The characteristics of insoluble softwood substrates affect fungal morphology, secretome composition, and hydrolytic efficiency of enzymes produced by Trichoderma reesei

Background: On-site enzyme production using Trichoderma reesei can improve yields and lower the overall cost of lignocellulose saccharification by exploiting the fungal gene regulatory mechanism that enables it to continuously adapt enzyme secretion to the substrate used for cultivation. To harness this, the interrelation between substrate characteristics and fungal response must be understood. However, fungal morphology or gene expression studies often lack structural and chemical substrate characterization. Here, T. reesei QM6a was cultivated on three softwood substrates: northern bleached softwood Kraft pulp (NBSK) and lodgepole pine pretreated either by dilute-acid-catalyzed steam pretreatment (LP-STEX) or mild alkaline oxidation (LP-ALKOX). With different pretreatments of similar starting materials, we presented the fungus with systematically modified substrates. This allowed the elucidation of substrate-induced changes in the fungal response and the testing of the secreted enzymes’ hydrolytic strength towards the same substrates. Results: Enzyme activity time courses correlated with hemicellulose content and cellulose accessibility. Specifically, increased amounts of side-chain-cleaving hemicellulolytic enzymes in the protein produced on the complex substrates (LP-STEX; LP-ALKOX) was observed by secretome analysis. Confocal laser scanning micrographs showed that fungal micromorphology responded to changes in cellulose accessibility and initial culture viscosity. The latter was caused by surface charge and fiber dimensions, and likely restricted mass transfer, resulting in morphologies of fungi in stress. Supplementing a basic cellulolytic enzyme mixture with concentrated T. reesei supernatant improved saccharification efficiencies of the three substrates, where cellulose, xylan, and mannan conversion was increased by up to 27, 45, and 2800%, respectively. The improvement was most pronounced for proteins produced on LP-STEX and LP-ALKOX on those same substrates, and in the best case, efficiencies reached those of a state-of-the-art commercial enzyme preparation. Conclusion: Cultivation of T. reesei on LP-STEX and LP-ALKOX produced a protein mixture that increased the hydrolytic strength of a basic cellulase mixture to state-of-the-art performance on softwood substrates. This suggests that the fungal adaptation mechanism can be exploited to achieve enhanced performance in enzymatic hydrolysis without a priori knowledge of specific substrate requirements

Activity and components of the granulocyte colony‐stimulating factor pathway in hidradenitis suppurativa*

Background Hidradenitis suppurativa (HS) is a chronic inflammatory disease, characterized by painful, purulent and destructive skin alterations in intertriginous areas. Objectives We investigated the expression and role in HS of granulocyte colony-stimulating factor (G-CSF), the regulator of neutrophil biology, as clinical signs of a neutrophilic granulocyte-driven inflammation are distinctive in the disease. Methods Skin and blood samples obtained from different cohorts of patients with HS and control individuals were assessed by RNA sequencing, quantitative polymerase chain reaction on reverse transcribed mRNA, and/or enzyme-linked immunosorbent assay. Mechanistic studies using keratinocytes, dermal fibroblasts, immune cell populations and skin biopsies were performed. Results G-CSF was abundant in HS skin, particularly in inflamed nodules and abscesses. Its levels even exceeded those found in other inflammatory skin diseases. Interleukin (IL)-1 and IL-17, respectively, induced G-CSF production by fibroblasts and keratinocytes. These effects were enhanced by tumour necrosis factor (TNF)-alpha and IL-36. Accordingly, fibroblasts separated from HS lesions expressed G-CSF, and IL-1 receptor antagonist reduced G-CSF levels in explanted HS skin. G-CSF blood levels positively correlated with severity of HS. Elevated lesional G-CSF receptor levels were linked to upregulation of molecules that contribute to prolonged activation of neutrophils by components of bacteria and damaged host cells [formyl peptide receptor 1 (FPR1), FPR2 and free fatty acid receptor 2 (FFAR2)], neutrophil survival [TNF receptor superfamily member 10C (TNFRSF10C/TRAIL-R3) and TNF receptor superfamily member 6B], kinases (tyrosine-protein kinase HCK and hexokinase 3), and skin destruction [MMP25 (matrix metalloproteinase 25) and ADAM8 (disintegrin and metalloproteinase domain-containing protein 8)]. G-CSF elevated the expression of FPR1, FFAR2, and TNFRSF10C/TRAIL-R3 in neutrophils and synergized with bacterial components to induce skin-destructive enzymes. Conclusions The G-CSF pathway engages both tissue and immune cells, is strongly activated in HS lesions, and offers the opportunity to target the neutrophil-driven inflammation

Maintaining friendships in early stage dementia: Factors to consider

Friendships and the importance of social connectiveness play a critical role in aging well, regardless of gender, race, social class, or impairment. Yet, dementia takes its toll on social relationships, and many friends withdraw and ‘disappear’, because they can no longer bear to see the changes that are taking place in their diagnosed friend. The dementia care literature documents this abandonment; however, this study examines the opposite occurrence. In order to understand more clearly the role of long-term friendships and how such friendships remain and continue, despite the diagnosis of dementia, this qualitative study examines in depth eight people in the early stages of dementia who have been able to maintain strong friendships despite the diagnosis. Factors that seem to play important roles are: (1) the importance of the friendships, (2) factors affecting the quality of the relationships, (3) mutually beneficial relationships, (4) core values, (5) acceptance and disclosure, and (6) recognition of strengths and understanding of limitations

Identification of Escherichia coli strains from water vending machines of Kelantan, Malaysia using 16S rRNA gene sequence analysis

Water vending machines provide an alternative source of clean and safe drinking water to the consumers. However, the quality of drinking water may alter due to contamination from lack of hygienic practices and maintenance of the machines. Hence, this study was conducted to determine the microbiological quality of water from vending machines and associated contact surfaces. Seventeen water samples and 85 swab samples (nozzles, drip trays, coin slots, buttons and doors) from 3 locations in Kelantan were collected. Polymerase chain reaction amplification and 16S ribosomal ribonucleic acid (rRNA) sequencing were carried out and sequences obtained were compared against the sequences available in the National Centre for Biotechnology Information database using the basic local alignment search tool programme. Coliform counts were observed in 94.12 % of water samples, 76.47 % of nozzles and 82.35 % of drip tray swabs. Furthermore, results of 16S rRNA sequence analysis indicated that two gram-negative isolates were identified as Escherichia coli U 5/41 (Accession no. NR_024570.1) and E. coli O157:H7 EDL933 (Accession no. CP008957.1) with similarity value of 100 %, respectively. The results from this study further improve our understanding of the potential microorganisms in drinking water. Regular maintenance and cleaning of water vending machines are important to reduce bacterial growth and the presence of waterborne pathogens

Copper effect on the protein composition of photosystem II

The definitive version is available at: http://www.blackwell-synergy.com/doi/full/10.1111/j.1399-3054.2000.1100419.xWe provide data from in vitro experiments on the polypeptide composition, photosynthetic electron transport and oxygen evolution activity of intact photosystem II (PSII) preparations under Cu(II) toxicity conditions. Low Cu(II) concentrations (Cu(II) per PSII reaction centre unit≤230) that caused around 50% inhibition of variable chlorophyll a fluorescence and oxygen evolution activity did not affect the polypeptide composition of PSII. However, the extrinsic proteins of 33, 24 and 17 kDa of the oxygen-evolving complex of PSII were removed when samples were treated with 300 μM CuCl2 (Cu(II) per PSII reaction centre unit=1 400). The LHCII antenna complex and D1 protein of the reaction centre of PSII were not affected even at these Cu(II) concentrations. The results indicated that the initial inhibition of the PSII electron transport and oxygen-evolving activity induced by the presence of toxic Cu(II) concentrations occurred before the damage of the oxygen-evolving complex. Indeed, more than 50% inhibition could be achieved in conditions where its protein composition and integrity was apparently preserved.This work was supported by the Dirección General de Investigación Científica y Técnica (Grant PB98-1632).Peer reviewe

The dynamic changes of dominant clones of Staphylococcus aureus causing bloodstream infections in the European region : Results of a second structured survey

Staphylococcus aureus is one of the most important human pathogens and meticillin-resistant S. aureus (MRSA) presents a major cause of healthcare- and community-acquired infections. This study investigated the spatial and temporal changes of S. aureus causing bacteraemia in Europe over a five-year interval and explored the possibility of integrating pathogen-based typing data with epidemiological and clinical information at a European level. Between January 2011 and July 2011, 350 laboratories serving 453 hospitals in 25 countries collected 3,753 isolates (meticillin-sensitive S. aureus (MSSA) and MRSA) from patients with S. aureus bloodstream infections. All isolates were sent to the national staphylococcal reference laboratories and characterised by quality-controlled spa typing. Data were uploaded to an interactive web-based mapping tool. A wide geographical distribution of spa types was found, with some prevalent in all European countries. MSSA was more diverse than MRSA. MRSA differed considerably between countries with major international clones expanding or receding when compared to a 2006 survey. We provide evidence that a network approach of decentralised typing and visualisation of aggregated data using an interactive mapping tool can provide important information on the dynamics of S. aureus populations such as early signalling of emerging strains, cross-border spread and importation by travel.Peer reviewe

Behind the stiff upper lip: war narratives of older men with dementia.

The concept of the stiff upper lip stands as a cultural metaphor for the repression and figurative ¿biting back¿ of traumatic experience, particularly in military contexts. For men born in the first half of the 20th century, maintaining a stiff upper lip involved the ability to exert high levels of cognitive control over the subjective, visceral and emotional domains of experience. In the most common forms of dementia, which affect at least one in five men now in their 80s and 90s, this cognitive control is increasingly lost. One result is that, with the onset of dementia, men who have in the intervening years maintained a relative silence about their wartime experiences begin to disclose detailed memories of such events, in some cases for the first time. This article draws on narrative biographical data from three men with late-onset dementia who make extensive reference to their experience of war. The narratives of Sid, Leonard and Nelson are used to explore aspects of collective memory of the two World Wars, and the socially constructed masculinities imposed on men who grew up and came of age during those decades. The findings show that in spite of their difficulties with short term memory, people with dementia can contribute rich data to cultural studies research. Some aspects of the narratives discussed here may also be considered to work along the line of the counter-hegemonic, offering insights into lived experiences of war that have been elided in popular culture in the post-War years

Redox-Dependent Stability, Protonation, and Reactivity of Cysteine-Bound Heme Proteins

Cysteine-bound hemes are key components of many enzymes and biological sensors. Protonation (deprotonation) of the Cys ligand often accompanies redox transformations of these centers. To characterize these phenomena, we have engineered a series of Thr78Cys/Lys79Gly/Met80X mutants of yeast cytochrome c (cyt c) in which Cys78 becomes one of the axial ligands to the heme. At neutral pH, the protonation state of the coordinated Cys differs for the ferric and ferrous heme species, with Cys binding as a thiolate and a thiol, respectively. Analysis of redox-dependent stability and alkaline transitions of these model proteins, as well as comparisons to Cys binding studies with the minimalist heme peptide microperoxidase-8, demonstrate that the protein scaffold and solvent interactions play important roles in stabilizing a particular Cys–heme coordination. The increased stability of ferric thiolate compared with ferrous thiol arises mainly from entropic factors. This robust cyt c model system provides access to all four forms of Cys-bound heme, including the ferric thiol. Protein motions control the rates of heme redox reactions, and these effects are amplified at low pH, where the proteins are less stable. Thermodynamic signatures and redox reactivity of the model Cys-bound hemes highlight the critical role of the protein scaffold and its dynamics in modulating redox-linked transitions between thiols and thiolates

Increased Prevalence of Metabolic Syndrome in Patients with Acne Inversa

BACKGROUND: Acne inversa (AI; also designated as Hidradenitis suppurativa) is a common chronic inflammatory skin disease, localized in the axillary, inguinal and perianal skin areas that causes painful, fistulating sinuses with malodorous purulence and scars. Several chronic inflammatory diseases are associated with the metabolic syndrome and its consequences including arteriosclerosis, coronary heart disease, myocardial infraction, and stroke. So far, the association of AI with systemic metabolic alterations is largely unexplored. METHODS AND FINDINGS: A hospital-based case-control study in 80 AI patients and 100 age- and sex-matched control participants was carried out. The prevalence of central obesity (odds ratio 5.88), hypertriglyceridemia (odds ratio 2.24), hypo-HDL-cholesterolemia (odds ratio 4.56), and hyperglycemia (odds ratio 4.09) in AI patients was significantly higher than in controls. Furthermore, the metabolic syndrome, previously defined as the presence of at least three of the five alterations listed above, was more common in those patients compared to controls (40.0% versus 13.0%; odds ratio 4.46, 95% confidence interval 2.02 to 9.96; P<0.001). AI patients with metabolic syndrome also had more pronounced metabolic alterations than controls with metabolic syndrome. Interestingly, there was no correlation between the severity or duration of the disease and the levels of respective parameters or the number of criteria defining the metabolic syndrome. Rather, the metabolic syndrome was observed in a disproportionately high percentage of young AI patients. CONCLUSIONS: This study shows for the first time that AI patients have a high prevalence of the metabolic syndrome and all of its criteria. It further suggests that the inflammation present in AI patients does not have a major impact on the development of metabolic alterations. Instead, evidence is given for a role of metabolic alterations in the development of AI. We recommend monitoring of AI patients in order to correct their modifiable cardiovascular risk factors