity. Notably, exogenous acetate rescues defective M2 polarization and histone acetylation following mitochondrial pyruvate carrier 1 (MPC1) inhibition or ACLY deficiency. Lastly, M2 macrophage­dependent tumor progression is impaired by conditional macrophage ACLY deficiency, further supporting a dominant role for glucose/lactate mitochondrial metabolism and histone acetylation in driving immune evasion. This work adds to our understanding of how mitochondrial metabolism affects macrophage functional phenotypes and identifies a unique tumor microenvironment (TME)­driven metabolic-epigenetic link in M2 macrophages.

Pre-attentive Visual Processing in Alzheimer's Disease: An Event-related Potential Study.

INTRODUCTION: While identifying Alzheimer's Disease (AD) in its early stages is crucial, traditional neuropsychological tests tend to lack sensitivity and specificity for its diagnosis. Neuropsychological studies have reported visual processing deficits of AD, and event-related potentials (ERPs) are suitable to investigate pre-attentive processing with superior temporal resolution. OBJECTIVE: This study aimed to investigate visual attentional characteristics of adults with AD, from pre-attentive to attentive processing, using a visual oddball task and ERPs. METHODS: Cognitively normal elderly controls (CN) and patients with probable AD (AD) were recruited. Participants performed a three-stimulus visual oddball task and were asked to press a designated button in response to the target stimuli. The amplitudes of 4 ERPs were analyzed. Mismatchnegativity (vMMN) was analyzed around the parieto-occipital and temporo-occipital regions. P3a was analyzed around the fronto-central regions, whereas P3b was analyzed around the centro-parietal regions. RESULTS: Late vMMN amplitudes of the AD group were significantly smaller than those of the CN group, while early vMMN amplitudes were comparable. Compared to the CN group, P3a amplitudes of the AD group were significantly smaller for the infrequent deviant stimuli, but the amplitudes for the standard stimuli were comparable. Lastly, the AD group had significantly smaller P3b amplitudes for the target stimuli compared to the CN group. CONCLUSION: Our findings imply that AD patients exhibit pre-attentive visual processing deficits, known to affect later higher-order brain functions. In a clinical setting, the visual oddball paradigm could be used to provide helpful diagnostic information since pre-attentive ERPs can be induced by passive exposure to infrequent stimuli.

Design and Construction of Vibrio cholerae Strains That Harbor Various CTX Prophage Arrays.

Toxigenic Vibrio cholerae strains arise upon infection and integration of the lysogenic cholera toxin phage, the CTX phage, into bacterial chromosomes. The V. cholerae serogroup O1 strains identified to date can be broadly categorized into three main groups: the classical biotype strains, which harbor CTX-cla; the prototype El Tor strains (Wave 1 strains), which harbor CTX-1; and the atypical El Tor strains, which harbor CTX-2 (Wave 2 strains) or CTX-3~6 (Wave 3 strains). The efficiencies of replication and transmission of CTX phages are similar, suggesting the possibility of existence of more diverse bacterial strains harboring various CTX phages and their arrays in nature. In this study, a set of V. cholerae strains was constructed by the chromosomal integration of CTX phages into strains that already harbored CTX phages or those that did not harbor any CTX phage or RS1 element. Strains containing repeats of the same kind of CTX phage, strains containing the same kind of CTX phage in each chromosome, strains containing alternative CTX phages in one chromosome, or containing different CTX phages in each chromosome have been constructed. Thus, strains with any CTX array can be designed and constructed. Moreover, the strains described in this study contained the toxT-139F allele, which enhances the expression of TcpA and cholera toxin. These characteristics are considered to be important for cholera vaccine development. Once their capacity to provoke immunity in human against V. cholerae infection is evaluated, some of the generated strains could be developed further to yield cholera vaccine strains.

Temporal Metagenomic and Metabolomic Characterization of Fresh Perennial Ryegrass Degradation by Rumen Bacteria.

Understanding the relationship between ingested plant material and the attached microbiome is essential for developing methodologies to improve ruminant nutrient use efficiency. We have previously shown that perennial ryegrass (PRG) rumen bacterial colonization events follow a primary (up to 4 h) and secondary (after 4 h) pattern based on the differences in diversity of the attached bacteria. In this study, we investigated temporal niche specialization of primary and secondary populations of attached rumen microbiota using metagenomic shotgun sequencing as well as monitoring changes in the plant chemistry using mid-infrared spectroscopy (FT-IR). Metagenomic Rapid Annotation using Subsystem Technology (MG-RAST) taxonomical analysis of shotgun metagenomic sequences showed that the genera Butyrivibrio, Clostridium, Eubacterium, Prevotella, and Selenomonas dominated the attached microbiome irrespective of time. MG-RAST also showed that Acidaminococcus, Bacillus, Butyrivibrio, and Prevotella rDNA increased in read abundance during secondary colonization, whilst Blautia decreased in read abundance. MG-RAST Clusters of Orthologous Groups (COG) functional analysis also showed that the primary function of the attached microbiome was categorized broadly within "metabolism;" predominantly amino acid, carbohydrate, and lipid metabolism and transport. Most sequence read abundances (51.6, 43.8, and 50.0% of COG families pertaining to amino acid, carbohydrate and lipid metabolism, respectively) within these categories were higher in abundance during secondary colonization. Kyoto encyclopedia of genes and genomes (KEGG) pathways analysis confirmed that the PRG-attached microbiota present at 1 and 4 h of rumen incubation possess a similar functional capacity, with only a few pathways being uniquely found in only one incubation time point only. FT-IR data for the plant residues also showed that the main changes in plant chemistry between primary and secondary colonization was due to increased carbohydrate, amino acid, and lipid metabolism. This study confirmed primary and secondary colonization events and supported the hypothesis that functional changes occurred as a consequence of taxonomical changes. Sequences within the carbohydrate metabolism COG families contained only 3.2% of cellulose activities, on average across both incubation times (1 and 4 h), suggesting that degradation of the plant cell walls may be a key rate-limiting factor in ensuring the bioavailability of intra-plant nutrients in a timely manner to the microbes and ultimately the animal. This suggests that a future focus for improving ruminant nutrient use efficiency should be altering the recalcitrant plant cell wall components and/or improving the cellulolytic capacity of the rumen microbiota.

X-linked Charcot-Marie-Tooth disease type 6 (CMTX6) patients with a p.R158H mutation in the pyruvate dehydrogenase kinase isoenzyme 3 gene.

Charcot-Marie-Tooth disease (CMT) is the most common inherited peripheral neuropathy. Mutations in the pyruvate dehydrogenase kinase isoenzyme 3 (PDK3) gene have been found to cause X-linked dominant CMT type 6 (CMTX6). This study identified the p.R158H PDK3 mutation after screening 67 probable X-linked CMT families. The mutation fully segregated with the phenotype, and genotyping the family indicated the mutation arose on a different haplotype compared with the original Australian CMTX6 family. Results of bisulphite sequencing suggest that methylated deamination of a CpG dinucleotide may cause the recurrent p.R158H mutation. The frequency of the p.R158H PDK3 mutation in Koreans is very rare. Magnetic resonance imaging revealed fatty infiltration involving distal muscles in the lower extremities. In addition, fatty infiltrations were predominantly observed in the soleus muscles, with a lesser extent in tibialis anterior muscles. This differs from demyelinating CMT1A patients and is similar to axonal CMT2A patients. The clinical, neuroimaging, and electrophysiological findings from a second CMTX6 family with the p.R158H PDK3 mutation were similar to the axonal neuropathy reported in the Australian family.

Betahistine reduces postoperative nausea and vomiting after laparoscopic gynecological surgery.

BACKGROUND: Patients undergoing laparoscopic gynecological surgery are at high risk of postoperative nausea and vomiting (PONV). We compared the antiemetic efficacy of ondansetron plus betahistine with that of ondansetron alone in this patient population. METHODS: In this randomized, double-blinded study, 168 patients were randomly allocated to receive placebo (O group) or betahistine 18 mg (OB group) orally 3 hours before surgery and 24 hours thereafter. In both groups, ondansetron 4 mg was administered at the end of surgery and 8 mg were added to an intravenous patient-controlled analgesia (IV-PCA) fentanyl solution. The primary outcome was complete response (no PONV and no rescue antiemetics) during the first 48 hours after surgery. The severity of nausea, pain score, and adverse events were assessed. RESULTS: The incidence of complete response was significantly higher in OB group than in O group (69% vs. 46%, P=0.004). The severity of nausea was lower in OB group than in O group during 30 minutes to 6 hours and 6 to 24 hours after surgery (P=0.001 and P<0.001). Pain score was similar between the groups. The incidence of dizziness was lower in OB group than in O group (13% vs. 40%, P < 0.001). Six patients (7%) in OB group and 15 patients (18%) in O group required early IV-PCA discontinuation, primarily because of PONV and/or dizziness (P=0.038). CONCLUSIONS: Compared to ondansetron alone, ondansetron plus betahistine was more effective to prevent PONV and dizziness in high-risk patients undergoing laparoscopic gynecological surgery.

Chemical tools to explore nutrient-driven O-GlcNAc cycling.

Posttranslational modifications (PTM) including glycosylation, phosphorylation, acetylation, methylation and ubiquitination dynamically alter the proteome. The evolutionarily conserved enzymes O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) and O-GlcNAcase are responsible for the addition and removal, respectively, of the nutrient-sensitive PTM of protein serine and threonine residues with O-GlcNAc. Indeed, the O-GlcNAc modification acts at every step in the "central dogma" of molecular biology and alters signaling pathways leading to amplified or blunted biological responses. The cellular roles of OGT and the dynamic PTM O-GlcNAc have been clarified with recently developed chemical tools including high-throughput assays, structural and mechanistic studies and potent enzyme inhibitors. These evolving chemical tools complement genetic and biochemical approaches for exposing the underlying biological information conferred by O-GlcNAc cycling.

Versatile O-GlcNAc transferase assay for high-throughput identification of enzyme variants, substrates, and inhibitors.

The dynamic glycosylation of serine/threonine residues on nucleocytoplasmic proteins with a single N-acetylglucosamine (O-GlcNAcylation) is critical for many important cellular processes. Cellular O-GlcNAc levels are highly regulated by two enzymes: O-GlcNAc transferase (OGT) is responsible for GlcNAc addition and O-GlcNAcase (OGA) is responsible for removal of the sugar. The lack of a rapid and simple method for monitoring OGT activity has impeded the efficient discovery of potent OGT inhibitors. In this study we describe a novel, single-well OGT enzyme assay that utilizes 6 × His-tagged substrates, a chemoselective chemical reaction, and unpurified OGT. The high-throughput Ni-NTA Plate OGT Assay will facilitate discovery of potent OGT-specific inhibitors on versatile substrates and the characterization of new enzyme variants.

Ruminal Prevotella spp. may play an important role in the conversion of plant lignans into human health beneficial antioxidants.

Secoisolariciresinol diglucoside (SDG), the most abundant lignan in flaxseed, is metabolized by the ruminal microbiota into enterolignans, which are strong antioxidants. Enterolactone (EL), the main mammalian enterolignan produced in the rumen, is transferred into physiological fluids, with potentially human health benefits with respect to menopausal symptoms, hormone-dependent cancers, cardiovascular diseases, osteoporosis and diabetes. However, no information exists to our knowledge on bacterial taxa that play a role in converting plant lignans into EL in ruminants. In order to investigate this, eight rumen cannulated cows were used in a double 4 × 4 Latin square design and fed with four treatments: control with no flax meal (FM), or 5%, 10% and 15% FM (on a dry matter basis). Concentration of EL in the rumen increased linearly with increasing FM inclusion. Total rumen bacterial 16S rRNA concentration obtained using Q-PCR did not differ among treatments. PCR-T-RFLP based dendrograms revealed no global clustering based on diet indicating between animal variation. PCR-DGGE showed a clustering by diet effect within four cows that had similar basal ruminal microbiota. DNA extracted from bands present following feeding 15% FM and absent with no FM supplementation were sequenced and it showed that many genera, in particular Prevotella spp., contributed to the metabolism of lignans. A subsequent in vitro study using selected pure cultures of ruminal bacteria incubated with SDG indicated that 11 ruminal bacteria were able to convert SDG into secoisolariciresinol (SECO), with Prevotella spp. being the main converters. These data suggest that Prevotella spp. is one genus playing an important role in the conversion of plant lignans to human health beneficial antioxidants in the rumen.

Optimizing the selectivity of DIFO-based reagents for intracellular bioorthogonal applications.

One of the most commonly employed bioorthogonal reactions with azides is copper-catalyzed azide-alkyne [3+2] cycloaddition (CuAAC, a 'click' reaction). More recently, the strain-promoted azide-alkyne [3+2] cycloaddition (SPAAC, a copper-free 'click' reaction) was developed, in which an alkyne is sufficiently strained to promote rapid cycloaddition with an azide to form a stable triazole conjugate. In this report, we show that an internal alkyne in a strained ring system with two electron-withdrawing fluorine atoms adjacent to the carbon-carbon triple bond reacts to yield covalent adducts not only with azide moieties but also reacts with free sulfhydryl groups abundant in the cytosol. We have identified conditions that allow the enhanced reactivity to be tolerated when using such conformationally strained reagents to enhance reaction rates and selectivity for bioorthogonal applications such as O-GlcNAc detection.

Fabrication and characterization of plasma-polymerized poly(ethylene glycol) film with superior biocompatibility.

A newly fabricated plasma-polymerized poly(ethylene glycol) (PP-PEG) film shows extremely low toxicity, low fouling, good durability, and chemical similarity to typical PEG polymers, enabling live cell patterning as well as various bioapplications using bioincompatible materials. The PP-PEG film can be overlaid on any materials via the capacitively coupled plasma chemical vapor deposition (CCP-CVD) method using nontoxic PEG200 as a precursor. The biocompatibility of the PP-PEG-coated surface is confirmed by whole blood flow experiments where no thrombi and less serum protein adsorption are observed when compared with bare glass, polyethylene (PE), and polyethylene terephthalate (PET) surfaces. Furthermore, unlike bare PE films, less fibrosis and inflammation are observed when the PP-PEG-coated PE film is implanted into subcutaneous pockets of mice groin areas. The cell-repellent property of PP-PEG is also verified via patterning of mammalian cells, such as fibroblasts and hippocampal neurons. These results show that our PP-PEG film, generated by the CCP-CVD method, is a biocompatible material that can be considered for broad applications in biomedical and functional materials fields.

Targeting somatostatin receptors using in situ-bioconjugated fluorescent nanoparticles.

AIM: The author's group report, for the first time, on the development of a quantum dot (QD)-based fluorescent somatostatin (somatotropin release-inhibiting factor [SRIF]) probe that enables specific targeting of somatostatin receptors. Receptor-mediated endocytosis of SRIF was imaged using this probe. MATERIALS & METHODS: Biotinylated SRIF-analog (SRIF-B) and streptavidin (Sav)-coated QDs were used for the probe synthesis. A dye-labeled streptavidin complex was used to evaluate the effect of Sav binding on the activity of SRIF-B. RESULTS: A preconjugated probe of the form SRIF-B:Sav-QD, was inactive and unable to undergo receptor-mediated endocytosis. An alternative in situ bioconjugation strategy, where SRIF-B and Sav-QD were added in two consecutive steps, enabled visualization of the receptor-mediated endocytosis. The process of Sav binding appeared to be responsible for the inactivity in the first case. CONCLUSION: The in situ two-step bioconjugation strategy allowed QDs to be targeted to somatostatin receptors. This strategy should enable flexible fluorescent tagging of SRIF for the investigation of molecular trafficking in cells and targeted delivery in live animals.

Chemical arsenal for the study of O-GlcNAc.

The concepts of both protein glycosylation and cellular signaling have been influenced by O-linked-ß-N-acetylglucosamine (O-GlcNAc) modification (O-GlcNAcylation) on the hydroxyl group of serine or threonine residues. Unlike conventional protein glycosylation, O-GlcNAcylation is localized in the nucleocytoplasm and its cycling is a dynamic process that operates in a highly regulated manner in response to various cellular stimuli. These characteristics render O-GlcNAcylation similar to phosphorylation, which has long been considered a major regulatory mechanism in cellular processes. Various efficient chemical approaches and novel mass spectrometric (MS) techniques have uncovered numerous O-GlcNAcylated proteins that are involved in the regulation of many important cellular events. These discoveries imply that O-GlcNAcylation is another major regulator of cellular signaling. However, in contrast to phosphorylation, which is regulated by hundreds of kinases and phosphatases, dynamic O-GlcNAc cycling is catalyzed by only two enzymes: uridine diphospho-N-acetyl-glucosamine:polypeptide ß-N-acetylglucosaminyl transferase (OGT) and ß-D-N-acetylglucosaminidase (OGA). Many useful chemical tools have recently been used to greatly expand our understanding of the extensive crosstalk between O-GlcNAcylation and phosphorylation and hence of cellular signaling. This review article describes the various useful chemical tools that have been developed and discusses the considerable advances made in the O-GlcNAc field.

As yet uncultured bacteria phylogenetically classified as Prevotella, Lachnospiraceae incertae sedis and unclassified Bacteroidales, Clostridiales and Ruminococcaceae may play a predominant role in ruminal biohydrogenation.

Microbial biohydrogenation of dietary poly-unsaturated fatty acids (PUFA) to saturated fatty acids (SFA) in the rumen results in the high ratio of SFA/PUFA in ruminant products, such as meat and milk. In vitro, Butyrivibrio proteoclasticus-related bacteria extensively biohydrogenate PUFA to SFA, yet their contribution in the rumen has not been confirmed. The aim of this study was to evaluate the role of Butyrivibrio proteoclasticus group bacteria in ruminal biohydrogenation and to assess the possible role of other bacteria. Fish oil at 0%, 1.5% and 3% dry matter intake was fed to eight Holstein × Friesian steers, in order to elicit changes in the extent of PUFA biohydrogenation. Fatty acid and B. proteoclasticus group 16S rRNA concentrations in rumen digesta were determined. Correlation between digesta 18:0 concentration and B. proteoclasticus group 16S rRNA concentration was low. Terminal restriction fragment length polymorphism and denaturing gradient gel electrophoresis (DGGE) coupled with multivariate statistics revealed that many terminal restriction fragments (T-RFs) and DGGE bands were linked to cis-9, trans-11 conjugated linoleic acid (CLA), 18:1 trans-11 and 18:0 ruminal concentrations. MiCA T-RF predictive identification software showed that these linked T-RFs were likely to originate from as yet uncultured bacteria classified as Prevotella, Lachnospiraceae incertae sedis, and unclassified Bacteroidales, Clostridiales and Ruminococcaceae. Sequencing of linked DGGE bands also revealed that as yet uncultured bacteria classified as Prevotella, Anaerovoax (member of the Lachnospiraceae incertae sedis family), and unclassified Clostridiales and Ruminococcaceae may play a role in biohydrogenation.

Plant-based strategies towards minimising 'livestock's long shadow'.

Ruminant farming is an important component of the human food chain. Ruminants can use offtake from land unsuitable for cereal crop cultivation via interaction with the diverse microbial population in their rumens. The rumen is a continuous flow fermenter for the digestion of ligno-cellulose, with microbial protein and fermentation end-products incorporated by the animal directly or during post-ruminal digestion. However, ruminal fermentation is inefficient in capturing the nutrient resource presented, resulting in environmental pollution and generation of greenhouse gases. Methane is generated as a consequence of ruminal fermentation and poor retention of ingested forage nitrogen causes nitrogenous pollution of water and land and contributes to the generation of nitrous oxide. One possible cause is the imbalanced provision of dietary substrates to the rumen micro-organisms. Deamination of amino acids by ammonia-producing bacteria liberates ammonia which can be assimilated by the rumen bacteria and used for microbial protein synthesis. However, when carbohydrate is limiting, microbial growth is slow, meaning low demand for ammonia for microbial protein synthesis and excretion of the excess. Protein utilisation can therefore be improved by increasing the availability of readily fermentable sugars in forage or by making protein unavailable for proteolysis through complexing with plant secondary products. Alternatively, realisation that grazing cattle ingest living cells has led to the discovery that plant cells undergo endogenous, stress-mediated protein degradation due to the exposure to rumen conditions. This presents the opportunity to decrease the environmental impact of livestock farming by using decreased proteolysis as a selection tool for the development of improved pasture grass varieties.

Transplantation of human neural stem cells transduced with Olig2 transcription factor improves locomotor recovery and enhances myelination in the white matter of rat spinal cord following contusive injury.

BACKGROUND: Contusive spinal cord injury is complicated by a delayed loss of oligodendrocytes, resulting in chronic progressive demyelination. Therefore, transplantation strategies to provide oligodendrocyte lineage cells and to enhance the extent of myelination appear to be justified for spinal cord repair. The present study investigated whether transplantation of human neural stem cells (NSCs) genetically modified to express Olig2 transcription factor, an essential regulator of oligodendrocyte development, can improve locomotor recovery and enhance myelination in a rat contusive spinal cord injury model. RESULTS: HB1.F3 (F3) immortalized human NSC line was transduced with a retroviral vector encoding Olig2, an essential regulator of oligodendrocyte development. Overexpression of Olig2 in human NSCs (F3.Olig2) induced activation of NKX2.2 and directed differentiation of NSCs into oligodendrocyte lineage cells in vitro. Introduction of Olig2 conferred higher proliferative activity, and a much larger number of F3.Olig2 NSCs were detected by 7 weeks after transplantation into contused spinal cord than that of parental F3 NSCs. F3.Olig2 NSCs exhibited frequent migration towards the white matter, whereas F3 NSCs were mostly confined to the gray matter or around the lesion cavities. Most of F3.Olig2 NSCs occupying the spared white matter differentiated into mature oligodendrocytes. Transplantation of F3.Olig2 NSCs increased the volume of spared white matter and reduced the cavity volume. Moreover, F3.Olig2 grafts significantly increased the thickness of myelin sheath around the axons in the spared white matter. Finally, animals with F3.Olig2 grafts showed an improvement in the quality of hindlimbs locomotion. CONCLUSION: Transplantation of NSCs genetically modified to differentiate into an oligodendrocytic lineage may be an effective strategy to improve functional outcomes following spinal cord trauma. The present study suggests that molecular factors governing cell fate decisions can be manipulated to enhance reparative potential of the cell-based therapy.

Rumen protozoa are rich in polyunsaturated fatty acids due to the ingestion of chloroplasts.

Within this study, we investigated whether the polyunsaturated fatty acids (PUFA)-rich nature of rumen protozoa is a consequence of ingestion of PUFA-rich chloroplasts. Four Hereford x Friesian steers were offered hay [low 18:3 (n-3) and low chlorophyll concentration] followed by freshly cut perennial ryegrass [high 18:3 (n-3) and high chlorophyll concentration] for 16 days. On the 14th and 16th days, rumen protozoa as well as attached and planktonic bacteria were fractionated 1 h before (-1 h), 2 and 6 h postfeeding, and their fatty acid concentrations determined. Protozoa fractionated from fresh grass-fed steers were richer (P<0.05) in PUFA, except conjugated linoleic acid, for all time points compared with those from hay-fed steers. Protozoal density was higher (P<0.05) for grass compared with hay. Entodinomorphid abundance was 3.4 times higher on fresh grass (P<0.01) compared with hay. Confocal microscopy and transmission electron microscopy confirmed that Epidinium spp. were commonly saturated with intracellular cytoplasmic chloroplasts. These data suggest that engulfment of chloroplasts is a major contributor to the high 18:3 (n-3) concentration of protozoa.

Preconditioning mediated by sublethal oxygen-glucose deprivation-induced cyclooxygenase-2 expression via the signal transducers and activators of transcription 3 phosphorylation.

The signal transducers and activators of transcription (STATs) were found to be essential for cardioprotection. However, their role in preconditioning (PC) neuroprotection remains undefined. Previously, our studies showed that PC mediated a signaling cascade that involves activation of epsilon protein kinase C (varepsilonPKC), extracellular signal-regulated kinase (ERK1/2), and cyclooxygenase-2 (COX-2) pathways. However, the intermediate pathway by which ERK1/2 activates COX-2 was not defined. In this study, we investigated whether the PC-induced signaling pathway requires phosphorylation of STAT isoforms for COX-2 expression. To mimic PC or lethal ischemia, mixed cortical neuron/astrocyte cell cultures were subjected to 1 and/or 4 h of oxygen-glucose deprivation (OGD), respectively. The results indicated serine phosphorylation of STAT3 after PC or varepsilonPKC activation. Inhibition of either varepsilonPKC or ERK1/2 activation abolished PC-induced serine phosphorylation of STAT3. Additionally, inhibition of STAT3 prevented PC-induced COX-2 expression and neuroprotection against OGD. Therefore, our findings suggest that PC signaling cascade involves STAT3 activation after varepsilonPKC and ERK1/2 activation. Finally, we show that STAT3 activation mediates COX-2 expression and ischemic tolerance.

Fish oil increases the duodenal flow of long chain polyunsaturated fatty acids and trans-11 18:1 and decreases 18:0 in steers via changes in the rumen bacterial community.

Ruminant fat is rich in SFA, partly due to the biohydrogenation of dietary PUFA to SFA in the rumen. This process can be inhibited by the dietary inclusion of fish oil. The only bacteria isolated from the rumen capable of converting PUFA to SFA are closely related to Clostridium proteoclasticum. The aim of this study was to investigate if a correlation could be found in vivo between dietary fish oil inclusions and the composition of the ruminal bacterial community and specifically of C. proteoclasticum. Six Hereford x Friesian steers, prepared with ruminal and duodenal cannulae, received grass silage plus 1 of 3 concentrates resulting in total dietary fish oil contents of 0, 1, or 3% of dry matter. A dual flow marker technique was employed to estimate the relative flow of fatty acids. Steers fed the 3% fish oil diet had 100% increases in trans 18:1 flow, whereas 18:0 flow declined to 39% of steers fed the control diet. 16S ribosomal RNA-based denaturing gradient gel electrophoresis profiles obtained from ruminal digesta showed major changes in the bacterial community within steers fed the 3% fish oil diet. Quantitative PCR indicated only a weak relation between numbers of C. proteoclasticum and 18:0 flow between treatments and in individual steers (P < 0.05, but the percentage variance accounted for only 22.8) and did not provide unambiguous evidence that numbers of C. proteoclasticum in the rumen dictate the ratios of SFA:PUFA available for absorption by the animal. Understanding which microbes biohydrogenate PUFA in the rumen is key to developing novel strategies to improve the quality of ruminant products.