Expression of lignocellulolytic enzymes in Pichia pastoris

BACKGROUND: Sustainable utilization of plant biomass as renewable source for fuels and chemical building blocks requires a complex mixture of diverse enzymes, including hydrolases which comprise the largest class of lignocellulolytic enzymes. These enzymes need to be available in large amounts at a low price to allow sustainable and economic biotechnological processes. Over the past years Pichia pastoris has become an attractive host for the cost-efficient production and engineering of heterologous (eukaryotic) proteins due to several advantages. RESULTS: In this paper codon optimized genes and synthetic alcohol oxidase 1 promoter variants were used to generate Pichia pastoris strains which individually expressed cellobiohydrolase 1, cellobiohydrolase 2 and beta-mannanase from Trichoderma reesei and xylanase A from Thermomyces lanuginosus. For three of these enzymes we could develop strains capable of secreting gram quantities of enzyme per liter in fed-batch cultivations. Additionally, we compared our achieved yields of secreted enzymes and the corresponding activities to literature data. CONCLUSION: In our experiments we could clearly show the importance of gene optimization and strain characterization for successfully improving secretion levels. We also present a basic guideline how to correctly interpret the interplay of promoter strength and gene dosage for a successful improvement of the secretory production of lignocellulolytic enzymes in Pichia pastoris

Using bi-spectral imaging technology for simulated online-weed control in winter wheat and maize

In spring 2011, two field trials on site-specific weed control in winter wheat and maize were carried out at Ihinger Hof research station of the University of Hohenheim. For the image acquisition, bi-spectral cameras were mounted on a vehicle. These cameras are able to take images free from disturbances by soil, mulch and stones. Images and the corresponding GPS-data were stored on-the-go. Afterwards, the images were analyzed by a weed recognition software. Weed infestation was mapped in consideration of weed species and weeds grouped according to their herbicide sensitivity. In order to simulate an online herbicide application, a onesided moving average of order five was used for the weed mapping. This kind of rearward calculation uses only the data of weed infestation which were already assessed behind or directly in the current position of the vehicle. The calculated weed distribution maps were checked by visual grid sampling. Herbicide application maps were generated by applying weed thresholds on the weed distribution maps. The herbicide application based on the maps was conducted by a multiple sprayer which allows the application of up to three herbicides independently from each other in a single pass across the field. Later on, the performance of the herbicide application was controlled again by visual grid sampling. Compared to a uniform herbicide application, the sitespecific weed control saved 83 % and 58 % herbicides respectively in winter wheat and 66 % in maize. The average efficacy of the site-specific herbicide application system in winter wheat was 70 % of the conventional herbicide application. Keywords: Bi-spectral cameras, herbicide application, image analysis, site-specific, weed controlSimulation einer online Unkrautkontrolle in Winterweizen und Mais unter Verwendung von BispektralkamerasIm Frühjahr 2011 wurden auf der Versuchsstation Ihinger Hof der Universität Hohenheim Feldversuche zur teilschlagspezifischen online-Unkrautkontrolle in den Kulturen Winterweizen und Mais durchgeführt. Die Bildaufnahme für die Erfassung der Verunkrautung mit Hilfe von digitaler Bildverarbeitung erfolgte mit einem Kamerafahrzeug, auf welchem Bispektralkameras montiert waren. Die Verwendung dieser Kameras gewährleistet die Aufnahme von Bildern, die von Boden, Steinen und Mulch weitestgehend ungestört sind. Zusammen mit den Bildern wurden die korrespondierenden Geokoordinaten gespeichert. Nach der anschließenden Bildauswertung unter Verwendung digitaler Bildauswerteverfahren erfolgte eine Kartierung der ermittelten Verunkrautung nach Unkrautarten bzw. Unkräuter gruppiert gemäß der Herbizidempfindlichkeit. Für die Kartierung wurden einseitig gleitende Mittelwerte der fünften Ordnung berechnet. Die Bestimmung des Mittelwertes erfolgte jeweils mit den vier Messwerten die zeitlich gesehen hinter dem aktuellen Messwert lagen und dem aktuellen Messwert. Durch diese rückwärtsgewandte Mittelwertbildung wurde die online Bildverarbeitung simuliert. Zur Überprüfung der Plausibilität der berechneten Unkrautverteilungskarten wurden die aus einer visuellen Rasterbonitur stammenden Daten herangezogen.Unter Verwendung von Schadschwellen wurden die auf der Basis der Bildanalyse erstellten Unkrautverteilungskarten für die Berechnung von Applikationskarten benutzt. Die Umsetzung der Applikationskarten erfolgte mit einer Dreikammerspritze, welche eine simultane Applikation von bis zu drei Herbiziden unabhängig voneinander erlaubt. Eine Erfolgskontrolle der durchgeführten Herbizidapplikation erfolgte im zeitlichen Abstand wiederum anhand einer visuellen Rasterbonitur. Im Vergleich zur betriebsüblichen Variante wurde durch die teilschlagspezifische Herbizidapplikation im Winterweizen eine Herbizideinsparung von 83 % bzw. 58 % und im Mais von 66 % realisiert. Die durchschnittliche Wirksamkeit der teilschlagspezifischen Herbizidmaßnahme im Winterweizen betrug 70 % der betriebsüblichen Variante. Stichwörter: Bildverarbeitung, Bispektralkameras, Herbizidapplikation, teilschlagspezifisch, Unkrautkontroll

Glucocorticoid-endocannabinoid interaction in cardiac surgical patients: relationship to early cognitive dysfunction and late depression

Background: Endocannabinoids (ECs) are rapidly acting immune-modulatory lipid-signaling molecules that are important for adaptation to stressful and aversive situations. They are known to interact with glucocorticoids and other stress-responsive systems. Maladaptation to acute or chronic stress represents a major risk factor for the development of psychiatric disorders. In the present study, we administered stress doses of hydrocortisone in a prospective, randomized, placebo-controlled double-blind study in patients undergoing cardiac surgery (CS) to examine the relationship between the use of glucocorticoids, plasma EC levels, and the occurrence of early postoperative cognitive dysfunction (delirium) and of later development of depression. Methods: We determined plasma levels of the ECs anandamide and 2-arachidonoylglycerol (2-AG) in CS patients of the hydrocortisone (n=56) and the placebo group (n=55) preoperatively, at postoperative day (POD) 1, at intensive care unit discharge, and at 6 months after CS (n=68). Postoperative delirium was diagnosed according to Diagnostic and Statistical Manual of the American Psychiatric Association IVth Edition (DSM-IV) criteria, and depression was determined by validated questionnaires and a standardized psychological interview (Structured Clinical Interview for DSM-IV). Results: Stress doses of hydrocortisone did not affect plasma EC levels and the occurrence of delirium or depression. However, patients who developed delirium on POD 1 had significantly lower preoperative 2-AG levels of the neuroprotective EC 2-AG (median values, 3.8 vs. 11.3 ng/ml; p=0.03). Preoperative 2-AG concentrations were predictive of postoperative delirium (sensitivity=0.70; specificity=0.69; cutoff value=4.9 ng/ml; receiver operating characteristic curve area=0.70; 95% confidence interval=0.54-0.85). Patients with depression at 6 months after CS (n=16) had significantly lower anandamide and 2-AG levels during the perioperative period. Conclusions: A low perioperative EC response may indicate an increased risk for early cognitive dysfunction and long-term depression in patients after CS. Glucocorticoids do not seem to influence this relationship

Enzymes from Higher Eukaryotes for Industrial Biocatalysis

The industrial production of fine chemicals, feed and food ingredients, pharmaceuticals, agrochemicals and their respective intermediates relies on an increasing application of biocatalysis, i.e. on enzyme or whole-cell catalyzed conversions of molecules. Simple procedures for discovery, cloning and over-expression as well as fast growth favour fungi, yeasts and especially bacteria as sources of biocatalysts. Higher eukaryotes also harbour an almost unlimited number of potential biocatalysts, although to date the limited supply of enzymes, the high heterogeneity of enzyme preparations and the hazard of infectious contaminants keep some interesting candidates out of reach for industrial bioprocesses. In the past only a few animal and plant enzymes from agricultural waste materials were employed in food processing. The use of bacterial expression strains or non-conventional yeasts for the heterologous production of efficient eukaryotic enzymes can overcome the bottleneck in enzyme supply and provide sufficient amounts of homogenous enzyme preparations for reliable and economically feasible applications at large scale. Ideal enzymatic processes represent an environmentally friendly, »near-to-completion« conversion of (mostly non-natural) substrates to pure products. Recent developments demonstrate the commercial feasibility of large-scale biocatalytic processes employing enzymes from higher eukaryotes (e.g. plants, animals) and also their usefulness in some small-scale industrial applications

Inhibition of HIV-1 in Cell Culture by Synthetic Humate Analogues Derived from Hydroquinone: Mechanism of Inhibition

AbstractHumic acids are natural constituents of soil and ground water and mainly consist of mixtures of polycyclic phenolic compounds. A similar complex of compounds with a mean size of about 1000 Da, designated HS-1500, was synthesized by oxidation of hydroquinone. HS-1500 inhibited HIV-1 infection of MT-2 cells with an IC50of 50–300 ng/ml and showed a mean cell toxicity of about 600 μg/ml. Inhibition of HIV-induced syncytium formation was observed at 10–50 μg/ml. Treatment of free and cell-attached HIV with HS-1500 irreversibly reduced its infectivity, whereas the susceptibility of target cells for the virus was not impaired by treatment prior to infection. The HIV envelope protein gp120SU bound to sepharose-coupled HS-1500 and could be eluted by high salt and detergent. HS-1500 interfered with the CD4-induced proteolytic cleavage of the V3 loop of virion gp120SU. Furthermore, binding of V3 loop-specific antibodies was irreversibly inhibited, whereas binding of soluble CD4 to gp120SU on virus and infected cells was not affected. In conclusion, our data suggest, that the synthetic humic acid analogue inhibits the infectivity of HIV particles by interference with a V3 loop-mediated step of virus entry

Nuclear-physical methods of analysis using spectrometric radiation sources

To analyze the nuclear-physical methods of elemental composition and distribution of impurities by the method of Rutherford backscattering, the 238Pu source was used as the source of ionizing radiation. It was shown that in the absence of an accelerator this method is promising, since it allows one to monitor the composition and distribution of elements during technological processes

Correlation between Age, Emergency Department Length of Stay and Hospital Admission Rate in Emergency Department Patients Aged ≥70 Years

Background: Interdisciplinary emergency departments (EDs) are confronted with trauma and nontrauma patients of any age group. Length of stay (LOS) and admission rates reflect both disease complexity and severity. Objective: To evaluate LOS and admission rates in different age groups according to traumatic and nontraumatic etiologies. Patients and Methods: During May 2011 a total of 4,653 adult patients (defined as ≥18 years old) seen in the ED of our municipal hospital were evaluated for their primary problem, Emergency Severity Index, LOS and admission rate. 1,841 trauma patients (mean age: 51.9 years; SD 22.5 years) and 2,812 nontrauma patients (mean age: 60.0 years; SD 20.4 years) were included. Results: Median LOS in the ED was 1:41 h (trauma) and 1:52 h (nontrauma). Trauma patients aged ≥70 years spent more time in the ED than nontrauma patients of this age group (patients aged ≥70 years median: 2:08 vs. 1:56 h; p < 0.0001). However, no significant difference was found in patients aged <70 years (1:33 vs. 1:48 h; p = 0.64). Comparing older with younger patients, median LOS within the ED was about 8 min longer in nontrauma patients aged ≥70 years (p = 0.22) and about 35 min longer in trauma patients aged ≥70 years (p < 0.00001). Conclusions: The correlation between age and LOS is stronger for trauma patients, which might indicate a special need for geriatric expertise in elderly trauma ED patients. Thus an interdisciplinary approach including surgical and geriatric expertise may be advantageous

Outcomes and Complication Rates of Cuff Downsizing in the Treatment of Worsening or Persistent Incontinence After Artificial Urinary Sphincter Implantation

Purpose This study investigated the functional outcomes and complication rates of cuff downsizing for the treatment of recurrent or persistent stress urinary incontinence (SUI) in men after the implantation of an artificial urinary sphincter (AUS). Methods Data from our institutional AUS database spanning the period from 2009 to 2020 were retrospectively analyzed. The number of pads per day was determined, a standardized quality of life (QoL) questionnaire and the International Consultation on Incontinence Questionnaire (ICIQ) were administered, and postoperative complications according to the Clavien-Dindo classification were analyzed. Results Out of 477 patients who received AUS implantation during the study period, 25 (5.2%) underwent cuff downsizing (median age, 77 years; interquartile range [IQR], 74–81 years; median follow-up, 4.4 years; IQR, 3–6.9 years). Before downsizing, SUI was very severe (ICIQ score 19–21) or severe (ICQ score 13–18) in 80% of patients, moderate (ICIQ score 6–12) in 12%, and slight (ICIQ score 1–5) in 8%. After downsizing, 52% showed an improvement of >5 out of 21 points. However, 28% still had very severe or severe SUI, 48% had moderate SUI, and 20% had slight SUI. One patient no longer had SUI. In 52% of patients, the use of pads per day was reduced by ≥50%. QoL improved by >2 out of 6 points in 56% of patients. Complications (infections/urethral erosions) requiring device explantation occurred in 36% of patients, with a median time to event of 14.5 months. Conclusions Although cuff downsizing carries a risk of AUS explantation, it can be a valuable treatment option for selected patients with persistent or recurrent SUI after AUS implantation. Over half of patients experienced improvements in symptoms, satisfaction, ICIQ scores, and pad use. It is important to inform patients about the potential risks and benefits of AUS to manage their expectations and assess individual risks

Unusual multisystemic involvement and a novel BAG3 mutation revealed by NGS screening in a large cohort of myofibrillar myopathies

Myofibrillar myopathies (MFM) are a group of phenotypically and genetically heterogeneous neuromuscular disorders, which are characterized by protein aggregations in muscle fibres and can be associated with multisystemic involvement.Methods We screened a large cohort of 38 index patients with MFM for mutations in the nine thus far known causative genes using Sanger and next generation sequencing (NGS). We studied the clinical and histopathological characteristics in 38 index patients and five additional relatives (n = 43) and particularly focused on the associated multisystemic symptoms.Results We identified 14 heterozygous mutations (diagnostic yield of 37%), among them the novel p.Pro209Gln mutation in the BAG3 gene, which was associated with onset in adulthood, a mild phenotype and an axonal sensorimotor polyneuropathy, in the absence of giant axons at the nerve biopsy. We revealed several novel clinical phenotypes and unusual multisystemic presentations with previously described mutations: hearing impairment with a FLNC mutation, dysphonia with a mutation in DES and the first patient with a FLNC mutation presenting respiratory insufficiency as the initial symptom. Moreover, we described for the first time respiratory insufficiency occurring in a patient with the p.Gly154Ser mutation in CRYAB. Interestingly, we detected a polyneuropathy in 28% of the MFM patients, including a BAG3 and a MYOT case, and hearing impairment in 13%, including one patient with a FLNC mutation and two with mutations in the DES gene. In four index patients with a mutation in one of the MFM genes, typical histological findings were only identified at the ultrastructural level (29%).Conclusions We conclude that extraskeletal symptoms frequently occur in MFM, particularly cardiac and respiratory involvement, polyneuropathy and/or deafness. BAG3 mutations should be considered even in cases with a mild phenotype or an adult onset. We identified a genetic defect in one of the known genes in less than half of the MFM patients, indicating that more causative genes are still to be found. Next generation sequencing techniques should be helpful in achieving this aim