Characterization of polycyclic aromatic compounds in historically contaminated soil by targeted and non-targeted chemical analysis combined with in vitro bioassay

Soil samples from a contaminated site in Sweden were analyzed to identify the presence of 78 polycyclic aromatic compounds (PACs) using gas chromatography coupled with mass spectrometry (GC-MS). The target analysis revealed large contributions not only from polycyclic aromatic hydrocarbons (PAHs), but also from alkylated- and oxygenated-PAHs (alkyl- and oxy-PAHs, respectively), and N-heterocyclics (NPACs). PAC profiles indicated primarily pyrogenic sources, although contribution of petrogenic sources was also observed in one sample as indicated by a high ratio of alkylated naphthalene compared to naphthalene. The aryl hydrocarbon receptor (AhR)-activity of the soil extracts was assessed using the H4IIe-pGudluc 1.1 cells bioassay. When compared with the calculated total AhR-activity of the PACs in the target list, 35-97% of the observed bioassay activity could be explained by 62 PACs with relative potency factors (REPs). The samples were further screened using GC coupled with OrbitrapTM high resolution MS (GC-HRMS) to investigate the presence of other PACs that could potentially contribute to the AhR-activity of the extracts. 114 unique candidate compounds were tentatively identified and divided into four groups based on their AhR-activity and environmental occurrence. Twelve substances satisfied all the criteria, and these compounds are suggested to be included in regular screening in future studies, although their identities were not confirmed by standards in this study. High unexplained bio-TEQ fractions in three of the samples may be explained by tentatively identified compounds (n = 35) with high potential of being toxic. This study demonstrates the benefit of combining targeted and non-targeted chemical analysis with bioassay analysis to assess the diversity and effects of PACs at contaminated sites. The applied prioritization strategy revealed a number of tentatively identified compounds, which likely contributed to the overall bioactivity of the soil extracts

An exploration of the mechanisms behind peripheral nerve injury

Despite surgical innovation, the sensory and motor outcome after peripheral nerve injury is incomplete. In this thesis, the biological pathways potentially responsible for the poor functional recoveries were investigated in both the distal nerve stump/target organ, spinal motoneurons and dorsal root ganglia (DRG). The effect of delayed nerve repair was determined in a rat sciatic nerve transection model. There was a dramatic decline in the number of regenerating motoneurons and myelinated axons found in the distal nerve stumps of animals undergoing nerve repair after a delay of 3 and 6 months. RT-PCR of the distal nerve stumps showed a decline in expression of Schwann cells (SC) markers, with a progressive increase in fibrotic and proteoglycan scar markers, with increased delayed repair time. Furthermore, the yield of SC which could be isolated from the distal nerve segments progressively fell with increased delay in repair time. Consistent with the impaired distal nerve stumps the target medial gastrocnemius (MG) muscles at 3- and 6-months delayed repair were atrophied with significant declines in wet weights (61% and 27% compared with contralateral sides). The role of myogenic transcription factors, muscle specific microRNAs and musclespecific E3 ubiquitin ligases in the muscle atrophy was investigated in both gastrocnemius and soleus muscles following either crush or nerve transection injury. In the crush injury model, the soleus muscle showed significantly increased recovery in wet weight at days 14 and 28 (compared with day 7) which was not the case for the gastrocnemius muscle which continued to atrophy. There was a significantly more pronounced up-regulation of MyoD expression in the denervated soleus muscle compared with the gastrocnemius muscle. Conversely, myogenin was more markedly elevated in the gastrocnemius versus soleus muscles. The muscles also showed significantly contrasting transcriptional regulation of the microRNAs miR-1 and miR-206. MuRF1 and Atrogin-1 showed the highest levels of expression in the denervated gastrocnemius muscle. Morphological and molecular changes in spinal motoneurons were compared after L4-L5 ventral root avulsion (VRA) and distal peripheral nerve axotomy (PNA). Neuronal degeneration was indicated by decreased immunostaining for microtubule-associated protein-2 in dendrites and synaptophysin in presynaptic boutons after both VRA and PNA. Immunostaining for ED1-reactive microglia and GFAPpositive astrocytes was significantly elevated in all experimental groups. qRT-PCR analysis and Western blotting of the ventral horn from L4-L5 spinal cord segments revealed a significant upregulation of apoptotic cell death mediators including caspases-3 and -8 and a range of related death receptors following VRA. In contrast, following PNA, only caspase-8 was moderately upregulated. The mechanisms of primary sensory neuron degeneration were also investigated in the DRG following peripheral nerve axotomy, where several apoptotic pathways including those involving the endoplasmic reticulum were shown to be upregulated. In summary, these results show that the critical time point after which the outcome of regeneration becomes too poor appears to be 3-months. Both proximal and distal injury affect spinal motoneurons morphologically, but VRA induces motoneuron degeneration mediated through both intrinsic and extrinsic apoptotic pathways. Primary sensory neuron degeneration involves several different apoptotic pathways, including the endoplasmic reticulum

Water jet-assisted lipoaspiration and Sepax cell separation system for the isolation of adipose stem cells with high adipogenic potential

Introduction: Water jet-assisted liposuction has gained popularity due to favourable fat grafting outcomes. In this study, we compared stem cells obtained from fat isolated with manual or the water jet-assisted procedure. Methods: Liposuction of abdominal fat was performed using the two methods on each donor (n = 10). Aspirate samples were collagenase digested and the isolated cells seeded in vitro prior to proliferation, adipogenic differentiation and angiogenic activity analyses. Results: Cells from either procedure proliferated at similar rates and exhibited a similar colony-forming ability. The cells expressed stem cell markers CD73, CD90 and CD105. In the water jet cell preparations, there were higher numbers of cells expressing CD146. Robust adipogenic differentiation was observed in cultures expanded from both manual and water jet lipoaspirates. Gene analysis showed higher expression of the adipocyte markers aP2 and GLUT4 in the adipocyte-differentiated water jet cell preparations, and ELISA indicated increased secretion of adiponectin from these cells. Both cell groups expressed vasculogenic factors and the water jet cells promoted the highest levels of in vitro angiogenesis. Given these positive results, we further characterised the water jet cells when prepared using an automated closed cell processing unit, the Sepax-2 system (Cytiva). The growth and stem cell properties of the Sepax-processed cells were similar to the standard centrifugation protocol, but there was evidence for greater adipogenic differentiation in the Sepax-processed cells. Conclusions: Water jet lipoaspirates yield cells with high adipogenic potential and angiogenic activity, which may be beneficial for use in cell-assisted lipotransfers

Development and validation of an in vitro model system to study peripheral sensory neuron development and injury

The ability to discriminate between diverse types of sensation is mediated by heterogeneous populations of peripheral sensory neurons. Human peripheral sensory neurons are inaccessible for research and efforts to study their development and disease have been hampered by the availability of relevant model systems. The in vitro differentiation of peripheral sensory neurons from human embryonic stem cells therefore provides an attractive alternative since an unlimited source of biological material can be generated for studies that specifically address development and injury. The work presented in this study describes the derivation of peripheral sensory neurons from human embryonic stem cells using small molecule inhibitors. The differentiated neurons express canonical- and modality-specific peripheral sensory neuron markers with subsets exhibiting functional properties of human nociceptive neurons that include tetrodotoxin-resistant sodium currents and repetitive action potentials. Moreover, the derived cells associate with human donor Schwann cells and can be used as a model system to investigate the molecular mechanisms underlying neuronal death following peripheral nerve injury. The quick and efficient derivation of genetically diverse peripheral sensory neurons from human embryonic stem cells offers unlimited access to these specialised cell types and provides an invaluable in vitro model system for future studies

Investigation of the Expression of Myogenic Transcription Factors, microRNAs and Muscle-Specific E3 Ubiquitin Ligases in the Medial Gastrocnemius and Soleus Muscles following Peripheral Nerve Injury

Despite surgical innovation, the sensory and motor outcome after a peripheral nerve injury remains incomplete. One contributing factor to the poor outcome is prolonged denervation of the target organ, leading to apoptosis of both mature myofibres and satellite cells with subsequent replacement of the muscle tissue with fibrotic scar and adipose tissue. In this study, we investigated the expression of myogenic transcription factors, muscle specific microRNAs and muscle-specific E3 ubiquitin ligases at several time points following denervation in two different muscles, the gastrocnemius (containing predominantly fast type fibres) and soleus (slow type) muscles, since these molecules may influence the degree of atrophy following denervation. Both muscles exhibited significant atrophy (compared with the contra-lateral sides) at 7 days following either a nerve transection or crush injury. In the crush model, the soleus muscle showed significantly increased muscle weights at days 14 and 28 which was not the case for the gastrocnemius muscle which continued to atrophy. There was a significantly more pronounced up-regulation of MyoD expression in the denervated soleus muscle compared with the gastrocnemius muscle. Conversely, myogenin was more markedly elevated in the gastrocnemius versus soleus muscles. The muscles also showed significantly contrasting transcriptional regulation of the microRNAs miR-1 and miR-206. MuRF1 and Atrogin-1 showed the highest levels of expression in the denervated gastrocnemius muscle. This study provides further insights regarding the intracellular regulatory molecules that generate and maintain distinct patterns of gene expression in different fibre types following peripheral nerve injury

Long-Term Effects of Fibrin Conduit with Human Mesenchymal Stem Cells and Immunosuppression after Peripheral Nerve Repair in a Xenogenic Model

Introduction: Previously we showed that a fibrin glue conduit with human mesenchymal stem cells (hMSCs) and cyclosporine A (CsA) enhanced early nerve regeneration. In this study long term effects of this conduit are investigated. Methods: In a rat model, the sciatic nerve was repaired with fibrin conduit containing fibrin matrix, fibrin conduit containing fibrin matrix with CsA treatment and fibrin conduit containing fibrin matrix with hMSCs and CsA treatment, and also with nerve graft as control. Results: At 12 weeks 34% of motoneurons of the control group regenerated axons through the fibrin conduit. CsA treatment alone or with hMSCs resulted in axon regeneration of 67% and 64% motoneurons respectively. The gastrocnemius muscle weight was reduced in the conduit with fibrin matrix. The treatment with CsA or CsA with hMSCs induced recovery of the muscle weight and size of fast type fibers towards the levels of the nerve graft group. Discussion: The transplantation of hMSCs for peripheral nerve injury should be optimized to demonstrate their beneficial effects. The CsA may have its own effect on nerve regeneration

Prophylactic mastectomy – Correlation between skin flap thickness and residual glandular tissue evaluated postoperatively by imaging

Background: Women with an increased hereditary risk of breast cancer can undergo risk-reducing prophylactic mastectomy. However, there is a balance between how much subcutaneous tissue should be resected to achieve maximal reduction of glandular tissue, while leaving viable skin flaps. Methods: Forty-five women previously operated with prophylactic mastectomy underwent magnetic resonance tomography (MRT) and ultrasound (US) to investigate the correlation between skin flap thickness and residual glandular tissue. Residual glandular tissue was documented as being present or not present, but not quantified, as the amount of residual glandular tissue in many cases was considered too small to make reliable volume quantifications with available tools. Since a mastectomy skin flap thickness of 5 mm is discussed as an oncologically safe thickness in the literature, this was used as a cut-off. Results: Following prophylactic mastectomy, residual glandular tissue was detected in 39.3% of all breasts and 27.9% of all the breast quadrants examined by MRT, and 44.1% of all breasts and 21.7% of all the breast quadrants examined by US. Residual glandular tissue was detected in 6.9% of the quadrants in skin flaps ≤ 5 mm and in 37.5% of the quadrants in skin flaps > 5 mm (OR 3.07; CI = 1.41–6.67; p = 0.005). Furthermore, residual glandular tissue increased significantly already when the skin flap thickness exceeded 7 mm. Conclusions: This study highlights that complete removal of glandular breast tissue during a mastectomy is difficult and suggests that this is an unattainable goal. We demonstrate that residual glandular tissue is significantly higher in skin flaps > 5 mm in comparison to skin flaps ≤ 5 mm, and that residual glandular tissue increases significantly already when the flap thickness exceeds 7 mm

Ecological risk assessment for the Western Australian offshore crustacean resource.

In December 2022, the Department of Primary Industries and Regional Development (DPIRD) convened an ecological risk assessment (ERA) of the fisheries that access the Offshore Crustacean Resource (Resource). The Western Australian commercial fisheries that access the Resource are the West Coast Deep Sea Crustacean Managed Fishery, South Coast Crustacean Managed Fishery and West Coast Rock Lobster Managed Fishery. Due to the predominantly offshore distribution of the Resource, there is only minor recreational and customary access of this Resource related to harvesting of southern rock lobster on the south coast. The ERA considered the potential ecological impacts of harvesting the Resource. The assessment focused on evaluating the impact of the commercial fishing sector on all relevant retained and bycatch species, endangered, threatened and protected (ETP) species, habitats and the broader environment. A broad range of stakeholders were invited to participate in the ERA workshop, including representatives of the commercial, recreational, customary and aquaculture fishing sectors, State and Commonwealth Government agencies, the conservation sector, universities and DPIRD staff including fisheries management, research, compliance and biosecurity personnel. Risk scores were determined based on available scientific information and expert knowledge. The assessment conforms to the AS/NZS ISO 31000 risk management standard, and to the methodology adopted by DPIRD which uses a consequence-likelihood analysis for estimating risk. Forty three ecological components were scored for risk. The majority (34) of components were evaluated as low or negligible risks, which do not require any specific control measures. Four components were evaluated as medium risks, which were assessed as acceptable under the current monitoring and control measures already in place. There were four high risks and one severe risk, which all related to stocks of the primary target species. These stocks had been formally assessed by DPIRD prior to this ERA and the pre-existing risk scores from those assessments were adopted in this ERA. Management changes have already been implemented that are expected to reduce the risk for each stock to an acceptable level. It is recommended that all risks be reviewed in five years

Outcome following soft tissue coverage with a medial gastrocnemius flap of an exposed or infected total knee arthroplasty

BACKGROUND: Soft tissue defects or periprosthetic infections after total knee arthroplasty (TKA) are severe complications that may lead to loss of the arthroplasty or the limb. Reconstructions with medial gastrocnemius flaps (MGF) are occasionally used to provide soft tissue coverage around the knee. AIMS: The study aimed to establish the rate of implant survivorship after MGF reconstruction for soft tissue coverage in the treatment of exposed or infected TKA and to establish functional outcome. METHODS: A retrospective analysis was performed on all patients who received soft tissue coverage with an MGF of an exposed or infected TKA between 2000 and 2017 at the Department of Hand and Plastic Surgery at Umeå University Hospital. The outcomes were implant survivorship and patient-reported outcome measures (PROMs) using the five-level EQ-5D version and The Knee Injury and Osteoarthritis Outcome Score. RESULTS: Forty-seven patients (mean age = 67 years, 30 women) were included. The mean time between flap coverage and follow-up was 6.7 (±3.4) years. Implant survivorship was observed in 28 of 47 (59.6%) patients at follow-up. Flap failure was rare, with only 3 of 47 (6.4%) cases. Of the 20 patients who answered the PROMs, 10 of 20 experienced moderate to severe pain or discomfort. CONCLUSIONS: Due to unfavorable underlying conditions, MGF reconstruction after TKA is often associated with a compromised functional outcome. Because donor site morbidity is limited and flap failure is unusual, the procedure can be considered prophylactically in a small subset of patients with risk factors to prevent soft tissue defects and periprosthetic joint infection

Interaction of adipose-derived stem cells with active and dormant breast cancer cells

Background: Although autologous fat grafting is considered a successful method for the management of contour deformities, the fat graft could potentially induce cancer reappearance by fueling dormant breast cancer cells. Our aim was to characterize the role of adipose-derived stem cells on active and dormant breast cancer cell growth. Methods: Cobalt chloride was used to induce dormancy in MCF-7 cancer cells. Proliferation of active and dormant cancer cells was determined in the presence of adipose-derived stem cells. A proteome array was used to detect cancer-related protein expression in the cell-conditioned medium. The migration of cancer cells was measured in response to conditioned medium from the adipose-derived stem cells. Results: The adipose-derived stem cells showed variable effects on active MCF-7 cells growth and inhibited MCF-7 proliferation after the withdrawal of cobalt chloride. Of the 84 different proteins measured in the conditioned medium, only tenascin-C was differentially expressed in the co-cultures. MCF-7 cells alone did not express tenascin-C, whereas co-cultures between MCF-7 and adipose-derived stem cells expressed more tenascin-C versus adipose-derived stem cells alone. The conditioned medium from co-cultures significantly increased the migration of the cancer cells. Conclusions: Adipose-derived stem cells themselves neither increased the growth or migration of cancer cells, suggesting that autologous fat grafting may be oncologically safe if reconstruction is postponed until there is no evidence of active disease. However, interactions between adipose-derived stem cells and MCF-7 cancer cells could potentially lead to the production of factors, which further promote cancer cell migration