A lateral-displacement flume for fish behavior and stranding studies during simulated pulsed flows

In regulated rivers, fluctuating water depths associated with pulsed discharges may strand small fish in side channels and pools. Quantitative assessments of stranded fish are difficult in field studies (e.g., due to unknown effects of avian and terrestrial vertebrate predators). To assess such lateral displacement and stranding on juvenile stream fishes, we designed, constructed, and tested (with three species) a 2 × 1-m, lateral-displacement flume. The flume featured a main channel that never drained and a raised, wide “floodplain” channel that alternately flooded, with a simulated pulse, and became dewatered. The floodplain contained four pools, with different shapes and draining capacities, in which fish could become stranded as the water level subsided. Fish-stranding rates (8%) in this relatively compact laboratory flume, after exposure to simulated pulsed stream flows, were comparable to those observed in past investigations using larger, artificial streams.     

Diel variability of mercury phase and species distributions in the Florida Everglades

Preliminary studies of mercury (Hg) cycling in the Everglades revealed that dissolved gaseous mercury (DGM), total mercury (Hg_T), and reactive mercury (Hg_R) show reproducible, diel trends. Peak water-column DGM concentrations were observed on or about noon, with a 3 to 7 fold increase over night-time concentrations. Production of DGM appears to cease during dark periods, with nearly constant water column concentrations that were at or near saturation with respect to the overlying air. A simple mass balance shows that the flux of Hg to the atmosphere from diel DGM production and evasion represents about 10% of the annual input from atmospheric deposition. Production of DGM is likely the result of an indirect photolysis reaction that involves the production of reductive species and/or reduction by electron transfer. Diel variability in Hg_T and Hg_R appears to be controlled by two factors: inputs from rainfall and photolytic sorption/desorption processes. A possible mechanism involves photolysis of chromophores on the surface of a solid substrate (e.g., the periphyton mat) giving rise to destabilization of sorbed mercury and net desorption during daylight. At night, the sorption reactions predominate and the water-column Hg_T decreases. Methylmercury (MeHg) also showed diel trends in concentration but were not clearly linked to the solar cycle or rainfall at the study site.     

Disparate distributions of chemolithotrophs containing form IA or IC large subunit genes for ribulose-1,5-bisphosphate carboxylase/oxygenase in intertidal marine and littoral lake sediments

The distributions of bacterial form IA and form IC ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) were investigated using Lowes Cove intertidal mudflat and Damariscotta Lake littoral sediments by PCR amplification of 492-495 bp fragments of the large subunit RuBisCO gene, cbbL. Genomic extracts for amplification were obtained from lake surface (upper 2 mm), mudflat surface (upper 2 mm), subsurface (5-7 cm), and soft-shell clam (Mya arenaria) burrow-wall sediments, as well as from a sulfide-oxidizing mat. Phylogenetic analyses of cbbL clone libraries revealed that Lowes Cove sediments were dominated by form IA cbbL-containing sequences most closely related to cbbL genes of sulfur-oxidizing bacteria or sulfide-oxidizing mats. In contrast, Damariscotta Lake cbbL clones contained primarily form IC cbbL sequences, which typify aerobic CO- and hydrogen-oxidizing facultative chemolithotrophs. Statistical analyses supported clear differentiation of intertidal and lake chemolithotroph communities, and provided evidence for some differentiation among intertidal communities. amova and libshuff analyses of Lowes Cove libraries suggested that M. arenaria burrow-wall sediments did not harbour distinct communities compared with surface and subsurface sediments, but that surface and subsurface libraries displayed moderate differences. The results collectively support a conceptual model in which the relative distribution of form IA- and IC-containing bacterial chemolithotrophs depends on sulfide availability, which could reflect the role of sulfate reduction in sediment organic matter metabolism, or the presence of geothermal sulfide sources.     

Mitochondrial oxidative phosphorylation compensation may preserve vision in patients with OPA1-linked autosomal dominant optic atrophy

Autosomal Dominant Optic Atrophy (ADOA) is the most common inherited optic atrophy where vision impairment results from specific loss of retinal ganglion cells of the optic nerve. Around 60% of ADOA cases are linked to mutations in the OPA1 gene. OPA1 is a fission-fusion protein involved in mitochondrial inner membrane remodelling. ADOA presents with marked variation in clinical phenotype and varying degrees of vision loss, even among siblings carrying identical mutations in OPA1. To determine whether the degree of vision loss is associated with the level of mitochondrial impairment, we examined mitochondrial function in lymphoblast cell lines obtained from six large Australian OPA1-linked ADOA pedigrees. Comparing patients with severe vision loss (visual acuity [VA]<6/36) and patients with relatively preserved vision (VA>6/9) a clear defect in mitochondrial ATP synthesis and reduced respiration rates were observed in patients with poor vision. In addition, oxidative phosphorylation (OXPHOS) enzymology in ADOA patients with normal vision revealed increased complex II+III activity and levels of complex IV protein. These data suggest that OPA1 deficiency impairs OXPHOS efficiency, but compensation through increases in the distal complexes of the respiratory chain may preserve mitochondrial ATP production in patients who maintain normal vision. Identification of genetic variants that enable this response may provide novel therapeutic insights into OXPHOS compensation for preventing vision loss in optic neuropathies.     

Induction of anti-tumor immunity by vaccination with dendritic cells pulsed with anti-CD44 IgG opsonized tumor cells

Due to the pivotal role that dendritic cells (DC) play in eliciting and maintaining functional anti-tumor T cell responses, these APC have been exploited against tumors. DC express several receptors for the Fc portion of IgG (Fcγ receptors) that mediate the internalization of antigen-IgG complexes and promote efficient MHC class I and II restricted antigen presentation. In this study, the efficacy of vaccination with DC pulsed with apoptotic B16 melanoma cells opsonized with an anti-CD44 IgG (B16-CD44) was explored. Immature bone marrow derived DC grown in vitro with IL-4 and GM-CSF were pulsed with B16-CD44. After 48 h of pulsing, maturation of DC was demonstrated by production of IL-12 and upregulation of CD80 and CD40 expression. To test the efficacy of vaccination with DC+B16-CD44, mice were vaccinated subcutaneously Lymphocytes from mice vaccinated with DC+B16-CD44 produced IFN-γ in response to B16 melanoma lysates as well as an MHC class I restricted B16 melanoma-associated peptide, indicating B16 specific CD8 T cell activation. Upon challenge with viable B16 cells, all mice vaccinated with DC alone developed tumor compared to 40% of mice vaccinated with DC+B16-CD44; 60% of the latter mice remained tumor free for at least 8 months. In addition, established lung tumors and distant metastases were significantly reduced in mice treated with DC+B16-CD44. Lastly, delayed growth of established subcutaneous tumors was induced by combination therapy with anti-CD44 antibodies followed by DC injection. This study demonstrates the efficacy of targeting tumor antigens to DC via Fcγ receptors.     

Development of a new bioprocess scheme using frozen seed train intermediates to initiate CHO cell culture manufacturing campaigns

Agility to schedule and execute cell culture manufacturing campaigns quickly in a multi‐product facility will play a key role in meeting the growing demand for therapeutic proteins. In an effort to shorten campaign timelines, maximize plant flexibility and resource utilization, we investigated the initiation of cell culture manufacturing campaigns using CHO cells cryopreserved in large volume bags in place of the seed train process flows that are conventionally used in cell culture manufacturing. This approach, termed FASTEC (Frozen Accelerated Seed Train for Execution of a Campaign), involves cultivating cells to high density in a perfusion bioreactor, and cryopreserving cells in multiple disposable bags. Each run for a manufacturing campaign would then come from a thaw of one or more of these cryopreserved bags. This article reviews the development and optimization of individual steps of the FASTEC bioprocess scheme: scaling up cells to greater than 70 × 10⁶ cells/mL and freezing in bags with an optimized controlled rate freezing protocol and using a customized rack configuration. Flow cytometry analysis was also employed to understand the recovery of CHO cells following cryopreservation. Extensive development data were gathered to ensure that the quantity and quality of the drug manufactured using the FASTEC bioprocess scheme was acceptable compared to the conventional seed train process flow. The result of offering comparable manufacturing options offers flexibility to the cell culture manufacturing network. Biotechnol. Bioeng. 2013; 110: 1376–1385. © 2012 Wiley Periodicals, Inc.     

New evidence for hybrid zones of forest and savanna elephants in Central and West Africa

The African elephant consists of forest and savanna subspecies. Both subspecies are highly endangered due to severe poaching and habitat loss, and knowledge of their population structure is vital to their conservation. Previous studies have demonstrated marked genetic and morphological differences between forest and savanna elephants, and despite extensive sampling, genetic evidence of hybridization between them has been restricted largely to a few hybrids in the Garamba region of northeastern Democratic Republic of Congo (DRC). Here, we present new genetic data on hybridization from previously unsampled areas of Africa. Novel statistical methods applied to these data identify 46 hybrid samples – many more than have been previously identified – only two of which are from the Garamba region. The remaining 44 are from three other geographically distinct locations: a major hybrid zone along the border of the DRC and Uganda, a second potential hybrid zone in Central African Republic and a smaller fraction of hybrids in the Pendjari–Arli complex of West Africa. Most of the hybrids show evidence of interbreeding over more than one generation, demonstrating that hybrids are fertile. Mitochondrial and Y chromosome data demonstrate that the hybridization is bidirectional, involving males and females from both subspecies. We hypothesize that the hybrid zones may have been facilitated by poaching and habitat modification. The localized geography and rarity of hybrid zones, their possible facilitation from human pressures, and the high divergence and genetic distinctness of forest and savanna elephants throughout their ranges, are consistent with calls for separate species classification.     

Electrophysiological and immunocytochemical characterization of DRG neurons on an organosilane surface in serum-free medium

We are attempting to recreate a stretch reflex circuit on a patterned Bio-MEMS (bio-microelectromechanical systems) chip with deflecting micro-cantilevers. The first steps to recreate this system is to be able to grow individual components of the circuit (sensory neuron, motoneuron, skeletal muscle, and muscle spindle) on a patternable, synthetic substrate coating the MEMS device. Sensory neurons represent the afferent portion of the stretch reflex arc and also play a significant role in transmitting the signal from the muscle spindle to the spinal cord motoneurons. We have utilized a synthetic silane substrate N-1[3-(trimethoxysilyl) propyl) diethylenetriamine (DETA) on which to grow and pattern the cells. DETA forms a self-assembled monolayer on a variety of silicon substrates, including glass, and can be patterned using photolithography. In this paper, we have evaluated the growth of sensory neurons on this synthetic silane substrate. We have investigated the immunocytochemical and electrophysiological properties of the sensory neurons on DETA and compared the resultant properties with a biological control substrate (ornithine/laminin). Immunocytochemical studies revealed the survival and growth of all three subtypes of sensory neurons: trkA, trkB, and trkC on both surfaces. Furthermore, whole-cell patch clamp recordings were used to study the electrophysiological properties of the sensory neurons on the two surfaces. There were no significant differences in the electrical properties of the neurons grown on either surface. This is the first study analyzing the immunocytochemical and electrophysiological properties of sensory neurons grown long-term in a completely defined environment and on a nonbiological substrate.     

Persistence and Expression of the Herpes Simplex Virus Genome in the Absence of Immediate-Early Proteins

The immediate-early (IE) proteins of herpes simplex virus (HSV) function on input genomes and affect many aspects of host cell metabolism to ensure the efficient expression and regulation of the remainder of the genome and, subsequently, the production of progeny virions. Due to the many and varied effects of IE proteins on host cell metabolism, their expression is not conducive to normal cell function and viability. This presents a major impediment to the use of HSV as a vector system. In this study, we describe a series of ICP4 mutants that are defective in different subsets of the remaining IE genes. One mutant, d109, does not express any of the IE proteins and carries a green fluorescent protein (GFP) transgene under the control of the human cytomegalovirus IE promoter (HCMVIEp). d109 was nontoxic to Vero and human embryonic lung (HEL) cells at all multiplicities of infection tested and was capable of establishing persistent infections in both of these cell types. Paradoxically, the genetic manipulations that were required to eliminate toxicity and allow the genome to persist in cells for long periods of time also dramatically lowered the level of transgene expression. Efficient expression of the HCMVIEp-GFP transgene in the absence of ICP4 was dependent on the ICP0 protein. In d109-infected cells, the level of transgene expression was very low in most cells but abundant in a small subpopulation of cells. However, expression of the transgene could be induced in cells containing quiescent d109 genomes weeks after the initial infection, demonstrating the functionality of the persisting genomes.