Molecular and morphological examination of Cyrtobagous sp. collected from Argentina, Paraguay, Brazil, Australia, and Florida

Two members of the floating fern genus Salvinia (Salviniaceae), S. minima Baker and S. molesta Mitchell, have established in the United States. Cyrtobagous salviniae Calder and Sands (Coleoptera: Curculionidae), long established on Florida S. minima, was released in Texas and Louisiana as a biocontrol agent for both species. Subsequently, sequence analysis of the 28S rRNA D2 expansion domain suggested that the Florida and Brazilian populations (used worldwide for biocontrol) of C. salviniae might constitute two cryptic species. In response, the Brazilian weevil was imported from Australia and released instead onto S. molesta. We sampled C. singularis Hustache and C. salviniae from their native ranges in Brazil, Argentina, and Paraguay and sequenced them (D2) along with Australian and Florida samples. The genetic distance between C. singularis and C. salviniae samples is much greater (almost 5×) than the distance between either the Florida and Brazilian samples or the Brazilian and Argentinean/ Paraguayan C. salviniae samples. Since C. singularis and C. salviniae are cryptic species, the Florida and Brazilian populations (or for that matter Brazilian and Argentinean/Paraguayan) could reasonably be described as demes or ecotypes. Occurrence data indicates that, in parts of their ranges, C. salviniae and C. singularis are not only sympatric but also feed on the same plant species at the same site. While host adaptation (species preferences) likely occurs within local demes, both species seem capable of adapting to the available resource (Salvinia species). Finally, a polymerase chain reaction (PCR) primer was developed to distinguish the Florida and Brazilian/Australian types.     

N-glycans on Nipah virus fusion protein protect against neutralization but reduce membrane fusion and viral entry

Nipah virus (NiV) is a deadly emerging paramyxovirus. The NiV attachment (NiV-G) and fusion (NiV-F) envelope glycoproteins mediate both syncytium formation and viral entry. Specific N-glycans on paramyxovirus fusion proteins are generally required for proper conformational integrity and biological function. However, removal of individual N-glycans on NiV-F had little negative effect on processing or fusogenicity and has even resulted in slightly increased fusogenicity. Here, we report that in both syncytium formation and viral entry assays, removal of multiple N-glycans on NiV-F resulted in marked increases in fusogenicity (>5-fold) but also resulted in increased sensitivity to neutralization by NiV-F-specific antisera. The mechanism underlying the hyperfusogenicity of these NiV-F N-glycan mutants is likely due to more-robust six-helix bundle formation, as these mutants showed increased fusion kinetics and were more resistant to neutralization by a fusion-inhibitory reagent based on the C-terminal heptad repeat region of NiV-F. Finally, we demonstrate that the fusogenicities of the NiV-F N-glycan mutants were inversely correlated with the relative avidities of NiV-F's interactions with NiV-G, providing support for the attachment protein "displacement" model of paramyxovirus fusion. Our results indicate that N-glycans on NiV-F protect NiV from antibody neutralization, suggest that this "shielding" role comes together with limiting cell-cell fusion and viral entry efficiencies, and point to the mechanisms underlying the hyperfusogenicity of these N-glycan mutants. These features underscore the varied roles that N-glycans on NiV-F play in the pathobiology of NiV entry but also shed light on the general mechanisms of paramyxovirus fusion with host cells.     

Toxic activity from cultures of Karlodinium micrum (=Gyrodinium galatheanum) (Dinophyceae)—a dinoflagellate associated with fish mortalities in an estuarine aquaculture facility

The goal of this study was to test for, and partially characterize, toxic activity associated with the dinoflagellate Karlodinium micrum. Since 1996, three fish kill events associated with blooms of K. micrum have occurred at HyRock Fish Farm, an estuarine pond aquaculture facility raising hybrid striped bass on the Chesapeake Bay, MD, USA. Using an assay based on the lysis of rainbow trout erythrocytes, cultures of a Chesapeake Bay isolate of K. micrum have been shown to produce toxic substances which are released upon cell disturbance or damage. The LC₅₀ for hemolysis of a sonicated cell suspension was 2.4×10⁴ cells ml⁻¹, well within the range of cell concentrations observed associated with fish kills. The toxic activity from K. micrum cells and culture filtrates was traced to two distinct fractions that co-elute with polar lipids. The LC₅₀ for hemolysis of the larger of these two fractions (Tox A) was 284 ng ml⁻¹ while the LC₅₀ of the second, smaller, fraction (Tox B) was 600 ng ml⁻¹. For comparison, the LC₅₀ for the standard hemolysin saponin was 3203 ng ml⁻¹. At concentrations of 800 and 2000 ng ml⁻¹, respectively, Tox A was further shown to be ichthyotoxic to zebrafish (Danio rerio) larvae (80% mortality), and cytotoxic to a mammalian GH(4)C(1) cell line (100% LDH release). At a concentration of 600 ng ml⁻¹ Tox B was shown to be cytotoxic to a mammalian GH(4)C(1) cell line (>30% LDH release), but not ichthyotoxic to zebrafish (D. rerio) larvae up to a concentration of 250 ng ml⁻¹. Although treatment with either algicidal copper or potassium permanganate caused significant lysis of K. micrum cells (>70%), toxic activity was released after treatment with copper and eliminated following treatment with potassium permanganate. This observation in cultures is consistent with observations made at HyRock Fish Farm where significantly higher mortality was observed following treatment of a K. micrum bloom with copper sulfate compared to treatment with potassium permanganate. This study represents the first direct evidence of the toxicity of K. micrum isolated from the Chesapeake Bay.     

Isolation and transformation of rice aleurone protoplasts

Summary Protoplasts isolated from the aleurone have been used extensively in molecular studies focusing on hormone-mediated regulation of gene expression in barley seed. To extend the use of aleurone protoplasts to other species, we have determined the conditions necessary for the isolation of protoplasts from rice aleurone layers of germinated seed. Many of the common cell wall degrading enzymes used in making protoplasts were tested for their ability to release protoplasts from rice aleurone layers. Cellulysin was found to be the most effective. Transformation of these aleurone protoplasts was accomplished using polyethylene glycol and DNA constructs containing the firefly luciferase reporter gene under the control of two different promoters were tested. Luciferase expression was 24-fold greater when the reporter gene was under the control of the CaMV 35S promoter than when the promoter from the alcohol dehydrogenase 1 gene was used. With the isolation and transformation of aleurone protoplasts from rice, it is now possible to investigate molecular events occurring in this tissue during germination.     

Recruitment and larval connectivity of a remnant Acropora community in the Arabian Gulf,United Arab Emirates

Coral cover and community structure in the Arabian Gulf have changed considerably in recent decades. Recurrent bleaching events have dramatically reduced the abundance of previously dominant Acropora corals and have given space to other more thermally resistant coral taxa. The loss of Acropora spp. has reduced reef structural complexity and associated biodiversity. Sir Bu Nair Island (SBN) is a nature reserve in the United Arab Emirates that sustains some of the last dense and extensive Acropora stands in the southern Gulf. This study investigated coral recruitment at a southern coral reef on SBN and examined larval dispersal and reef connectivity between SBN and other local and regional reefs through an agent-based model coupled with a 3D hydrodynamic model. Recruitment was surveyed with settlement tiles deployed from April to September 2019. Contrary to other reefs in the Gulf, we found that Acropora is indeed the major coral recruiter settling at SBN reefs, followed by Porites. The models indicate that SBN reefs are mostly self-seeding but also connected to other reefs in the Gulf. SBN can supply coral larvae to the neighbouring islands Siri and Abu Musa, and nearby reefs along with the north-eastern Emirates, Iranian coast and Strait of Hormuz. Findings highlight the importance of SBN to protect remnant populations of the locally almost extinct Acropora in a region where natural coral recovery is increasingly sparse.

     

Correlations between local strains and tissue phenotypes in an experimental model of skeletal healing

Defining how mechanical cues regulate tissue differentiation during skeletal healing can benefit treatment of orthopaedic injuries and may also provide insight into the influence of the mechanical environment on skeletal development. Different global (i.e., organ-level) mechanical loads applied to bone fractures or osteotomies are known to result in different healing outcomes. However, the local stimuli that promote formation of different skeletal tissues have yet to be established. Finite element analyses can estimate local stresses and strains but require many assumptions regarding tissue material properties and boundary conditions. This study used an experimental approach to investigate relationships between the strains experienced by tissues in a mechanically stimulated osteotomy gap and the patterns of tissue differentiation that occur during healing. Strains induced by the applied, global mechanical loads were quantified on the mid-sagittal plane of the callus using digital image correlation. Strain fields were then compared to the distribution of tissue phenotypes, as quantified by histomorphometry, using logistic regression. Significant and consistent associations were found between the strains experienced by a region of the callus and the tissue type present in that region. Specifically, the probability of encountering cartilage increased, and that of encountering woven bone decreased, with increasing octahedral shear strain and, to a lesser extent, maximum principal strain. Volumetric strain was the least consistent predictor of tissue type, although towards the end of the four-week stimulation timecourse, cartilage was associated with increasingly negative volumetric strains. These results indicate that shear strain may be an important regulator of tissue fate during skeletal healing.     

Generation of a tumor vaccine candidate based on conjugation of a MUC1 peptide to polyionic papillomavirus virus-like particles

Virus-like particles (VLPs) are promising vaccine technology due to their safety and ability to elicit strong immune responses. Chimeric VLPs can extend this technology to low immunogenicity foreign antigens. However, insertion of foreign epitopes into the sequence of self-assembling proteins can have unpredictable effects on the assembly process. We aimed to generate chimeric bovine papillomavirus (BPV) VLPs displaying a repetitive array of polyanionic docking sites on their surface. These VLPs can serve as platform for covalent coupling of polycationic fusion proteins. We generated baculoviruses expressing chimeric BPV L1 protein with insertion of a polyglutamic-cysteine residue in the BC, DE, HI loops and the H4 helix. Expression in insect cells yielded assembled VLPs only from insertion in HI loop. Insertion in DE loop and H4 helix resulted in partially formed VLPs and capsomeres, respectively. The polyanionic sites on the surface of VLPs and capsomeres were decorated with a polycationic MUC1 peptide containing a polyarginine-cysteine residue fused to 20 amino acids of the MUC1 tandem repeat through electrostatic interactions and redox-induced disulfide bond formation. MUC1-conjugated fully assembled VLPs induced robust activation of bone marrow-derived dendritic cells, which could then present MUC1 antigen to MUC1-specific T cell hybridomas and primary naïve MUC1-specific T cells obtained from a MUC1-specific TCR transgenic mice. Immunization of human MUC1 transgenic mice, where MUC1 is a self-antigen, with the VLP vaccine induced MUC1-specific CTL, delayed the growth of MUC1 transplanted tumors and elicited complete tumor rejection in some animals.