p62 overexpression induces TDP-43 cytoplasmic mislocalisation, aggregation and cleavage and neuronal death.

Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) that exist on a spectrum of neurodegenerative disease. A hallmark of pathology is cytoplasmic TDP-43 aggregates within neurons, observed in 97% of ALS cases and ~ 50% of FTLD cases. This mislocalisation from the nucleus into the cytoplasm and TDP-43 cleavage are associated with pathology, however, the drivers of these changes are unknown. p62 is invariably also present within these aggregates. We show that p62 overexpression causes TDP-43 mislocalisation into cytoplasmic aggregates, and aberrant TDP-43 cleavage that was dependent on both the PB1 and ubiquitin-associated (UBA) domains of p62. We further show that p62 overexpression induces neuron death. We found that stressors (proteasome inhibition and arsenic) increased p62 expression and that this shifted the nuclear:cytoplasmic TDP-43 ratio. Overall, our study suggests that environmental factors that increase p62 may thereby contribute to TDP-43 pathology in ALS and FTLD.

A randomized trial of intravenous acetaminophen versus indomethacin for treatment of hemodynamically significant PDAs in VLBW infants.

Objective was to compare the rate of successful treatment of hsPDA based on echocardiogram criteria after use of IV acetaminophen or IV indomethacin in very low-birthweight infants. The study was a multi-center, randomized controlled trial. Infants born prior to 32 weeks with birthweight ≤ 1500 g were included if PDA treatment was indicated within the 21 days after birth. hsPDA was defined by strict echocardiogram criteria. Eligible infants were randomized to treatment with either IV acetaminophen or IV indomethacin. Of 86 eligible infants, 17 infants were randomized to acetaminophen and 20 to indomethacin. One (5.9%) hsPDA in the acetaminophen group had successful treatment compared to 11 (55%) in the indomethacin group (p = 0.002). Eight (47%) in the acetaminophen group and 3 (15%) in the indomethacin group received transcatheter PDA closure (p = 0.07). IV indomethacin was more effective than IV acetaminophen for treatment of hsPDAs. More infants in the acetaminophen group received transcatheter closure.

Substrate modulus of 3D-printed scaffolds regulates the regenerative response in subcutaneous implants through the macrophage phenotype and Wnt signaling.

The growing need for therapies to treat large cutaneous defects has driven recent interest in the design of scaffolds that stimulate regenerative wound healing. While many studies have investigated local delivery of biologics as a restorative approach, an increasing body of evidence highlights the contribution of the mechanical properties of implanted scaffolds to wound healing. In the present study, we designed poly(ester urethane) scaffolds using a templated-Fused Deposition Modeling (t-FDM) process to test the hypothesis that scaffolds with substrate modulus comparable to that of collagen fibers enhance a regenerative versus a fibrotic response. We fabricated t-FDM scaffolds with substrate moduli varying from 5 to 266 MPa to investigate the effects of substrate modulus on healing in a rat subcutaneous implant model. Angiogenesis, cellular infiltration, collagen deposition, and directional variance of collagen fibers were maximized for wounds treated with scaffolds having a substrate modulus (Ks = 24 MPa) comparable to that of collagen fibers. The enhanced regenerative response in these scaffolds was correlated with down-regulation of Wnt/ß-catenin signaling in fibroblasts, as well as increased polarization of macrophages toward the restorative M2 phenotype. These observations highlight the substrate modulus of the scaffold as a key parameter regulating the regenerative versus scarring phenotype in wound healing. Our findings further point to the potential use of scaffolds with substrate moduli tuned to that of the native matrix as a therapeutic approach to improve cutaneous healing.

Sources of inoculum for Phytophthora ramorum in a redwood forest.

ABSTRACT Sources of inoculum were investigated for dominant hosts of Phytophthora ramorum in a redwood forest. Infected trunks, twigs, and/or leaves of bay laurel (Umbellularia californica), tanoak (Lithocarpus densiflorus), and redwood (Sequoia sempervirens) were tested in the laboratory for sporangia production. Sporangia occurred on all plant tissues with the highest percentage on bay laurel leaves and tanoak twigs. To further compare these two species, field measurements of inoculum production and infection were conducted during the rainy seasons of 2003-04 and 2004-05. Inoculum levels in throughfall rainwater and from individual infections were significantly higher for bay laurel as opposed to tanoak for both seasons. Both measurements of inoculum production from bay laurel were significantly greater during 2004-05 when rainfall extended longer into the spring, while inoculum quantities for tanoak were not significantly different between the 2 years. Tanoak twigs were more likely to be infected than bay laurel leaves in 2003-04, and equally likely to be infected in 2004-05. These results indicate that the majority of P. ramorum inoculum in redwood forest is produced from infections on bay laurel leaves. Years with extended rains pose an elevated risk for tanoak because inoculum levels are higher and infectious periods continue into late spring.

Mechanical abrasion improves early incorporation of small intestinal submucosa.

It has been shown that gross incorporation of porcine-derived small intestinal submucosa (SiS) is limited at 2 weeks. This study evaluates a technique for improving the early incorporation of implanted eight-ply SiS. Six pigs underwent implantation of SiS on the peritoneal surface using three techniques: suture fixation of stock-perforated SiS, suture fixation of manually perforated SiS, and suture fixation of stock-perforated SiS to mechanically abraded peritoneum. Gross incorporation was evaluated and random samples harvested for tensiometric analysis 2 weeks after implantation. SiS placed onto mechanically abraded peritoneum demonstrated significantly greater gross incorporation than both stock-perforated SiS (100% versus 42%, P = 0.015) and manually perforated SiS (100% versus 50%, P = 0.042). There was no difference in gross incorporation between stock and manually perforated SiS. Using tensiometric analysis, the force required to separate the peritoneum from the SiS implant was significantly greater for the SiS placed onto mechanically abraded peritoneum (4.4 +/- 1.7 kg . f/cm2) than for both the stock-perforated SiS samples (1.0 +/- 0.5 kg x f/cm2) and the needle-perforated SiS samples (1.4 +/- 0.9 kg x f/cm2; P < 0.001). There was no difference between stock and manually perforated SiS at 2 weeks. Mechanical abrasion of the peritoneum before SiS onlay leads to improved gross incorporation 2 weeks after implantation in a porcine model of herniorrhaphy. Long-term studies and histologic analysis are needed to validate this method as a means for improving early incorporation of SiS.

Expression of the TAL1/SCL transcription factor in physiological and pathological vascular processes.

The TAL1/SCL transcription factor is essential for haematopoietic commitment and vascular remodelling during embryonic development. To help clarify its role in postnatal vascular processes, we characterized the expression of mouse Tal1 protein by immunocytochemistry in several experimental models of blood vessel formation. In adult mice, Tal1 protein was expressed in rare microvascular endothelial cells and in extravascular cells provisionally identified as endothelial progenitors from their morphology, proximity to vessels and expression of vascular endothelial growth factor receptor-2. The number of Tal1-expressing endothelial cells increased significantly but transiently in all the models-hormone-induced ovulation, wound healing and tumour development. Finally, Tal1 protein was detected in the nuclei of newly formed lymphatic endothelial cells in tumour-bearing animals. These results show that TAL1 is expressed by vascular endothelial cells and endothelial progenitors at sites of physiological and pathological neovascularization and suggest a role for this transcription factor in adult vasculogenesis. This work also provides the first evidence for TAL1 expression in lymphangiogenesis.

The central ACL defect as a model for failure of intra-articular healing.

Intra-articular soft tissues, such as the anterior cruciate ligament (ACL), fail to heal in contrast to the extra-articular medial collateral ligament (MCL), which undergoes classic healing. The goal of this study was to validate a model for failure of intra-articular healing that could be used in the future to test new repair strategies. We conducted a two-part experiment, the first part ex vivo, and the second in vivo. Our initial ex vivo experiments were used to determine the optimal width of the central defect in the canine ACL that would produce reproducible structural properties at time zero. The second experimental series used this optimal scalpel blade width to create a central defect in the canine ACL followed by measurement of structural properties in the ACL after either a 3- or 6-week in vivo healing period. A 3.5-mm beaver blade resulted in a maximum tolerated load of 56.8 +/- 4.7% (mean +/- SEM) of control at time zero. After the 3- and 6-week in vivo healing periods, the maximum load was 74.6 +/- 5.3 at 3 weeks and 64.9 +/- 3.8% at 6 weeks compared to control. Thus, biomechanical parameters tested at 6 weeks after creation of a defect showed no significant gains from defects tested immediately after the creation of injury. The centrally placed ACL defect in this canine model demonstrates failure to mechanically heal, which should prove suitable for future in vivo evaluation of the biomechanical and histological response to tissue engineering repair strategies for intra-articular soft tissues.

Posterior resin-based composite: review of the literature.

The use of direct posterior resin-based composite has increased primarily due to patient esthetic desires and product improvements. Other factors (substantiated or not) contributing to increased use of resin-based composite are environmental and health concerns with dental amalgam. New visible light cured resin-based composite products are introduced yearly, as manufacturers continue to improve this tooth-colored restorative material. This paper will characterize current posterior resin-based composite materials (hybrid, microfill, flowable, and packable), review recent in vitro and clinical research, and recommend indications for these materials. In addition, the literature on compomers will be reviewed and recommendation made for their use. The data indicates that composite resin is a technique sensitive restorative material that can be used in large preparations if proper manipulation and isolation can be maintained. Compomers may also be used as an esthetic posterior restorative if proper isolation is provided.

Collagen expression and biomechanical response to human recombinant transforming growth factor beta (rhTGF-beta2) in the healing rabbit MCL.

We investigated biomechanical and collagen expression in a healing bilateral rabbit medial collateral ligament (MCL) model to human recombinant transforming growth factor beta (rhTGF-beta2) at three and six weeks. Each rabbit had rhTGF-beta2 in a bioabsorbable pellet administered in one side, with the contralateral side serving as control (no rhTGF-beta2). All MCL healed with rhTGF-beta2 producing a profoundly increased scar mass at three weeks which decreased in size toward control at six weeks. In-situ hybridization demonstrated collagen expression (type I and III) no different than control at three weeks, but by six weeks elevated expression of type I was seen. Biomechanical analysis at three weeks showed no effect of rhTGF-beta2 on structural properties. However, at six weeks rhTGF-beta2 significantly inhibited both the maximum load (p < 0.05) and energy absorbed (p < 0.05) with no change in stiffness. Despite increased type I collagen expression and profound increase in early scar mass, rhTGF-beta2 did not improve the structural properties. Whether the dose or mode of delivery is responsible for decline in structural properties cannot be determined in this design. We hypothesize investigations of healing ligaments to cytokines should have biologic and biomechanical properties correlated in the same study at a minimum of two time points.

First Report of Phytophthora ramorum on Douglas-Fir in California.

Phytophthora ramorum S. Werres & A.W.A.M. de Cock was isolated from three Douglas-fir (Pseudotsuga menziesii) saplings in a mixedevergreen forest in Sonoma County, California. Symptoms on these saplings included cankers on small branches (0.5 to 1 cm in diameter) resulting in wilting of new shoots, dieback of branches, and loss of leaves as much as 15 cm from the twig tip. Symptoms were observed on most saplings growing in the same area. On several smaller saplings (<1 m tall), P. ramorum infection resulted in the death of the leader and the top several whorls of branches. Isolates were identified as P. ramorum by their abundant chlamydospores and caducous, semi-papillate sporangia (2) and internal transcribed spacer rDNA sequences identical to those of isolates of P. ramorum from Quercus spp., Lithocarpus densiflorus, and Rhododendron (1,2). To test for pathogenicity, foliage inoculations were conducted on seedlings in two trials by misting 30 leaves per trial (five leaves per seedling plus controls) with sterile distilled water and pinning inoculum plugs, taken from the margin of P. ramorum cultures, to the upper surface of leaves. Inoculation resulted in lesions ranging between 1 and 12 mm long, and P. ramorum was recovered from 47% of inoculated leaves. Symptoms were not restricted to inoculated leaves, and in 26 single-leaf inoculations, lesions 17 to 85 mm long developed on branches (five mm in diameter) adjacent to the inoculated leaf. Isolation success from branch lesions was 50%, despite the fact that such lesions were apparently disjunct from the small 1-mm lesions developing on inoculated leaves. Stems of Douglas-fir seedlings (approximately 1 cm in diameter) were wound inoculated (1) in two trials consisting of 10 inoculated seedlings per trial plus 10 controls. After 6 weeks, lesion lengths in the cambium averaged 38 mm (range 12 to 62 mm), and three seedlings were completely girdled. P. ramorum was recovered from 75% of inoculated stems. Mean lesion lengths on seedlings inoculated with P. ramorum were significantly greater (P < 0.05) in both trials than those of control inoculations (mean 9 mm) based on analysis of variance. We have not observed unusual mortality or disease symptoms on overstory Douglas-fir trees in natural forests. The importance of P. ramorum branch tip dieback for growth and reproduction of Douglas-fir is unknown. Douglas-fir is present in many forests in California and Oregon already infested by P. ramorum, yet we have found infection of plants at only one location. At this site, symptomatic Douglas-fir saplings were surrounded by bay laurel (Umbellularia californica) trees with extremely high levels of P. ramorum infection. P. ramorum is known to sporulate prolifically on bay laurel leaves. More studies are necessary to determine if the incidence of P. ramorum in Douglas-fir extends to other locations or if it is limited to this one locale. References: (1) D. M. Rizzo et al. Plant Disease 86:205, 2002. (2) S. Werres et al. Mycol. Res. 105:1155, 2001.

Phytophthora ramorum as the Cause of Extensive Mortality of Quercus spp. and Lithocarpus densiflorus in California.

A new canker disease, commonly known as sudden oak death, of Lithocarpus densiflorus, Quercus agrifolia, Q. kelloggii, and Q. parvula var. shrevei in California is shown to be caused by Phytophthora ramorum. The pathogen is a recently described species that previously was known only from Germany and the Netherlands on Rhododendron spp. and a Viburnum sp. This disease has reached epidemic proportions in forests along approximately 300 km of the central coast of California. The most consistent and diagnostic symptoms on trees are cankers that develop before foliage symptoms become evident. Cankers have brown or black discolored outer bark and seep dark red sap. Cankers occur on the trunk at the root crown up to 20 m above the ground, but do not enlarge below the soil line into the roots. Individual cankers are delimited by thin black lines in the inner bark and can be over 2 m in length. In L. densiflorus saplings, P. ramorum was isolated from branches as small as 5 mm in diameter. L. densiflorus and Q. agrifolia were inoculated with P. ramorum in the field and greenhouse, and symptoms similar to those of naturally infected trees developed. The pathogen was reisolated from the inoculated plants, which confirmed pathogenicity.

An in vivo comparison of topical agents on wound repair.

Selection of the ideal antiseptic or antimicrobial treatment for contaminated wounds remains a controversial decision. Clinical decisions are often made on the basis of in vitro studies and personal preference. Although topical solutions are widely used, their comparative in vivo effects on wound healing are largely unreported.A porcine wound model was used to compare five commonly used topical agents-5% mafenide acetate (Sulfamylon solution), 10% povidone with 1% free iodine (Betadine), 0.25% sodium hypochlorite ("half-strength" Dakin), 3% hydrogen peroxide, and 0.25% acetic acid-with a control group. Reepithelialization, angiogenesis, neodermal regeneration, fibroblast proliferation, collagen production, and bacterial colony counts were analyzed at 4 and 7 days after wounding (n = 4). Reepithelialization was not significantly influenced among the various treatment modalities tested. Sulfamylon and Dakin solutions significantly increased neodermal thickness (p < 0.05), whereas hydrogen peroxide and acetic acid significantly inhibited neodermal formation (p < 0.001). All treatments except hydrogen peroxide significantly increased fibroblast proliferation. Sulfamylon and Betadine significantly enhanced angiogenesis (p < 0.05). Sulfamylon proved most effective in maintaining an aseptic environment while concomitantly increasing angiogenesis, fibroblast proliferation, and dermal thickness compared with control. These data show that selection of a particular topical treatment can affect various aspects of wound repair in an animal model. These results suggest that the selection of topical treatments in the clinical setting should be carefully tailored to match unique wound situations and therapeutic endpoints.

Towards retrievable vascularized bioartificial pancreas: induction and long-lasting stability of polymeric mesh implant vascularized with the help of acidic and basic fibroblast growth factors and hydrogel coating.

We seek to improve existing methodologies for allogenic grafting of pancreatic islets. The lack of success of encapsulated transplanted islets inside the peritoneal cavity is presently attributed to poor vascularization of the implant. A thick, fibrotic capsule often surrounds the graft, limiting survival. We have tested the hypothesis that neovascularization of the graft material can be induced by the addition of proper angiogenic factors embedded within a polymeric coat. Biocompatible and nonresorbable meshes coated with hydrophilic polymers were implanted in rats and harvested after 1-, 6-, and 12-week intervals. The implant response was assessed by histological observations on the degree of vascularity, fibrosis, and inflammation. Macrostructural geometry of meshes was conducive to tissue ingrowth into the interstitial space between the mesh filaments. Hydrogel coating with incorporated acidic or basic FGF in an electrostatic complex with polyelectrolytes and/or with heparin provided a sustained slow release of the angiogenic growth factor. Anti-factor VIII and anti-collagen type IV antibodies and a GSL I-B4 lectin were used to measure the extent of vascularization. Vigorous and persistent vascularization radiated several hundred microns from the implant. The level of vascularization should provide a sufficient diffusion of nutrients and oxygen to implanted islets. Based on our observations, stable vascularization may require a sustained angiogenic signal to allow for the development of a permanent implant structure.

Delayed wound healing in aged rats is associated with increased collagen gel remodeling and contraction by skin fibroblasts, not with differences in apoptotic or myofibroblast cell populations.

Aging has been anecdotally reported to result in prolonged wound healing. Measurement of punch biopsy wound closure in young (4 month old) and old (36 month old) rats indicated there was a significant delay in wound closure by old rats during the early phase of repair, after which closure rates were equivalent. The delay in granulation tissue accumulation in older animals could involve premature programmed cell death (apoptosis); however, apoptotic fibroblasts in sponge granulation tissue and tissue culture were less abundant in samples from old rats relative to young rats. Myofibroblasts express alpha-smooth muscle actin, and they are believed to be important in wound contraction. There were no significant differences in overall abundance or distribution of alpha-smooth muscle actin containing myofibroblasts in granulation tissue and in cultured granulation tissue fibroblasts regardless of the age of the donor rat. The spatial distribution of myofibroblasts and apoptotic cells was distinct. Fibroblasts from granulation tissue and skin explants were placed in a collagen gel contraction assay prior to the 5th passage to determine their in vitro contractility. While granulation tissue fibroblasts from young and old rats showed similar collagen gel contractility, skin fibroblasts from old rats displayed greater collagen gel contractile behavior than young skin fibroblasts. Greater gel contractility of fibroblasts from old rats appeared to result, in large part, from the ability of those cells to cause generalized gel degradation. Gelatin zymography indicated a greater abundance of matrix metalloproteinase-2 in supernatants from gels containing skin fibroblasts from old rats. Taken together, these results suggest that the age-associated healing delay in the rat may not be related to the appearance or abundance of distinct myofibroblast or apoptotic cell populations. Proteolysis may have a significant role in delayed wound healing in aged animals.

Wound-healing defects in mice lacking fibrinogen.

In addition to its key role in the control of blood loss following injury, fibrin(ogen) has been proposed to play an important role in tissue repair by providing an initial matrix that can stabilize wound fields and support local cell proliferation and migration. To test directly these concepts, the effect of fibrinogen deficiency on cutaneous tissue repair in mice was investigated using incisional and excisional wounds. The time required to overtly heal wounds was similar in fibrinogen-deficient and control mice, but histologic evaluation revealed distinct differences in the repair process, including an altered pattern of epithelial cell migration and increased epithelial hyperplasia. Furthermore, granulation tissue in fibrinogen-deficient mice failed to adequately close the wound gap, resulting in persistent open wounds or partially covered sinus tracts. The tensile strength of these wounds was also reduced compared with control mice. The most profound defect in wound tissue organization was observed in fibrinogen-deficient mice following the subcutaneous implantation of a porous tubing chamber. Cells migrated into the wall of the implants at a similar rate as control mice, but cells from fibrinogen-deficient animals were unable to efficiently organize and migrate into wound fluid-filled dead space within the center of the implants. These studies show that re-epithelialization, granulation tissue formation, including the establishment of neovasculature, and the formation of fibrotic scar tissue can proceed in the absence of fibrin(ogen) and all of its proteolytic derivatives. However, fibrin (ogen) is important for appropriate cellular migration and organization within wound fields and in initially establishing wound strength and stability. (Blood. 2001;97:3691-3698)

Graduate status and age at entry to medical school as predictors of doctors' choice of long-term career.

OBJECTIVE: To determine whether graduate entry to medical school, taking an intercalated degree during medical school, and age at entry to medical school are related to choice of eventual career. DESIGN: Postal questionnaires. SETTING: United Kingdom (UK). SUBJECTS: All doctors who qualified in the UK in 1993 or 1996. RESULTS: We analysed whether graduate status and age on entry to medical school, and taking an intercalated degree during medical school, were predictors of the choice of eventual career, adjusting for differences by sex, year of qualification and medical school. General practice was the career choice of 27.0% (79/293) of graduate entrants and 21.6% (1095/5073) of non-graduate entrants, a difference of borderline significance (P=0.04). Of the non-graduate entrants, general practice was the career choice of 15.3% (319/2081) of doctors who took an intercalated degree and 25.9% (776/2992) of doctors who did not (P < 0.001). Within the hospital specialties, those who took an intercalated degree were more likely than others to choose the hospital medical specialties or pathology. Age alone was not a predictor for choice of any area of practice. CONCLUSIONS: There was no evidence of an association between age at entry to medical school and choice of eventual career. Graduates at entry to medical school were a little more likely than non-graduates to choose general practice but the relationship was not a strong one. In these respects, changing the entry profile of medical students is unlikely to result in major shifts of career choice towards general practice.

Loss of British-trained doctors from the medical workforce in Great Britain.

OBJECTIVE: To summarize trends over time in the percentage of British medical graduates who subsequently practise in Great Britain in the National Health Service (NHS), in Great Britain outside the NHS, outside Great Britain, or do not practise medicine. DESIGN: Questionnaire-based postal surveys and government employment records. SETTING: Great Britain. SUBJECTS: All British medical graduates of 1974, 1977, 1983, 1988, and 1993. MAIN OUTCOME MEASURES: Type and location of employment at successive years after graduation. RESULTS: Differences in career destination between cohorts were generally small. Combining data from all cohorts studied, 85% of doctors were working in the NHS 2 years after graduation, 82% after 5 and 10 years, 81% after 15 years, and 77% after 20 years. Part-time working was much higher among women than among men. Allowing for loss and part-time working, the whole-time equivalent available to the NHS at 15 years after graduation was 60% of women and 80% of men. More men than women worked in medicine outside the NHS in Britain and abroad. Loss from medicine altogether was small, and higher among women. CONCLUSIONS: Medical workforce planning in Great Britain should assume that 15-20% of home-trained doctors will not be working in the NHS within a few years of graduation. Comparing cohorts at the same career stage showed no evidence of increased loss from the NHS in recent times. Although a higher percentage of women than men were not working in medicine at all, recent trends suggest that this percentage is falling.

Non-random chromosomal rearrangements in pancreatic cancer cell lines identified by spectral karyotyping.

The molecular events involved in pancreatic cancer are becoming increasingly well characterized, with mutations in the dominant oncogene KRAS and the tumour suppressor genes TP53, CDKN2A and MADH4 being typically observed. However, other genetic abnormalities remain to be identified and molecular cytogenetics may be useful to detect chromosomal loci involved in recurrent rearrangements. We have used spectral karyotyping to characterize cytogenetic aberrations in a panel of 20 human pancreatic carcinoma cell lines and confirmed their identities by dual and triple color fluorescence in situ hybridization. The most common partial or whole-arm gains involved 5p, 7q, 12p, 1q, 7p, 5q, 9p, 9q and 11p. The most common partial or whole-arm losses affected 9p, 11q, 18q, 3p, 2q and 1p, as well as the short arms of the acrocentric chromosomes. Spectral karyotyping allowed us to identify a number of recurrent structural aberrations, all of them unbalanced: most frequently i(5)(p10), del(11)(q23), i(12)(p10), i(1)(q10), del(7)(q22) and del(10)(p11). Spectral karyotyping mapped the complex aberrations occurring in pancreatic cancer cell lines and identified non-random patterns of chromosomal rearrangement. This comprehensive characterization should be useful to direct future investigation. The observation that loss at 11q and gains at 5p with i(5)(p10) and 12p with i(12)(p10) are more frequent changes than previously reported would justify more intensive investigation of these chromosomal regions.