Validation and discussion of clinical practicability of the 2022 graded prognostic assessment for NSCLC adenocarcinoma patients with brain metastases in a routine clinical cohort.

Introduction: The goal of this analysis is to validate the 2022 graded prognostic assessment (GPA) for patients with brain metastases from adenocarcinoma of the lung and to discuss its clinical practicability. Methods/material: 137 patients with adenocarcinoma of the lung were included in this analysis. The disease specific GPA for NSCLC, Lung-molGPA and the GPA for NSCLC adenocarcinoma were calculated. Overall survival was calculated for each GPA group. Additionally, expected and actual OS in the prognostic groups of the GPA available at the time of the patients' diagnosis was compared. Results: Median overall survival (OS) from diagnosis of brain metastases was 15 months (95% confidence interval (CI) 9.7-20.3 months). The median OS in the three individual prognostic groups was 7 months for GPA 0-1, 16 months for GPA 1.5-2, 33 months for GPA 2.5-3 and not reached for GPA 3.5-4 (p<0.001). Median survival times for the individual groups were similar to those published in the original GPA publication. Regarding the expected and actual OS when using the available GPA at the time of diagnosis there was an underestimation of survival of more than 3 months for all except the worst prognosis group. Conclusion: We were able to validate the 2022 GPA for NSCLC adenocarcinoma patients with brain metastases in a similar cohort from a non-academic center. However, the practical applicability regarding the expected median OS might be limited due to the constantly evolving treatment landscape and the consecutive improvement in overall survival.

PET/CT radiomics for prediction of hyperprogression in metastatic melanoma patients treated with immune checkpoint inhibitors.

Purpose: This study evaluated pretreatment 2[18F]fluoro-2-deoxy-D-glucose (FDG)-PET/CT-based radiomic signatures for prediction of hyperprogression in metastatic melanoma patients treated with immune checkpoint inhibition (ICI). Material and method: Fifty-six consecutive metastatic melanoma patients treated with ICI and available imaging were included in the study and 330 metastatic lesions were individually, fully segmented on pre-treatment CT and FDG-PET imaging. Lesion hyperprogression (HPL) was defined as lesion progression according to RECIST 1.1 and doubling of tumor growth rate. Patient hyperprogression (PD-HPD) was defined as progressive disease (PD) according to RECIST 1.1 and presence of at least one HPL. Patient survival was evaluated with Kaplan-Meier curves. Mortality risk of PD-HPD status was assessed by estimation of hazard ratio (HR). Furthermore, we assessed with Fisher test and Mann-Whitney U test if demographic or treatment parameters were different between PD-HPD and the remaining patients. Pre-treatment PET/CT-based radiomic signatures were used to build models predicting HPL at three months after start of treatment. The models were internally validated with nested cross-validation. The performance metric was the area under receiver operating characteristic curve (AUC). Results: PD-HPD patients constituted 57.1% of all PD patients. PD-HPD was negatively related to patient overall survival with HR=8.52 (95%CI 3.47-20.94). Sixty-nine lesions (20.9%) were identified as progressing at 3 months. Twenty-nine of these lesions were classified as hyperprogressive, thereby showing a HPL rate of 8.8%. CT-based, PET-based, and PET/CT-based models predicting HPL at three months after the start of treatment achieved testing AUC of 0.703 +/- 0.054, 0.516 +/- 0.061, and 0.704 +/- 0.070, respectively. The best performing models relied mostly on CT-based histogram features. Conclusions: FDG-PET/CT-based radiomic signatures yield potential for pretreatment prediction of lesion hyperprogression, which may contribute to reducing the risk of delayed treatment adaptation in metastatic melanoma patients treated with ICI.

Characterization of the rumen and fecal microbiome in bloated and non-bloated cattle grazing alfalfa pastures and subjected to bloat prevention strategies.

Frothy bloat is an often fatal digestive disorder of cattle grazing alfalfa pastures. The aim of this study was to investigate ruminal and fecal microbiota dynamics associated with development of alfalfa-induced frothy bloat and to further explore how bloat prevention strategies influence the composition of these microbial communities. In a 3 × 3 crossover experiment, twelve rumen-cannulated steers were sequentially subjected to: (1) pure alfalfa pasture, (2) pure alfalfa pasture supplemented with the pluronic detergent ALFASURE, and (3) alfalfa - sainfoin mixed pasture. Eleven out of 12 steers in pure alfalfa pasture developed clinical bloat, whereas ALFASURE treatment prevented the development of bloat in all 12 steers and alfalfa - sainfoin prevented bloat in 5 out of 11 steers. Development of bloat was associated with considerable shifts in the microbiota profile of rumen contents. In particular, the microbiota of solid rumen contents from bloated steers contained higher species richness and diversity. Streptococcus, Succinivibrio and unclassified Myxococcales were enriched in the rumen microbiota of bloated steers, whereas Fibrobacter and Ruminococcus were overrepresented in the rumen contents of non-bloated steers. Our results provide novel insights into bloat-associated shifts in the composition and predicted functional properties of the rumen microbiota of cattle grazing alfalfa pasture.

Isolation of High Quality RNA for Metatranscriptomic Analysis of Lignocellulose Digestion in the Rumen.

Metatranscriptomics can be used to examine both the composition of a microbial community as well as its metabolic activity under a particular set of conditions and complement metagenomic studies. The availability of low-cost, high-throughput next-generation sequencing has led to a rapid increase in the number of metatranscriptomic studies being undertaken. One of the primary difficulties when conducting transcriptomics is the ability to isolate high-quality RNA from samples of interest. The application of metatranscriptomics in rumen microbiology is still relatively novel but there is a significant push toward applying this technology in this field. In this protocol, we outline the method that is used routinely in our laboratory to purify high quality RNA from rumen contents that are suitable for metatranscriptomic sequencing using RNA-seq.

Electrochemically Triggered Release of Reagent to the Proximal Leaflet of a Microcavity Supported Lipid Bilayer.

A novel and versatile approach to electrichemically triggering the release of a reagent, ß-cyclodextrin (ß-CD), selectively to the proximal leaflet of a supported lipid bilayer is described. Selective delivery is achieved by creating a spanning lipid bilayer across a microcavity array and exploiting the irreversible redox disassembly of the host-guest complex formed between thiolated ferrocene (Fc) and ß-cyclodextrin (ß-CD) in the presence of chloride. Self-assembled monolayers of the ferrocene-alkanethiols were formed regioselectively on the interior surface of highly ordered 2.8 µm cavities while the exterior top surface of the array was blocked with a monolayer of mercaptoethanol. The Fc monolayers were complexed with ß-CD or ß-CD-conjugated to streptavidin (ß-CD-SA). Phospholipid bilayers were then assembled across the array via combined Langmuir-Blodgett/vesicle fusion leading to a spanning bilayer suspended across the aqueous filled microcavities. Upon application of a positive potential, ferrocene is oxidized to ferrocinium cation, disrupting the inclusion complex and leading to the release of the ß-CD into the microcavity solution where it diffuses to the lower leaflet of the suspended bilayer. Disassembly of the supramolecular complex within the cavities and binding of the ß-CD-SA to a biotinylated bilayer was followed by voltammetry and impedance spectroscopy where it caused a large increase in membrane resistance. For unmodified ß-CD, the extraction of cholesterol from a cholesterol containing bilayer was evident in a decrease in the bilayer resistance. For the first time, this direct approach to targeted delivery of a reagent to the proximal layer of a lipid bilayer offers the potential to build models of bidirectional signaling (inside-out vs outside-in) in cell membrane model systems.

Age and feeding system (supplemental feeding versus grazing) modulates colonic bacterial succession and host mucosal immune maturation in goats.

The gut microbiome plays important roles in the regulation of gastrointestinal tract functional development and host mucosal immune maturation. This study was conducted to test the hypothesis that age and feeding system (supplemental feeding [Sup] vs. grazing [G]) could alter colonic bacterial diversity and host mucosal immune maturation. Thirty Liuyang black goat kids ( = 4) were slaughtered on d 0, d 7 (nonrumination), d 28, d 42 (transition), and d 70 (rumination). The colonic microbiota was profiled by Miseq sequencing of the 16S rRNA gene. Host colonic mucosal immune maturation was examined using mRNA level expression of Toll-like receptors (TLR), proinflammatory cytokines, and the Toll-IL-1R (TIR) domain-containing adaptor. A correlation analysis was conducted to elucidate the relationship between bacterial diversity and fermentation parameters and host immune maturation variables. The results showed that α diversity indexes ( < 0.05), abundances of genera ( = 0.003) and ( = 0.024), ( = 0.004), and ( = 0.046) mRNA expressions were lower for Sup than for G, whereas the abundance of genera and ( < 0.05) was greater for Sup than for G. Regardless of the feeding system, bacterial 16S rRNA gene copy number and α diversity indexes increased ( < 0.05), whereas Proteobacteria abundance decreased linearly from d 0 to 70 after birth ( = 0.026). At the genus level, dominated the first week and declined sharply afterward, whereas abundance was greatest on d 7. abundance decreased linearly ( = 0.021), whereas abundances of , , , , and increased with age ( < 0.05). These findings coincided with increased , , and myeloid differentiation factor 88 () mRNA expressions with age ( < 0.05). Finally, correlation analysis revealed that different genera participated in different roles in fermentation capacity and host mucosal immune maturation. Collectively, colonic bacterial diversity and host mucosal immune maturation are age related, and concentrate supplement could alter bacterial diversity and alleviate overinflammation responses.

Endovascular repair or open repair for ruptured abdominal aortic aneurysm: a Cochrane systematic review.

OBJECTIVES: Emergency endovascular aneurysm repair (eEVAR) may improve outcomes for patients with ruptured abdominal aortic aneurysm (RAAA). The study aim was to compare the outcomes for eEVAR with conventional open surgical repair for the treatment of RAAA. SETTING: A systematic review of relevant publications was performed. Randomised controlled trials (RCTs) comparing eEVAR with open surgical repair for RAAA were included. PARTICIPANTS: 3 RCTs were included, with a total of 761 patients with RAAA. INTERVENTIONS: Meta-analysis was performed with fixed-effects models with ORs and 95% CIs for dichotomous data and mean differences with 95% CIs for continuous data. PRIMARY AND SECONDARY OUTCOME MEASURES: Primary outcome was short-term mortality. Secondary outcome measures included aneurysm-specific and general complication rates, quality of life and economic analysis. RESULTS: Overall risk of bias was low. There was no difference between the 2 interventions on 30-day (or in-hospital) mortality, OR 0.91 (95% CI 0.67 to 1.22; p=0.52). 30-day complications included myocardial infarction, stroke, composite cardiac complications, renal complications, severe bowel ischaemia, spinal cord ischaemia, reoperation, amputation and respiratory failure. Reporting was incomplete, and no robust conclusion was drawn. For complication outcomes that did include at least 2 studies in the meta-analysis, there was no clear evidence to support a difference between eEVAR and open repair. Longer term outcomes and cost per patient were evaluated in only a single study, thus precluding definite conclusions. CONCLUSIONS: Outcomes between eEVAR and open repair, specifically 30-day mortality, are similar. However, further high-quality trials are required, as the paucity of data currently limits the conclusions.

Non-clinical safety evaluation of repeated intramuscular administration of the AS15 immunostimulant combined with various antigens in rabbits and cynomolgus monkeys.

Combination of tumor antigens with immunostimulants is a promising approach in cancer immunotherapy. We assessed animal model toxicity of AS15 combined with various tumor antigens: WT1 (rabbits), or p501, dHER2 and recPRAME (cynomolgus monkeys), administered in seven or 20 dose regimens versus a saline control. Clinical and ophthalmological examinations, followed by extensive post-mortem pathological examinations, were performed on all animals. Blood hematology and biochemistry parameters were also assessed. Antigen-specific antibody titers were determined by enzyme-linked immunosorbent assay. Additional assessments in monkeys included electrocardiography and immunohistochemical evaluations of the p501 expression pattern. Transient increases in body temperature were observed 4 h or 24 h after injections of recPRAME + AS15 and dHER2 + AS15. Edema and erythema were observed up to 1 week after most injections of recPRAME + AS15 and all injections of dHER2 + AS15. No treatment-related effects were observed for electrocardiography parameters. Mean fibrinogen levels were significantly higher in all treated groups compared to controls, but no differences could be observed at the end of the treatment-free period. Transient but significant differences in biochemistry parameters were observed post-injection: lower albumin/globulin ratios (p501 + AS15), and higher bilirubin, urea and creatinine (dHER2 + AS15). Pathology examinations revealed significant increases in axillary lymph node mean weights (recPRAME + AS15) compared to controls. A 100% seroconversion rate was observed in all treated groups, but not in controls. p501 protein expression was observed in prostates of all monkeys from studies assessing p501 + AS15. These results suggest a favorable safety profile of the AS15-containing candidate vaccines, supporting the use of AS15 for clinical development of potential anticancer vaccines.

Mck2-dependent infection of alveolar macrophages promotes replication of MCMV in nodular inflammatory foci of the neonatal lung.

Infection with cytomegalovirus (CMV) shows a worldwide high prevalence with only immunocompromised individuals or newborns to become symptomatic. The host's constitution and the pathogen's virulence determine whether disease occurs after infection. Mouse CMV (MCMV) is an appreciated pathogen for in vivo investigation of host-pathogen interactions. It has recently been reported that a single base pair deletion can spontaneously occur in the open reading frame of MCMV-encoded chemokine 2 (MCK2), preventing the expression of the full-length gene product. To study the consequences of this mutation, we compared the Mck2-defective reporter virus MCMV-3D with the newly generated repaired Mck2(+) mutant MCMV-3DR. Compared with MCMV-3D, neonatal mice infected with MCMV-3DR showed severe viral disease after lung infection. Viral disease coincided with high viral activity in multiple organs and increased virus replication in previously described nodular inflammatory foci (NIF) in the lung. Notably, MCMV-3DR showed tropism for alveolar macrophages in vitro and in vivo, whereas MCMV-3D did not infect this cell type. Moreover, in vivo depletion of alveolar macrophages reduced MCMV-3DR replication in the lung. We proposed an Mck2-mediated mechanism by which MCMV exploits alveolar macrophages to increase replication upon first encounter with the host's lung mucosa.

Nonclinical reproductive and developmental safety evaluation of the MAGE-A3 Cancer Immunotherapeutic, a therapeutic vaccine for cancer treatment.

We assessed potential toxic effects of the MAGE-A3 Cancer Immunotherapeutic on female fertility and embryo-fetal, pre- and post-natal development in rats and on male fertility in rats and monkeys. Three groups of 48 female (Study 1) or 22 male (Study 2) CD rats received 5 or 3 injections of 100µL of saline, AS15 immunostimulant, or MAGE-A3 Cancer Immunotherapeutic (MAGE-A3 recombinant protein combined with AS15) at various timepoints pre- or post-mating. Male Cynomolgus monkeys (Study 3) received 8 injections of 500µL of saline (n=2) or the MAGE-A3 Cancer Immunotherapeutic (n=6) every 2 weeks. Rats were sacrificed on gestation day 20 or lactation day 25 (Study 1) or 9 weeks after first injection (Study 2) and monkeys, 3 days or 8 weeks after last injection. Injections were well tolerated. Female rat mating performance or fertility, pre- and post-natal survival, offspring development up to 25 days of age, and male mating performance (rats) or fertility parameters (rats and monkeys) were unaffected.

The application of water soluble, mega-Stokes-shifted BODIPY fluorophores to cell and tissue imaging.

BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) fluorophores are widely used in bioimaging to label proteins, lipids and nucleotides, but in spite of their attractive optical properties they tend to be prone to self-quenching because of their notably small Stokes shift. Herein, we compare two BODIPY compounds from a recently developed family of naphthyridine substituted BODIPY derivatives, one a visible emitting derivative (BODIPY-VIS) and one a near-infrared emitting fluorophore with a Stokes shift of approximately 165 nm as contrast reagents for live mammalian cells and murine brain tissue. The compounds were rendered water soluble by their conjugation to polyethylene glycol (PEG). Both PEGylated compounds exhibited good cell uptake compared with their parent compounds and confocal fluorescence microscopy revealed all dyes explored to be nuclear excluding, localizing predominantly within the lipophilic organelles; the endoplasmic reticulum and mitochondria. Cytotoxicity studies revealed that these BODIPY derivatives are modestly cytotoxic at concentrations exceeding 10 µM where they induce apoptosis and necrosis. Although the quantum yield of emission of the visible emitting fluorophore was over an order of magnitude greater than the Mega-Stokes shifted probe, the latter showed considerably reduced tendency to self quench and less interference from autofluorescence. The near-infrared probe also showed good penetrability and staining in live tissue samples. In the latter case similar tendency to exclude the nucleus and to localize in the mitochondria and endoplasmic reticulum was observed as in live cells. This to our knowledge is the first demonstration of such a Mega-Stokes BODIPY probe applied to cell and tissue imaging.

Fluorescence in situ hybridization probing of protozoal Entodinium spp. and their methanogenic colonizers in the rumen of cattle fed alfalfa hay or triticale straw.

AIMS: To develop and test a fluorescence in situ hybridization (FISH) based technique and to identify and quantify simultaneously those methanogenic populations colonizing Entodinium spp. in the rumen of cows fed different forages. METHODS AND RESULTS: New FISH probes targeting protozoal Entodinium spp. were designed and used together with FISH probes for methanogens in the cow rumen. The composition and relative abundance of methanogenic populations colonizing Entodinium simplex-, E. caudaum- and Entodinium furca-related populations were similar. Methanogens including Methanobrevibacter thaueri, Methanobrevibacter millerae and Methanobrevibacter smithii, and members of Methanomicrobium and Methanosphaera were generally the predominant colonizers of protozoa, regardless of the forage fed to cattle. Individual animals appeared to differ in which ruminal methanogenic populations colonized each of the individual Entodinium spp. CONCLUSIONS: Simultaneous FISH probing is shown here to be a reliable and effective approach to investigate the dynamics of symbiotic relationships between ruminal protozoa and methanogens at a single cell level. Phylogenetically closely related Entodinium spp. were colonized by similar methanogenic populations regardless of the forage fed. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report of the methanogenic archaeal populations that specifically colonize Entodinium spp. as identified using simultaneous FISH probing.

Biochemical and kinetic characterization of the multifunctional ß-glucosidase/ß-xylosidase/α-arabinosidase, Bgxa1.

Functional screening of a metagenomic library constructed with DNA extracted from the rumen contents of a grass/hay-fed dairy cow identified a protein, ß-glucosidase/ß-xylosidase/α-arabinosidase gene (Bgxa1), with high levels of ß-glucosidase activity. Purified Bgxa1 was highly active against p-nitrophenyl-ß-D-glucopyranoside (pNPG), cellobiose, p-nitrophenyl-ß-D-xylopyranoside (pNPX) and p-nitrophenyl-α-D-arabinofuranoside (pNPAf), suggesting it is a multifunctional ß-glucosidase/ß-xylosidase/α-arabinosidase. Kinetic analysis of the protein indicated that Bgxa1 has the greatest catalytic activity against pNPG followed by pNPAf and pNPX, respectively. The catalytic efficiency of ß-glucosidase activity was 100× greater than ß-xylosidase or α-arabinosidase. The pH and temperature optima for the hydrolysis of selected substrates also differed considerably with optima of pH 6.0/45 °C and pH 8.5/40 °C for pNPG and pNPX, respectively. The pH dependence of pNPAf hydrolysis displayed a bimodal distribution with maxima at both pH 6.5 and pH 8.5. The enzyme exhibited substrate-dependent responses to changes in ionic strength. Bgxa1 was highly stable over a broad pH range retaining at least 70 % of its relative catalytic activity from pH 5.0-10.0 with pNPG as a substrate. Homology modelling was employed to probe the structural basis of the unique specificity of Bgxa1 and revealed the deletion of the PA14 domain and insertions in loops adjacent to the active site. This domain has been found to be an important determinant in the substrate specificity of proteins related to Bgxa1. It is postulated that these indels are, in part, responsible for the multifunctional activity of Bgxa1. Bgxa1 acted synergistically with endoxylanase (Xyn10N18) when incubated with birchwood xylan, increasing the release of reducing sugars by 168 % as compared to Xyn10N18 alone. Examination of Bgxa1 and Xyn10N18 synergy with a cellulase for the saccharification of alkali-treated straw revealed that synergism among the three enzymes enhanced sugar release by 180 % as compared to cellulase alone. Our results suggest that Bgxa1 has a number of properties that make it an interesting candidate for the saccharification of lignocellulosic material.

Changes in the rumen epimural bacterial diversity of beef cattle as affected by diet and induced ruminal acidosis.

Little is known about the nature of the rumen epithelial adherent (epimural) microbiome in cattle fed different diets. Using denaturing gradient gel electrophoresis (DGGE), quantitative real-time PCR (qPCR), and pyrosequencing of the V3 hypervariable coding region of 16S rRNA, epimural bacterial communities of 8 cattle were profiled during the transition from a forage to a high-concentrate diet, during acidosis, and after recovery. A total of 153,621 high-quality gene sequences were obtained, with populations exhibiting less taxonomic variability among individuals than across diets. The bacterial community composition exhibited clustering (P < 0.03) by diet, with only 14 genera, representing >1% of the rumen epimural population, differing (P ≤ 0.05) among diets. During acidosis, levels of Atopobium, Desulfocurvus, Fervidicola, Lactobacillus, and Olsenella increased, while during the recovery, Desulfocurvus, Lactobacillus, and Olsenella reverted to levels similar to those with the high-grain diet and Sharpea and Succinivibrio reverted to levels similar to those with the forage diet. The relative abundances of bacterial populations changed during diet transition for all qPCR targets except Streptococcus spp. Less than 5% of total operational taxonomic units (OTUs) identified exhibited significant variability across diets. Based on DGGE, the community structures of epithelial populations differed (P ≤ 0.10); segregation was most prominent for the mixed forage diet versus the grain, acidotic challenge, and recovery diets. Atopobium, cc142, Lactobacillus, Olsenella, RC39, Sharpea, Solobacterium, Succiniclasticum, and Syntrophococcus were particularly prevalent during acidosis. Determining the metabolic roles of these key genera in the rumens of cattle fed high-grain diets could define a clinical microbial profile associated with ruminal acidosis.

Biochemical analysis of a highly specific, pH stable xylanase gene identified from a bovine rumen-derived metagenomic library.

A metagenomic library was generated using microbial DNA extracted from the rumen contents of a grass hay-fed dairy cow using a bacterial artificial chromosome-based vector system. Functional screening of the library identified a gene encoding a potent glycoside hydrolase, xyn10N18, localised within a xylanolytic gene cluster consisting of four open-reading frames (ORFs). The ORF, xyn10N18, encodes an endo-ß-1,4-xylanase with a glycosyl hydrolase family 10 (GH10) catalytic domain, adopts a canonical α8/ß8-fold and possesses conserved catalytic glutamate residues typical of GH10 xylanases. Xyn10N18 exhibits optimal catalytic activity at 35 °C and pH 6.5 and was highly stable to pH changes retaining at least 85 % relative catalytic activity over a broad pH range (4.0-12.0). It retained 25 % of its relative activity at both low (4 °C) and high (55 °C) temperatures, however the stability of the enzyme rapidly decreased at temperatures of >40 °C. The specific activity of Xyn10N18 is enhanced by the divalent cations Mn(2+) and Co(2+) and is dramatically reduced by Hg(2+) and Cu(2+). Interestingly, EDTA had little effect on specific activity indicating that divalent cations do not function mechanistically. The enzyme was highly specific for xylan containing substrates and showed no catalytic activity against cellulose. Analysis of the hydrolysis products indicated that Xyn10N18 was an endoxylanase. Through a combination of structural modelling and in vitro enzyme characterisation this study provides an understanding of the mechanism and the substrate specificity of this enzyme serving as a starting point for directed evolution of Xyn10N18 and subsequent downstream use in industry.

Characterization of rumen bacterial diversity and fermentation parameters in concentrate fed cattle with and without forage.

AIMS: To determine the effects of the removal of forage in high-concentrate diets on rumen fermentation conditions and rumen bacterial populations using culture-independent methods. METHODS AND RESULTS: Detectable bacteria and fermentation parameters were measured in the solid and liquid fractions of digesta from cattle fed two dietary treatments, high concentrate (HC) and high concentrate without forage (HCNF). Comparison of rumen fermentation conditions showed that duration of time spent below pH 5·2 and rumen osmolality were higher in the HCNF treatment. Simpson's index of 16S PCR-DGGE images showed a greater diversity of dominant species in the HCNF treatment. Real-time qPCR showed populations of Fibrobacter succinogenes (P = 0·01) were lower in HCNF than HC diets. Ruminococcus spp., F. succinogenes and Selenomonas ruminantium were at higher (P ≤ 0·05) concentrations in the solid vs the liquid fraction of digesta regardless of diet. CONCLUSIONS: The detectable bacterial community structure in the rumen is highly diverse. Reducing diet complexity by removing forage increased bacterial diversity despite the associated reduction in ruminal pH being less conducive for fibrolytic bacterial populations. Quantitative PCR showed that removal of forage from the diet resulted in a decline in the density of some, but not all fibrolytic bacterial species examined. SIGNIFICANCE AND IMPACT OF THE STUDY: Molecular techniques such as DGGE and qPCR provide an increased understanding of the impacts of dietary changes on the nature of rumen bacterial populations, and conclusions derived using these techniques may not match those previously derived using traditional laboratory culturing techniques.

Isolation and characterization of a ferulic acid esterase (Fae1A) from the rumen fungus Anaeromyces mucronatus.

AIMS: A novel ferulic acid esterase gene from rumen fungus Anaeromyces mucronatus was cloned, heteroexpressed in Escherichia coli and characterized. METHODS AND RESULTS: A total of 30 clones exhibiting activity on α-naphthyl acetate (α-NA) were isolated from an A. mucronatus YE505 cDNA library. Sequence analysis revealed that these clones represented two esterase-coding sequences. The gene, fae1A, showed highest amino acid sequence identity to CE family 1 esterases from anaerobic micro-organisms such as Orpinomyces sp., Ruminococcus albus and Clostridium thermocellum. The gene comprised 828 nucleotides encoding a polypeptide of 275 amino acids. The coding sequence was cloned into the pET30a expression vector and overexpressed in E. coli BL21 (DE3). Gene product Fae1A was found to exhibit activity against a number of substrates including naphthyl fatty acid esters, p-nitrophenyl fatty acid esters and hydroxylcinnamic acid esters. CONCLUSIONS: Fae1A exhibited a lower K(m) and higher catalytic efficiency (k(cat) /K(m) ) on ferulic acid esters than on α-NA or p-nitrophenyl acetate, suggesting that it has a higher affinity for ethyl and methyl ferulate than for the acetyl esters. It releases ferulic acid and p-coumaric acid from barley straw. Activity of Fae1A was inhibited by the serine-specific protease inhibitor, phenylmethylsulfonyl fluoride, indicating that a serine residue plays a role in its activity. SIGNIFICANCE AND IMPACT OF THE STUDY: To our knowledge, this is the first report of characterization of carbohydrate esterase gene from the genus of Anaeromyces.

Effects of lauric and myristic acids on ruminal fermentation, production, and milk fatty acid composition in lactating dairy cows.

The objectives of this experiment were to investigate the effects of lauric (LA) and myristic (MA) acids on ruminal fermentation, production, and milk fatty acid (FA) profile in lactating dairy cows and to identify the FA responsible for the methanogen-suppressing effect of coconut oil. The experiment was conducted as a replicated 3×3 Latin square. Six ruminally cannulated cows (95±26.4 DIM) were subjected to the following treatments: 240 g/cow per day each of stearic acid (SA, control), LA, or MA. Experimental periods were 28 d and cows were refaunated between periods. Lauric acid reduced protozoal counts in the rumen by 96%, as well as acetate, total VFA, and microbial N outflow from the rumen, compared with SA and MA. Ruminal methane production was not affected by treatment. Dry matter intake was reduced 35% by LA compared with SA and MA, which resulted in decreased milk yield. Milk fat content also was depressed by LA compared with SA and MA. Treatment had no effect on milk protein content. All treatments increased milk concentration of the respective treatment FA. Concentration of C12:0 was more than doubled by LA, and C14:0 was increased (45%) by MA compared with SA. Concentration of milk FAC16 FA and MUFA were increased, by LA compared with the other treatments. In this study, LA had profound effects on ruminal fermentation, mediated through inhibited microbial populations, and decreased DMI, milk yield, and milk fat content. Despite the significant decrease in protozoal counts, however, LA had no effect on ruminal methane production. Thus, the antimethanogenic effect of coconut oil, observed in related studies, is likely due to total FA application level, the additive effect of LA and MA, or a combination of both. Both LA and MA modified milk FA profile significantly.

In vitro evaluation of cashew nut shell liquid as a methane-inhibiting and propionate-enhancing agent for ruminants.

Cashew nut shell liquid (CNSL) containing antibacterial phenolic compounds was evaluated for its potency as a feed additive for ruminants. In experiment 1, ruminal responses to CNSL supplementation were assessed using a batch culture system. Rumen fluid from cattle was diluted with artificial saliva and incubated for 18h in a batch culture with a mixed diet containing a 30:70 hay:concentrate diet to which raw or heated CNSL was added at a final concentration of 500 µg/mL. In experiment 2, a Rusitec, using rumen fluid from the same cattle, was operated over a period of 7 d during which only raw CNSL was tested at concentrations of 0, 50, 100, or 200 µg/mL, and variations in fermentation and bacterial population were assessed. In experiment 3, a pure culture study was conducted using selected bacteria to determine their susceptibility to CNSL. In experiment 1, methane production was inhibited by raw CNSL (56.9% inhibition) but not by heated CNSL. Total volatile fatty acid concentration was not affected, whereas increased concentrations of propionate and decreased concentrations of acetate and butyrate were observed using either raw or heated CNSL. These changes were more obvious when raw CNSL was tested. In experiment 2, raw CNSL inhibited methanogenesis and increased propionate production in a dose-dependent manner, showing maximum methane inhibition (70.1%) and propionate enhancement (44.4%) at 200 µg/mL supplementation. Raw CNSL increased total volatile fatty acid concentration and dry matter digestibility. Raw CNSL also appeared to induce a dramatic shift in the population of rumen microbiota, based on decreased protozoa numbers and changes in quantitative PCR assay values for representative bacterial species. In experiment 3, using pure cultures, raw CNSL prevented the growth of hydrogen-, formate-, and butyrate-producing rumen bacteria, but not the growth of bacteria involved in propionate production. Based on these data, raw CNSL, rich in the antibacterial phenolic compound anacardic acid, is a potential candidate feed additive with selective activity against rumen microbes, leading to fermentation that results in decreased methane and enhanced propionate production.

[Advancement of the medical doctorate]. / Weiterentwicklung des Promotionsverfahrens in der Medizin.

The medical doctorate and the subsequent advanced research qualification in medicine have an exceptional position within the natural sciences. While, in the German system, graduation to the degree of a medical doctor is often an initiation into scientific practice, the in-depth scientific education of medical doctors may be achieved in various configurations. In recent years, structured programs for doctorates in medicine and natural sciences have found increasing acceptance, following recommendations of national scientific councils ("Deutsche Forschungsgemeinschaft" and "Hochschulrat"). Hannover Medical School has been offering such programs for a number of years. The StrucMed program increases the quality of medical doctorate studies, typically performed in the third and fourth years of university studies. The Hannover Biomedical Research School (HBRS) combines several programs for a doctorate in natural sciences, creating a platform for an internationally oriented education of post-graduates in various disciplines of life sciences. Evaluating the achievements and career paths of the trainees will contribute to the successful integration of research work in an efficiency-oriented clinical environment.