Rapid removal of glycerol from frozen‐thawed red blood cells

The storage of red blood cells (RBCs) in a refrigerated state allows a shelf life of a few weeks, whereas RBCs frozen in 40% glycerol have a shelf life of 10 years. Despite the clear logistical advantages of frozen blood, it is not widely used in transfusion medicine. One of the main reasons is that existing post‐thaw washing methods to remove glycerol are prohibitively time consuming, requiring about an hour to remove glycerol from a single unit of blood. In this study, we have investigated the potential for more rapid removal of glycerol. Using published biophysical data for human RBCs, we mathematically optimized a three‐step deglycerolization process, yielding a procedure that was less than 32 s long. This procedure was found to yield 70% hemolysis, a value that was much higher than expected. Consequently, we systematically evaluated three‐step deglycerolization procedures, varying the solution composition and equilibration time in each step. Our best results consisted of less than 20% hemolysis for a deglycerolization time of 3 min, and it is expected that even further improvements could be made with a more thorough optimization and more reliable biophysical data. Our results demonstrate the potential for significantly reducing the deglycerolization time compared with existing methods. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:609–620, 2013     

RIG-I Detects Triphosphorylated RNA of Listeria monocytogenes during Infection in Non-Immune Cells

The innate immune system senses pathogens by pattern recognition receptors in different cell compartments. In the endosome, bacteria are generally recognized by TLRs; facultative intracellular bacteria such as Listeria, however, can escape the endosome. Once in the cytosol, they become accessible to cytosolic pattern recognition receptors, which recognize components of the bacterial cell wall, metabolites or bacterial nucleic acids and initiate an immune response in the host cell. Current knowledge has been focused on the type I IFN response to Listeria DNA or Listeria-derived second messenger c-di-AMP via the signaling adaptor STING. Our study focused on the recognition of Listeria RNA in the cytosol. With the aid of a novel labeling technique, we have been able to visualize immediate cytosolic delivery of Listeria RNA upon infection. Infection with Listeria as well as transfection of bacterial RNA induced a type-I-IFN response in human monocytes, epithelial cells or hepatocytes. However, in contrast to monocytes, the type-I-IFN response of epithelial cells and hepatocytes was not triggered by bacterial DNA, indicating a STING-independent Listeria recognition pathway. RIG-I and MAVS knock-down resulted in abolishment of the IFN response in epithelial cells, but the IFN response in monocytic cells remained unaffected. By contrast, knockdown of STING in monocytic cells reduced cytosolic Listeria-mediated type-I-IFN induction. Our results show that detection of Listeria RNA by RIG-I represents a non-redundant cytosolic immunorecognition pathway in non-immune cells lacking a functional STING dependent signaling pathway.     

Darkfield Adapter for Whole Slide Imaging: Adapting a Darkfield Internal Reflection Illumination System to Extend WSI Applications

We present a new method for whole slide darkfield imaging. Whole Slide Imaging (WSI), also sometimes called virtual slide or virtual microscopy technology, produces images that simultaneously provide high resolution and a wide field of observation that can encompass the entire section, extending far beyond any single field of view. For example, a brain slice can be imaged so that both overall morphology and individual neuronal detail can be seen. We extended the capabilities of traditional whole slide systems and developed a prototype system for darkfield internal reflection illumination (DIRI). Our darkfield system uses an ultra-thin light-emitting diode (LED) light source to illuminate slide specimens from the edge of the slide. We used a new type of side illumination, a variation on the internal reflection method, to illuminate the specimen and create a darkfield image. This system has four main advantages over traditional darkfield: (1) no oil condenser is required for high resolution imaging (2) there is less scatter from dust and dirt on the slide specimen (3) there is less halo, providing a more natural darkfield contrast image, and (4) the motorized system produces darkfield, brightfield and fluorescence images. The WSI method sometimes allows us to image using fewer stains. For instance, diaminobenzidine (DAB) and fluorescent staining are helpful tools for observing protein localization and volume in tissues. However, these methods usually require counter-staining in order to visualize tissue structure, limiting the accuracy of localization of labeled cells within the complex multiple regions of typical neurohistological preparations. Darkfield imaging works on the basis of light scattering from refractive index mismatches in the sample. It is a label-free method of producing contrast in a sample. We propose that adapting darkfield imaging to WSI is very useful, particularly when researchers require additional structural information without the use of further staining.     

Head-to-Head Comparison of Two Multi-Locus Sequence Typing (MLST) Schemes for Characterization of Acinetobacter baumannii Outbreak and Sporadic Isolates

To compare the two Acinetobacter baumannii multi-locus sequence typing (MLST) schemes and to assess their suitability to aid in outbreak analysis we investigated the molecular epidemiology of 99 Acinetobacter baumannii isolates representing outbreak-related and sporadic isolates from 24 hospitals in four different countries (Germany, Poland, Sweden, and Turkey). Pulsed-field gel electrophoresis (PFGE) was used as the reference method to determine the epidemiologic relatedness of isolates and compared to MLST using both the Oxford and Pasteur scheme. Rep-PCR was used to define international clonal lineages (IC). We identified 26 unique outbreak strains and 21 sporadic strains. The majority of outbreaks were associated with carbapenem-resistant A. baumannii harbouring oxacillinase OXA-23-like and corresponding to IC 2. Sequence types (STs) obtained from the Oxford scheme correlate well with PFGE patterns, while the STs of the Pasteur scheme are more in accordance with rep-PCR grouping, but neither one is mirroring completely the results of the comparator. On two occasions the Oxford scheme identified two different STs within a single outbreak where PFGE patterns had only one band difference. The CCs of both MLST schemes were able to define clonal clusters that were concordant with the ICs determined by rep-PCR. IC4 corresponds to the previously described CC15 Pasteur (= CC103 Oxford). It can be concluded that both MLST schemes are valuable tools for population-based studies. In addition, the higher discriminatory power of the Oxford scheme that compares with the resolution obtained with PFGE can often aid in outbreak analysis.     

Auditory anatomy of beaked whales and other odontocetes: Potential for cochlear stimulation via a “vibroacoustic duct mechanism”

Computed tomography (CT) and microcomputed tomography (microCT) were used to examine the structures involved in cochlear stimulation in odontocetes and terrestrial mammals. Cranial CT examined the osseous attachment of the skull to the tympanoperiotic complex (TPC) and the path of the endocranial foramen of the vestibulocochlear nerve (EFVN), which was assumed to contain the perilymphatic duct. Additional CTs of TPC were taken postextraction to examine the gross morphology of this structure. MicroCT was used to examine the acoustic windows of the cochlea, including the round and oval windows and the apertures of the cochlear and vestibular aqueducts. Cranial CT scans demonstrated an osseous connection between the skull and TPC in beaked whales and Physeter macrocephalus. EFVN traveled through a greater length of cranial bone and communicated more closely with the periotic bone in beaked whales than in other species. Ziphius cavirostris was observed to have a reduced medial sulcus of the mallear ridge (MSMR) and tympanic plate and an enlarged aperture of the cochlear aqueduct, respectively. The potential significance of these findings, including the role of the perilymphatic duct as a novel route of cochlear stimulation referred to as the “vibroacoustic duct mechanism,” are discussed.     

The role of fish-poultry integration on fish growth performance,yields and economic benefits among smallholder farmers in sub-Saharan Africa,Tanzania

Aquaculture practices from sub-Saharan Africa are characterised by low production, owing to improper technology. Production can be increased through integrating fish farming with other existing on-farm activities, particularly livestock husbandry. We assessed the role of fish-poultry integration on all male Nile tilapia, Oreochromis niloticus growth performance, yields and economic benefits among smallholder farmers in sub-Saharan Africa, Tanzania. The study also compared phytoplankton species composition, abundance and biomass between the fish-poultry integration and non-integrated system. After 180 days of the experiment, all male O. niloticus cultured under fish-poultry integration exhibited significantly higher growth rates than those in the non-integrated system (p < 0.05). Gross fish yield (GFY), net fish yield (NFY) and net annual yields (NAY) obtained from fish-poultry integration were significantly higher than those from non-integrated system (p < 0.05). Partial enterprise budget analysis revealed that fish-poultry integration was more profitable than the non-integrated system. Moreover, fish-poultry integrated system produced significantly higher phytoplankton abundance and biomass than those from the non-integrated system. Results demonstrate that rural smallholder farmers can achieve higher growth rate, farm net yields and income by integrating all male O. niloticus with other on-farm activities than practising a stand-alone fish culture system.     

Regulation of human cytidine triphosphate synthetase 1 by glycogen synthase kinase 3

Cytidine triphosphate synthetase (CTPS) catalyzes the rate-limiting step in the de novo synthesis of CTP, and both the yeast and human enzymes have been reported to be regulated by protein kinase A or protein kinase C phosphorylation. Here, we provide evidence that stimulation or inhibition of protein kinase A and protein kinase C does not alter the phosphorylation of endogenous human CTPS1 in human embryonic kidney 293 cells under the conditions tested. Unexpectedly, we found that low serum conditions increased phosphorylation of endogenous CTPS1 and this phosphorylation was inhibited by the glycogen synthase kinase 3 (GSK3) inhibitor indirubin-3'-monoxime and GSK3beta short interfering RNAs, demonstrating the involvement of GSK3 in phosphorylation of endogenous human CTPS1. Separating tryptic peptides from [(32)P]orthophosphate-labeled cells and analyzing the phosphopeptides by mass spectrometry identified Ser-574 and Ser-575 as phosphorylated residues. Mutation of Ser-571 demonstrated that Ser-571 was the major site phosphorylated by GSK3 in intact human embryonic kidney 293 cells by GSK3 in vitro. Furthermore, mutation of Ser-575 prevented the phosphorylation of Ser-571, suggesting that phosphorylation of Ser-575 was necessary for priming the GSK3 phosphorylation of Ser-571. Low serum was found to decrease CTPS1 activity, and incubation with the GSK3 inhibitor indirubin-3'-monoxime protected against this decrease in activity. Incubation with an alkaline phosphatase increased CTPS1 activity in a time-dependent manner, demonstrating that phosphorylation inhibits CTPS1 activity. This is the first study to investigate the phosphorylation and regulation of human CTPS1 in human cells and suggests that GSK3 is a novel regulator of CTPS activity.     

Identification and structural basis of binding to host lung glycogen by streptococcal virulence factors

The ability of pathogenic bacteria to recognize host glycans is often essential to their virulence. Here we report structure-function studies of previously uncharacterized glycogen-binding modules in the surface-anchored pullulanases from Streptococcus pneumoniae (SpuA) and Streptococcus pyogenes (PulA). Multivalent binding to glycogen leads to a strong interaction with alveolar type II cells in mouse lung tissue. X-ray crystal structures of the binding modules reveal a novel fusion of tandem modules into single, bivalent functional domains. In addition to indicating a structural basis for multivalent attachment, the structure of the SpuA modules in complex with carbohydrate provides insight into the molecular basis for glycogen specificity. This report provides the first evidence that intracellular lung glycogen may be a novel target of pathogenic streptococci and thus provides a rationale for the identification of the streptococcal alpha-glucan-metabolizing machinery as virulence factors.