Professional master's degree programs in regulatory affairs and biomedical quality systems

Two new programs are designed to supply biotechnology professionals with industry-specific skills required for successful biomedical product development.     

Calcium bursts induced by nanosecond electric pulses

We report here real-time imaging of calcium bursts in human lymphocytes exposed to nanosecond, megavolt-per-meter pulsed electric fields. Ultra-short (less than 30 ns), high-field (greater than 1 MV/m), electric pulses induce increases in cytosolic calcium concentration and translocation of phosphatidylserine (PS) to the outer layer of the plasma membrane in Jurkat T lymphoblasts. Pulse-induced calcium bursts occur within milliseconds and PS externalization within minutes. Caspase activation and other indicators of apoptosis follow these initial symptoms of nanosecond pulse exposure. Pulse-induced PS translocation is observed even in the presence of caspase inhibitors. Ultra-short, high-field, electroperturbative pulse effects differ substantially from those associated with electroporation, where pulses of a few tens of kilovolts-per-meter lasting a few tens of microseconds open pores in the cytoplasmic membrane. Nanosecond pulsed electric fields, because their duration is less than the plasma membrane charging time, develop voltages across intracellular structures without porating the cell.     

A role for cytoplasmic dynein and LIS1 in directed cell movement

Cytoplasmic dynein has been implicated in numerous aspects of intracellular movement. We recently found dynein inhibitors to interfere with the reorientation of the microtubule cytoskeleton during healing of wounded NIH3T3 cell monolayers. We now find that dynein and its regulators dynactin and LIS1 localize to the leading cell cortex during this process. In the presence of serum, bright diffuse staining was observed in regions of active ruffling. This pattern was abolished by cytochalasin D, and was not observed in cells treated with lysophosphatidic acid, conditions which allow microtubule reorientation but not forward cell movement. Under the same conditions, using total internal reflection fluorescence microscopy, clear punctate dynein/dynactin containing structures were observed along the sides and at the tips of microtubules at the leading edge. Overexpression of dominant negative dynactin and LIS1 cDNAs or injection of antidynein antibody interfered with the rate of cell migration. Together, these results implicate a leading edge cortical pool of dynein in both early and persistent steps in directed cell movement.     

Identification of novel roles of the cytochrome p450 system in early embryogenesis: effects on vasculogenesis and retinoic Acid homeostasis

The cytochrome P450-dependent monooxygenase system catalyzes the metabolism of xenobiotics and endogenous compounds, including hormones and retinoic acid. In order to establish the role of these enzymes in embryogenesis, we have inactivated the system through the deletion of the gene for the electron donor to all microsomal P450 proteins, cytochrome P450 reductase (Cpr). Mouse embryos homozygous for this deletion died in early to middle gestation (approximately 9.5 days postcoitum [dpc]) and exhibited a number of novel phenotypes, including the severe inhibition of vasculogenesis and hematopoiesis. In addition, defects in the brain, limbs, and cell types where CPR was shown to be expressed were observed. Some of the observed abnormalities have been associated with perturbations in retinoic acid homeostasis in later embryogenesis. Consistent with this possibility, embryos at 9.5 dpc had significantly elevated levels of retinoic acid and reduced levels of retinol. Further, some of the observed phenotypes could be either reversed or exacerbated by decreasing or increasing maternal retinoic acid exposure, respectively. Detailed analysis demonstrated a close relationship between the observed phenotype and the expression of genes controlling vasculogenesis. These data demonstrate that the cytochrome P450 system plays a key role in early embryonic development; this process appears to be, at least in part, controlled by regional concentrations of retinoic acid and has profound effects on blood vessel formation.     

Development of 22 new microsatellite loci for fishers (Martes pennanti) with variability results from across their range

We developed 22 new microsatellite loci for the fisher (Martes pennanti), a North American mesocarnivore. The loci were developed with samples from the southern Sierra Nevada Mountains in California, and were screened with samples from this population and four other populations. We observed a range of six to 21 polymorphic loci per population, with the Sierra Nevada population exhibiting markedly lower levels of variation compared to the other four.     

Conjugation of methoxypolyethylene glycol to the surface of bovine red blood cells

Methoxypolyethylene glycol (mPEG) covalently bound to the surface of human red blood cells (hRBCs) has been shown to decrease immunological recognition of hRBC surface antigens (Bradley et al., 2002). However, there is an increasing shortage of hRBC donations, thus making hRBCs scarce and expensive (Davey, 2004; Riess, 2001). The goal of this study is to similarly PEGylate the surface of bovine RBCs (bRBCs) with the aim of reducing the demand on human blood donations needed for blood transfusions. This study investigates the feasibility of modifying the surface of bRBCs with the succinimidyl ester of methoxypolyethylene glycol propionic acid (SPA-mPEG) for use as a potential blood substitute. The oxygen binding affinity of PEGylated bRBCs was moderately increased with increasing initial SPA-mPEG concentrations up to 4 mM when reacted with bRBCs at a hematocrit of 12%. Oxygen transport simulations verified that SPA-mPEG conjugated bRBCs could still transport oxygen to pancreatic islet tissues even under extreme conditions. PEGylated bRBCs reconstituted to a hematocrit of 40% exhibited viscosities on the order of approximately 3 cp, similar to hRBCs at the same hematocrit. Taken together, the results of this study demonstrate the success of PEGylating bRBCs to yield modified cells with oxygen binding, transport and flow properties similar to that of hRBCs.     

Correction to: Vertical Redistribution of Soil Organic Carbon Pools After Twenty Years of Nitrogen Addition in Two Temperate Coniferous Forests

Ecosystems - In the article by Forstner et al. (2018), the surnames of co-authors Katharina M. Keiblinger and Patrick Schleppi were misspelled. We apologize and ask readers to cite the corrected...     

Accessorizing and anchoring the LINC complex for multifunctionality

A migrating cell must establish front-to-back polarity in order to move. In this issue, Juanes-Garcia et al. (2015. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201407059) report that a short serine-rich motif in nonmuscle myosin IIB is required to establish the cell’s rear. This motif represents a new paradigm for what determines directional cell migration.Directed cell movement is instrumental for organismal development, immune responses, and the progression of diseases, such as cancer (Gardel et al., 2010). To achieve directed movement, an individual cell must establish front-to-back polarity, where there is coordinated protrusion of its front and retraction of its back (Fig. 1 A; Vicente-Manzanares et al., 2007). How polymerizing actin filaments drive protrusion of the front is understood in exquisite detail (Pollard and Borisy, 2003; Pollard, 2007). The mechanisms defining how actin filament contraction defines the back of the cell (Yam et al., 2007) have been more difficult to elucidate. Contraction of actin filaments in crawling cells is driven by nonmuscle myosin II (NMII; Vicente-Manzanares et al., 2009). NMII has three isoforms, NMII-A, NMII-B, and NMII-C, all of which can bind and contract actin filaments to generate force. Importantly, NMII-A and NMII-B have different cellular localizations, which could drive their functions (Kolega, 1998; Vicente-Manzanares et al., 2007). NMII-A localizes primarily to the front, protrusive edge and is required for adhesion maturation. In contrast, NMII-B localizes behind NMII-A, primarily to large and stable actin stress fibers in the middle and back of the cell (Kolega, 1998; Vicente-Manzanares et al., 2007). NMII-B is required for front-to-back polarity, as cells lacking NMII-B lose large stress fibers and fail to define their rear (Vicente-Manzanares et al., 2007). The major question of what drives the polarized localization of NMII-B is unknown. In this issue, Juanes-Garcia et al. report that a short serine-rich motif in NMII-B is responsible for both its localization and the establishment of front-to-back cellular polarity.Open in a separate windowFigure 1.Nonmuscle myosins in cell migration. (A) Schematic showing a top view of a crawling cell. The front of the cell is protruding (top arrow), and the back of the cell is retracting (bottom arrow). The protrusion of the edge is driven by polymerization of actin filament networks in the lamellipodium (gray hash marks). NMII-containing stress fibers (SF, dark blue lines) are assembled behind the lamellipodium. SFs are connected to focal adhesions (gray ovals) either directly or indirectly through non-NMII–containing actin bundles (Dorsal SF, light blue lines; Naumanen et al., 2008). Moving away from the cell’s front, there is a decreasing and increasing gradient of NMII-A (red wedge) and NMII-B (green wedge), respectively (Kolega, 1998). (B) Schematic of NMII-A and NMII-B isoforms. A single NMII molecule is a hexamer of two heavy chains (i.e., NMII-A, NMII-B, or NMIIC), two regulatory light chains, and two essential light chains (Vicente-Manzanares et al., 2009). The overall structure of NMII-A and NMII-B molecules is similar, with two motor domains, a coiled-coil rod domain, and a short nonhelical tail domain. The serine-rich motif is unique to NMII-B, and the role for this motif in SF contraction and the ability of the cell to apply forces to its environment are yet to be determined.Though NMII-A and NMII-B are genetically and structurally very similar, Juanes-Garcia et al. (2015) identified a serine-rich sequence (SFSSSRS) in the C terminus of NMII-B (Fig. 1 B). The authors effectively used cells depleted of NMII-B (Vicente-Manzanares et al., 2007) to test the role of this serine motif in front-to-back polarity. Although expression of wild-type NMII-B rescued front-to-back polarity, expressing NMII-B lacking the serine motif did not. Interestingly, simply inserting the serine-rich motif from NMII-B into NMII-A (NMII-A5S) conferred the ability to rescue front-to-back polarity. In addition, NMII-A5S did not localize to the front of the cell or play a role in adhesion maturation like wild-type NMII-A. Mass spectrometry analysis revealed three of the residues in the serine motif of NMII-B were phosphorylated in cells, and one of these, serine 1935, was found to be crucial for the wild-type kinetics and localization of NMII-B. A phosphomimetic point mutation, S1935D, failed to rescue front-to-back polarity in NMII-B–depleted cells. In contrast, expression of the nonphosphorylatable mutant, S1935A, localized normally to large actin stress fibers and did rescue front-to-back polarity.To provide further evidence that serine 1935 is a regulatory element of front-to-back polarity, Juanes-Garcia et al. (2015) investigated the role of PKC, which acts upstream of NMII in cell polarization (Gomes et al., 2005; Even-Faitelson and Ravid, 2006), in NMII-B–generated stable actin bundles. Cells expressing constitutively active PKC produced isotropic protrusions at the perimeter of the cell, while also failing to produce large, stable NMII-B decorated actin bundles. This isotropic protrusive phenotype was blocked when nonphosphorylatable NMII-B S1935A was expressed in cells but not with wild type or S1935D. Thus, PKC was implicated as the likely upstream regulator of NMII-B activity, which negatively regulates stable actin stress fiber formation by phosphorylating NMII-B at serine 1935. Taken together, the data presented strongly suggest that a small regulatory motif on NMII-B controls cellular front-to-back polarity in migrating cells.The findings presented in this issue by Juanes-Garcia et al. (2015) shine a bright spotlight on a family of motors that has already taken “center stage” in cellular research (Vicente-Manzanares et al., 2009). Some exciting new questions as to how NMII-B functions in the establishment of asymmetric cellular shape and function can now be addressed, including but clearly not limited to: How does the unique enzymatic activity of NMII-B’s motor domain synergize with the serine motif to drive front-to-back polarity (Billington et al., 2013)? What are the structural and dynamic implications for homo- and/or hetero-NMII filament formation (Ricketson et al., 2010; Beach et al., 2014; Shutova et al., 2014)? Does the NMII-B serine motif play a role in the establishment of more complex 3D cellular shapes? Is the serine motif required for directional cell migration through physiological environments?     

Reinforcing the LINC complex connection to actin filaments: the role of FHOD1 in TAN line formation and nuclear movement

Positioning the nucleus is critical for many cellular processes including cell division, migration and differentiation. The linker of nucleoskeleton and cytoskeleton (LINC) complex spans the inner and outer nuclear membranes and has emerged as a major factor in connecting the nucleus to the cytoskeleton for movement and positioning. Recently, we discovered that the diaphanous formin family member FHOD1 interacts with the LINC complex component nesprin-2 giant (nesprin-2G) and that this interaction plays essential roles in the formation of transmembrane actin-dependent nuclear (TAN) lines and nuclear movement during cell polarization in fibroblasts. We found that FHOD1 strengthens the connection between nesprin-2G and rearward moving dorsal actin cables by providing a second site of interaction between nesprin-2G and the actin cable. These results indicate that the LINC complex connection to the actin cytoskeleton can be enhanced by cytoplasmic factors and suggest a new model for TAN line formation. We discuss how the nesprin-2G-FHOD1 interaction may be regulated and its possible functional significance for development and disease.     

Postcranial stem tetrapod remains from the Devonian of Scat Craig, Morayshire, Scotland

The postcranial stem tetrapod remains from Scat Craig include a neural arch, humerus, tibia, femur, and incomplete pectoral girdles and ilia. These elements are all large or very large compared with the corresponding bones of other stem tetrapods. They correlate well in size with the proportions of Elginerpeton , the known stem tetrapod from Scat Craig, and probably belong to this genus. The neural arch has weak zygapophyses, and the ilia and shoulder girdles resemble those of Ichthyostega . The femur is strongly twisted, with the intercondylar fossa facing anteroventrally, so the hind limb probably functioned as a paddle. The tibia is broad, as in Acanthostega and Ichthyostega . The humerus is approximately intermediate in shape between those of osteolepiforms and later stem tetrapods, but seems to have a ventral radial facet like Ichthyostega . Overall, the postcranial bones combine apparent synapomorphies with Ichthyostega and characters which are uniquely primitive among stemgroup tetrapods. This character combination is incongruent. A recently discovered postorbital bone from the site is, strictly speaking, indeterminable but may belong to Elginerpeton ; it broadly resembles the postorbitals of Ichthyostega and Acanthostega , and demonstrates that the typical stem tetrapod facial morphology had evolved before the end of the Frasnian.