Abortive vaccinia virus replication and maturation in SCCF1 cells.

<p><b>A)</b> At 48 hours after infection, the replication centre (viral factory) is inconspicuous, only a few immature viruses (IV) were found around it. <b>B)</b> Two IVs were visible, one IV showed nucleoid inside thus can be identified as IVN. Neither mature viruses nor enveloped viruses were detected throughout all infected cells. <b>C)</b> there are two small immature viruses (IV) located within cell and in the edge of an abnormal granular virosome (VS) region. No MVs were visible there. <b>D)</b> The immature viruses (IV) migrate to peripheral of the cell, underneath cell membrane. They still fail to get membranous envelope even at this late stage of nearly being released out.</p

Vitamin D Metabolites and Their Association with Calcium, Phosphorus, and PTH Concentrations, Severity of Illness, and Mortality in Hospitalized Equine Neonates

<div><p>Background</p><p>Hypocalcemia is a frequent abnormality that has been associated with disease severity and outcome in hospitalized foals. However, the pathogenesis of equine neonatal hypocalcemia is poorly understood. Hypovitaminosis D in critically ill people has been linked to hypocalcemia and mortality; however, information on vitamin D metabolites and their association with clinical findings and outcome in critically ill foals is lacking. The goal of this study was to determine the prevalence of vitamin D deficiency (hypovitaminosis D) and its association with serum calcium, phosphorus, and parathyroid hormone (PTH) concentrations, disease severity, and mortality in hospitalized newborn foals.</p><p>Methods and Results</p><p>One hundred newborn foals ≤72 hours old divided into hospitalized (n = 83; 59 septic, 24 sick non-septic [SNS]) and healthy (n = 17) groups were included. Blood samples were collected on admission to measure serum 25-hydroxyvitamin D₃ [25(OH)D₃], 1,25-dihydroxyvitamin D₃ [1,25(OH) ₂D₃], and PTH concentrations. Data were analyzed by nonparametric methods and univariate logistic regression. The prevalence of hypovitaminosis D [defined as 25(OH)D₃ <9.51 ng/mL] was 63% for hospitalized, 64% for septic, and 63% for SNS foals. Serum 25(OH)D₃ and 1,25(OH) ₂D₃ concentrations were significantly lower in septic and SNS compared to healthy foals (P<0.0001; P = 0.037). Septic foals had significantly lower calcium and higher phosphorus and PTH concentrations than healthy and SNS foals (P<0.05). In hospitalized and septic foals, low 1,25(OH)₂D₃ concentrations were associated with increased PTH but not with calcium or phosphorus concentrations. Septic foals with 25(OH)D₃ <9.51 ng/mL and 1,25(OH) ₂D₃ <7.09 pmol/L were more likely to die (OR=3.62; 95% CI = 1.1-12.40; OR = 5.41; 95% CI = 1.19-24.52, respectively).</p><p>Conclusions</p><p>Low 25(OH)D₃ and 1,25(OH)₂D₃ concentrations are associated with disease severity and mortality in hospitalized foals. Vitamin D deficiency may contribute to a pro-inflammatory state in equine perinatal diseases. Hypocalcemia and hyperphosphatemia together with decreased 1,25(OH)₂D₃ but increased PTH concentrations in septic foals indicates that PTH resistance may be associated with the development of these abnormalities.</p></div

Production of virus in SCC-F1 cells.

<p><b>A)</b> Infected SCC-F1 cells constantly produce infectious particles to the supernatant. <b>B)</b> Virus released to the supernatant by infected SCCF1 cells was able to re-infect both A549 and SCC-F1 cells. <b>C)</b> Viral particles released to the supernatant were completely neutralized with anti-VV antibody.</p

Vaccinia virus replication and maturation in A549 cells.

<p><b>A-B</b>: After enter A549 cells, at 48 hours post infection, vaccinia viral particles begin their normal life cycle quickly: form replication centre (viral factory) in juxta-nuclear region manifested as a homogeneous, low-to-middle electron density, void of any organelles. The replication begins with formation of crescent (Cr), then immature virus (IV), immature virus with nucleoids (IVN), mature virus (MV). Many MV are wrapped by intracellular membrane from Trans-Golgi cistern, form intracellular enveloped virus (IEV). IEVs move towards cell periphery, fuss with cell membrane and obtained another envelope from cell membrane, finally they are released to extracellular space to form extracellular enveloped virus (EEV). Some EEV remain attached on cell membrane as cell associated extracellular enveloped virus (CEV). <b>C-D</b> Different intermediate particles from cells other than cells in a-b in high magnification.</p

Electron microscopy of negative stained, purified vaccinia viral particles released from cultured cells.

<p>Supernatant from infected SCCF1 or A549 cells was collected, purified and negative staining electron microscopy was performed. <b>A-C:</b> viral particles produced by A549 cells: mature particles of brick-shaped, with outer envelope (OE) derived from cell membrane. <b>D-E</b>: immature vaccinia viral particles produced by SCCF1 cells: the particles are irregular in shape and in electron density. Their internal contents are not tightly packed, no clear boundary or membranous outer envelope is visible.</p

In vitro cytotoxicity and and transduction of vvdd in SCCF1 cells.

<p><b>A)</b> SCCF1 cells were infected with vvdd with 0.01, 0.1 or 1 pfu/cell. Viability of cells was measured on day 3. <b>B)</b> Compared to A549 cells, SCCF1 cells maintained their viability significantly better on day 3 <b>C)</b> When SCCF1 cells formed a tight monolayer before infection, the cells were still alive 10 days after infection. <b>D)</b> SCCF1 cell were infected with 0.04, 0.2, 1 or 5 pfu per cell and luciferase expression was measured 4 h postinfection in relative light units.</p

Serum 25(OH)D₃ and 1,25(OH)₂D₃ concentrations in healthy, SNS, and septic foals.

<p>Values are expressed as median and 95% CI. (A) Septic and SNS foals had significantly lower serum 25(OH)D_<b>3</b> concentrations compared to healthy foals (P < 0.0001). (B) Septic and SNS foals had significantly lower serum 1,25(OH)_<b>2</b>D_<b>3</b> concentrations compared to healthy foals (P = 0.037). * indicates a statistically significant difference from healthy foals.</p

Serum total calcium and phosphorus concentrations in healthy, SNS, and septic foals.

<p>Values are expressed as median and 95% CI. (A) Septic foals had significantly lower serum total calcium concentrations compared to healthy and SNS foals (P = 0.01). (B) Septic foals had significantly higher serum phosphorus concentrations compared to healthy and SNS foals (P = 0.02). * indicates a statistically significant difference from healthy and SNS foals.</p

Percent distribution of serum 25(OH)D₃, total calcium, and phosphorus concentrations in hospitalized, septic, and SNS foals, based on 95% CI values from healthy foals.

<p>*P < 0.05</p><p>**P < 0.01; SNS—sick non-septic foals</p><p>For vitamin D, * = statistically different from hypervitaminosis D; ** = statistically different from normovitaminosis D and hypervitaminosis D in all foal groups.</p><p>For calcium, ** = statistically different from hypercalcemia in hospitalized and septic foals, and statistically different from hypocalcemia in SNS foals.</p><p>For phosphorus, ** = statistically different from normophosphatemia and hypophosphatemia.</p><p>Percent distribution of serum 25(OH)D₃, total calcium, and phosphorus concentrations in hospitalized, septic, and SNS foals, based on 95% CI values from healthy foals.</p

Loss of Myoferlin Redirects Breast Cancer Cell Motility towards Collective Migration

<div><p>Cell migration plays a central role in the invasion and metastasis of tumors. As cells leave the primary tumor, they undergo an epithelial to mesenchymal transition (EMT) and migrate as single cells. Epithelial tumor cells may also migrate in a highly directional manner as a collective group in some settings. We previously discovered that myoferlin (MYOF) is overexpressed in breast cancer cells and depletion of MYOF results in a mesenchymal to epithelial transition (MET) and reduced invasion through extracellular matrix (ECM). However, the biomechanical mechanisms governing cell motility during MYOF depletion are poorly understood. We first demonstrated that lentivirus-driven shRNA-induced MYOF loss in MDA-MB-231 breast cancer cells (MDA-231^MYOF-KD) leads to an epithelial morphology compared to the mesenchymal morphology observed in control (MDA- 231^LTVC) and wild-type cells. Knockdown of MYOF led to significant reductions in cell migration velocity and MDA- 231^MYOF-KD cells migrated directionally and collectively, while MDA-231^LTVC cells exhibited single cell migration. Decreased migration velocity and collective migration were accompanied by significant changes in cell mechanics. MDA-231^MYOF-KD cells exhibited a 2-fold decrease in cell stiffness, a 2-fold increase in cell-substrate adhesion and a 1.5-fold decrease in traction force generation. <i>In vivo</i> studies demonstrated that when immunocompromised mice were implanted with MDA- 231^MYOF-KD cells, tumors were smaller and demonstrated lower tumor burden. Moreover, MDA- 231^MYOF-KD tumors were highly circularized and did not invade locally into the adventia in contrast to MDA- 231^LTVC-injected animals. Thus MYOF loss is associated with a change in tumor formation in xenografts and leads to smaller, less invasive tumors. These data indicate that MYOF, a previously unrecognized protein in cancer, is involved in MDA-MB-231 cell migration and contributes to biomechanical alterations. Our results indicate that changes in biomechanical properties following loss of this protein may be an effective way to alter the invasive capacity of cancer cells.</p></div