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The authors discuss the cataloging of electronic journals in the Zeitschriftendatenbank (ZDB), the German serials database. Some of the matters yet to be resolved regarding their cataloging, such as the placement of URLs, the ascertaining of meaningful dates of publication, and the use of successive vs. latest entry cataloging, are examined. The HTML catalog of electronic journals, which is derived from the ZDB, is described. [ABSTRACT FROM AUTHOR]

Background: The use of patient-specific instruments (PSIs) for osteotomies is becoming more popular in orthopaedic surgery for correcting mechanical axis and posttraumatic deformities. However, the PSI reduction guides have great potential for intraoperative deformation, which adversely affects the accuracy of the procedure., Purpose: To conduct a finite element analysis (FEA) to analyze different design parameters to improve the intraoperative stability of the reduction guides., Study Design: Descriptive laboratory study., Methods: A reduction guide with a rectangular cross section and four 4-mm K-wire slots was simplified, and the following parameters were modified: width, height, profile design, K-wire thickness, and positions. Bending and torsional moments were applied to the guide construct and guide deformation and equivalent stress were determined using FEA., Results: Increasing the profile height by 25% resulted in a 44% reduction in guide deformation for bending (37% for torsion). A 25% increase in profile width led to an 18% deformation reduction for bending (22% for torsion). Transverse K-wire slots resulted in 51% less deformation in torsion compared with longitudinally oriented slots. Placing the central K-wire slots 25% closer to the osteotomy reduced guide deformation by 20% for bending and 11% for torsion., Conclusion: The most effective methods to increase reduction guide stability are to increase the guide height and reduce the central K-wire distance to the osteotomy., Clinical Relevance: When performing opening or closing wedge osteotomies, which mainly involve bending of the guide, a high-profile guide and longitudinally oriented K-wire slots should be used. When torque is expected as in rotational osteotomies, the K-wire holes in guides should be oriented transversely to reduce intraoperative deformation., Competing Interests: One or more of the authors has declared the following potential conflict of interest or source of funding: S.F.F. has received consulting fees from Medacta International SA, Zimmer Biomet, and Karl Storz and is a board member for ESSKA-EKA Osteotomy Committee. AOSSM checks author disclosures against the Open Payments Database (OPD). AOSSM has not conducted an independent investigation on the OPD and disclaims any liability or responsibility relating thereto., (© The Author(s) 2024.)

Background: Failure rates in the management of recurrent posterior shoulder instability remain a concern. Cadaveric studies have established that posterior capsulolabral tears, glenoid retroversion, and posterior glenoid bone loss result in increased posterior humeral head translation in the setting of a posteriorly directed force. A high and flat acromion has recently been associated with posterior instability. Therefore, the purpose of this study was to evaluate a potential stabilizing effect of the acromion against posterior humeral head displacement., Methods: Eight fresh-frozen human cadaveric shoulders were biomechanically tested in a shoulder simulator in the load-and-shift and Jerk test positions. Prior to testing, CT scans were performed to measure native glenoid width, glenoid retroversion, posterior acromial coverage (PAC), sagittal acromial tilt (SAT) and posterior acromial height (PAH). Each specimen underwent four testing conditions using pre-planned and 3D printed cutting and reduction guides: (1) Intact joint, native acromion; (2) Intact joint, severe acromial malalignment (SAT 69°, PAC 47°, PAH 26mm); (3) Intact joint, moderate acromial malalignment (SAT 59°, PAC 57°, PAH 20mm); (4) Intact joint, corrected acromial alignment (SAT 48°, PAC 70°, PAH 11mm). The degree of acromial malalignment and acromial reorientation was chosen based on a previous study that defined acromial anatomy in patients with posterior instability. The humeral head was translated posteriorly until reaching either (1) a peak force of 150N or (2) a maximum posterior displacement of 50% of the glenoid width. Forces (N), displacement (mm) and acromiohumeral contact pressures (kPA) were simultaneously recorded., Results: The force needed to displace the humeral head by 50% of the glenoid width decreased between 23% and 60% in moderate to severe acromial malalignment (high and flat acromion) and increased up to 122% following surgical correction of acromial alignment (low and steep acromion) when compared to the native condition. Correction of acromial alignment significantly increased stability compared to all other scenarios after ≥5% of displacement (p<0.05 for all comparisons). Furthermore, it increased acromiohumeral contact pressures compared with severe malalignment in 30° flexion and with moderate and severe acromial malalignment in 60° flexion (p<0.05 for all comparisons)., Conclusion: The acromion acts as a mechanical buttress to posterior humeral head displacement. Surgical correction of acromial malalignment cannot only effectively restore but increase glenohumeral joint stability. Future studies are needed to define the quantitative relevance of the different factors contributing to posterior shoulder instability and assist in defining the optimal amount of correction needed in an individual situation., (Copyright © 2024. Published by Elsevier Inc.)

Infectious laryngotracheitis (ILT) is a respiratory disease affecting chickens worldwide. Unlike many countries, Switzerland does not vaccinate against ILT. This study analysed ILT samples from 21 natural outbreaks in Switzerland using restriction fragment length polymorphism (RFLP) and multiple gene sequencing. Chicken embryo origin (CEO) and tissue culture origin (TCO) vaccine strains were included as references. Both vaccine strains were distinguishable, and 14 out of 21 samples resembled the CEO vaccine. Additionally, four distinct non-vaccine-like groups were identified. Sequencing of three genes from selected Swiss samples and those from neighbouring countries revealed four phylogenetic clades. Notably, four Swiss field strains formed two unique clades, not closely related to vaccine strains or ILTV from neighbouring countries. Overall, RFLP results were supported by sequencing data. This study demonstrates the presence of both vaccine-like and wild-type ILT viruses in Switzerland, where vaccination is de facto prohibited., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The current clinical gold standard to assess the condition and detect loosening of pedicle screw implants is radiation-emitting medical imaging. However, solely based on medical imaging, clinicians are not able to reliably identify loose implants in a substantial amount of cases. To complement medical imaging for pedicle screw loosening detection, we propose a new methodology and paradigm for the radiation-free, non-destructive, and easy-to-integrate loosening detection based on vibroacoustic sensing. For the detection of a loose implant, we excite the vertebra of interest with a sine sweep vibration at the spinous process and use a custom highly sensitive piezo vibration sensor attached directly at the screw head to capture the propagated vibration characteristics which are analyzed using a detection pipeline based on spectrogram features and a SE-ResNet-18. To validate the proposed approach, we propose a novel, biomechanically validated simulation technique for pedicle screw loosening, conduct experiments using four human cadaveric lumbar spine specimens, and evaluate our algorithm in a cross-validation experiment. The proposed method reaches a sensitivity of 91.50 ± 6.58 % and a specificity of 91.10 ± 2.27 % for pedicle screw loosening detection., Competing Interests: Declarations Ethics approval An ethical approval for all ex-vivo experiments (Kantonale Ethikkommission Zurich, protocol number: 2020-01913) as well as informed consent from all subjects involved in this study and/or their legal guardians has been obtained. All experiments presented within this paper were carried out in accordance with relevant guidelines and regulations. Conflict of interest The authors declare no competing interests., (© 2024. The Author(s).)

3D preoperative planning for high tibial osteotomies (HTO) has increasingly replaced 2D planning but is complex, time-consuming and therefore expensive. Several interdependent clinical objectives and constraints have to be considered, which often requires multiple rounds of revisions between surgeons and biomedical engineers. We therefore developed an automated preoperative planning pipeline, which takes imaging data as an input to generate a ready-to-use, patient-specific planning solution. Deep-learning based segmentation and landmark localization was used to enable the fully automated 3D lower limb deformity assessment. A 2D-3D registration algorithm allowed the transformation of the 3D bone models into the weight-bearing state. Finally, an optimization framework was implemented to generate ready-to use preoperative plannings in a fully automated fashion, using a genetic algorithm to solve the multi-objective optimization (MOO) problem based on several clinical requirements and constraints. The entire pipeline was evaluated on a large clinical dataset of 53 patient cases who previously underwent a medial opening-wedge HTO. The pipeline was used to automatically generate preoperative solutions for these patients. Five experts blindly compared the automatically generated solutions to the previously generated manual plannings. The overall mean rating for the algorithm-generated solutions was better than for the manual solutions. In 90% of all comparisons, they were considered to be equally good or better than the manual solution. The combined use of deep learning approaches, registration methods and MOO can reliably produce ready-to-use preoperative solutions that significantly reduce human workload and related health costs.

Purpose: Primary glenohumeral osteoarthritis is commonly associated with static posterior subluxation of the humeral head. Scapulae with static/dynamic posterior instability feature a superiorly and horizontally oriented acromion. We investigated whether the acromion acts as a restraint to posterior humeral translation., Methods: Five three-dimensional (3D) printed scapula models were biomechanically tested. A statistical shape mean model (SSMM) of the normal scapula of 40 asymptomatic shoulders was fabricated. Next, a SSMM of scapular anatomy associated with posterior subluxation was generated using data of 20 scapulae ("B1"). This model was then used to generate three models of surgical correction: glenoid version, acromial orientation, and acromial and glenoid orientation. With the joint axially loaded (100N) and the humerus stabilized, an anterior translation force was applied to the scapula in 35°, 60° and 75° of glenohumeral flexion. Translation (mm) was measured., Results: In the normal scapula, the humerus translates significantly less to contact with the acromion compared to all other configurations (p < .000 for all comparisons; i.e. 35°: "normal" 8,1 mm (± 0,0) versus "B1" 11,9 mm (± 0,0) versus "B1 Acromion Correction" 12,2 mm (± 0,2) versus "B1 Glenoid Correction" 13,3 mm (± 0,1)). Restoration of normal translation was only achieved with correction of glenoid and acromial anatomy (i.e. 75°: "normal" 11 mm (± 0,8) versus "B1 Acromion Correction" 17,5 mm (± 0,1) versus "B1 Glenoid Correction" 19,7 mm (± 1,3) versus "B1 Glenoid + Acromion Correction" 11,5 mm (± 1,1))., Conclusions: Persistence or recurrence of static/dynamic posterior instability after correction of glenoid version alone may be related to incomplete restoration of the intrinsic stability that is conferred by a normal acromial anatomy., Level of Evidence V: biomechanical study., (© 2023. The Author(s).)

Case: A 40-year-old man presented with progressive shoulder pain, associated with static posterior subluxation and mild eccentric glenohumeral osteoarthritis. Compared with a mean statistical shape model of a normal shoulder, the patient's acromion was abnormally high and horizontal, and the glenoid abnormally inclined inferiorly and minimally retroverted. Restoration of normal scapular anatomy using 3-dimensional planned acromial and glenoid osteotomies led to recentering of the joint and full shoulder function up to 24 months postoperatively., Conclusion: The correction of associated acromial and glenoid malformation can revert early static posterior subluxation of the shoulder. Whether successful recentering prevents progression of osteoarthritis remains to be established., Competing Interests: Disclosure: The Disclosure of Potential Conflicts of Interest forms are provided with the online version of the article (http://links.lww.com/JBJSCC/C92)., (Copyright © 2023 The Authors. Published by The Journal of Bone and Joint Surgery, Incorporated. All rights reserved.)

Avian orthoavulavirus-1 (AOAV-1) is the causative agent of Newcastle disease in poultry. This highly infectious disease causes large economic losses annually and worldwide. AOAV-1 does not only infect poultry, but it has a very broad host range and has been detected in over 230 bird species to date. A distinct group of viral strains within AOAV-1 are pigeon-adapted strains, also named pigeon paramyxovirus-1 (PPMV-1). AOAV-1 is transmitted through the feces of infected birds and secretions from the nasal and oral cavities and eyes. It is worth mentioning that wild birds can transmit the virus to captive birds, especially feral pigeons to poultry. Therefore, early and sensitive detection of this virus-including the monitoring of pigeons-is of utmost importance. A variety of molecular methods for the detection of AOAV-1 already exist, but the detection of the F gene cleavage site of currently circulating PPMV-1 strains has not proven to be particularly sensitive or suitable. As presented here, by modifying the primers and probe of an already established real-time reverse-transcription PCR, the sensitivity could be increased, allowing for a more reliable detection of the AOAV-1 F gene cleavage site. Furthermore, it becomes clear how important it is to constantly monitor and, if necessary, adapt existing diagnostic procedures.

Pigeon paramyxovirus-1 (PPMV-1) is predominantly isolated from pigeons or doves and forms a separate group of viral strains within Avian Orthoavulavirus-1, the causative agent of Newcastle disease in poultry. Since the introduction of PPMV-1 into Europe in 1981, these strains have rapidly spread all over Europe, and are nowadays considered to be enzootic in feral and hobby pigeons ( Columba livia domestica ). Infections with PPMV-1 can range from asymptomatic to fatal. To assess whether PPMV-1 continuously circulates in healthy feral pigeons, 396 tissue samples of pigeons from the city of Zurich were tested by reverse transcriptase real-time PCR over the period of one year. PPMV-1-RNA was detected in 41 feral pigeons (10.35%), determined as the dominant European genotype VI.2.1.1.2.2. In 38 of the 41 pigeons where organ samples tested positive, PPMV-1-RNA was also detected in either choana or cloaca swabs. There were no significant differences in positivity rates between seasons, age, and sex. The current study shows that feral pigeons without clinical signs of disease can harbour and most likely excrete PPMV-1. Spill-over into free-range holdings of chickens are therefore possible, as observed in a recent outbreak of Newcastle disease in laying hens due to PPMV-1 genotype VI.2.1.1.2.2. in the canton of Zurich in January 2022.

The forage maturation hypothesis (FMH) assumes that herbivores cope with the trade-off between digestibility and biomass in forage by selecting vegetation at intermediate growth. The green wave hypothesis (GWH) extends the FMH to suggest how spatiotemporal heterogeneity in plant quality shapes migratory movements of herbivores. Growing empirical support for these hypotheses mainly comes from studies in vast landscapes with large-scale habitat heterogeneity. It is unclear, however, to what extent ungulates surf green waves in human-altered landscapes with small-scale heterogeneity in terms of land use and topography. We used plant phenological proxies derived from Sentinel 2 satellite data to analyze the habitat selection of 93 collared red deer ( Cervus elaphus ) in montane and alpine habitats. Using a step selection analysis, we investigated how plant phenology, that is, the instantaneous rate of green-up (IRG) and normalized difference vegetation index (NDVI), and a set of variables describing topography and human presence influenced red deer resource selection in open habitats. We learned that red deer selected areas with high biomass at green-up and avoided habitats with possible exposure to human activity. Additionally, landscape structure and topography strongly influenced spatial behavior of red deer. We further compared cumulative access to high-quality forage across migrant strategies and found migrants gained better access than residents. Many migratory individuals surfed the green wave, and their surfing behavior, however, became less pronounced with decreasing distance to settlements. Within the constraints of topography and human land use, red deer track spring green-up on a fine spatiotemporal scale and follow the green wave across landscapes in migration movements. Thus, they benefit from high-quality forage even in human-dominated landscapes with small-scale heterogeneity and vegetation emerging in a heterogenic, dynamic mosaic., Competing Interests: We declare no conflict of interest., (© 2022 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd.)

Tetratrichomonas gallinarum and Trichomonas gallinae are pathogenic avian parasites that infect a wide range of bird species. The pathologic potential of T. gallinarum is controversial, whereas T. gallinae causes disease in many avian species. Infections are often asymptomatic in doves and pigeons; thus, columbids are presumed to represent the natural hosts for trichomonads. The detection of T. gallinarum and T. gallinae is based on direct microscopic observation or a conventional PCR assay. Microscopy is not very sensitive, and identification of the trichomonads at the genus or species level is not possible. Conventional PCR assays have been developed primarily for phylogenetic studies, which detect a wide range of Trichomonas spp. but do not allow their differentiation. We developed a duplex real-time PCR (rtPCR) assay for the simultaneous detection and differentiation of T. gallinarum and T. gallinae . We found that the rtPCR assay detected 10 2 plasmid DNA copies of T. gallinarum and as few as 10 1 plasmid DNA copies of T. gallinae.

Background: Although the utility of patient-specific instruments (PSI) has been well established for complex osteotomies in orthopedic surgery, it is yet to be comparatively analyzed for complex spinal deformity correction, such as pedicle subtraction osteotomy (PSO)., Methods: Six thoracolumbar human cadavers were used to perform nine PSOs using the free-hand (FH) technique and nine with PSI (in total 18 PSOs). Osteotomy planes were planned on the basis of preoperative computed tomography (CT). A closing-wedge angle of 30° was targeted for each PSO. Postoperative CT scans were obtained to measure segmental lordosis correction and the deviation from the planned 30° correction as well as the osseous gap of posterior elements., Results: The time required to perform a PSO was 18:22 (range 10:22-26:38) min and 14:14 (range 10:13-22:16) min in the PSI and FH groups, respectively (p = 0.489). The PSI group had a significantly higher lordosis gain (29°, range 23-31° vs. 21°, range 13-34°; p = 0.015). The lordosis gain was significantly more accurate with PSI (deviation angle: 1°; range 0-7°) than with the FH technique (9°; range 4-17°; p = 0.003). PSI achieved a significantly smaller residual osseous gap of the posterior elements (5 mm; range 0-9 mm) than the FH group (11 mm; range 3-27 mm; p = 0.043).With PSI, an angular difference of 3° (range 1-12°), a translational offset of 1 (range 0-6) mm at the level of the lamina, and a vertebral body entry point deviation of 1 (range 0-4) mm was achieved in the osteotomies., Conclusions: PSI-guided PSO can be a more feasible and accurate approach in achieving a planned lordosis angle than the traditional FH technique in a cadaver model. This approach further reduced osseous gaps, potentially promoting higher fusion rates in vivo., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 The Authors. Published by Elsevier Ltd on behalf of North American Spine Society.)

Avian bornaviruses were first described in 2008 as the causative agents of proventricular dilatation disease (PDD) in parrots and their relatives (Psittaciformes). To date, 15 genetically highly diverse avian bornaviruses covering at least five viral species have been discovered in different bird orders. Currently, the primary diagnostic tool is the detection of viral RNA by conventional or real-time RT-PCR (rRT-PCR). One of the drawbacks of this is the usage of either specific assays, allowing the detection of one particular virus, or of assays with a broad detection spectrum, which, however, do not allow for the simultaneous specification of the detected virus. To facilitate the simultaneous detection and specification of avian bornaviruses, a multiplex real-time RT-PCR assay was developed. Whole-genome sequences of various bornaviruses were aligned. Primers were designed to recognize conserved regions within the overlapping X/P gene and probes were selected to detect virus species-specific regions within the target region. The optimization of the assay resulted in the sensitive and specific detection of bornaviruses of Psittaciformes, Passeriformes, and aquatic birds. Finally, the new rRT-PCR was successfully employed to detect avian bornaviruses in field samples from various avian species. This assay will serve as powerful tool in epidemiological studies and will improve avian bornavirus detection.

Background: Individual constitutional differences in glenoid shape and bone remodeling require a patient-specific and longitudinal approach to evaluate the biomechanical effects of glenoid bone grafting in patients with anterior shoulder instability., Purpose: To quantify the longitudinal, in vivo, biomechanical effects of bone grafting, bone graft remodeling, and glenoid shape in patients with anterior shoulder instability by means of patient-specific finite element models., Study Design: Descriptive laboratory study., Methods: In total, 25 shoulders of 24 patients with anterior shoulder instability and anterior glenoid bone loss underwent an arthroscopic iliac crest bone graft transfer (ICBGT) procedure with either autologous or allogenic bone. Patient-specific finite element simulations based on preoperative, postoperative, and follow-up computed tomography scans were used to quantify the bone-mediated stability ratio (SR) and the distance to dislocation. Additionally, the relationship between glenoid morphological parameters and the SR was assessed., Results: The ICBGT procedure significantly increased the SR and distance to dislocation in the 2-, 3-, and 4-o ' clock directions immediately after the surgical intervention ( P < .01) in both the autograft and the allograft groups. Although the SR and distance to dislocation decreased subsequently, autografts showed long-term effects on SR and dislocation distance in the 3-o'clock direction ( P < .01) and on SR in the 4-o'clock direction ( P < .01). Allografts showed no significant effect on SR and dislocation distance in long-term follow-up ( P > .05). Overall, glenoid retroversion as well as cavity depth predicted stability in all 4 dislocation directions, with glenoid cavity depth showing the highest correlation coefficients ( R = 0.71, 0.8, 0.73, and 0.7 for 2-, 3-, 4-, and 5-o'clock, respectively)., Conclusion: The autologous ICBGT procedure biomechanically improved anterior shoulder stability in long-term follow-up, whereas the use of allografts did not show any bone-mediated biomechanical effect at follow-up due to resorption. Furthermore, in addition to measurements of defect extent, the glenoid depth and version seem to be useful parameters to determine the biomechanical effect and need for glenoid bone grafting in patients with shoulder instability., Clinical Relevance: This study proposes the use of autologous bone grafts for a successful long-term stabilization effect. Additionally, this study proposes additional glenoid morphological measures to predict shoulder stability.

Gallid alphaherpesvirus 1 (syn. infectious laryngotracheitis virus; ILTV) is the causative agent of infectious laryngotracheitis, a respiratory disease of chickens causing substantial economic losses in the poultry industry every year. Currently, the most efficient way to achieve protection against infection is immunization with live-attenuated vaccines. However, this vaccination strategy entails the risk of generating new pathogenic viruses resulting from spontaneous mutations or from recombination with field strains. This work presents a new approach based on virus-like particles (VLPs) displaying ILTV glycoproteins B (gB) or G (gG) on their surface. The main focus of this pilot study was to determine the tolerability of VLPs delivered in ovo and intramuscularly (i.m.) into chickens and to investigate the nature of the immune response elicited. The study revealed that the new vaccines were well tolerated in hybrid layer chicks independent of the administration method (in ovo or i.m.). Upon in ovo injection, vaccination with VLP-gG led to an antibody response, while a cellular immune response in VLP-gB-immunized chickens was hardly detectable. Since the administration of VLPs had no visible side effects in vivo and was shown to elicit an antibody-based immune response, we anticipate that VLPs will become a valuable platform for the development of new safe vaccines for poultry.

Between 2008 and 2012, commercial Swiss layer and layer breeder flocks experiencing problems in laying performance were sampled and tested for infection with Duck adenovirus A (DAdV-A; previously known as Egg drop syndrome 1976 virus). Organ samples from birds sent for necropsy as well as blood samples from living animals originating from the same flocks were analyzed. To detect virus-specific DNA, a newly developed quantitative real-time polymerase chain reaction method was applied, and the presence of antibodies against DAdV-A was tested using a commercially available enzyme-linked immunosorbent assay. In 5 out of 7 investigated flocks, viral DNA was detected in tissues. In addition, antibodies against DAdV-A were detected in all of the flocks.

Infectious bronchitis, a disease of chickens caused by Avian coronavirus infectious bronchitis virus (IBV), leads to severe economic losses for the poultry industry worldwide. Various attempts to control the virus based on vaccination strategies are performed. However, due to the emergence of novel genotypes, an effective control of the virus is hindered. In 1996, a novel viral genotype named IBV-QX was reported for the first time in Qingdao, Shandong province, China. The first appearance of an IBV-QX isolate in Europe was reported between 2003 and 2004 in The Netherlands. Subsequently, infections with this genotype were found in several other European countries such as France, Italy, Germany, United Kingdom, Slovenia, and Sweden. The present report describes the use of a new set of degenerate primers that amplify a 636-bp fragment within the S1 gene by reverse transcription polymerase chain reaction to detect the occurrence of IBV-QX infection in Switzerland.

To aid in the rapid diagnosis of myxomatosis in rabbits, a real-time polymerase chain reaction (PCR) for the specific detection of Myxoma virus is described. Primers and probe were designed to amplify a 147-bp fragment within the Serp2 gene. The assay was able to detect 23 copies of a synthesized oligo indicating a reliable sensitivity. In addition, the real-time PCR did not detect the Rabbit fibroma virus used in myxomatosis vaccines. The novel PCR was shown to be able to detect Myxoma virus in fresh and paraffin-embedded rabbit tissues originating from myxomatosis cases from various regions in Switzerland.

Glucocorticoid (GC) action depends on GC plasma concentration, cellular GC receptor expression, and the pre-receptor hormone metabolism catalyzed by 11β-hydroxysteroid dehydrogenase (11β-HSD). 11β-Hydroxysteroid dehydrogenase exists in 2 isoforms; 11β-HSD1 converts inactive cortisone to cortisol, and 11β-HSD2 converts cortisol to cortisone. Increasing evidence in humans and experimental animals suggests that altered tissue cortisol metabolism may predispose to diabetes mellitus (DM). Once DM is established, hyperglycemia and hyperlipidemia may further maintain the abnormal metabolism of cortisol. To gain further insight in this regard, healthy cats were infused for 10 d with lipids (n = 6) or saline (n = 5). At the end of the infusion period, tissue samples from adipose tissue (visceral, subcutaneous), liver, and muscle were collected to determine mRNA expression of 11β-HSD1, 11β-HSD2, and GC receptor by real-time reverse-transcriptase polymerase chain reaction; blood samples were collected to determine plasma cortisol and leptin concentrations. Lipid infusion resulted in greater 11β-HSD1 expression and lower GC receptor expression in visceral and subcutaneous adipose tissue, and lower 11β-HSD2 expression in visceral adipose tissue and liver. Plasma cortisol did not differ. Leptin and body weight increased in lipid-infused cats. In spite of comparable circulating cortisol levels, up-regulation of 11β-HSD1 and down-regulation of 11β-HSD2 expression may result in increased tissue cortisol concentrations in fat depots of hyperlipidemic cats. Down-regulation of GC receptor may represent a self-protective mechanism against increased tissue cortisol levels. In conclusion, hyperlipidemia has a profound effect on 11β-HSD expression and supports the connection between high lipid concentrations and tissue cortisol metabolism., (Copyright © 2010 Elsevier Inc. All rights reserved.)

The cat has recently been proposed as a valuable model for type 2 diabetes mellitus (T2DM), because feline diabetes shares several similarities with the disease in humans. Impaired beta-cell function, decreased beta-cell mass, insulin resistance that is often related to obesity, and pancreatic amyloid deposition, are among these common features. In this study, and to further develop the cat as a model of T2DM, feline pancreatic islets were isolated and real-time PCR quantification of mRNA transcripts of genes central to beta-cell function and survival established. In particular, mRNA quantification systems were determined for insulin, the insulin enhancer pancreatic duodenal homeobox-1 (PDX-1), the insulin suppressor CCAAT/enhancer binding protein-beta (C/EBPbeta), glucose transporter isoform 2 (GLUT2), Fas receptor, the caspase-8 inhibitor FLIP (FLICE [caspase-8]-inhibitory protein) and two chemokines, interleukin (IL)-8 and monocyte chemoattractant protein-1 (MCP-1). Pancreatic islets were isolated by collagenase digestion from healthy cat donors. Partial feline mRNA sequences were determined for PDX-1, C/EBPbeta, GLUT2 and FLIP using primers identified from conserved regions of human, dog and rat mRNA. These novel and the previously available sequences (insulin, Fas receptor, IL-8 and MCP-1) were used to design feline-specific primers suitable for real-time PCR in isolated pancreatic islets. The adopted protocol of collagenase digestion yielded pancreatic islets that were frequently surrounded by acinar cells. Quantification of mRNA transcripts was simple and reproducible in healthy cats. Characterisation of genes related to insulin signalling in cats will prove useful to better understand the pathogenesis of feline diabetes and possibly of human T2DM., (2008 Elsevier Ltd. All rights reserved.)

Malignant pleural mesotheliomas (MPMs) are usually wild type for the p53 gene but contain homozygous deletions in the INK4A locus that encodes p14(ARF), an inhibitor of p53-MDM2 interaction. Previous findings suggest that lack of p14(ARF) expression and the presence of SV40 large T antigen (L-Tag) result in p53 inactivation in MPM. We did not detect SV40 L-Tag mRNA in either MPM cell lines or primary cultures, and treatment of p14(ARF)-deficient cells with cisplatin (CDDP) increased both total and phosphorylated p53 and enhanced p53 DNA-binding activity. On incubation with CDDP, levels of positively regulated p53 transcriptional targets p21(WAF), PIG3, MDM2, Bax, and PUMA increased in p14(ARF)-deficient cells, whereas negatively regulated survivin decreased. Significantly, p53-induced apoptosis was activated by CDDP in p14(ARF)-deficient cells, and treatment with p53-specific siRNA rendered them more CDDP-resistant. p53 was also activated by: 1) inhibition of MDM2 (using nutlin-3); 2) transient overexpression of p14(ARF); and 3) targeting of survivin using antisense oligonucleotides. However, it is noteworthy that only survivin downregulation sensitized cells to CDDP-induced apoptosis. These results suggest that p53 is functional in the absence of p14(ARF) in MPM and that targeting of the downstream apoptosis inhibitor survivin can sensitize to CDDP-induced apoptosis.

The p73 gene codes for various different protein isoforms. They include proteins expressed under the control of the P1 promoter that contain a transactivation domain and are similar in function to p53 (TAp73 isoforms), as well as proteins regulated by the P2 promoter that lack this domain and function as dominant negative inhibitors of TAp73 and p53 (DeltaNp73 isoforms). Whereas TAp73 functions as a tumor suppressor with pro-apoptotic function, DeltaNp73 is likely to prevent the induction of apoptosis in tumor cells and to participate in oncogenesis. Here we used a loss-of-function strategy to assess the role of DeltaNp73 in SH-SY5Y neuroblastoma cells. An antisense oligonucleotide designed to target DeltaNp73 mRNA, but not TAp73, was used to effectively downregulate this transcript. DeltaNp73 downregulation was accompanied by increased levels of the pro-apoptotic BH3 family member PUMA at the mRNA and protein level, and by conformational activation of BAX which translocated to mitochondria. These DeltaNp73 antisense-mediated alterations led to the induction of apoptosis as detected by decreased cell viability, augmented DNA fragmentation and increased caspase-3 activity in cell lysates. Our results demonstrate the cytoprotective role of DeltaNp73 in neuroblastoma and suggest its use as a target for molecular intervention therapy.

We previously reported the Bcl-2/Bcl-xL-bispecific activity of the 2'-O-(2-methoxy)ethyl (2'-MOE)-modified gapmer antisense oligonucleotide 4625. This oligonucleotide has 100% complementarity to Bcl-2 and three mismatches to Bcl-xL. In the present study, the isosequential locked nucleic acid (LNA)-modified oligonucleotide 5005 was generated, and its ability to further improve the downregulation of the two antiapoptotic targets in tumor cells was examined. We demonstrate that compared with 4625, 5005 more effectively decreased the expression of the mismatching Bcl-xL target gene in MDA-MB-231 breast and H125 lung cancer cells. In both cell lines, antisense activity caused decreased cell viability by induction of apoptosis. Moreover, in combination with various anticancer agents, 5005 reduced tumor cell viability more effectively than 4625. We describe for the first time the functional comparison of isosequential Bcl-2/Bcl-xL-bispecific 2'-MOE and LNA-modified antisense oligonucleotides and report that the LNA analog more effectively downregulated the two apoptosis inhibitors overexpressed in human tumors. Our data underscore the ability of LNA modifications to enhance the efficacy and favorably modulate the target specificity of antisense oligonucleotides.

Recent clinical studies have shown the promise of bcl-2 antisense therapy in patients with melanoma. To further demonstrate the importance of bcl-2 and validate the related antiapoptotic protein bcl-xL as targets for antisense therapy in melanoma, their implication as survival factors in melanoma cells of different clinical stages as well as in normal melanocytes was investigated. Primary cell cultures derived from 17 melanomas, the cell line A375, and normal melanocytes from healthy donors were treated with antisense oligonucleotides targeting either the bcl-xL mRNA or the bcl-2 and the bcl-xL mRNAs simultaneously. Bcl-2 and bcl-xL expression in cells was analyzed by real-time polymerase chain reaction and Western blotting. Cell viability was assessed in 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide and apoptosis assays. Bcl-2 expression was low in melanoma cells of stages I, II, and III, hardly detectable in A375 cells, but high in normal melanocytes. Bcl-xL expression was high in all cell types tested. As shown in A375 cells and the stage III melanoma cells 0513, both the bcl-xL monospecific oligonucleotide 4259 and the bcl-2/bcl-xL bispecific oligonucleotide 4625 effectively reduced tumor cell viability by induction of apoptosis with IC50 values ranging from 200 to 350 nM. Oligonucleotide 4625 proved to be superior to 4259, as it significantly reduced the viability of cells from all melanoma stages. Both oligonucleotides reduced also the viability of normal melanocytes. Our data suggest that bcl-2 and bcl-xL are promising targets for antisense therapy of melanoma, and that the simultaneous downregulation of their expression may provide additional clinical benefit.

Feline leukaemia virus (FeLV) infection in domestic cats can vary in its outcome (persistent, transient, no infection) for reasons that are not entirely known. It was hypothesized that the initial virus and provirus load could significantly influence the course of retrovirus infection. To determine the role of provirus loads, two methods of PCR, a nested PCR and a fluorogenic probe-based (TaqMan) real-time quantitative PCR, which were specific to the U3 region of FeLV-A were established. FeLV provirus in naturally and experimentally infected cats was then measured. Only 3 weeks after experimental FeLV-A infection, persistently infected cats demonstrated higher provirus loads and lower humoral immune responses than cats that had overcome antigenaemia. Lower initial provirus loads were associated with successful humoral immune responses. Unexpectedly, provirus in the buffy-coat cells of two cats that tested negative for the p27 antigen (a marker for viraemia) was also detected. In 597 Swiss cats, comparison of p27 antigen levels with PCR results revealed broad agreement. However, similar to the experimental situation, a significant number of animals (10%) was negative for the p27 antigen and FeLV-positive by PCR. These cats had a mean provirus load 300-fold lower than that of animals testing positive for the p27 antigen. In conclusion, an association between the provirus load and the outcome of FeLV infection was found. Detection of provirus carriers should contribute to further the control of FeLV. In addition, quantification of provirus loads will lead to a better understanding of FeLV pathogenesis and anti-retrovirus protective mechanisms.