Pleurotus ostreatus (the oyster mushroom) and other white rot filamentous basidiomycetes are key players in the global carbon cycle. P. ostreatus is also a commercially important edible fungus with medicinal properties and is important for biotechnological and environmental applications. Efficient gene targeting via homologous recombination (HR) is a fundamental tool for facilitating comprehensive gene function studies. Since the natural HR frequency in Pleurotus transformations is low (2.3%), transformed DNA is predominantly integrated ectopically. To overcome this limitation, a general gene targeting system was developed by producing a P. ostreatus PC9 homokaryon Δku80 strain, using carboxin resistance complemented by the development of a protocol for hygromycin B resistance protoplast-based DNA transformation and homokaryon isolation. The Δku80 strain exhibited exclusive (100%) HR in the integration of transforming DNA, providing a high efficiency of gene targeting. Furthermore, the Δku80 strains produced showed a phenotype similar to that of the wild-type PC9 strain, with similar growth fitness, ligninolytic functionality, and capability of mating with the incompatible strain PC15 to produce a dikaryon which retained its resistance to the corresponding selection and was capable of producing typical fruiting bodies. The applicability of this system is demonstrated by inactivation of the versatile peroxidase (VP) encoded by mnp4. This enzyme is part of the ligninolytic system of P. ostreatus, being one of the nine members of the manganese-peroxidase (MnP) gene family, and is the predominantly expressed VP in Mn2+-deficient media. mnp4 inactivation provided a direct proof that mnp4 encodes a key VP responsible for the Mn2+-dependent and Mn2+-independent peroxidase activity under Mn2+-deficient culture conditions.

Multiple myeloma (MM) is a largely incurable plasma cell malignancy with a poorly understood and heterogeneous clinical course. To identify potential, functionally relevant somatic mutations in MM, we performed whole-exome sequencing of five primary MM, corresponding germline DNA and six MM cell lines, and developed a bioinformatics strategy that also integrated published mutational data of 38 MM patients. Our analysis confirms that identical, recurrent mutations of single genes are infrequent in MM, but highlights that mutations cluster in important cellular pathways. Specifically, we show enrichment of mutations in adhesion molecules of MM cells, emphasizing the important role for the interaction of the MM cells with their microenvironment. We describe an increased rate of mutations in receptor tyrosine kinases (RTKs) and associated signaling effectors, for example, in EGFR, ERBB3, KRAS and MAP2K2, pointing to a role of aberrant RTK signaling in the development or progression of MM. The diversity of mutations affecting different nodes of a particular signaling network appears to be an intrinsic feature of individual MM samples, and the elucidation of intra- as well as interindividual redundancy in mutations that affect survival pathways will help to better tailor targeted therapeutic strategies to the specific needs of the MM patient.

The versatile-peroxidase (VP) encoded by mnp4 is one of the nine members of the manganese-peroxidase (MnP) gene family that constitutes part of the ligninolytic system of the white-rot basidiomycete Pleurotus ostreatus (oyster mushroom). VP enzymes exhibit dual activity on a wide range of substrates. As Mn2+ supplement to P. ostreatus cultures results in enhanced degradation of recalcitrant compounds and lignin, we examined the effect of Mn2+ on the expression profile of the MnP gene family. In P. ostreatus (monokaryon PC9), mnp4 was found to be the predominantly expressed mnp in Mn2+-deficient media, whereas strongly repressed (to approximately 1%) in Mn2+-supplemented media. Accordingly, in-vitro Mn2+-independent activity was found to be negligible. We tested whether release of mnp4 from Mn2+ repression alters the activity of the ligninolytic system. A transformant over-expressing mnp4 (designated OEmnp4) under the control of the β-tubulin promoter was produced. Now, despite the presence of Mn2+ in the medium, OEmnp4 produced mnp4 transcript as well as VP activity as early as 4 days after inoculation. The level of expression was constant throughout 10 days of incubation (about 0.4-fold relative to β-tubulin) and the activity was comparable to the typical activity of PC9 in Mn2+-deficient media. In-vivo decolorization of the azo dyes Orange II, Reactive Black 5, and Amaranth by OEmnp4 preceded that of PC9. OEmnp4 and PC9 were grown for 2 weeks under solid-state fermentation conditions on cotton stalks as a lignocellulosic substrate. [14C]-lignin mineralization, in-vitro dry matter digestibility, and neutral detergent fiber digestibility were found to be significantly higher (about 25%) in OEmnp4-fermented substrate, relative to PC9. We conclude that releasing Mn2+ suppression of VP4 by over-expression of the mnp4 gene in P. ostreatus improved its ligninolytic functionality.

OBJECTIVE

To evaluate the glucose dependency of glucose-dependent insulinotropic polypeptide (GIP) effects on insulin and glucagon release in 10 healthy male subjects ([means ± SEM] aged 23 ± 1 years, BMI 23 ± 1 kg/m2, and HbA1c 5.5 ± 0.1%).

RESEARCH DESIGN AND METHODS

Saline or physiological doses of GIP were administered intravenously (randomized and double blinded) during 90 min of insulin-induced hypoglycemia, euglycemia, or hyperglycemia.

RESULTS

During hypoglycemia, GIP infusion caused greater glucagon responses during the first 30 min compared with saline (76 ± 17 vs. 28 ± 16 pmol/L per 30 min, P < 0.008), with similar peak levels of glucagon reached after 60 min. During euglycemia, GIP infusion elicited larger glucagon responses (62 ± 18 vs. −11 ± 8 pmol/L per 90 min, P < 0.005). During hyperglycemia, comparable suppression of plasma glucagon (−461 ± 81 vs. −371 ± 50 pmol/L per 90 min, P = 0.26) was observed with GIP and saline infusions. In addition, during hyperglycemia, GIP more than doubled the insulin secretion rate (P < 0.0001).

CONCLUSIONS

In healthy subjects, GIP has no effect on glucagon responses during hyperglycemia while strongly potentiating insulin secretion. In contrast, GIP increases glucagon levels during fasting and hypoglycemic conditions, where it has little or no effect on insulin secretion. Thus, GIP seems to be a physiological bifunctional blood glucose stabilizer with diverging glucose-dependent effects on the two main pancreatic glucoregulatory hormones.

Introduction

In addition to the lipid-lowering effect of bile acid sequestrants (BASs), they also lower blood glucose and, therefore, could be beneficial in the treatment of patients with type 2 diabetes mellitus (T2DM). Three oral BASs are approved by the US Food and Drug Administration (FDA) for the treatment of hypercholesterolaemia: colestipol, cholestyramine and colesevelam. The BAS colestimide/colestilan is used in Japan. Colesevelam was recently approved by the FDA for the treatment of T2DM. We plan to provide a systematic review with meta-analysis of the glucose-lowering effect of BASs with the aim to evaluate their potential as glucose-lowering agents in patients with T2DM.

Methods and analysis

In accordance with the preferred reporting items for systematic reviews and meta-analyses statement, a systematic review with meta-analysis of randomised clinical trials of BASs (vs placebo, oral antidiabetes drugs or insulin), reporting measures of glycaemic control in adult patients with T2DM, will be performed. Change in glycated haemoglobin constitutes the primary endpoint, and secondary endpoints include changes in fasting plasma glucose, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, total cholesterol, triglycerides, body weight and body mass index and adverse events. Electronic searches will be performed in The Cochrane Library, MEDLINE and EMBASE, along with manual searches in the reference lists of relevant papers. The analyses will be performed based on individual patient data and summarised data. The primary meta-analysis will be performed using random effects models owing to expected intertrial heterogeneity. Dichotomous data will be analysed using risk difference and continuous data using weighted mean differences, both with 95% CIs.

Ethics and dissemination

The study will evaluate the potential of BASs as glucose-lowering agents and possibly contribute to the clinical management of patients with T2DM.

Results

The study will be disseminated by peer-review publication and conference presentation.

Protocol registration

PROSPERO CRD42012002552.

Graphical abstract

PdIn intermetallic phases can be switched in methanol steam reforming between a CO2-selective multilayer and an In-diluted phase by annealing at 453 K or 623 K.

Highlights

► A multilayer Pd1In1 phase in MSR is highly CO2-selective between 493 and 623 K. ► An In-diluted PdIn intermetallic phase yields CO formation via full methanol dehydrogenation. ► Higher reaction temperatures are needed for comparable CO2-TOF values as on supported PdIn/In2O3. ► A bimetal-oxide synergism for efficient CO2 formation is operative.

In situ X-ray photoelectron spectroscopy (in situ XPS) was used to study the structural and catalytic properties of Pd–In near-surface intermetallic phases in correlation with previously studied PdZn and PdGa.

Room temperature deposition of ∼4 monolayer equivalents (MLEs) of In metal on Pd foil and subsequent annealing to 453 K in vacuum yields a ∼1:1 Pd/In near-surface multilayer intermetallic phase. This Pd1In1 phase exhibits a similar “Cu-like” electronic structure and indium depth distribution as its methanol steam reforming (MSR)-selective multilayer Pd1Zn1 counterpart.

Catalytic characterization of the multilayer Pd1In1 phase in MSR yielded a CO2-selectivity of almost 100% between 493 and 550 K. In contrast to previously studied In2O3-supported PdIn nanoparticles and pure In2O3, intermediate formaldehyde is only partially converted to CO2 using this Pd1In1 phase. Strongly correlated with PdZn, on an In-diluted PdIn intermetallic phase with “Pd-like” electronic structure, prepared by thermal annealing at 623 K, methanol steam reforming is suppressed and enhanced CO formation via full methanol dehydrogenation is observed.

To achieve CO2-TOF values on the isolated Pd1In1 intermetallic phase as high as on supported PdIn/In2O3, at least 593 K reaction temperature is required. A bimetal-oxide synergism, with both bimetallic and oxide synergistically contributing to the observed catalytic activity and selectivity, manifests itself by accelerated formaldehyde-to-CO2 conversion at markedly lowered temperatures as compared to separate oxide and bimetal. Combination of suppression of full methanol dehydrogenation to CO on Pd1In1 inhibited inverse water–gas-shift reaction on In2O3 and fast water activation/conversion of formaldehyde is the key to the low-temperature activity and high CO2-selectivity of the supported catalyst.

Abstract

Recombinant adeno-associated virus (rAAV) vectors offer promise for the gene therapy of α1-antitrypsin (AAT) deficiency. In our prior trial, an rAAV vector expressing human AAT (rAAV1-CB-hAAT) provided sustained, vector-derived AAT expression for >1 year. In the current phase 2 clinical trial, this same vector, produced by a herpes simplex virus complementation method, was administered to nine AAT-deficient individuals by intramuscular injection at doses of 6.0×1011, 1.9×1012, and 6.0×1012 vector genomes/kg (n=3 subjects/dose). Vector-derived expression of normal (M-type) AAT in serum was dose dependent, peaked on day 30, and persisted for at least 90 days. Vector administration was well tolerated, with only mild injection site reactions and no serious adverse events. Serum creatine kinase was transiently elevated on day 30 in five of six subjects in the two higher dose groups and normalized by day 45. As expected, all subjects developed anti-AAV antibodies and interferon-γ enzyme-linked immunospot responses to AAV peptides, and no subjects developed antibodies to AAT. One subject in the mid-dose group developed T cell responses to a single AAT peptide unassociated with any clinical effects. Muscle biopsies obtained on day 90 showed strong immunostaining for AAT and moderate to marked inflammatory cell infiltrates composed primarily of CD3-reactive T lymphocytes that were primarily of the CD8+ subtype. These results support the feasibility and safety of AAV gene therapy for AAT deficiency, and indicate that serum levels of vector-derived normal human AAT >20 μg/ml can be achieved. However, further improvements in the design or delivery of rAAV-AAT vectors will be required to achieve therapeutic target serum AAT concentrations.

Flotte and colleagues report on a phase 2 trial in which the same α1-antitrypsin (AAT) AAV vector as in phase 1 is administered intramuscularly to nine AAT-deficient individuals at one of three doses. Vector-derived expression of normal (M-type) AAT in serum is shown to be dose dependent, peaks on day 30, and persists for at least 90 days, although AAT levels were sub-therapeutic.

Background

Antipsychotic-induced weight gain constitutes a major unresolved clinical problem which may ultimately be associated with reducing life expectancy by 25 years. Overweight is associated with brain deterioration, cognitive decline and poor quality of life, factors which are already compromised in normal weight patients with schizophrenia.

Here we outline the current strategies against antipsychotic-induced weight gain, and we describe peripheral and cerebral effects of the gut hormone glucagon-like peptide-1 (GLP-1). Moreover, we account for similarities in brain changes between schizophrenia and overweight patients.

Discussion

Current interventions against antipsychotic-induced weight gain do not facilitate a substantial and lasting weight loss. GLP-1 analogs used in the treatment of type 2 diabetes are associated with significant and sustained weight loss in overweight patients. Potential effects of treating schizophrenia patients with antipsychotic-induced weight gain with GLP-1 analogs are discussed.

Conclusions

We propose that adjunctive treatment with GLP-1 analogs may constitute a new avenue to treat and prevent metabolic and cerebral deficiencies in schizophrenia patients with antipsychotic-induced weight gain. Clinical research to support this idea is highly warranted.

Graphical abstract

Highlights

► A Pd1Ga1 surface without Ga2O3 contact is inactive for CO2 formation in methanol steam reforming. ► In situ XPS spectroscopy showed that water activation is blocked on Pd1Ga1. ► The valence band electronic structure of Pd1Ga1 favors selective dehydrogenation to H2CO. ► In oxidative steam reforming, the Pd1Ga1 surface behaves like extended Pd. ► Thus, total methanol oxidation by O2 at low temperatures is predominant.

In situ X-ray photoelectron spectroscopy and low-energy ion scattering were used to study the preparation, (thermo)chemical and catalytic properties of 1:1 PdGa intermetallic near-surface phases. Deposition of several multilayers of Ga metal and subsequent annealing to 503–523 K led to the formation of a multi-layered 1:1 PdGa near-surface state without desorption of excess Ga to the gas phase. In general, the composition of the PdGa model system is much more variable than that of its PdZn counterpart, which results in gradual changes of the near-surface composition with increasing annealing or reaction temperature.

In contrast to near-surface PdZn, in methanol steam reforming, no temperature region with pronounced CO2 selectivity was observed, which is due to the inability of purely intermetallic PdGa to efficiently activate water. This allows to pinpoint the water-activating role of the intermetallic/support interface and/or of the oxide support in the related supported PdxGa/Ga2O3 systems, which exhibit high CO2 selectivity in a broad temperature range. In contrast, corresponding experiments starting on the purely bimetallic model surface in oxidative methanol reforming yielded high CO2 selectivity already at low temperatures (∼460 K), which is due to efficient O2 activation on PdGa. In situ detected partial and reversible oxidative Ga segregation on intermetallic PdGa is associated with total oxidation of intermediate C1 oxygenates to CO2.

Abstract

Recombinant adeno-associated virus (rAAV) vectors offer promise for gene therapy of alpha-1 antitrypsin (AAT) deficiency. A toxicology study in mice evaluated intramuscular injection of an rAAV vector expressing human AAT (rAAV-CB-hAAT) produced using a herpes simplex virus (HSV) complementation system or a plasmid transfection (TFX) method at doses of 3 × 1011 vg (1.2 × 1013 vg/kg) for both vectors and 2 × 1012 vg (8 × 1013 vg/kg) for the HSV-produced vector. The HSV-produced vector had favorable in vitro characteristics in terms of purity, efficiency of transduction, and hAAT expression. There were no significant differences in clinical findings or hematology and clinical chemistry values between test article and control groups and no gross pathology findings. Histopathological examination demonstrated minimal to mild changes in skeletal muscle at the injection site, consisting of focal chronic interstitial inflammation and muscle degeneration, regeneration, and vacuolization, in vector-injected animals. At the 3 × 1011 vg dose, serum hAAT levels were higher with the HSV-produced vector than with the TFX-produced vector. With the higher dose of HSV-produced vector, the increase in serum hAAT levels was dose-proportional in females and greater than dose-proportional in males. Vector copy numbers in blood were highest 24 hr after dosing and declined thereafter, with no detectable copies present 90 days after dosing. Antibodies to hAAT were detected in almost all vector-treated animals, and antibodies to HSV were detected in most animals that received the highest vector dose. These results support continued development of rAAV-CB-hAAT for treatment of AAT deficiency.

Chulay and colleagues generate an AAV1 vector encoding human alpha-1 antitrypsin (AAV.hAAT) using a recombinant herpes simplex virus complementation system. They evaluate the efficacy of this vector, compared to an AAV.hAAT vector generated using the triple transfection method, in mice.

Gibberellin (GA) signalling during pumpkin male flower development is highly regulated, including biosynthetic, perception, and transduction pathways. GA 20-oxidases, 3-oxidases, and 2-oxidases catalyse the final part of GA synthesis. Additionally, 7-oxidase initiates this part of the pathway in some cucurbits including Cucurbita maxima L. (pumpkin). Expression patterns for these GA-oxidase-encoding genes were examined by competitive reverse transcription-PCR (RT-PCR) and endogenous GA levels were determined during pumpkin male flower development. In young flowers, GA20ox3 transcript levels are high in stamens, followed by high levels of the GA precursor GA9. Later, just before flower opening, transcript levels for GA3ox3 and GA3ox4 increase in the hypanthium and stamens, respectively. In the stamen, following GA3ox4 expression, bioactive GA4 levels rise dramatically. Accordingly, catabolic GA2ox2 and GA2ox3 transcript levels are low in developing flowers, and increase in mature flowers. Putative GA receptor GID1b and DELLA repressor GAIPb transcript levels do not change in developing flowers, but increase sharply in mature flowers. Emasculation arrests floral development completely and leads to abscission of premature flowers. Application of GA4 (but not of its precursors GA12-aldehyde or GA9) restores normal growth of emasculated flowers. These results indicate that de novo GA4 synthesis in the stamen is under control of GA20ox3 and GA3ox4 genes just before the rapid flower growth phase. Stamen-derived bioactive GA is essential and sufficient for male flower development, including the petal and the pedicel growth.

Studying the physiological properties and synaptic connections of specific neurons in the intact tissue is a challenge for those cells that lack conspicuous morphological features or show a low population density. This applies particularly to retinal amacrine cells, an exceptionally multiform class of interneurons that comprise roughly 30 subtypes in mammals1. Though being a crucial part of the visual processing by shaping the retinal output2, most of these subtypes have not been studied up to now in a functional context because encountering these cells with a recording electrode is a rare event.

Recently, a multitude of transgenic mouse lines is available that express fluorescent markers like green fluorescent protein (GFP) under the control of promoters for membrane receptors or enzymes that are specific to only a subset of neurons in a given tissue3,4. These pre-labeled cells are therefore accessible to directed microelectrode targeting under microscopic control, permitting the systematic study of their physiological properties in situ. However, excitation of fluorescent markers is accompanied by the risk of phototoxicity for the living tissue. In the retina, this approach is additionally hampered by the problem that excitation light causes appropriate stimulation of the photoreceptors, thus inflicting photopigment bleaching and transferring the retinal circuits into a light-adapted condition. These drawbacks are overcome by using infrared excitation delivered by a mode-locked laser in short pulses of the femtosecond range. Two-photon excitation provides energy sufficient for fluorophore excitation and at the same time restricts the excitation to a small tissue volume minimizing the hazards of photodamage5. Also, it leaves the retina responsive to visual stimuli since infrared light (>850 nm) is only poorly absorbed by photopigments6.

In this article we demonstrate the use of a transgenic mouse retina to attain electrophysiological in situ recordings from GFP-expressing cells that are visually targeted by two-photon excitation. The retina is prepared and maintained in darkness and can be subjected to optical stimuli which are projected through the condenser of the microscope (Figure 1). Patch-clamp recording of light responses can be combined with dye filling to reveal the morphology and to check for gap junction-mediated dye coupling to neighboring cells, so that the target cell can by studied on different experimental levels.

The second, internet-based multicenter study (MCSII) of the Spine Study Group of the German Association of Trauma Surgery (Deutsche Gesellschaft für Unfallchirurgie) is a representative patient collection of acute traumatic thoracolumbar (T1–L5) injuries. The MCSII results are an update of those obtained with the first multicenter study (MCSI) more than a decade ago. The aim of the study was to assess and bring into focus: the (1) epidemiologic data, (2) surgical and radiological outcome, and (3) 2-year follow-up (FU) results of these injuries. According to the Magerl/AO classification, there were 424 (57.8%) compression fractures (A type), 178 (24.3%) distractions injuries (B type), and 131 (17.9%) rotational injuries (C type). B and C type injuries carried a higher risk for neurological deficits, concomitant injuries, and multiple vertebral fractures. The level of injury was located at the thoracolumbar junction (T11–L2) in 67.0% of the case. 380 (51.8%) patients were operated on by posterior stabilization and instrumentation alone (POSTERIOR), 34 (4.6%) had an anterior procedure (ANTERIOR), and 319 (43.5%) patients were treated with combined posteroanterior surgery (COMBINED). 65% of patients with thoracic (T1–T10) and 57% with lumbar spinal (L3–L5) injuries were treated with a single posterior approach (POSTERIOR). 47% of the patients with thoracolumbar junction (T11–L2) injuries were either operated from posterior or with a combined posterior–anterior surgery (COMBINED) each. Short angular stable implant systems have replaced conventional non-angular stable instrumentation systems to a large extent. The posttraumatic deformity was restored best with COMBINED surgery. T-spine injuries were accompanied by a higher number and more severe neurologic deficits than TL junction or L-spine injuries. At the same time T-spine injuries showed less potential for neurologic recovery especially in paraplegic (Frankel/AISA A) patients. 5% of all patients required revision surgery for perioperative complications. Follow-up data of 558 (76.1%) patients were available and collected during a 30-month period from 1 January 2004 until 31 May 2006. On average, a posterior implant removal was carried out in a total of 382 COMBINED and POSTERIOR patients 12 months after the initial surgery. On average, the rehabilitation process required 3–4 weeks of inpatient treatment, followed by another 4 months of outpatient therapy and was significantly shorter when compared with MCSI in the mid-1990s. From the time of injury until FU, 80 (60.6%) of 132 patients with initial neurological deficits improved at least one grade on the Frankel/ASIA Scale; 8 (1.3%) patients deteriorated. A higher recovery rate was observed for incomplete neurological injuries (73%) than complete neurological injuries (44%). Different surgical approaches did not have a significant influence on the neurologic recovery until FU. Nevertheless, neurological deficits are the most important factors for the functional outcome and prognosis of TL spinal injuries. POSTERIOR patients had a better functional and subjective outcome at FU than COMBINED patients. However, the posttraumatic radiological deformity was best corrected in COMBINED patients and showed significantly less residual kyphotic deformity (biseg GDW −3.8° COMBINED vs. −6.1° POSTERIOR) at FU (p = 0.005). The sagittal spinal alignment was better maintained when using vertebral body replacement implants (cages) in comparison to iliac strut grafts. Additional anterior plate systems did not have a significant influence on the radiological FU results. In conclusion, comprehensive data of a large patient population with acute thoracolumbar spinal injuries has been obtained and analyzed with this prospective internet-based multicenter study. Thus, updated results and the clinical outcome of the current operative treatment strategies in participating German and Austrian trauma centers have been presented. Nevertheless, it was not possible to answer all remaining questions to contradictory findings of the subjective, clinical outcome and corresponding radiological findings between different surgical subgroups. Randomized-controlled long-term investigations seem mandatory and the next step in future clinical research of Spine Study Group of the German Trauma Society.

Gene tagging facilitates systematic genomic and proteomic analyses but chromosomal tagging typically disrupts gene regulatory sequences. Here we describe a seamless gene tagging approach that preserves endogenous gene regulation and is potentially applicable in any species with efficient DNA double-strand break repair by homologous recombination. We implement seamless tagging in Saccharomyces cerevisiae and demonstrate its application for protein tagging while preserving simultaneously upstream and downstream gene regulatory elements. Seamless tagging is compatible with high-throughput strain construction using synthetic genetic arrays (SGA), enables functional analysis of transcription antisense to open reading frames and should facilitate systematic and minimally-invasive analysis of gene functions.

OBJECTIVE

Glucagon-like peptide 1 (GLP-1) exerts beneficial antidiabetic actions via effects on pancreatic β- and α-cells. Previous studies have focused on the improvements in β-cell function, while the inhibition of α-cell secretion has received less attention. The aim of this research was to quantify the glucagonostatic contribution to the glucose-lowering effect of GLP-1 infusions in patients with type 2 diabetes.

RESEARCH DESIGN AND METHODS

Ten male patients with well-regulated type 2 diabetes (A1C 6.9 ± 0.8%, age 56 ± 10 years, BMI 31 ± 3 kg/m2 [means ± SD]) were subjected to five 120-min glucose clamps at fasting plasma glucose (FPG) levels. On day 1, GLP-1 was infused to stimulate endogenous insulin release and suppress endogenous glucagon. On days 2–5, pancreatic endocrine clamps were performed using somatostatin infusions of somatostatin and/or selective replacement of insulin and glucagon; day 2, GLP-1 plus basal insulin and glucagon (no glucagon suppression or insulin stimulation); day 3, basal insulin only (glucagon deficiency); day 4, basal glucagon and stimulated insulin; and day 5, stimulated insulin. The basal plasma glucagon levels were chosen to simulate portal glucagon levels.

RESULTS

Peptide infusions produced the desired hormone levels. The amount of glucose required to clamp FPG was 24.5 ± 4.1 (day 1), 0.3 ± 0.2 (day 2), 10.6 ± 1.1 (day 3), 11.5 ± 2.7 (day 4), and 24.5 ± 2.6 g (day 5) (day 2 was lower than days 3 and 4, which were both similar and lower than days 1 and 5).

CONCLUSIONS

We concluded that insulin stimulation (day 4) and glucagon inhibition (day 3) contribute equally to the effect of GLP-1 on glucose turnover in patients with type 2 diabetes, and these changes explain the glucose-lowering effect of GLP-1 (day 5 vs. day 1).

Abstract

The ability of recombinant adeno-associated viral (rAAV) vectors to exhibit minimal immunogenicity and little to no toxicity or inflammation while eliciting robust, multiyear gene expression in vivo are only a few of the salient features that make them ideally suited for many gene therapy applications. A major hurdle for the use of rAAV in sizeable research and clinical applications is the lack of efficient and versatile large-scale production systems. Continued progression toward flexible, scalable production techniques is a prerequisite to support human clinical evaluation of these novel biotherapeutics. This review examines the current state of large-scale production methods that employ the herpes simplex virus type 1 (HSV) platform to produce rAAV vectors for gene delivery. Improvements have substantially advanced the HSV/AAV hybrid method for large-scale rAAV manufacture, facilitating the generation of highly potent, clinical-grade purity rAAV vector stocks. At least one human clinical trial employing rAAV generated via rHSV helper-assisted replication is poised to commence, highlighting the advances and relevance of this production method.

The authors report on a prospectively followed series of 35 patients with injuries of the thoracolumbar spine from T7 to L3. The radiological course after combined posterior–anterior surgery with anterior column reconstruction with a distractible vertebral body replacing implant demonstrated a stable reconstruction technique with almost no re-kyphosing. In 18/18 patients with CT follow-up intervertebral fusion was observed as bony bridging lateral to the VBR implant. The functional/clinical outcome of the patients was analysed with a set of eight validated outcome scales. After an average follow-up period of 2½ years encouraging results were noticed. The neurological improvement rate (≥1 Frankel/ASIA grade) was 8/12 patients (67%) with a complete recovery in 6 cases. 17/29 patients returned to former occupation; 20/29 patients returned to former leisure activities; 24/28 patients rated their general outcome as “unlimited and pain free” or “occasionally and/or mild complaints” with a VAS score of >80 (scale 0–100). The psychometric questionnaires revealed good results with strong correlation comparing the different scoring systems statistically: mean McGill Pain Questionnaire 12.5 (0–40); mean Oswestry Disability Index 20% (0–51). 13/29 patients scored <4 in the Roland and Morris Disability Questionnaire. The German back pain questionnaire (Funktionsfragebogen Hannover Rücken) showed a mean “functional capacity” of 75%, corresponding with moderate restriction. We concluded the presented method as highly effective to completely reduce and maintain an anatomic spinal alignment. The outcome tended to be better in comparison with non-operatively treated patients as well as with norm populations with low back pain.

Type 2 diabetes mellitus is a metabolic disease associated with low quality of life and early death. The goal in diabetes treatment is to prevent these outcomes by tight glycemic control and minimizing vascular risk factors. So far, even intensified combination regimen with the traditional antidiabetes agents have failed to obtain these goals. Incretin mimetics are a new class of antidiabetes drugs which involve modulation of the incretin system. They bind to and activate glucagon-like peptide-1 (GLP-1) receptors on pancreatic beta-cells following which insulin secretion and synthesis are initiated. Since the compounds have no insulinotropic activity at lower glucose concentrations the risk of hypoglycemia – a well-known shortcoming of existing antidiabetes treatments – is low. Additionally, incretin mimetics have been shown to be associated with beneficial effects on cardiovascular risk factors such as weight loss, decrease in blood pressure and changes in lipid profile. Current clinical data on the two available incretin mimetics, exenatide and liraglutide, are evaluated in this review, focusing on pharmacology, efficacy, safety and tolerability. The review is built on a systematic PubMed and Medline search for publications with the key words GLP-1 receptor agonist, exenatide, liraglutide and type 2 diabetes mellitus up to January 2009.

Worldwide agriculture is one of the main drivers of biodiversity decline. Effective conservation strategies depend on the type of relationship between biodiversity and land-use intensity, but to date the shape of this relationship is unknown. We linked plant species richness with nitrogen (N) input as an indicator of land-use intensity on 130 grasslands and 141 arable fields in six European countries. Using Poisson regression, we found that plant species richness was significantly negatively related to N input on both field types after the effects of confounding environmental factors had been accounted for. Subsequent analyses showed that exponentially declining relationships provided a better fit than linear or unimodal relationships and that this was largely the result of the response of rare species (relative cover less than 1%). Our results indicate that conservation benefits are disproportionally more costly on high-intensity than on low-intensity farmland. For example, reducing N inputs from 75 to 0 and 400 to 60 kg ha−1 yr−1 resulted in about the same estimated species gain for arable plants. Conservation initiatives are most (cost-)effective if they are preferentially implemented in extensively farmed areas that still support high levels of biodiversity.

SUMMARY

Gene transfer has potential as a once-only treatment that reduces viral load, preserves the immune system, and avoids lifetime highly active antiretroviral therapy. This study, the first randomized, double-blind, placebo-controlled, phase II cell-delivered gene transfer clinical trial, was conducted in 74 HIV-1 infected adults who received a tat/vpr specific anti-HIV ribozyme (OZ1) or placebo delivered in autologous CD34+ hematopoietic progenitor cells. There were no OZ1-related adverse events. There was no statistical difference in viral load between the OZ1 and placebo group at the primary end-point (average at weeks 47 and 48) but time weighted areas under the curve from weeks 40-48 and 40-100 were significantly lower in the OZ1 group. Throughout the 100 weeks, CD4+ lymphocyte counts were higher in the OZ1 group. This study provides the first indication that cell-delivered gene transfer is safe and biologically active in HIV patients and can be developed as a conventional therapeutic product.

The Carney triad is a rare syndrome of unknown aetiology, with synchronous or metachronous appearance of rare neoplasms: gastrointestinal stromal tumours (GISTs), pulmonary chondromas and extra‐adrenal paragangliomas. In most cases, the Carney triad is incomplete. The combination encountered typically, GISTs and pulmonary chondromas, was also seen in our patient, a 22‐year‐old woman. She was diagnosed with the triad after Billroth II gastrectomy for histologically proved gastric GISTs. The diagnosis of pulmonary chondromas was confirmed by transthoracic, computed tomography‐guided needle biopsy. An oesophageal leiomyoma was resected 2 years after the initial diagnosis, on suspicion of paraganglioma. The clinical course of the patient has been uneventful since. The last follow‐up was carried out 6 years after the initial diagnosis. On histological examination, the cells of gastric GIST were partly positive for CD34, whereas CD117 was expressed in all areas in variable intensity and S‐100 protein was negative. The oesophageal tumour was classified as leiomyoma due to strong immunopositivity for smooth muscle actin and desmin, being negative for CD34 and CD117. Two different gastric GIST lesions as well as the oesophageal leiomyoma and normal tissue were analysed for activating mutations in common hot spots of KIT (exon 9 and 11) and platelet‐derived growth factor receptor α (exon 18), but in all probes wild‐type sequences were found. These results are in accordance with the first published analyses of GIST lesions from Carney patients.

A clinical trial was initiated to evaluate the recommended dose of cyclophosphamide in combination with bortezomib and dexamethasone as induction treatment before stem cell transplantation for younger patients with newly diagnosed multiple myeloma (MM). Thirty patients were treated with three 21-day cycles of bortezomib 1.3 mg/m2 on days 1, 4, 8, and 11 plus dexamethasone 40 mg on the day of bortezomib injection and the day after plus cyclophosphamide at 900, 1,200, or 1,500 mg/m2 on day 1. The maximum tolerated dose of cyclophosphamide was defined as 900 mg/m2. At this dose level, 92% of patients achieved at least a partial response. The overall response rate [complete response (CR) plus partial response (PR)] across all dose levels was 77%, with a 10% CR rate. No patient experienced progressive disease. The most frequent adverse events were hematological and gastrointestinal toxicities as well as neuropathy. The results suggest that bortezomib in combination with cyclophosphamide at 900 mg/m2 and dexamethasone is an effective induction treatment for patients with newly diagnosed MM that warrants further investigation.