Melanoma cells adopt features of both mesenchymal and amoeboid migration within confining channels.

For metastasis to occur, cancer cells must traverse a range of tissue environments. In part, this is accomplished by cells adjusting their migration mode to one that is best suited to the environment. Melanoma cells have been shown to be particularly plastic, frequently using both mesenchymal and amoeboid (bleb-based) modes of migration. It has been demonstrated that 2D confinement will promote the transition from mesenchymal to bleb-based migration. However, if melanoma cells similarly transition to bleb-based migration in response to 3D confinement, such as within narrow channels, is unknown. Here, using micro-fabricated channels, we demonstrate that metastatic, A375-M2, melanoma cells adopt features of both mesenchymal and bleb-based migration. In narrow (8 µm; height and width) channels coated with fibronectin, ~ 50% of melanoma cells were found to use either mesenchymal or bleb-based migration modes. In contrast, the inhibition of Src family kinases or coating channels with BSA, completely eliminated any features of mesenchymal migration. Detailed comparisons of migration parameters revealed that blebbing cells, particularly in the absence of adhesions, were faster than mesenchymal cells. In contrast to what has been previously shown under conditions of 2D confinement, pharmacologically inhibiting Arp2/3 promoted a fast filopodial-based mode of migration. Accordingly, we report that melanoma cells adopt a unique range of phenotypes under conditions of 3D confinement.

A Meta-Analysis of Autologous Microsurgical Breast Reconstruction and Timing of Adjuvant Radiation Therapy.

BACKGROUND: Postmastectomy radiation therapy (PMRT) decreases loco-regional recurrence and improves survival in patients with locally advanced breast cancer. Autologous free flap reconstruction, while more durable in the setting of radiation than alloplastic reconstruction, is still susceptible to radiation-induced fibrosis, contracture, fat necrosis, volume loss, and distortion of breast shape. Options for reconstruction timing (immediate vs. delayed) have been discussed to mitigate these effects, but a clear optimum is not known. METHODS: A systematic review of the literature was conducted according to PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) guidelines using search terms "breast reconstruction AND (radiation OR irradiation OR radiotherapy)" were used. Inclusion criteria consisted of studies reporting complications for free flap breast reconstruction in the setting of PMRT. Patients who underwent PMRT were pooled into two groups: those who underwent immediate free flap reconstruction prior to PMRT and those who underwent delayed reconstruction after PMRT. RESULTS: Out of the 23 studies, 12 focused on immediate reconstruction, seven focused on delayed reconstruction, and four studies included both groups. Overall, 729 patients underwent immediate reconstruction, while 868 underwent delayed reconstruction. Complete and partial flap loss rates were significantly higher in patients undergoing delayed reconstruction, while infection and wound-healing complication rates were higher in those undergoing immediate reconstructions. Rates of unplanned reoperations, vascular complications, hematoma/seroma, and fat necrosis did not differ significantly between the two groups. However, rates of planned revision surgeries were higher in the delayed reconstruction group. CONCLUSION: Immediate free flap breast reconstruction is associated with superior flap survival compared with delayed reconstruction. Rates of complications are largely comparable, and rates of revision surgeries are equivalent. The differences in long-term aesthetic outcomes are not, however, clearly assessed by the available literature. Even in the face of PMRT, immediate free flap breast reconstruction is an effective approach.

Analysis of shunted hydrocephalus follow-up: What do routine clinic visits yield? What factors affect revision surgery presentation and outcomes?

Frequency and duration of outpatient clinic follow-up for patients with shunted hydrocephalus varies among clinicians and assessment of follow-up regimens is lacking. The aim of this study is to investigate whether routine clinic visits alter care and whether they identify patients requiring shunt revision surgery, as well as, to better understand how patients utilize the outpatient clinic and present for shunt revision evaluation. This is a single-centered retrospective study of 154 patients requiring shunt revision surgery from 2009 to 2018 who had at least one prior clinic evaluation. The median age for shunt placement and revision were 3 months and 11 years old, respectively. Routine clinic visits led to a change in care for 16 patients (10.4%); including additional imaging, follow-up, or a combination of the two. With regards to revision surgery, days from prior shunt surgery, Chiari II/myelomeningocele pathology, and shunt type (p < 0.01) did affect time to presentation. Four patients (2.6%) requiring revision surgery were identified at routine clinic follow-up, while 92 (59.7%) and 47 (30.5%) presented to the emergency department and clinic sick visit, respectively. Presentation to clinic resulted in a statistically significant decrease in shunt revision surgery length-of-stay compared to presentation to the emergency department or inpatient admission for another condition. Even with increased emergency room utilization, increased clinic connectivity, and improved patient education, routine clinic visits remain an important component in the follow-up of patients with shunted hydrocephalus by helping to guide clinical care and identify patients requiring shunt revision surgery.

NOTCH3 is non-enzymatically fragmented in inherited cerebral small-vessel disease.

The small-vessel disorder cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) arises from mutations in the human gene encoding NOTCH3 and results in vascular smooth muscle cell degeneration, stroke, and dementia. However, the structural changes in NOTCH3 involved in CADASIL etiology are unclear. Here, we discovered site-specific fragmentation of NOTCH3 protein in pathologically affected vessels of human CADASIL-affected brains. EM-based experiments to pinpoint NOTCH3 localization in these brains indicated accumulation of NOTCH3 fragmentation products in the basement membrane, collagen fibers, and granular osmiophilic material within the cerebrovasculature. Using antibodies generated against a disease-linked neo-epitope found in degenerating vascular medium of CADASIL brains, we mapped the site of fragmentation to the NOTCH3 N terminus at the peptide bond joining Asp80 and Pro81 Cleavage at this site was predicted to separate the first epidermal growth factor (EGF)-like domain from the remainder of the protein. We found that the cleavage product from this fragmentation event is released into the conditioned medium of cells expressing recombinant NOTCH3 fragments. Mutagenesis of Pro81 abolished the fragmentation, and low pH and reducing conditions enhanced NOTCH3 proteolysis. Furthermore, substitution of multiple cysteine residues of the NOTCH3 N terminus activated proteolytic release of the first EGF-like repeat, suggesting that the elimination of multiple disulfide bonds in NOTCH3 accelerates its fragmentation. These characteristics link the signature molecular genetic alterations present in individuals with CADASIL to a post-translational protein alteration in degenerating brain arteries. The cellular consequences of these pathological NOTCH3 fragments are an important area for future investigation.