Recovery of cellulase activity after ethanol stripping in a novel pilot-scale unit

Recycling of enzymes has a potential interest during cellulosic bioethanol production as purchasing enzymes is one of the largest expenses in the process. By recycling enzymes after distillation, loss of sugars and ethanol are avoided, but depending on the distillation temperature, there is a potential risk of enzyme degradation. Studies of the rate of enzyme denaturation based on estimation of the denaturation constant K _D was performed using a novel distillation setup allowing stripping of ethanol at 50–65 °C. Experiments were performed in a pilot-scale stripper, where the effect of temperature (55–65 °C) and exposure to gas–liquid and liquid–heat transmission interfaces were tested on a mesophilic and thermostable enzyme mixture in fiber beer and buffer. Lab-scale tests were included in addition to the pilot-scale experiments to study the effect of shear, ethanol concentration, and PEG on enzyme stability. When increasing the temperature (up to 65 °C) or ethanol content (up to 7.5 % w/v), the denaturation rate of the enzymes increased. Enzyme denaturation occurred slower when the experiments were performed in fiber beer compared to buffer only, which could be due to PEG or other stabilizing substances in fiber beer. However, at extreme conditions with high temperature (65 °C) and ethanol content (7.5 % w/v), PEG had no enzyme stabilizing effect. The novel distillation setup proved to be useful for maintaining enzyme activity during ethanol extraction.     

Fine tuning of the catalytic activity of colicin E7 nuclease domain by systematic N‐terminal mutations

The nuclease domain of colicin E7 (NColE7) promotes the nonspecific cleavage of nucleic acids at its C‐terminal HNH motif. Interestingly, the deletion of four N‐terminal residues (446–449 NColE7 = KRNK) resulted in complete loss of the enzyme activity. R447A mutation was reported to decrease the nuclease activity, but a detailed analysis of the role of the highly positive and flexible N‐terminus is still missing. Here, we present the study of four mutants, with a decreased activity in the following order: NColE7  >> KGNK > KGNG ～ GGNK > GGNG. At the same time, the folding, the metal‐ion, and the DNA‐binding affinity were unaffected by the mutations as revealed by linear and circular dichroism spectroscopy, isothermal calorimetric titrations, and gel mobility shift experiments. Semiempirical quantum chemical calculations and molecular dynamics simulations revealed that K446, K449, and/or the N‐terminal amino group are able to approach the active centre in the absence of the other positively charged residues. The results suggested a complex role of the N‐terminus in the catalytic process that could be exploited in the design of a controlled nuclease.     

Revisiting the structure of the Vps10 domain of human sortilin and its interaction with neurotensin

Sortilin is a multifunctional receptor involved in sorting and apoptosis. We have previously reported a 2.0‐Å structure of the Vps10 ectodomain in complex with one of its ligands, the tridecapeptide neurotensin. Here we set out to further characterize the structural properties of sortilin and its interaction with neurotensin. To this end, we have determined a new 2.7 Å structure using a crystal grown with a 10‐fold increased concentration of neurotensin. Here a second peptide fragment was observed within the Vps10 β‐propeller, which may in principle either represent a second molecule of neurotensin or the N‐terminal part of the molecule bound at the previously identified binding site. However, in vitro binding experiments strongly favor the latter hypothesis. Neurotensin thus appears to bind with a 1:1 stoichiometry, and whereas the N‐terminus does not bind on its own, it enhances the affinity in context of full‐length neurotensin. We conclude that the N‐terminus of neurotensin probably functions as an affinity enhancer for binding to sortilin by engaging the second binding site. Crystal packing differs partly from the previous structure, which may be due to variations in the degree and pattern of glycosylations. Consequently, a notable hydrophobic loop, not modeled previously, could now be traced. A computational analysis suggests that this and a neighboring loop may insert into the membrane and thus restrain movement of the Vps10 domain. We have, furthermore, mapped all N‐linked glycosylations of CHO‐expressed human sortilin by mass spectrometry and find that their locations are compatible with membrane insertion of the hydrophobic loops.     

Genomic evaluation of both purebred and crossbred performances

Background

For a two-breed crossbreeding system, Wei and van der Werf presented a model for genetic evaluation using information from both purebred and crossbred animals. The model provides breeding values for both purebred and crossbred performances. Genomic evaluation incorporates marker genotypes into a genetic evaluation system. Among popular methods are the so-called single-step methods, in which marker genotypes are incorporated into a traditional animal model by using a combined relationship matrix that extends the marker-based relationship matrix to non-genotyped animals. However, a single-step method for genomic evaluation of both purebred and crossbred performances has not been developed yet.

Results

An extension of the Wei and van der Werf model that incorporates genomic information is presented. The extension consists of four steps: (1) the Wei van der Werf model is reformulated using two partial relationship matrices for the two breeds; (2) marker-based partial relationship matrices are constructed; (3) marker-based partial relationship matrices are adjusted to be compatible to pedigree-based partial relationship matrices and (4) combined partial relationship matrices are constructed using information from both pedigree and marker genotypes. The extension of the Wei van der Werf model can be implemented using software that allows inverse covariance matrices in sparse format as input.

Conclusions

A method for genomic evaluation of both purebred and crossbred performances was developed for a two-breed crossbreeding system. The method allows information from crossbred animals to be incorporated in a coherent manner for such crossbreeding systems.     

Evolution of Acyl-Substrate Recognition by a Family of Acyl-Homoserine Lactone Synthases

Members of the LuxI protein family catalyze synthesis of acyl-homoserine lactone (acyl-HSL) quorum sensing signals from S-adenosyl-L-methionine and an acyl thioester. Some LuxI family members prefer acyl-CoA, and others prefer acyl-acyl carrier protein (ACP) as the acyl-thioester substrate. We sought to understand the evolutionary history and mechanisms mediating this substrate preference. Our phylogenetic and motif analysis of the LuxI acyl-HSL synthase family indicates that the acyl-CoA-utilizing enzymes evolved from an acyl-ACP-utilizing ancestor. To further understand how acyl-ACPs and acyl-CoAs are recognized by acyl-HSL synthases we studied BmaI1, an octanoyl-ACP-dependent LuxI family member from Burkholderia mallei, and BjaI, an isovaleryl-CoA-dependent LuxI family member from Bradyrhizobium japonicum. We synthesized thioether analogs of their thioester acyl-substrates to probe recognition of the acyl-phosphopantetheine moiety common to both acyl-ACP and acyl-CoA substrates. The kinetics of catalysis and inhibition of these enzymes indicate that they recognize the acyl-phosphopantetheine moiety and they recognize non-preferred substrates with this moiety. We find that CoA substrate utilization arose through exaptation of acyl-phosphopantetheine recognition in this enzyme family.     

An Extract of Pomegranate Fruit and Galangal Rhizome Increases the Numbers of Motile Sperm: A Prospective,Randomised, Controlled,Double-Blinded Trial

Pomegranate fruit (Punica granatum) and galangal (Alpinia galanga) have separately been shown to stimulate spermatogenesis and to increase sperm counts and motility in rodents. Within traditional medicine, pomegranate fruit has long been used to increase fertility, however studies on the effect on spermatogenesis in humans have never been published. With this study we investigated whether oral intake of tablets containing standardised amounts of extract of pomegranate fruit and powder of greater galangal rhizome (Punalpin) would increase the total number of motile spermatozoa. The study was designed as a prospective, randomized, controlled, double-blinded trial. Enrolment was based on the mean total number of motile spermatozoa of two ejaculates. The participants delivered an ejaculate after 4–8 days of tablet intake and two ejaculates just before they stopped taking the tablets. Seventy adult men with a semen quality not meeting the standards for commercial application at Nordic Cryobank, but without azoospermia, were included in the study. Participants were randomized to take tablets containing extract of pomegranate fruit (standardised with respect to punicalagin A+B, punicalin and ellagic acid) and freeze-dried rhizome of greater galangal (standardised with respect to 1′S-1′-acetoxychavicol acetate) or placebo on a daily basis for three months. Sixty-six participants completed the intervention (active treatment: n = 34; placebo: n = 32). After the intervention the total number of motile spermatozoa was increased in participants treated with plant extracts compared with the placebo group (p = 0.026). After three months of active treatment, the average total number of motile sperm increased by 62% (from 23.4 to 37.8 millions), while for the placebo group, the number of motile sperm increased by 20%. Sperm morphology was not affected by the treatment. Our findings may help subfertile men to gain an improved amount of motile ejaculated sperm by taking tablets containing preparations of pomegranate fruit extract and rhizome of greater galangal.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01357044","term_id":"NCT01357044"}}NCT01357044     

Fasting Increases Human Skeletal Muscle Net Phenylalanine Release and This Is Associated with Decreased mTOR Signaling

Aim

Fasting is characterised by profound changes in energy metabolism including progressive loss of body proteins. The underlying mechanisms are however unknown and we therefore determined the effects of a 72-hour-fast on human skeletal muscle protein metabolism and activation of mammalian target of rapamycin (mTOR), a key regulator of cell growth.

Methods

Eight healthy male volunteers were studied twice: in the postabsorptive state and following 72 hours of fasting. Regional muscle amino acid kinetics was measured in the forearm using amino acid tracers. Signaling to protein synthesis and breakdown were assessed in skeletal muscle biopsies obtained during non-insulin and insulin stimulated conditions on both examination days.

Results

Fasting significantly increased forearm net phenylalanine release and tended to decrease phenylalanine rate of disappearance. mTOR phosphorylation was decreased by ∼50% following fasting, together with reduced downstream phosphorylation of 4EBP1, ULK1 and rpS6. In addition, the insulin stimulated increase in mTOR and rpS6 phosphorylation was significantly reduced after fasting indicating insulin resistance in this part of the signaling pathway. Autophagy initiation is in part regulated by mTOR through ULK1 and fasting increased expression of the autophagic marker LC3B-II by ∼30%. p62 is degraded during autophagy but was increased by ∼10% during fasting making interpretation of autophagic flux problematic. MAFbx and MURF1 ubiquitin ligases remained unaltered after fasting indicating no change in protesomal protein degradation.

Conclusions

Our results show that during fasting increased net phenylalanine release in skeletal muscle is associated to reduced mTOR activation and concomitant decreased downstream signaling to cell growth.     

Human FK506 binding protein 65 is associated with colorectal cancer

We initiated the present study to identify new genes associated with colorectal cancer. In a previously published microarray study an EST (W80763), later identified as the gene hFKBP10 (NM_021939), was found to be strongly expressed in tumors while absent in the normal mucosa. Here we describe this gene hFKBP10 together with its encoded protein hFKBP65 as a novel marker associated with colorectal cancer. Analysis of 31 colorectal adenocarcinomas and 14 normal colorectal mucosa by RealTime PCR for hFKBP10 showed a significant up-regulation in tumors, when compared with normal mucosa. Immunohistochemical analysis of 26 adenocarcinomas and matching normal mucosa, as well as benign hyperplastic polyps and adenomas, using a monoclonal anti-hFKBP65 antibody, showed that the protein was not present in normal colorectal epithelial cells, but strongly expressed in the tumor cells of colorectal cancer. The protein was also expressed in fibroblasts of both normal mucosa and tumor tissue. Western blot analysis of matched tumors and normal mucosa supported the finding of increased hFKBP65 expression in tumors compared with normal mucosa, in addition to identifying the molecular mass of hFKBP65 to approximately 72 kDa. Cellular localization and glycosylation studies revealed the hFKBP65 protein to be localized in the endoplasmic reticulum, and to be N-glycosylated. In conclusion, the protein hFKBP65 is associated with colorectal cancer, and we hypothesize the protein to be involved in fibroblast and transformed epithelial cell-specific protein synthesis in the endoplasmic reticulum.     

Metabolic network analysis of Bacillus clausii on minimal and semirich medium using (13)C-labeled glucose

Using (13)C-labeled glucose fed to the facultative alkalophilic Bacillus clausii producing the alkaline serine protease Savinase, the intracellular fluxes were quantified in continuous cultivation and in batch cultivation on a minimal medium. The flux through the pentose phosphate pathway was found to increase with increasing specific growth rate but at a much lower level than previously reported for Bacillus subtilis. Two futile cycles in the pyruvate metabolism were included in the metabolic network. A substantial flux in the futile cycle involving malic enzyme was estimated, whereas only a very small or zero flux through PEP carboxykinase was estimated, indicating that the latter enzyme was not active during growth on glucose. The uptake of the amino acids in a semirich medium containing 15 of the 20 amino acids normally present in proteins was estimated using fully labeled glucose in batch cultivations. It was found that leucine, isoleucine, and phenylalanine were taken up from the medium and not synthesized de novo from glucose. In contrast, serine and threonine were completely synthesized from other metabolites and not taken up from the medium. Valine, proline, and lysine were partly taken up from the medium and partly synthesized from glucose. The metabolic network analysis was extended to include analysis of growth on the semirich medium containing amino acids, and the metabolic flux distribution on this medium was estimated and compared with growth on minimal medium.     

A program against bacterial bioterrorism: improved patient management and acquisition of new knowledge on infectious diseases

In 2002 it was decided to establish laboratory facilities in Denmark for diagnosing agents associated with bioterrorism in order to make an immediate appropriate response to the release of such agents possible. Molecular assays for detection of specific agents and molecular and proteomic techniques for identification of bacteria were introduced as part of the program. All assays and techniques were made accessible for use in diagnosing patients, even when an intentional release was not suspected. Medical expertise on different diseases was established at the department as an integrated part of the program. The analyses included PCR assays for specific bacteria, identification of isolated bacteria by DNA sequencing, detection and identification of bacteria in clinical sample material by universal bacterial PCR and DNA sequencing, and identification of bacteria by mass spectrometry. The established analyses formed a basis on which a series of further developments was built. In addition to reducing the time for obtaining diagnoses and improving the accuracy of diagnosis of individual infected patients, the analyses provided new knowledge on the frequency and distribution of some bacterial infections, including Q fever, tularemia, trench fever, brucellosis, and melioidosis. The implementation of an antibioterrorism program in a clinical diagnostic setting improved the diagnostic possibilities for patients in Denmark and provided new epidemiologic information. It also introduced a number of diagnostic assays for bacterial infections not associated with bioterrorism that are difficult to culture or identify.