Vascular endothelial growth factor (VEGF) is a critical regulator of angiogenesis that stimulates proliferation, migration, and proteolytic activity of endothelial cells. Although the mitogenic activity of VEGF is endothelial cell specific, recent reports indicate VEGF is able to stimulate chemotaxis and tissue factor production in monocytes. VEGF-stimulated activity in monocytes is mediated by the VEGF receptor flt-1. The purpose of the present study was to investigate the effects of VEGF on another major cell type in the vascular wall, namely, the vascular smooth muscle cell (SMC). Using cultured cells, we showed that VEGF has a minimal mitogenic effect on SMCs, which is in accordance with published data. However, VEGF treatment significantly enhanced production of matrix metalloproteinase (MMP)-1, -3, and -9 by human SMCs. The upregulation of MMP-1 and MMP-9 was pronounced, and the stimulation for MMP-3 was less prominent. Stimulation could be demonstrated at both protein and mRNA levels, as reflected by ELISA, zymography, and Northern blot analysis. To explore the signal transduction pathway for the effect of VEGF on SMCs, we studied the expression of 2 high-affinity VEGF receptors, the kinase insert domain-containing receptor (KDR) and flt-1, in human SMCs. Both reverse transcriptase-polymerase chain reaction and immunoblotting revealed the expression of flt-1. Immunoprecipitation followed by immunoblotting illustrated phosphorylation of the flt-1 receptor after VEGF treatment. Similar methodology failed to detect expression of KDR in human SMCs. These data suggest the role of flt-1 in mediating VEGF-stimulated MMP expression of SMCs. The physiological relevance of MMP upregulation was studied by examining VEGF-stimulated SMC migration through 2 synthetic extracellular matrix barriers, Matrigel and Vitrogen. Our results indicate that VEGF treatment accelerated SMC migration through both barriers, and that this response was blocked by MMP inhibition in Matrigel, which supports a permissive role of MMP in SMC migration. These data are the first to show a direct effect of VEGF on SMCs. SMC-derived MMPs may be an additional source of proteases to digest vascular basement membrane, which is a crucial step in the initial stage of angiogenesis. The MMPs may also contribute to SMC migration in angiogenesis and atherogenesis.

Recently a striking elevation of the activity of chitotriosidase, an endo beta-glucosaminidase distinct from lysozyme, was found in plasma from patients with Gaucher type I disease (McKusick 230800). Plasma chitotriosidase originates from activated macrophages and this elevation is secondary to the basic defect in Gaucher disease. To investigate the specificity of this phenomenon, we have investigated 24 different lysosomal storage diseases. In 11 different diseases increased chitotriosidase activity in plasma was found (in 28% of the patients). None of these diseases showed elevations as high as in Gaucher disease. Chitotriosidase was not significantly elevated in plasma from 20 different non-lysosomal enzymopathies or in plasma from patients with infectious diseases associated with hepatomegaly. The results show that marked elevation of chitotriosidase activity in plasma appears to be specific for Gaucher disease. The data further suggest that elevated levels of chitotriosidase activity in plasma from patients with unexplained diseases may be indicative for a lysosomal disorder.

In eukaryotic cells, newly synthesized secretory proteins require COPII (coat protein complex II) to exit the endoplasmic reticulum (ER). COPII contains five core components: SAR1, SEC23, SEC24, SEC13, and SEC31. SEC23 is a GTPase-activating protein that activates the SAR1 GTPase and also plays a role in cargo recognition. Missense mutations in the human COPII paralogues SEC23A and SEC23B result in craniolenticulosutural dysplasia and congenital dyserythropoietic anemia type II, respectively. We now report that mice completely deficient for SEC23B are born with no apparent anemia phenotype, but die shortly after birth, with degeneration of professional secretory tissues. In SEC23B-deficient embryonic pancreas, defects occur in exocrine and endocrine tissues shortly after differentiation. Pancreatic acini are completely devoid of zymogen granules, and the ER is severely distended. Similar ultrastructural alterations are also observed in salivary glands, but not in liver. Accumulation of proteins in the ER lumen activates the proapoptotic pathway of the unfolded protein response, suggesting a central role for apoptosis in the degeneration of these tissues in SEC23B-deficient embryos. Although maintenance of the secretory pathway should be required by all cells, our findings reveal a surprising tissue-specific dependence on SEC23B for the ER exit of highly abundant cargo, with high levels of SEC23B expression observed in professional secretory tissues. The disparate phenotypes in mouse and human could result from residual SEC23B function associated with the hypomorphic mutations observed in humans, or alternatively, might be explained by a species-specific shift in function between the closely related SEC23 paralogues. mammalian embryo abnormalities | vesicular transport protein | genetics | secretory granules | pancreatitis I n eukaryotic cells, secreted proteins and proteins that are targeted to the plasma membrane and internal organelles are synthesized in the endoplasmic reticulum (ER) and sorted through the secretory pathway. This process has been extensively studied, particularly in budding yeast (1). Proteins destined to traffic from the ER to the Golgi are packaged into COPII (coat protein complex II)-coated vesicles (2-4). COPII is composed of at least five proteins, a small GTPase SAR1 and two cytosolic protein complexes, SEC23-SEC24 and SEC13-SEC31 (5). The GTP-bound form of SAR1 binds to the ER membrane and recruits the SEC23-SEC24 heterodimer to form the "prebudding complex," which in turn recruits the outer coat composed of SEC13-SEC31 heterotetramers to complete the COPII coat structure (6).The COPII complex captures cargo into vesicles and mediates vesicle budding from the ER. Cargo recognition appears to be mediated primarily by the SEC24 subunit, which recognizes divergent export signals located in the cytosolic domain of cargo proteins (7,8). SEC23 and SAR1 also play a role in the recognition of at least a subset of cargos (9, 10). SEC23 is a GTPaseactivating protein (GAP) that activates the SAR1...

The secretory pathway of eukaryotic cells packages cargo proteins into COPII-coated vesicles for transport from the endoplasmic reticulum (ER) to the Golgi. We now report that complete genetic deficiency for the COPII component SEC24A is compatible with normal survival and development in the mouse, despite the fundamental role of SEC24 in COPII vesicle formation and cargo recruitment. However, these animals exhibit markedly reduced plasma cholesterol, with mutations in Apoe and Ldlr epistatic to Sec24a, suggesting a receptor-mediated lipoprotein clearance mechanism. Consistent with these data, hepatic LDLR levels are up-regulated in SEC24A-deficient cells as a consequence of specific dependence of PCSK9, a negative regulator of LDLR, on SEC24A for efficient exit from the ER. Our findings also identify partial overlap in cargo selectivity between SEC24A and SEC24B, suggesting a previously unappreciated heterogeneity in the recruitment of secretory proteins to the COPII vesicles that extends to soluble as well as trans-membrane cargoes.DOI: http://dx.doi.org/10.7554/eLife.00444.001

BACKGROUND: Secondary neoplasms of the thyroid gland (SNTGs) are uncommon, and it is important to recognize them in thyroid fine-needle aspiration biopsy (FNAB). METHODS: The authors report a cohort of 62 SNTGs from 7 institutions in the United States and Europe. Patients were identified retrospectively by searching through medical records of the respective institutions. All initial diagnoses were rendered by FNAB. RESULTS: SNTGs represented 0.16% of all thyroid FNABs and were more frequent among women (ratio of women to men, 1.2:1.0). The mean patient age was of 59 years (range, 7-84 years), the mean tumor size was 3 cm (range, 0.9-7 cm), and the mean interval from diagnosis of the primary tumor was 45 months (range, 0-156 months). Eighty-seven percent of SNTGs were diagnosed as malignant by FNAB, and there was a specific SNTG diagnosis in 93% of patients. Immunocytochemistry and flow cytometry, which were used in 30% of patients, were useful ancillary studies. Adenocarcinomas (n 5 23; 37%) and squamous cell carcinomas (SCCs) (n 5 22; 35.5%) represented the majority of SNTGs, followed by lymphoma (n 5 5; 8%), melanoma (n 5 5; 8%), adenoid cystic carcinoma (n 5 3; 5%), and various sarcomas (n 5 3; 5%). Adenocarcinomas originated from the kidney (n 5 9; 39%), lung (n 5 6; 26%), breast (n 5 5; 22%), and colon (n 5 3; 13%). SCCs originated mostly from the head and neck (n 5 13; 59%), followed by lung (n 5 3; 13%), esophagus (n 5 3; 14%), and unknown primary sites (n 5 3; 14%). CONCLUSIONS:Adenocarcinomas from the kidney, lung, breast, and colon along with SCCs represent the majority of SNTGs. The current results indicate that FNAB is a sensitive and accurate method for diagnosing SNTG; however, diagnostic difficulties can occur. Knowledge of clinical history and the judicious application of ancillary studies can increase the sensitivity and accuracy of FNAB for detecting SNTGs. Cancer (Cancer Cytopathol) 2015;123:19-29. V C 2014 American Cancer Society.KEY WORDS: thyroid; secondary neoplasm; metastasis; renal cell carcinoma; squamous cell carcinoma; adenocarcinoma; lymphoma; adenoid cystic carcinoma; fine-needle aspiration; cytology. INTRODUCTIONSecondary neoplasms of the thyroid gland (SNTGs), representing either metastases or direct extension of tumors from adjacent anatomic structures, are uncommon. Their reported incidence varies substantially, however, ranging from 0.1% to 3% in clinical series. 1-15 They have been reported as incidental findings in autopsy studies [16][17][18] with a frequency of 4.4% to 24% in patients with a known primary cancer or widespread Original Article malignancy in which clinically occult thyroid micrometastases may be detected. 4,[19][20][21][22] In the United States and Europe, the most commonly reported primary tumor resulting in symptomatic SNTG is renal cell carcinoma (RCC), closely followed by carcinomas of the breast, lung, and colon. 5,23-25 Secondary lymphoma, melanoma, sarcoma, and head and neck squamous cell carcinoma (SCC) also account for a significant proportion of SNT...

Centralized facilities for genetic engineering, or "biofoundries", offer the potential to design organisms to address emerging needs in medicine, agriculture, industry, and defense. The field has seen rapid advances in technology, but it is difficult to gauge current capabilities or identify gaps across projects. To this end, our foundry was assessed via a timed "pressure test", in which 3 months were given to build organisms to produce 10 molecules unknown to us in advance. By applying a diversity of new approaches, we produced the desired molecule or a closely related one for six out of 10 targets during the performance period and made advances toward production of the others as well. Specifically, we increased the titers of 1-hexadecanol, pyrrolnitrin, and pacidamycin D, found novel routes to the enediyne warhead underlying powerful antimicrobials, established a cell-free system for monoterpene production, produced an intermediate toward vincristine biosynthesis, and encoded 7802 individually retrievable pathways to 540 bisindoles in a DNA pool. Pathways to tetrahydrofuran and barbamide were designed and constructed, but toxicity or analytical tools inhibited further progress. In sum, we constructed 1.2 Mb DNA, built 215 strains spanning five species ( Saccharomyces cerevisiae, Escherichia coli, Streptomyces albidoflavus, Streptomyces coelicolor, and Streptomyces albovinaceus), established two cell-free systems, and performed 690 assays developed in-house for the molecules.

BackgroundSpinal cord ischemia-reperfusion (I/R) involves two-phase injury, including an initial acute ischemic insult and subsequent inflammatory reperfusion injury, resulting in blood-spinal cord barrier (BSCB) dysfunction involving the TLR4 pathway. However, the correlation between TLR4/MyD88-dependent and TLR4/TRIF-dependent pathways in BSCB dysfunction is not fully understood. The aim of this study is to characterize inflammatory responses in spinal cord I/R and the events that define its clinical progression with delayed neurological deficits, supporting a bimodal mechanism of injury.MethodsRats were intrathecally pretreated with TAK-242, MyD88 inhibitory peptide, or Resveratrol at a 12 h interval for 3 days before undergoing 14-minute occlusion of aortic arch. Evan’s Blue (EB) extravasation and water content were detected at 6, 12, 18, 24, 36, 48, and 72 h after reperfusion. EB extravasation, water content, and NF-κB activation were increased with time after reperfusion, suggesting a bimodal distribution, as maximal increasing were detected at both 12 and 48 h after reperfusion. The changes were directly proportional to TLR4 levels determined by Western blot. Double-labeled immunohistochemical analysis was also used to detect the relationship between different cell types of BSCB with TLR4. Furthermore, NF-κB and IL-1β were analyzed at 12 and 48 h to identify the correlation between MyD88-dependent and TRIF-dependent pathways.ResultsRats without functional TLR4 and MyD88 attenuated BSCB leakage and inflammatory responses at 12 h, suggesting the ischemic event was largely mediated by MyD88-dependent pathway. Similar protective effects observed in rats with depleted TLR4, MyD88, and TRIF receptor at 48 h infer that the ongoing inflammation which occurred in late phase was mainly initiated by TRIF-dependent pathway and such inflammatory response could be further amplified by MyD88-dependent pathway. Additionally, microglia appeared to play a major role in early phase of inflammation after I/R injury, while in late responding phase both microglia and astrocytes were necessary.ConclusionsThese findings indicate the relevance of TLR4/MyD88-dependent and TLR4/TRIF-dependent pathways in bimodal phases of inflammatory responses after I/R injury, corresponding with the clinical progression of injury and delayed onset of symptoms. The clinical usage of TLR4 signaling inhibitors at different phases may be a therapeutic option for the prevention of delayed injury.

Acrolein, an α,β-unsaturated aldehyde and a reactive product of lipid peroxidation, has been suggested as a key factor in neural post-traumatic secondary injury in SCI, mainly based on in vitro and ex vivo evidence. Here we demonstrate an increase of acrolein up to 300%; the elevation lasted at least two weeks in a rat SCI model. More importantly, hydralazine, a known acrolein scavenger can provide neuroprotection when applied systemically. Besides effectively reducing acrolein, hydralazine treatment also resulted in significant amelioration of tissue damage, motor deficits, and neuropathic pain. This effect was further supported by demonstrating the ability of hydralazine to reach spinal cord tissue at a therapeutic level following intraperitoneal application. This suggests that hydralazine is an effective neuroprotective agent not only in vitro, but in a live animal model of SCI as well. Finally, the role of acrolein in SCI was further validated by the fact that acrolein injection into the spinal cord caused significant SCI-like tissue damage and motor deficits. Taken together, available evidence strongly suggests a critical causal role of acrolein in the pathogenesis of spinal cord trauma. Since acrolein has been linked to a variety of illness and conditions, we believe that acrolein-scavenging measures have the potential to be expanded significantly ensuring a broad impact on human health.