Functional Human Liver Preservation and Recovery by Means of Subnormothermic Machine Perfusion

There is currently a severe shortage of liver grafts available for transplantation. Novel organ preservation techniques are needed to expand the pool of donor livers. Machine perfusion of donor liver grafts is an alternative to traditional cold storage of livers and holds much promise as a modality to expand the donor organ pool. We have recently described the potential benefit of subnormothermic machine perfusion of human livers. Machine perfused livers showed improving function and restoration of tissue ATP levels. Additionally, machine perfusion of liver grafts at subnormothermic temperatures allows for objective assessment of the functionality and suitability of a liver for transplantation. In these ways a great many livers that were previously discarded due to their suboptimal quality can be rescued via the restorative effects of machine perfusion and utilized for transplantation. Here we describe this technique of subnormothermic machine perfusion in detail. Human liver grafts allocated for research are perfused via the hepatic artery and portal vein with an acellular oxygenated perfusate at 21 °C.     

Human immunodeficiency virus type 1 assembly, budding, and cell-cell spread in T cells take place in tetraspanin-enriched plasma membrane domains

Human immunodeficiency virus type-1 (HIV-1) egress from infected CD4+ T cells is thought to be via assembly and budding at the plasma membrane and may involve components of the T-cell secretory apparatus, including tetraspanins. However, many studies on HIV-1 assembly have examined the trafficking of viral proteins in isolation, and most have used immortalized epithelial, fibroblastic, or hematopoietic cell lines that may not necessarily reflect natural infection of susceptible T cells. Here we have used immunofluorescence and cryoimmunoelectron microscopy (CEM) to examine protein transport during HIV-1 assembly in productively infected Jurkat CD4+ T cells and primary CD4+ T cells. The HIV-1 envelope glycoprotein (Env) and the core protein (Gag) colocalize strongly with CD63 and CD81 and less strongly with CD9, whereas no colocalization was seen between Env or Gag and the late endosome/lysosomal marker Lamp2. CEM revealed incorporation of CD63 and CD81 but not Lamp2 into virions budding at the plasma membrane, and this was supported by immunoprecipitation studies, confirming that HIV-1 egress in T cells is trafficked via tetraspanin-enriched membrane domains (TEMs) that are distinct from lysosomal compartments. CD63, CD81, and, to a lesser extent, CD9 were recruited to the virological synapse (VS), and antibodies against these tetraspanins reduced VS formation. We propose that HIV-1 promotes virus assembly and cell-cell transfer in T cells by targeting plasma membrane TEMs.     

Out of the frying pan: Reintroduction of toad‐smart northern quolls to southern Kakadu National Park

Invasive species are a leading cause of native biodiversity loss. In Australia, the toxic, invasive cane toad Rhinella marina has caused massive and widespread declines of northern quolls Dasyurus hallucatus. Quolls are fatally poisoned if they mistakenly prey on adult toads. To prevent the extinction of this native dasyurid from the Top End, an insurance population was set up in 2003 on two toad‐free islands in Arnhem Land. In 2015, quolls were collected from one of these islands (Astell) for reintroduction. We used conditioned taste aversion to render 22 of these toad‐naïve quolls toad averse. Seven quolls received no taste aversion training. The source island was also predator‐free, so all quolls received very basic predator‐aversion training. In an attempt to re‐establish the mainland population, we reintroduced these 29 northern quolls into Kakadu National Park in northern Australia where cane toads have been established for 13 years. The difference in survival between toad‐averse and toad‐naive quolls was immediately apparent. Toad‐naive quolls were almost all killed by toads within 3 days. Toad‐averse quolls, on the other hand, not only survived longer but also were recorded mating. Our predator training, however, was far less effective. Dingo predation accounted for a significant proportion of toad‐smart quoll mortality. In Kakadu, dingoes have been responsible for high levels of quoll predation in the past and reintroduced animals are often vulnerable to predation‐mediated population extinction. Dingoes may also be more effective predators in fire degraded landscapes. Together, these factors could explain the extreme predation mortality that we witnessed. In addition, predator aversion may have been lost from the predator‐free island populations. These possibilities are not mutually exclusive but need to be investigated because they have clear bearing on the long‐term recovery of the endangered northern quoll.     

A replication-deficient murine gamma-herpesvirus blocked in late viral gene expression can establish latency and elicit protective cellular immunity

The human gamma-herpesviruses, EBV and Kaposi's sarcoma-associated herpesvirus, are widely disseminated and are associated with the onset of a variety of malignancies. Thus, the development of prophylactic and therapeutic vaccination strategies is an important goal. The experimental mouse gamma-herpesvirus, gammaHV68 (or MHV-68), has provided an in vivo model for studying immune control of these persistent viruses. In the current studies, we have examined infectivity, immunogenicity, and protective efficacy following infection with a replication-deficient gammaHV68 blocked in late viral gene expression, ORF31STOP. The data show that ORF31STOP was able to latently infect B cells. However, the anatomical site and persistence of the infection depended on the route of inoculation, implicating a role for viral replication in viral spread but not the infectivity per se. Furthermore, i.p. infection with ORF31STOP elicited strong cellular immunity but a non-neutralizing Ab response. In contrast, intranasal infection was poorly immunogenic. Consistent with this, mice infected i.p. had enhanced control of both the lytic and latent viral loads following challenge with wild-type gammaHV68, whereas intranasal infected mice were not protected. These data provide important insight into mechanisms of infection and protective immunity for the gamma-herpesviruses and demonstrate the utility of replication-deficient mutant viruses in direct testing of "proof of principal" vaccination strategies.     

Measuring bidirectional mutation

The estimation of mutation rates is usually based on a model in which mutations are rare independent Poisson events. Back-mutation of mutants, an even rarer event, is ignored. In the hypermutating B cells of the immune system, mutation between phenotypes exhibiting, vs. not exhibiting, surface immunoglobulin is common in both directions. We develop three strategies for the estimation of mutation rates under circumstances such as these, where mutation rates in both directions are estimated simultaneously. Our model for the growth of a cell culture departs from the classical assumption of cell division as a memoryless (Poisson) event; we model cell division as giving rise to sequential generations of cells. On this basis, a Monte-Carlo simulation is developed. We develop also a numerical approach to calculating the probability distribution for the proportion of mutants in each culture as a function of forward- and backward-mutation rates. Although both approaches are too computationally intensive for routine laboratory use, they provide the insight necessary to develop and evaluate a third, 'hand-calculator' approach to extracting mutation rate estimators from experiments of this type.     

Experimental adhesion model: effect of viscosities of fluids put in the peritoneal cavity on preventing peritoneal adhesions.

In this study we assessed the effectiveness of fluid viscosities placed in the peritoneal cavity to prevent postoperative peritoneal adhesions. Thirty-six Wistar albino female rats (average weight: 160 +/- 30 g, average age: 6.5 months) were divided into three groups of equal number. A standard adhesion pattern was formed in each group. Then, 3 ml isotonic sodium chloride solution (relative viscosity value: 1) was added into the peritoneal cavity of group 1; 3 ml standard 6% hydroxy ethyl starch solution (HES) (relative viscosity value: 2.9) was added into the peritoneal cavity of group 2; and a standard HES solution that was concentrated by dehydration (relative viscosity value: 249.7) was added into the peritoneal cavity of group 3. All rats were sacrificed on postoperative day 10 and the adhesions that formed were graded. In group 1, grade-3 adhesions developed in 9 (75%) rats, and grade-2 developed in 3 (25%) rats. In group 2, grade-3 adhesions developed in 1 (8.3%) rat, grade-2 developed in 6 (50%) rats, and grade-1 developed in 5 (41.6%) rats; in group 3, grade-3 adhesions developed in 9 (75%) rats, and grade-2 developed in 3 (25%) rats. The adhesion scores of group 3 and group 1 were equal to each other (P=1), while the adhesion score of group 2 was significantly less (chi(2): 18.23, P<0.001). Increasing the viscosity of fluids that are inserted in the peritoneal cavity may reduce the formation of postoperative peritoneal adhesions till a critical value of unknown viscosity is achieved. The mechanism behind this process remains unclear.     

Sharp genetic break between Atlantic and English Channel populations of the polychaete Pectinaria koreni, along the North coast of France

This study uses enzymatic and mitochondrial genes to infer the relative importance of historical processes and contemporary hydrodynamic features on the observed patterns of genetic structure in subdivided populations of Pectinaria koreni (Polychaeta: Pectinariidae) along the coasts of Brittany and the English Channel. Nucleotide sequence variation of a 603-bp fragment of the mtDNA cytochrome oxidase subunit I gene revealed a surprisingly deep phylogeographic break of about 16% divergence separating the Brittany and Channel populations, which coincides with a biogeographic boundary along the western coast of Brittany. Deep sequence divergence with fixed haplotype differences and the inversion of allele frequencies at two enzyme loci suggests the occurrence of potential cryptic or sibling species of P. koreni. The two clades showed opposite features. Channel populations exhibited bimodal match-mismatch curves due to two highly divergent haplotypes occurring at high frequencies and no overall heterozygote deficiencies at enzyme loci, suggesting respectively, a historic secondary contact between two differentiated populations followed by contemporary panmixia. On the contrary, Brittany populations displayed unimodal curves with low nucleotide diversity and highly significant heterozygote deficiencies, probably reminiscent of a recent population expansion and recolonisation of Brittany with contemporary admixture of divergent populations.     

Modulating furin activity with designed mini-PDX peptides: synthesis and in vitro kinetic evaluation

A peptide was designed from reactive site loop structure of alpha1 Antitrypsin Portland known as alpha1 PDX as a novel mini-PDX inhibitor of furin. The sequence was derived from (367-394) that contains the crucial furin cleavage motif RIPR382. A P3 mutant replacing Ile380 by Leu was prepared as a first model peptide. A Cys residue was inserted at each terminal of the peptide for purpose of cyclisation which was accomplished by air or iodine-induced oxidation. This mini-PDX peptide both cyclic and acyclic form inhibited in vitro furin activity (IC50 in nM) when measured against either substrates Boc-RVRRdown double arrow MCA or QVEGF-C [Abz-QVHSIIRRdown double arrow SLP-Y(NO2)-A-CONH2, Abz=2-amino benzoic acid and Y(NO2)=3-nitro tyrosine], latter being derived from vascular endothelial growth factor-C (VEGF-C) processing site. The geometrically constrained structure mimicking PDX reactive loop is crucial for enzyme inhibition. Our study further revealed that both mini-PDX peptides inactivate furin in a slow tight binding manner, with disulfide-bridged cyclic form being slightly more potent. Unlike PDX, these peptides inhibit furin via a different mechanistic pathway. The study provides an alternate strategy for development of efficient peptide-based inhibitors of Proprotein Convertases including furin.