A novel method for the establishment of autologous skin cell suspensions: characterisation of cellular sub-populations, epidermal stem cell content and wound response-enhancing biological properties.

Introduction: Autologous cell suspension (ACS)-based therapy represents a highly promising approach for burns and chronic wounds. However, existing technologies have not achieved the desired clinical success due to several limitations. To overcome practical and cost-associated obstacles of existing ACS methods, we have established a novel methodology for rapid, enzymatic disaggregation of human skin cells and their isolation using a procedure that requires no specialist laboratory instrumentation and is performed at room temperature. Methods: Cells were isolated using enzymatic disaggregation of split-thickness human skin followed by several filtration steps for isolation of cell populations, and cell viability was determined. Individual population recovery was confirmed in appropriate culture medium types, and the presence of epidermal stem cells (EpSCs) within keratinocyte sub-populations was defined by flow cytometry via detection of CD49 and CD71. Positive mediators of wound healing secreted by ACS-derived cultures established on a collagen-based wound-bed mimic were detected by proteome arrays and quantified by ELISA, and the role of such mediators was determined by cell proliferation assays. The effect of ACS-derived conditioned-medium on myofibroblasts was investigated using an in-vitro model of myofibroblast differentiation via detection of α-SMA using immunoblotting and immunofluorescence microscopy. Results: Our methodology permitted efficient recovery of keratinocytes, fibroblasts and melanocytes, which remained viable upon long-term culture. ACS-derivatives comprised sub-populations with the CD49-high/CD71-low expression profile known to demarcate EpSCs. Via secretion of mitogenic factors and wound healing-enhancing mediators, the ACS secretome accelerated keratinocyte proliferation and markedly curtailed cytodifferentiation of myofibroblasts, the latter being key mediators of fibrosis and scarring. Discussion: The systematic characterisation of the cell types within our ACS isolates provided evidence for their superior cell viability and the presence of EpSCs that are critical drivers of wound healing. We defined the biological properties of ACS-derived keratinocytes, which include ability to secrete positive mediators of wound healing as well as suppression of myofibroblast cytodifferentiation. Thus, our study provides several lines of evidence that the established ACS isolates comprise highly-viable cell populations which can physically support wound healing and possess biological properties that have the potential to enhance not only the speed but also the quality of wound healing.

Biomolecular insights into North African-related ancestry, mobility and diet in eleventh-century Al-Andalus.

Historical records document medieval immigration from North Africa to Iberia to create Islamic al-Andalus. Here, we present a low-coverage genome of an eleventh century CE man buried in an Islamic necropolis in Segorbe, near Valencia, Spain. Uniparental lineages indicate North African ancestry, but at the autosomal level he displays a mosaic of North African and European-like ancestries, distinct from any present-day population. Altogether, the genome-wide evidence, stable isotope results and the age of the burial indicate that his ancestry was ultimately a result of admixture between recently arrived Amazigh people (Berbers) and the population inhabiting the Peninsula prior to the Islamic conquest. We detect differences between our sample and a previously published group of contemporary individuals from Valencia, exemplifying how detailed, small-scale aDNA studies can illuminate fine-grained regional and temporal differences. His genome demonstrates how ancient DNA studies can capture portraits of past genetic variation that have been erased by later demographic shifts-in this case, most likely the seventeenth century CE expulsion of formerly Islamic communities as tolerance dissipated following the Reconquista by the Catholic kingdoms of the north.

WODA Technical Guidance on Underwater Sound from Dredging.

The World Organization of Dredging Associations (WODA) has identified underwater sound as an environmental issue that needs further consideration. A WODA Expert Group on Underwater Sound (WEGUS) prepared a guidance paper in 2013 on dredging sound, including a summary of potential impacts on aquatic biota and advice on underwater sound monitoring procedures. The paper follows a risk-based approach and provides guidance for standardization of acoustic terminology and methods for data collection and analysis. Furthermore, the literature on dredging-related sounds and the effects of dredging sounds on marine life is surveyed and guidance on the management of dredging-related sound risks is provided.

Mosaic maternal ancestry in the Great Lakes region of East Africa.

The Great Lakes lie within a region of East Africa with very high human genetic diversity, home of many ethno-linguistic groups usually assumed to be the product of a small number of major dispersals. However, our knowledge of these dispersals relies primarily on the inferences of historical, linguistics and oral traditions, with attempts to match up the archaeological evidence where possible. This is an obvious area to which archaeogenetics can contribute, yet Uganda, at the heart of these developments, has not been studied for mitochondrial DNA (mtDNA) variation. Here, we compare mtDNA lineages at this putative genetic crossroads across 409 representatives of the major language groups: Bantu speakers and Eastern and Western Nilotic speakers. We show that Uganda harbours one of the highest mtDNA diversities within and between linguistic groups, with the various groups significantly differentiated from each other. Despite an inferred linguistic origin in South Sudan, the data from the two Nilotic-speaking groups point to a much more complex history, involving not only possible dispersals from Sudan and the Horn but also large-scale assimilation of autochthonous lineages within East Africa and even Uganda itself. The Eastern Nilotic group also carries signals characteristic of West-Central Africa, primarily due to Bantu influence, whereas a much stronger signal in the Western Nilotic group suggests direct West-Central African ancestry. Bantu speakers share lineages with both Nilotic groups, and also harbour East African lineages not found in Western Nilotic speakers, likely due to assimilating indigenous populations since arriving in the region ~3000 years ago.

Characterization of underwater sounds produced by hydraulic and mechanical dredging operations.

Sound recordings were made of two dredging operations at hydrophone depths of 3 and 9.1 m at distances up to 1.2 km from the source in shallow waters (<15 m) of New York Harbor. Sound sources included rock fracturing by a hydraulic cutterhead dredge and six distinct sources associated with a mechanical backhoe dredging operation during rock excavation. To place sound emitted from these dredges in perspective with other anthropogenic sounds, recordings were also made of several deep-draft commercial vessels. Results are presented as sound pressure levels (SPLs) in one-third octave versus range across the 20 Hz to 20 kHz frequency band. To address concerns for protection of fishery resource occupying the harbor, SPL were examined at frequency bands of 50-1000 Hz and 100-400 Hz, the ranges where the majority of fishes without hearing specializations detect sound and the range of greatest sensitivity, respectively. Source levels (dB re 1 µPa-1 m rms) were back calculated using fitted regression (15LogR). The strongest sound sources (180-188.9 dB) were emitted by commercial shipping. Rock fracturing produced a source level of 175 dB, whereas six distinct sources associated with rock excavation had source levels ranging from 164.2 to 179.4 dB re 1 µPa-1 m (rms).

Perlecan domain V inhibits amyloid-ß induced brain endothelial cell toxicity and restores angiogenic function.

In Alzheimer's disease (AD), amyloid-ß (Aß) deposits in the cerebrovasculature can result in neurovascular dysfunction and/or cerebral amyloid angiopathy. The accumulation of Aß in blood vessels can cause endothelial cell damage, resulting in impaired Aß clearance by the blood-brain barrier. Additionally, impaired endothelial cell function can result in decreased angiogenesis in the brains of AD patients, affecting cognitive function. VEGF is a crucial mediator of angiogenesis and is deficient in AD brains thus promoting angiogenesis could be an important component of successful AD treatment. The C-terminal portion of the extracellular matrix proteoglycan perlecan, Domain V (DV), promotes brain-derived endothelial cell proliferation and is proangiogenic in that it increases VEGFR2 expression and production of VEGF. In this study, we show that Aß25-35 reduces proliferation of a mouse brain microvascular endothelial cell line (MBEC) in vitro and that DV and mouse LG3 (C-terminal fragment of DV) block these effects of Aß25-35. Additionally, we show that DV restores the ability of MBECs to form tube-like structures on Matrigel in the presence of Aß25-35 and that this is α5ß1 dependent. Interestingly, the reduction in tube-like structure formation by Aß25-35 was not due to endothelial cell death, suggesting that Aß25-35 induces the downregulation of a cell surface molecule required for adhesion events critical to the angiogenic process. We propose a model suggesting that DV works through both the α5ß1 integrin receptor and VEGFR2 to increase VEGF production, causing competition with Aß25-35 for VEGFR2 binding, thus ultimately increasing VEGF expression and restoring angiogenesis. This supports DV as a potential anti-amyloid therapy.

Molecular biomarkers of vascular dysfunction in obstructive sleep apnea.

Untreated and long-lasting obstructive sleep apnea (OSA) may lead to important vascular abnormalities, including endothelial cell (EC) dysfunction, hypertension, and atherosclerosis. We observed a correlation between microcirculatory reactivity and endothelium-dependent release of nitric oxide in OSA patients. Therefore, we hypothesized that OSA affects (micro)vasculature and we aimed to identify vascular gene targets of OSA that could possibly serve as reliable biomarkers of severity of the disease and possibly of vascular risk. Using quantitative RT-PCR, we evaluated gene expression in skin biopsies of OSA patients, mouse aortas from animals exposed to 4-week intermittent hypoxia (IH; rapid oscillations in oxygen desaturation and reoxygenation), and human dermal microvascular (HMVEC) and coronary artery endothelial cells (HCAEC) cultured under IH. We demonstrate a significant upregulation of endothelial nitric oxide synthase (eNOS), tumor necrosis factor-alpha-induced protein 3 (TNFAIP3; A20), hypoxia-inducible factor 1 alpha (HIF-1α?? and vascular endothelial growth factor (VEGF) expression in skin biopsies obtained from OSA patients with severe nocturnal hypoxemia (nadir saturated oxygen levels [SaO2]<75%) compared to mildly hypoxemic OSA patients (SaO2 75%-90%) and a significant upregulation of vascular cell adhesion molecule 1 (VCAM-1) expression compared to control subjects. Gene expression profile in aortas of mice exposed to IH demonstrated a significant upregulation of eNOS and VEGF. In an in vitro model of OSA, IH increased expression of A20 and decreased eNOS and HIF-1α expression in HMVEC, while increased A20, VCAM-1 and HIF-1αexpression in HCAEC, indicating that EC in culture originating from distinct vascular beds respond differently to IH stress. We conclude that gene expression profiles in skin of OSA patients may correlate with disease severity and, if validated by further studies, could possibly predict vascular risk in OSA patients.

Fishery resource utilization of a restored estuarine borrow pit: a beneficial use of dredged material case study.

Numerous pits in coastal waters are subject to degraded water quality and benthic habitat conditions, resulting in degraded fish habitat. A pit in Barnegat Bay, New Jersey (USA) was partially filled with dredged sediment to increase flushing, alleviate hypoxia, and enhance benthic assemblages. Restoration objectives were assessed in terms of benthic community parameters and fishery resource occupation. Restoration resulted in increased benthic diversity (bottom samples) and the absence of water column stratification. Fisheries resources occupied the entire water column, unlike pre-restoration conditions where finfish tended to avoid the lower water column. The partial restoration option effectively reproduced an existing borrow pit configuration (Hole #5, control), by decreasing total depth from -11 m to -5.5 m, thereby creating a habitat less susceptible to hypoxic/anoxic conditions, while retaining sufficient vertical relief to maintain associations with juvenile weakfish and other forage fishes. Partially filling pits using dredged material represents a viable restoration alternative.

Perlecan Domain V induces VEGf secretion in brain endothelial cells through integrin α5ß1 and ERK-dependent signaling pathways.

Perlecan Domain V (DV) promotes brain angiogenesis by inducing VEGF release from brain endothelial cells (BECs) following stroke. In this study, we define the specific mechanism of DV interaction with the α(5)ß(1) integrin, identify the downstream signal transduction pathway, and further investigate the functional significance of resultant VEGF release. Interestingly, we found that the LG3 portion of DV, which has been suggested to possess most of DV's angio-modulatory activity outside of the brain, binds poorly to α(5)ß(1) and induces less BEC proliferation compared to full length DV. Additionally, we implicate DV's DGR sequence as an important element for the interaction of DV with α(5)ß(1). Furthermore, we investigated the importance of AKT and ERK signaling in DV-induced VEGF expression and secretion. We show that DV increases the phosphorylation of ERK, which leads to subsequent activation and stabilization of eIF4E and HIF-1α. Inhibition of ERK activity by U0126 suppressed DV-induced expression and secretion of VEGR in BECs. While DV was capable of phosphorylating AKT we show that AKT phosphorylation does not play a role in DV's induction of VEGF expression or secretion using two separate inhibitors, LY294002 and Akt IV. Lastly, we demonstrate that VEGF activity is critical for DV increases in BEC proliferation, as well as angiogenesis in a BEC-neuronal co-culture system. Collectively, our findings expand our understanding of DV's mechanism of action on BECs, and further support its potential as a novel stroke therapy.

Perlecan domain V inhibits α2 integrin-mediated amyloid-ß neurotoxicity.

Amyloid-ß (Aß) peptide is a key component of amyloid plaques, one of the pathological features of Alzheimer's disease. Another feature is pronounced cell loss in the brain leading to an enlargement of the ventricular area and a decrease in brain weight and volume. Aß plaque deposition and neuronal toxicity can be modeled by treating human cortical neuronal cultures with Aß and showing robust Aß deposition and neurotoxicity that is mediated by α2ß1 and αvß1 integrins. The current study expands on these findings by showing that the domain V of perlecan, a known α2 integrin ligand, inhibits Aß neurotoxicity in an α2 integrin-dependent manner. Additionally, Aß binds more efficiently to cells expressing activated α2 integrin. Finally the inhibition of Aß neurotoxicity with domain V is synergistic with inhibitors of αv integrin and ß1 integrin. We propose that domain V and potentially other α2 integrin ligands could be a new therapeutic approach for inhibiting the Aß plaque deposition and neurotoxicity observed in Alzheimer's disease.

Perlecan domain V is neuroprotective and proangiogenic following ischemic stroke in rodents.

Stroke is the leading cause of long-term disability and the third leading cause of death in the United States. While most research thus far has focused on acute stroke treatment and neuroprotection, the exploitation of endogenous brain self-repair mechanisms may also yield therapeutic strategies. Here, we describe a distinct type of stroke treatment, the naturally occurring extracellular matrix fragment of perlecan, domain V, which we found had neuroprotective properties and enhanced post-stroke angiogenesis, a key component of brain repair, in rodent models of stroke. In both rat and mouse models, Western blot analysis revealed elevated levels of perlecan domain V. When systemically administered 24 hours after stroke, domain V was well tolerated, reached infarct and peri-infarct brain vasculature, and restored stroke-affected motor function to baseline pre-stroke levels in these multiple stroke models in both mice and rats. Post-stroke domain V administration increased VEGF levels via a mechanism involving brain endothelial cell α5ß1 integrin, and the subsequent neuroprotective and angiogenic actions of domain V were in turn mediated via VEGFR. These results suggest that perlecan domain V represents a promising approach for stroke treatment.

Ancient voyaging and Polynesian origins.

The "Polynesian motif" defines a lineage of human mtDNA that is restricted to Austronesian-speaking populations and is almost fixed in Polynesians. It is widely thought to support a rapid dispersal of maternal lineages from Taiwan ~4000 years ago (4 ka), but the chronological resolution of existing control-region data is poor, and an East Indonesian origin has also been proposed. By analyzing 157 complete mtDNA genomes, we show that the motif itself most likely originated >6 ka in the vicinity of the Bismarck Archipelago, and its immediate ancestor is >8 ka old and virtually restricted to Near Oceania. This indicates that Polynesian maternal lineages from Island Southeast Asia gained a foothold in Near Oceania much earlier than dispersal from either Taiwan or Indonesia 3-4 ka would predict. However, we find evidence in minor lineages for more recent two-way maternal gene flow between Island Southeast Asia and Near Oceania, likely reflecting movements along a "voyaging corridor" between them, as previously proposed on archaeological grounds. Small-scale mid-Holocene movements from Island Southeast Asia likely transmitted Austronesian languages to the long-established Southeast Asian colonies in the Bismarcks carrying the Polynesian motif, perhaps also providing the impetus for the expansion into Polynesia.

Endostatin binds nerve growth factor and thereby inhibits neurite outgrowth and neuronal migration in-vitro.

Endostatin (ES), the C-terminal fragment of collagen XVIII known for its anti-angiogenic properties, is associated with neurological diseases in mammals. In this study, we investigated the effect of ES on nerve growth factor (NGF)-induced neuronal differentiation, migration, neuritogenesis, and neurite extension. ES partially inhibited PC12 cell differentiation and cerebellar granule cell migration. In addition, neurite outgrowth was inhibited in a concentration-dependent manner. This effect was also matrix-dependent, as we observed better inhibition on PC12 cells grown on collagen compared to laminin matrices. Furthermore, we observed partial NGF depletion by collagen and ES, but not by laminin suggesting that NGF-matrix interactions may be important for promoting neuritogenesis, competitive inhibition by ES or low affinity matrix impairs PC12 differentiation and neurite outgrowth. Finally, using a biosensor technique, we demonstrated a direct interaction between NGF and ES suggesting the mechanism of action of ES may involve NGF sequestration. In conclusion, our study demonstrates the inhibitory effect of ES on different steps of neurogenesis including cell differentiation and migration and neuritogenesis by NGF sequestration. Such sequestration may compromise brain repair following injury, but also may play important role in axon finding as well as a potent therapeutical target in diseases involving abnormal elevated neurotrophic growth factor levels. Taken together, this study raises the consideration of ES as a double-edge sword that carries both deleterious and putative therapeutical effects.

Propionibacterium acnes and Staphylococcus epidermidis isolated from refractory endodontic lesions are opportunistic pathogens.

The predominant cultivable microbiota from 20 refractory endodontic lesions (9 with abscesses and 11 without abscesses) were determined, and Propionibacterium acnes and Staphylococcus epidermidis were among the most predominant organisms. The number of species identified from lesions with abscesses (14.1 ± 2.6) was significantly greater (P < 0.001) than the number from lesions without abscesses (7.4 ± 5.9). Comparison of perioral isolates using repetitive extragenic palindromic PCR of the same species from the same subjects demonstrated that the endodontic and skin populations were significantly different. The P. acnes isolates were typed on the basis of recA gene sequence comparison, and only three types (types I, II, and III) were identified among 125 isolates examined. However, we found that type I (type IA and IB) isolates were primarily isolated from the skin, while types II and III were significantly more likely to be isolated from the endodontic lesions (P < 10(-10)). We found that the robustness of the recA phylotypes was not strong by comparing the partial gene sequences of six putative virulence determinants, PAmce, PAp60, PA-25957, PA-5541, PA-21293, and PA-4687. The resulting neighbor-joining trees were incongruent, and significant (phi test; P = 2.2 × 10(-7)) evidence of recombination was demonstrated, with significant phylogenetic heterogeneity being apparent within the clusters. P. acnes and S. epidermidis isolated from refractory endodontic infections, with or without periapical abscesses, are likely to be nosocomial infections.

Anterior thoracoscopic instrumentation in adolescent idiopathic scoliosis: a systematic review.

STUDY DESIGN: A Systematic Review of published series of patients with adolescent idiopathic scoliosis treated with anterior thoracoscopic instrumentation. OBJECTIVE: To conduct a systematic review of the results of thoracoscopic surgery and to compare them with those of open anterior and posterior spine instrumentation to enable surgeons judge the applicability of the method. SUMMARY OF BACKGROUND DATA: Instrumentation through video-assisted thoracoscopic surgery is an attractive alternative for the treatment of thoracic adolescent idiopathic scoliosis. The advantages claimed by its proponent over conventional instrumentations are better cosmesis and reduced morbidity due its minimal invasive nature. However, superiority of thoracoscopic instrumentation over conventional methods has not been proven so far. METHODS: Via Medline, Pubmed, and other literature searches, 8 articles met the inclusion criteria for our systematic review. The evaluations were made according to the parameters employed for evaluating spinal deformities. Instrumentation through video-assisted thoracoscopic surgery results were compared to those of open anterior or posterior surgeries. RESULTS: Mean number of instrumented levels was 7. The extent of disc excision was not indicated in any of the studies. The mean operative time was found to be approximately 5.2 hours. Average blood loss was 391.7 mL (100-1300 mL). The average Cobb curve correction was 64.6%. The mean preoperative kyphosis angle was 21.3 degrees ; the postoperative kyphosis angle was 25.2 degrees . These angular corrections were found to be comparable to posterior procedures using hooks, but less than with pedicle screws. Two studies reported on patient satisfaction favoring thoracoscopic instrumentation. Instrumentation-related complications were the most predominant. CONCLUSION: Anterior thoracoscopic instrumentation is comparable in terms of curve correction to anterior or posterior procedures. Theoretical advantages of better cosmesis and less aggressiveness seem to be offset by the increased operative and intensive care unit time, and complication rate. More prospective studies need to be conducted to determine the benefit and general applicability of this procedure.

A risk-informed decision framework for setting environmental windows for dredging projects.

Sediment dredging is necessary to sustain navigation infrastructure in ports and harbor areas. In the United States alone between 250 and 300 million cubic yards of sediment are dredged annually. Dredging activities may cause stress on aquatic biota by locally increasing turbidity and suspended sediment concentrations, physically disturbing habitat by elevated sedimentation rates, interfering in migratory behaviors, and hydraulically entraining bottom dwelling organisms. Environmental windows are a management practice used to alleviate such stresses on resident and transient biota by placing temporal restrictions on the conduct of dredging operations. Adherence to environmental windows can significantly inflate costs for project sponsors and local stakeholders. Since their inception following passage of NEPA in 1969 the process for setting environmental windows has not followed structured procedures and represents an example of the difficulty inherent in achieving a balance between biological resource protection and cost-effective construction and maintenance of navigation infrastructure. Recent developments in the fields of risk assessment for non-chemical stressors as well as experience in implementing structured risk-informed decision-making tools for sediment and natural resource management are summarized in this paper in relation to setting environmental windows. Combining risk assessment and multi-criteria decision analysis allows development of a framework for an objective process consistent with recommendations by the National Academy of Sciences for setting environmental windows. A hypothetical application of the framework for protection of Pacific herring (Clupea pallasii) in San Francisco Bay is discussed.

Catecholamine-induced cardiomyopathy.

OBJECTIVE: To review the pathogenesis as well as the clinical and laboratory features of catecholamine-induced cardiomyopathy associated with pheochromocytoma and other disorders and discuss the various treatment options available. METHODS: Materials used for this article were identified through MEDLINE, PubMed, and Google Scholar searches of the relevant literature from 1955 to the present. RESULTS: Catecholamines and their oxidation products cause a direct toxic effect on the myocardium. Catecholamines also exert a receptor-mediated effect on the myocardium. Catecholamine-mediated myocardial stunning has been implicated in the pathogenesis of stress-induced cardiomyopathy. Biopsy of the myocardium in patients with pheochromocytoma or those with stress-induced cardiomyopathy shows similar pathologic findings. The clinical features in pheochromocytoma-related cardiomyopathy include hypertension, dilated or hypertrophic cardiomyopathy, pulmonary edema due to cardiogenic and noncardiogenic factors, cardiac arrhythmias, and even cardiac arrest. Stress-related cardiomyopathy such as takotsubo cardiomyopathy occurs primarily in postmenopausal women. These patients may present with clinical features suggestive of an acute myocardial infarction or a hemodynamically compromised state. The definitive management of cardiomyopathy associated with pheochromocytoma includes medical treatment with alpha-adrenergic blockade, possibly along with angiotensin converting enzyme blockers and beta1-adrenergic receptor blockers, followed by excision of the tumor. Stress-induced cardiomyopathy is usually self-limiting; patients may require support with nonadrenergic inotropes. CONCLUSION: Recognition of catecholamine-induced cardiomyopathy, especially in patients with pheochromocytoma, before surgical treatment is important to minimize morbidity and mortality.

Responses of benthic macroinvertebrates to thin-layer disposal of dredged material in Mississippi Sound, USA.

Benthic community responses to thin-layer disposal of dredged material were assessed at three sites in Mississippi Sound, USA. Community composition differed between disposal and reference sites (ANOSIM) for each disposal event. Oweniid sand worms, Amphinomid fire worms, and brittle stars (Ophiuriodea) contributed the most to community distinctions between disposal and reference sites (SIMPER), with higher abundances at the disposal sites. Total infaunal abundance was similar to pre-disposal and reference conditions within 3-10 months of thin-layer disposal. Distinctions in community composition between disposal and reference sites (nMDS) persisted throughout the 16-month study period, but were less pronounced at the site where sediment composition was unchanged by disposal. Size distributions of some taxa (e.g., gastropod and hemichordate) suggest adults recolonized the newly deposited sediments either through vertical migration or lateral immigration from adjacent areas. Thin-layer disposal offers a viable alternative to conventional open-water disposal practices and warrants further exploration for other areas with deeper bathymetries and different hydrodynamic regimes.