Alkaloids in plants and root cultures of Atropa belladonna overexpressing putrescine N-methyltransferase

Putrescine N-methyltransferase (PMT) is the first alkaloid-specific enzyme for nicotine and tropane alkaloid formation. The pmt gene from Nicotiana tabacum was fused to the CaMV 35S promoter and integrated into the Atropa belladonna genome. Transgenic plants and derived root cultures were analysed for gene expression and for levels of alkaloids and their precursors. Scopolamine, hyoscyamine, tropine, pseudotropine, tropinone, and calystegines were found unaltered or somewhat decreased in pmt-overexpressing lines compared to controls. When root cultures were treated with 5% sucrose, calystegine levels were elevated in control roots, but were not affected in pmt-overexpressing roots. 1 microM auxin reduced calystegine levels in control roots, while in pmt-overexpressing roots all alkaloids remained unaltered. Expression level of pmt alone is apparently not limiting for tropane alkaloid formation in A. belladonna.     

T cell costimulus-independent and very efficacious inhibition of tumor growth in mice bearing subcutaneous or leukemic human B cell lymphoma xenografts by a CD19-/CD3- bispecific single-chain antibody construct

We have recently demonstrated that a recombinant single-chain bispecific Ab construct, bscCD19xCD3, in vitro induces rapid B lymphoma-directed cytotoxicity at picomolar concentrations with unstimulated peripheral T cells. In this study, we show that treatment of nonobese diabetic SCID mice with submicrogram doses of bscCD19xCD3 could prevent growth of s.c. human B lymphoma xenografts and essentially cured animals when given at an early tumor stage. The effect was dose dependent, dependent on E:T ratio and the time between tumor inoculation and administration of bscCD19xCD3. No therapeutic effect was seen in the presence of human lymphocytes alone, a vehicle control, or with a bispecific single-chain construct of identical T cell-binding activity but different target specificity. In a leukemic nonobese diabetic SCID mouse model, treatment with bscCD19xCD3 prolonged survival of mice in a dose-dependent fashion. The human lymphocytes used as effector cells in both animal models did not express detectable T cell activation markers at the time of coinoculation with tumor cells. The bispecific Ab therefore showed an in vivo activity comparable to that observed in cell culture with respect to high potency and T cell costimulus independence. These properties make bscCD19xCD3 superior to previously investigated CD19 bispecific Ab-based therapies.     

Solvent-tolerant bacteria for biotransformations in two-phase fermentation systems

Product removal from aqueous media poses a challenge in biotechnological whole-cell biotransformation processes in which substrates and/or products may have toxic effects. The assignment of an additional liquid solvent phase provides a solution, as it facilitates in situ product recovery from aqueous media. In such two-phase systems, toxic substrates and products are present in the aqueous phase in tolerable but still bioavailable amounts. As a matter of course, adequate organic solvents have to possess hydrophobicity properties akin to substrates and products of interest, which in turn involves intrinsic toxicity of the solvents used. The employment of bacteria being able to adapt to otherwise toxic solvents helps to overcome the problem. Adaptive mechanisms enabling such solvent tolerant bacteria to survive and grow in the presence of toxic solvents generally involve either modification of the membrane and cell surface properties, changes in the overall energy status, or the activation and/or induction of active transport systems for extruding solvents from membranes into the environment. It is anticipated that the biotechnological production of a number of important fine chemicals in amounts sufficient to compete economically with chemical syntheses will soon be possible by making use of solvent-tolerant microorganisms.     

FPF1 modulates the competence to flowering in Arabidopsis

During the transition to flowing the FPF1 gene is expressed in the peripheral zone of apical meristems and in floral meristems of Arabidopsis. Constitutive expression of FPF1 causes early flowering in Arabidopsis under both long-day and short-day conditions and leads to a shortened juvenile phase as measured by the trichome distribution on the abaxial leaf surface. In the classical late flowering mutants, overexpression of FPF1 compensates partially for the late flowering phenotype, indicating that FPF1 acts downstream or in a parallel pathway to the mutated genes. The co-overexpression of 35S::AP1 with 35S::FPF1 leads to a synergistic effect on the shortening of the time to flowering under short-day conditions. The co-overexpression of 35S::FPF1 and 35S::LFY, however, shows only an additive reduction of flowering time and the conversion of nearly every shoot meristem, except the inflorescence meristem, to a floral meristem under the same light conditions. In addition, the constitutive expression of FPF1 attenuates the severe lfy-1 phenotype under short days and phenocopies to a great extent the lfy-1 mutant grown under long-day conditions. Thus, we assume that FPF1 modulates the competence to flowering of apical meristems.     

Sex ratio of mirid populations shifts in response to hostplant co‐infestation or altered cytokinin signaling

Herbivore species sharing a host plant often compete. In this study, we show that host plant-mediated interaction between two insect herbivores-a generalist and a specialist-results in a sex ratio shift of the specialist's offspring. We studied demographic parameters of the specialist Tupiocoris notatus(Hemiptera: Miridae)when co-infesting the host plant Nicotiana attenuata(Solanaceae) with the generalist leafhopper Empoasca sp.(Hemiptera: Cicadellidae). We show that the usually female-biased sex ratio of T. notatus shifts toward a higher male proportion in the offspring on plants coinfested by Empoasca sp. This sex ratio change did not occur after oviposition, nor is it due differential mortality of female and male nymphs. Based on pyrosequencing and PCR of bacterial 16 S rRNA amplicons, we concluded that sex ratio shifts were unlikely to be due to infection with Wolbachia or other known sex ratio-distorting endosymbionts. Finally, we used transgenic lines of N.attenuata to evaluate if the sex ratio shift could be mediated by changes in general or specialized host plant metabolites. We found that the sex ratio shift occurred on plants deficient in two cytokinin receptors(irCHK2/3).Thus, cytokinin-regulated traits can alter the offspring sex ratio of the specialist T.notatus.     

Biochemical Characteristics and PrPSc Distribution Pattern in the Brains of Cattle Experimentally Challenged with H-type and L-type Atypical BSE

Besides the classical form of bovine spongiform encephalopathy (BSE) that has been known for almost three decades, two atypical forms designated H-type and L-type BSE have recently been described. While the main diagnostic feature of these forms is the altered biochemical profile of the accumulated PrP^Sc, it was also observed in the initial analysis that L-type BSE displays a distribution pattern of the pathological prion protein (PrP^Sc), which clearly differs from that observed in classical BSE (C-type). Most importantly, the obex region in the brainstem is not the region with the highest PrP^Sc concentrations, but PrP^Sc is spread more evenly throughout the entire brain. A similar distribution pattern has been revealed for H-type BSE by rapid test analysis. Based on these findings, we performed a more detailed Western blot study of the anatomical PrP^Sc distribution pattern and the biochemical characteristics (molecular mass, glycoprofile as well as PK sensitivity) in ten different anatomical locations of the brain from cattle experimentally challenged with H- or L-type BSE, as compared to cattle challenged with C-type BSE. Results of this study revealed distinct differences in the PrP^Sc deposition patterns between all three BSE forms, while the biochemical characteristics remained stable for each BSE type among all analysed brain areas.     

Accuracy of single-pass whole-body computed tomography for detection of injuries in patients with major blunt trauma

Background:

Contrast-enhanced whole-body computed tomography (also called “pan-scanning”) is considered to be a conclusive diagnostic tool for major trauma. We sought to determine the accuracy of this method, focusing on the reliability of negative results.

Methods:

Between July 2006 and December 2008, a total of 982 patients with suspected severe injuries underwent single-pass pan-scanning at a metropolitan trauma centre. The findings of the scan were independently evaluated by two reviewers who analyzed the injuries to five body regions and compared the results to a synopsis of hospital charts, subsequent imaging and interventional procedures. We calculated the sensitivity and specificity of the pan-scan for each body region, and we assessed the residual risk of missed injuries that required surgery or critical care.

Results:

A total of 1756 injuries were detected in the 982 patients scanned. Of these, 360 patients had an Injury Severity Score greater than 15. The median length of follow-up was 39 (interquartile range 7–490) days, and 474 patients underwent a definitive reference test. The sensitivity of the initial pan-scan was 84.6% for head and neck injuries, 79.6% for facial injuries, 86.7% for thoracic injuries, 85.7% for abdominal injuries and 86.2% for pelvic injuries. Specificity was 98.9% for head and neck injuries, 99.1% for facial injuries, 98.9% for thoracic injuries, 97.5% for abdominal injuries and 99.8% for pelvic injuries. In total, 62 patients had 70 missed injuries, indicating a residual risk of 6.3% (95% confidence interval 4.9%–8.0%).

Interpretation:

We found that the positive results of trauma pan-scans are conclusive but negative results require subsequent confirmation. The pan-scan algorithms reduce, but do not eliminate, the risk of missed injuries, and they should not replace close monitoring and clinical follow-up of patients with major trauma.Severe trauma is one of the 10 leading causes of burden of disease in North America and Europe.¹ According to a report from the Canadian National Trauma Registry, 14 065 major injuries with an Injury Severity Score above 12 occurred between 2008 and 2009 in the eight provinces that contribute to the National Trauma Registry.² Of these, there were 1605 (11%) deaths, and 212 098 hospital-days resulted.Priority-oriented management requires accurate imaging during resuscitation to avoid missed injuries.^3,4 Standardized algorithms, such as Advanced Trauma Life Support (ATLS), typically comprise physical examination, focused thoracoabdominal ultrasonography and plain radiography of the chest, spine and pelvis, followed by computed tomography (CT) of the head and other selected body areas. The effectiveness of this staged diagnostic approach, however, has been called into question in recent years.^5–9Contrast-enhanced whole-body CT scanning, often referred to as “pan-scanning,” was first proposed in the late 1990s as an alternative to sequential radiologic imaging in trauma settings.¹⁰ Pan-scan algorithms have been shown to accelerate diagnostic work-up, but their effect on survival is controversial.^11–14 Opponents have voiced concerns about the overexposure of patients to radiation with the increasing and often uncritical use of CT scanning.¹⁵We designed the PATRES (Pan-Scan for Trauma Resuscitation) study to assess the accuracy of the pan-scan in detecting injuries to different body regions in patients with suspected major blunt trauma.     

Human Langerhans Cells Control Th Cells via Programmed Death-Ligand 1 in Response to Bacterial Stimuli and Nickel-Induced Contact Allergy

Langerhans cells (LCs) are suspected to initiate inflammatory immune responses to contact allergens and pathogenic bacteria. In chronic infectious diseases, programmed death ligand (PD-L) 1 exhibits both inhibitory and costimulatory functions on T cell-mediated activation and tolerance. Here, we investigated the effects of contact allergens and bacterial stimuli on PD-L1 expression in LCs and the effects of altered PD-L1 expression on cytokine release of subsequently cocultured T cells. Monocyte-derived LCs (MoLCs), LCs, and skin sections of patients suffering from allergic contact dermatitis were challenged with nickel and then analyzed for PD-L1 expression by confocal laser scanning microscopy and flow cytometry. In blocking experiments, we found that the release of Th cell specific cytokines was dependent on both stimulation of LCs and inhibition of PD-L1-PD-1 interactions. Stimulation with peptidoglycan (PGN) or lipopolysaccharide (LPS) and blockage of PD-L1 with a specific antibody triggered the release of high levels of IL-17, IL-22, TNF-α, and IFN-γ in CD4⁺T cells. If nickel was used as a stimulus, blockage of PD-L1 led to high amounts of TNF-α and IL-22. A closer look revealed PD-L1-dependent upregulation of IL-17 secretion in FACS-sorted CCR6⁺/CCR4⁺ T memory cells. In the presence of anti-PD-L1, PGN induced secretion of IFN-γ and IL-17 in total CCR6⁺ cells, while nickel triggered secretion of IFN-γ and IL-17 exclusively in CCR6⁺/CCR4⁺ cells. Our findings suggest that PD-L1 on LCs plays a crucial role in type IV allergic reactions and in response to bacterial stimuli by controlling the nature of inflammatory Th cell responses.