Apical expansion of calvarial osteoblasts and suture patency is dependent on fibronectin cues.

The skull roof, or calvaria, is comprised of interlocking plates of bones that encase the brain. Separating these bones are fibrous sutures that permit growth. Currently, we do not understand the instructions for directional growth of the calvaria, a process which is error-prone and can lead to skeletal deficiencies or premature suture fusion (craniosynostosis, CS). Here, we identify graded expression of fibronectin (FN1) in the mouse embryonic cranial mesenchyme (CM) that precedes the apical expansion of calvaria. Conditional deletion of Fn1 or Wasl leads to diminished frontal bone expansion by altering cell shape and focal actin enrichment, respectively, suggesting defective migration of calvarial progenitors. Interestingly, Fn1 mutants have premature fusion of coronal sutures. Consistently, syndromic forms of CS in humans exhibit dysregulated FN1 expression, and we also find FN1 expression altered in a mouse CS model of Apert syndrome. These data support a model of FN1 as a directional substrate for calvarial osteoblast migration that may be a common mechanism underlying many cranial disorders of disparate genetic etiologies.

Risk Factors for Surgical Site Complications After Outpatient Lumbar Spine Surgery.

Background: With the recent increase of minor lumbar spine surgeries being performed in the outpatient setting, there is a need for information on factors that contribute to post-operative complications for these surgeries. Patients and Methods: This was a prospective observational study examining risk factors for self-reported post-operative drainage in patients who underwent lumbar spine surgery. Patient surveys and the hospital's electronic medical records were used to collect data on patient demographic, patient lifestyle, and surgical variables. Univariable and multivariable analyses in addition to a random forest classifier were performed. Results: A total of 146 patients were enrolled in the study with 111 patients included in the final analysis. The average age and body mass index (BMI) of these patients was 66 and 27.8, respectively. None of the 146 patients in this study developed surgical site infection. Older age, no steroid use, no pet ownership, and spine surgery involving two or more levels were all found to be risk factors for wound drainage. Conclusions: This study evaluated lifestyle, environmental, and traditional risk factors for surgical site drainage that have not been explored cohesively related to outpatient orthopedic surgery. Consistent with the existing literature, outpatient spine surgery involving two or more levels was most strongly associated with surgical site drainage after surgery.

Apical expansion of calvarial osteoblasts and suture patency is dependent on graded fibronectin cues.

The skull roof, or calvaria, is comprised of interlocking plates of bone. Premature suture fusion (craniosynostosis, CS) or persistent fontanelles are common defects in calvarial development. Although some of the genetic causes of these disorders are known, we lack an understanding of the instructions directing the growth and migration of progenitors of these bones, which may affect the suture patency. Here, we identify graded expression of Fibronectin (FN1) protein in the mouse embryonic cranial mesenchyme (CM) that precedes the apical expansion of calvarial osteoblasts. Syndromic forms of CS exhibit dysregulated FN1 expression, and we find FN1 expression is altered in a mouse CS model as well. Conditional deletion of Fn1 in CM causes diminished frontal bone expansion by altering cell polarity and shape. To address how osteoprogenitors interact with the observed FN1 prepattern, we conditionally ablate Wasl/N-Wasp to disrupt F-actin junctions in migrating cells, impacting lamellipodia and cell-matrix interaction. Neural crest-targeted deletion of Wasl results in a diminished actin network and reduced expansion of frontal bone primordia similar to conditional Fn1 mutants. Interestingly, defective calvaria formation in both the Fn1 and Wasl mutants occurs without a significant change in proliferation, survival, or osteogenesis. Finally, we find that CM-restricted Fn1 deletion leads to premature fusion of coronal sutures. These data support a model of FN1 as a directional substrate for calvarial osteoblast migration that may be a common mechanism underlying many cranial disorders of disparate genetic etiologies.

Addressing decontaminated respirators: Some methods appear to damage mask integrity and protective function.

Decontamination of N95 respirators is being used by clinicians in the face of a global shortage of these devices. Some treatments for decontamination, such as some vaporized hydrogen peroxide methods or ultraviolet methods, had no impact on respiratory performance, while other treatments resulted in substantial damage to masks.

Activation of antioxidant pathways in ras-mediated oncogenic transformation of human surface ovarian epithelial cells revealed by functional proteomics and mass spectrometry.

Cellular transformation is a complex process involving genetic alterations associated with multiple signaling pathways. Development of a transformation model using defined genetic elements has provided an opportunity to elucidate the role of oncogenes and tumor suppressor genes in the initiation and development of ovarian cancer. To study the cellular and molecular mechanisms of Ras-mediated oncogenic transformation of ovarian epithelial cells, we used a proteomic approach involving two-dimensional electrophoresis and mass spectrometry to profile two ovarian epithelial cell lines, one immortalized with SV40 T/t antigens and the human catalytic subunit of telomerase and the other transformed with an additional oncogenic ras(V12) allele. Of approximately 2200 observed protein spots, we have identified >30 protein targets that showed significant changes between the immortalized and transformed cell lines using peptide mass fingerprinting. Among these identified targets, one most notable group of proteins altered significantly consists of enzymes involved in cellular redox balance. Detailed analysis of these protein targets suggests that activation of Ras-signaling pathways increases the threshold of reactive oxidative species (ROS) tolerance by up-regulating the overall antioxidant capacity of cells, especially in mitochondria. This enhanced antioxidant capacity protects the transformed cells from high levels of ROS associated with the uncontrolled growth potential of tumor cells. It is conceivable that an enhanced antioxidation capability may constitute a common mechanism for tumor cells to evade apoptosis induced by oxidative stresses at high ROS levels.

Human telomerase reverse transcriptase mRNA is highly expressed in normal breast tissues and down-regulated in ductal carcinoma in situ.

Telomerase is a ribonuclear protein that maintains telomere length in eukaryotic cells. Activation of the human telomerase reverse transcriptase (hTERT), the catalytic subunit of telomerase, has been implicated in cellular immortalization and may play a critical role in carcinogenesis. Prior studies in DCIS have been small, and used polymerase chain reaction (PCR)-based methods on heterogeneous tissue extracts. In this study, we used in situ hybridization to determine the cell type and topographic expression pattern of hTERT mRNA in a large series of cased in normal breast ductal-lobular units and ductal carcinoma in situ (DCIS). A total of 120 breast samples were evaluated. High level of hTERT mRNA expression was observed in both normal breast ductal-lobular units and DCIS, which showed a very heterogeneous expression pattern. Overall, hTERT mRNA expression was significantly higher in normal cells compared to DCIS (p=0.000). These results suggest that hTERT not only plays an important role in breast cancer progression but it is also involved in regulating the cellular function of normal breast ductal-lobular units. This finding challenges the conventional view that hTERT mRNA expression is repressed in somatic cells but activated in neoplastic cells. Our results also suggest that telomerase reverse transcriptase mRNA expression may play an important role in the homeostasis of normal breast epithelial cells and neoplastic cells do not necessarily have increased level of expression of hTERT.

A genetically defined model for human ovarian cancer.

Disruptions of the p53, retinoblastoma (Rb), and RAS signaling pathways and activation of human telomerase reverse transcriptase (hTERT) are common in human ovarian cancer; however, their precise role in ovarian cancer development is not clear. We thus introduced the catalytic subunit of hTERT, the SV40 early genomic region, and the oncogenic alleles of human HRAS or KRAS into human ovarian surface epithelial cells and examined the phenotype and gene expression profile of those cells. Disruption of p53 and Rb pathway by SV40 early genomic region and hTERT immortalized but did not transform the cells. Introduction of HRAS(V12) or KRAS(V12) into the immortalized cells, however, allowed them to form s.c. tumors after injection into immunocompromised mice. Peritoneal injection of the transformed cells produced undifferentiated carcinoma or malignant mixed Mullerian tumor and developed ascites; the tumor cells are focally positive for CA125 and mesothelin. Gene expression profile analysis of transformed cells revealed elevated expression of several cytokines, including interleukin (IL)-1beta, IL-6, and IL-8, that are up-regulated by the nuclear factor-kappaB pathway, which is known to contribute to the tumor growth of naturally ovarian cancer cells. Incubation with antibodies to IL-1beta or IL-8 led to apoptosis in the ras-transformed cells and ovarian cancer cells but not in immortalized cells that had not been transformed. Thus, the transformed human ovarian surface epithelial cells recapitulated many features of natural ovarian cancer including a subtype of ovarian cancer histology, formation of ascites, CA125 expression, and nuclear factor-kappaB-mediated cytokine activation. These cells provide a novel model system to study human ovarian cancer.

Microsatellite instability and expression of hMLH1 and hMSH2 proteins in ovarian endometrioid cancer.

Microsatellite instability and loss of heterozygosity has been implicated in ovarian carcinogenesis. The reported frequency of microsatellite instability in human ovarian cancer varies significantly owing to the use of heterogeneous tumor histotypes and various microsatellite markers in different laboratories. In this study, we determined the frequency of microsatellite instability in 74 ovarian endometrioid carcinomas using four microsatellite markers (BAT25, BAT26, D5S346, D17S250), and examined hMLH1 and hMSH2 protein expression. In all, 20% of the tumors were microsatellite instability high (two or more markers showing instability) and 12% were microsatellite instability low (one marker showed instability). Loss of hMLH1 and/or hMSH2 expression was found in nine of 15 microsatellite instability-high tumors. The microsatellite instability-high phenotype tended to occur more frequently in low-grade tumors (P=0.053), but did not correlate with clinical stage. Totally, 38% of cases also displayed loss of heterozygosity at D17S250; this loss of heterozygosity was associated with high clinical stage (P=0.097). Our results indicate that both microsatellite and loss of heterozygosity at D17S250 are involved in the development of ovarian endometrioid carcinoma.

Inhibition of breast and ovarian tumor growth through multiple signaling pathways by using retrovirus-mediated small interfering RNA against Her-2/neu gene expression.

The Her-2/neu oncogene is overexpressed in approximately 30% of breast and ovarian cancer cases and often indicates a poor prognosis. Therapeutic agents against Her-2/neu have been intensively sought over the past decade. Here we show that small interfering RNA (siRNA) can silence the expression of Her-2/neu in models of human breast or ovarian cancer through retrovirus-mediated transfer of an siRNA against Her-2/neu. Cells infected with retrovirus expressing anti-Her-2/neu siRNA exhibit slower proliferation, increased apoptosis, increased G0/G1 arrest, and decreased tumor growth. Changes in cell cycle-associated factors included decreased levels of phosphatidylinositol 3-kinase, pAkt, and cyclin D1 and increased levels of p27 and phosphorylated retinoblastoma protein. Knockdown of Her-2/neu expression by siRNA is also associated with increased expression of the anti-angiogenic factor thrombospondin-1 and decreased expression of the pro-angiogenic vascular endothelial growth factor, suggesting that Her-2/neu stimulates tumor growth at least in part by regulating angiogenesis. siRNA-mediated gene silencing of Her-2/neu and increasing the expression of thrombospondin-1 may be a useful therapeutic strategy for Her-2/neu-over-expressing breast or ovarian cancer.