Besides intrinsic changes, malignant cells also release soluble signals that reshape their microenvironment. Among these signals is WNT1-inducible signaling pathway protein 1 (WISP1), a secreted ...matricellular protein whose expression is elevated in several cancers, including melanoma, and is associated with reduced survival of patients diagnosed with primary melanoma. Here, we found that WISP1 knockout increases cell proliferation and represses wound healing, migration, and invasion of mouse and human melanoma cells in multiple in vitro assays. Metastasis assays revealed that WISP1 knockout represses tumor metastasis of B16F10 and YUMM1.7 melanoma cells in both C57BL/6Ncrl and NOD-scid IL2Rγnull (NSG) mice. WT B16F10 cells having an invasion phenotype in a transwell assay possessed a gene expression signature similar to that observed in the epithelial–mesenchymal transition (EMT), including E-cadherin repression and fibronectin and N-cadherin induction. Upon WISP1 knockout, expression of these EMT signature genes went in the opposite direction in both mouse and human cell lines, and EMT-associated gene expression was restored upon exposure to media containing WISP1 or to recombinant WISP1 protein. In vivo, Wisp1 knockout–associated metastasis repression was reversed by the reintroduction of either WISP1 or snail family transcriptional repressor 1 (SNAI1). Experiments testing EMT gene activation and inhibition with recombinant WISP1 or kinase inhibitors in B16F10 and YUMM1.7 cells suggested that WISP1 activates AKT Ser/Thr kinase and that MEK/ERK signaling pathways shift melanoma cells from proliferation to invasion. Our results indicate that WISP1 present within the tumor microenvironment stimulates melanoma invasion and metastasis by promoting an EMT-like process.
Human disease pathophysiology commonly involves metabolic disruption at both the cellular and subcellular levels. Isolated mitochondria are a powerful model for separating global cellular changes ...from intrinsic mitochondrial alterations. However, common laboratory practices for isolating mitochondria (e.g., differential centrifugation) routinely results in organelle preparations with variable mitochondrial purity. To overcome this issue, we developed a mass spectrometry-based method that quantitatively evaluates sample-specific percent mitochondrial enrichment. Sample-specific mitochondrial enrichment was then used to correct various biochemical readouts of mitochondrial function to a 'fixed' amount of mitochondrial protein, thus allowing for intrinsic mitochondrial bioenergetics, relative to the underlying proteome, to be assessed across multiple mouse tissues (e.g., heart, brown adipose, kidney, liver). Our results support the use of mitochondrial-targeted nLC-MS/MS as a method to quantitate mitochondrial enrichment on a per-sample basis, allowing for unbiased comparison of functional parameters between populations of mitochondria isolated from metabolically distinct tissues. This method can easily be applied across multiple experimental settings in which intrinsic shifts in the mitochondrial network are suspected of driving a given physiological or pathophysiological outcome.
Obesity, characterized by chronic low-grade inflammation of the adipose tissue, is associated with adverse coronavirus disease 2019 (COVID-19) outcomes, yet the underlying mechanism is unknown. To ...explore whether severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection of adipose tissue contributes to pathogenesis, we evaluated COVID-19 autopsy cases and deeply profiled the response of adipose tissue to SARS-CoV-2 infection in vitro. In COVID-19 autopsy cases, we identified SARS-CoV-2 RNA in adipocytes with an associated inflammatory infiltrate. We identified two distinct cellular targets of infection: adipocytes and a subset of inflammatory adipose tissue-resident macrophages. Mature adipocytes were permissive to SARS-CoV-2 infection; although macrophages were abortively infected, SARS-CoV-2 initiated inflammatory responses within both the infected macrophages and bystander preadipocytes. These data suggest that SARS-CoV-2 infection of adipose tissue could contribute to COVID-19 severity through replication of virus within adipocytes and through induction of local and systemic inflammation driven by infection of adipose tissue-resident macrophages.
Summary Background Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease of upper and lower motor neurons, associated with frontotemporal dementia (FTD) in about 14% of ...incident cases. We assessed the frequency of the recently identified C9orf72 repeat expansion in familial and apparently sporadic cases of ALS and characterised the cognitive and clinical phenotype of patients with this expansion. Methods A population-based register of patients with ALS has been in operation in Ireland since 1995, and an associated DNA bank has been in place since 1999. 435 representative DNA samples from the bank were screened using repeat-primed PCR for the presence of a GGGGCC repeat expansion in C9orf72 . We assessed clinical, cognitive, behavioural, MRI, and survival data from 191 (44%) of these patients, who comprised a population-based incident group and had previously participated in a longitudinal study of cognitive and behavioural changes in ALS. Findings Samples from the DNA bank included 49 cases of known familial ALS and 386 apparently sporadic cases. Of these samples, 20 (41%) cases of familial ALS and 19 (5%) cases of apparently sporadic ALS had the C9orf72 repeat expansion. Of the 191 patients for whom phenotype data were available, 21 (11%) had the repeat expansion. Age at disease onset was lower in patients with the repeat expansion (mean 56·3 SD 8·3 years) than in those without (61·3 10·6 years; p=0·043). A family history of ALS or FTD was present in 18 (86%) of those with the repeat expansion. Patients with the repeat expansion had significantly more co-morbid FTD than patients without the repeat (50% vs 12%), and a distinct pattern of non-motor cortex changes on high-resolution 3 T magnetic resonance structural neuroimaging. Age-matched univariate analysis showed shorter survival (20 months vs 26 months) in patients with the repeat expansion. Multivariable analysis showed an increased hazard rate of 1·9 (95% 1·1–3·7; p=0·035) in those patients with the repeat expansion compared with patients without the expansion Interpretation Patients with ALS and the C9orf72 repeat expansion seem to present a recognisable phenotype characterised by earlier disease onset, the presence of cognitive and behavioural impairment, specific neuroimaging changes, a family history of neurodegeneration with autosomal dominant inheritance, and reduced survival. Recognition of patients with ALS who carry an expanded repeat is likely to be important in the context of appropriate disease management, stratification in clinical trials, and in recognition of other related phenotypes in family members. Funding Health Seventh Framework Programme, Health Research Board, Research Motor Neuron, Irish Motor Neuron Disease Association, The Motor Neurone Disease Association of Great Britain and Northern Ireland, ALS Association.
This study presents performance specifications of an in‐house developed human leukocyte antigen (HLA) typing assay using next‐generation sequencing (NGS) on the Illumina MiSeq platform. A total of ...253 samples, previously characterized for HLA‐A, ‐B, ‐C, ‐DRB1 and ‐DQB1 were included in this study, which were typed at high‐resolution using a combination of Sanger sequencing, sequence‐specific primer (SSP) and sequence‐specific oligonucleotide probe (SSOP) technologies and recorded at the two‐field level. Samples were selected with alleles that cover a high percentage of HLA specificities in each of five different race/ethnic groups: European, African‐American, Asian Pacific Islander, Hispanic and Native American. Sequencing data were analyzed by two software programs, Omixon's target and GenDx's NGSengine. A number of metrics including allele balance, sensitivity, specificity, precision, accuracy and remaining ambiguity were assessed. Data analyzed by the two software systems are shown independently. The majority of alleles were identical in the exonic sequences (third field) with both programs for HLA‐A, ‐B, ‐C and ‐DQB1 in 97.7% of allele determinations. Among the remaining discrepant genotype calls at least one of the analysis programs agreed with the reference typing. Upon additional manual analysis 100% of the 2530 alleles were concordant with the reference HLA genotypes; the remaining ambiguities did not exceed 0.8%. The results demonstrate the feasibility and significant benefit of HLA typing by NGS as this technology is highly accurate, eliminates virtually all ambiguities, provides complete sequencing information for the length of the HLA gene and forms the basis for utilizing a single methodology for HLA typing in the immunogenetics labs.
The purported migrations that have formed the peoples of Britain have been the focus of generations of scholarly controversy. However, this has not benefited from direct analyses of ancient genomes. ...Here we report nine ancient genomes (∼ 1 ×) of individuals from northern Britain: seven from a Roman era York cemetery, bookended by earlier Iron-Age and later Anglo-Saxon burials. Six of the Roman genomes show affinity with modern British Celtic populations, particularly Welsh, but significantly diverge from populations from Yorkshire and other eastern English samples. They also show similarity with the earlier Iron-Age genome, suggesting population continuity, but differ from the later Anglo-Saxon genome. This pattern concords with profound impact of migrations in the Anglo-Saxon period. Strikingly, one Roman skeleton shows a clear signal of exogenous origin, with affinities pointing towards the Middle East, confirming the cosmopolitan character of the Empire, even at its northernmost fringes.
Depressional wetlands of the extensive U.S. and Canadian Prairie Pothole Region afford numerous ecosystem processes that maintain healthy watershed functioning. However, these wetlands have been lost ...at a prodigious rate over past decades due to drainage for development, climate effects, and other causes. Options for management entities to protect the existing wetlands, and their functions, may focus on conserving wetlands based on spatial location vis-à-vis a floodplain or on size limitations (e.g., permitting smaller wetlands to be destroyed but not larger wetlands). Yet the effects of such management practices and the concomitant loss of depressional wetlands on watershed-scale hydrological, biogeochemical, and ecological functions are largely unknown. Using a hydrological model, we analyzed how different loss scenarios by wetland size and proximal location to the stream network affected watershed storage (i.e., inundation patterns and residence times), connectivity (i.e., streamflow contributing areas), and export (i.e., streamflow) in a large watershed in the Prairie Pothole Region of North Dakota, USA. Depressional wetlands store consequential amounts of precipitation and snowmelt. The loss of smaller depressional wetlands (< 3.0 ha) substantially decreased landscape-scale inundation heterogeneity, total inundated area, and hydrological residence times. Larger wetlands act as hydrologic “gatekeepers,” preventing surface runoff from reaching the stream network, and their modeled loss had a greater effect on streamflow due to changes in watershed connectivity and storage characteristics of larger wetlands. The wetland management scenario based on stream proximity (i.e., protecting wetlands 30 m and ~450 m from the stream) alone resulted in considerable landscape heterogeneity loss and decreased inundated area and residence times. With more snowmelt and precipitation available for runoff with wetland losses, contributing area increased across all loss scenarios. We additionally found that depressional wetlands attenuated peak flows; the probability of increased downstream flooding from wetland loss was also consistent across all loss scenarios. It is evident from this study that optimizing wetland management for one end goal (e.g., protection of large depressional wetlands for flood attenuation) over another (e.g., protecting of small depressional wetlands for biodiversity) may come at a cost for overall watershed hydrological, biogeochemical, and ecological resilience, functioning, and integrity.
Background and purpose
Primary lateral sclerosis (PLS) is a progressive upper motor neuron disorder associated with considerable clinical disability. Symptoms are typically exclusively linked to ...primary motor cortex degeneration and the contribution of pre‐motor, supplementary motor, cortico‐medullary and inter‐hemispheric connectivity alterations are less well characterized.
Methods
In a single‐centre, prospective, longitudinal neuroimaging study 41 patients with PLS were investigated. Patients underwent standardized neuroimaging, genetic profiling with whole exome sequencing, and comprehensive clinical assessments including upper motor neuron scores, tapping rates, mirror movements, spasticity assessment, cognitive screening and evaluation for pseudobulbar affect. Longitudinal neuroimaging data from 108 healthy controls were used for image interpretation. A standardized imaging protocol was implemented including 3D T1‐weighted structural, diffusion tensor imaging and resting‐state functional magnetic resonance imaging. Following somatotopic segmentation, cortical thickness analyses, probabilistic tractography, blood oxygenation level dependent signal analyses and brainstem volumetry were conducted to evaluate cortical, brainstem, cortico‐medullary and inter‐hemispheric connectivity alterations both cross‐sectionally and longitudinally.
Results
Our data confirm progressive primary motor cortex degeneration, considerable supplementary motor and pre‐motor area involvement, progressive brainstem atrophy, cortico‐medullary and inter‐hemispheric disconnection, and close associations between clinical upper motor neuron scores and somatotopic connectivity indices in PLS.
Discussion
Primary lateral sclerosis is associated with relentlessly progressive motor connectome degeneration. Clinical disability in PLS is likely to stem from a combination of intra‐ and inter‐hemispheric connectivity decline and primary, pre‐ and supplementary motor cortex degeneration. Simple ‘bedside’ clinical tools, such as tapping rates, are excellent proxies of the integrity of the relevant fibres of the contralateral corticospinal tract.
The rate of hiatal hernia (HH) repair during conversion bariatric surgery is largely unknown. We sought to determine this rate in 12,788 patients undergoing conversion surgery using the 2020 ...participant use file of the MBSAQIP database. Concurrent HH repair was performed in 24.1% of conversion cases; most commonly during SG to RYGB (33.1%), followed by AGB to SG conversion (20.2%). The remaining conversion pathways had a repair rate around 13%. Only 12.1% of HH repairs were performed using a mesh. GERD was the primary indication for conversion in 65% of the SG to RYGB cases. A much higher proportion of patients with concomitant HH repair reported GERD as the main reason for conversion than those without a HH repair (44.5% vs. 23.7%; p<0.001).
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