Hepatic Mitochondrial Function Analysis Using Needle Liver Biopsy Samples

Backgrounds and Aim

Current assessment of pre-operative liver function relies upon biochemical blood tests and histology but these only indirectly measure liver function. Mitochondrial function (MF) analysis allows direct measurement of cellular metabolic function and may provide an additional index of hepatic health. Conventional MF analysis requires substantial tissue samples (>100 mg) obtained at open surgery. Here we report a method to assess MF using <3 mg of tissue obtained by a Tru-cut® biopsy needle making it suitable for percutaneous application.

Methods

An 18G Bard® Max-core® biopsy instrument was used to collect samples. The optimal Tru-cut® sample weight, stability in ice-cold University of Wisconsin solution, reproducibility and protocol utility was initially evaluated in Wistar rat livers then confirmed in human samples. MF was measured in saponin-permeabilized samples using high-resolution respirometry.

Results

The average mass of a single rat and human liver Tru-cut® biopsy was 5.60±0.30 and 5.16±0.15 mg, respectively (mean; standard error of mean). Two milligram of sample was found the lowest feasible mass for the MF assay. Tissue MF declined after 1 hour of cold storage. Six replicate measurements within rats and humans (n = 6 each) showed low coefficient of variation (<10%) in measurements of State-III respiration, electron transport chain (ETC) capacity and respiratory control ratio (RCR). Ischemic rat and human liver samples consistently showed lower State-III respiration, ETC capacity and RCR, compared to normal perfused liver samples.

Conclusion

Consistent measurement of liver MF and detection of derangement in a disease state was successfully demonstrated using less than half the tissue from a single Tru-cut® biopsy. Using this technique outpatient assessment of liver MF is now feasible, providing a new assay for the evaluation of hepatic function.     

Evidence for Finely-Regulated Asynchronous Growth of Toxoplasma gondii Cysts Based on Data-Driven Model Selection

Toxoplasma gondii establishes a chronic infection by forming cysts preferentially in the brain. This chronic infection is one of the most common parasitic infections in humans and can be reactivated to develop life-threatening toxoplasmic encephalitis in immunocompromised patients. Host-pathogen interactions during the chronic infection include growth of the cysts and their removal by both natural rupture and elimination by the immune system. Analyzing these interactions is important for understanding the pathogenesis of this common infection. We developed a differential equation framework of cyst growth and employed Akaike Information Criteria (AIC) to determine the growth and removal functions that best describe the distribution of cyst sizes measured from the brains of chronically infected mice. The AIC strongly support models in which T. gondii cysts grow at a constant rate such that the per capita growth rate of the parasite is inversely proportional to the number of parasites within a cyst, suggesting finely-regulated asynchronous replication of the parasites. Our analyses were also able to reject the models where cyst removal rate increases linearly or quadratically in association with increase in cyst size. The modeling and analysis framework may provide a useful tool for understanding the pathogenesis of infections with other cyst producing parasites.     

Best linear unbiased prediction of host-range of the facultative parasite Colletotrichum gloeosporioides f. sp. salsolae, a potential biological control agent of Russian thistle

Russian thistle or tumbleweed (Salsola tragus L.) is an introduced invasive weed in N. America. It is widely distributed in the US and is a target of biological control efforts.The fungus Colletotrichum gloeosporioides (Penz.) Penz. & Sacc. in Penz. f. sp. salsolae (CGS) is a facultative parasite under evaluation for classical biological control of this weed. Host-range tests were conducted with CGS in quarantine to determine whether the fungus is safe to release in N. America. Ninetytwo accessions were analyzed from 19 families: Aizoaceae, Alliaceae, Amaranthaceae, Apiaceae, Asteraceae, Brassicaceae, Cactaceae, Campanulaceae, Chenopodiaceae, Cucurbitaceae, Cupressaceae, Fabaceae, Malvaceae, Nyctaginaceae, Phytolaccaceae, Poaceae, Polygonaceae, Sarcobataceae, and Solanaceae and 10 tribes within the Chenopodiaceae: Atripliceae, Beteae, Camphorosmeae, Chenopodieae, Corispermeae, Halopepideae, Polycnemeae, Salicornieae, Salsoleae, and Suaedeae. These included 62 genera and 120 species. To facilitate interpretation of results, disease reaction data were combined with a relationship matrix derived from internal transcribed spacer DNA sequences and analyzed with mixed model equations to produce Best Linear Unbiased Predictors (BLUPs) for each species. Twenty-nine species (30 accessions) from seven closely-related Chenopodiaceae tribes had significant levels of disease severity as indicated by BLUPs, compared to six species determined to be susceptible with least squares means estimates. The 29 susceptible species were: 1 from Atripliceae, 4 from Camphorosmeae, 1 from Halopepideae, 2 from Polycnemeae, 6 from Salicornieae, 8 from Salsolae, and 7 from Suaedeae. Most species in the genus Salsola, which are all introduced and weedy, were very susceptible and damaged by CGS. Statistical comparisons and contrasts of BLUPs indicated that these Salsola species were significantly more susceptible than non-target species, including 15 species from relatives in the closely-related genera Bassia (=Kochia), Nitrophila, Salicornia, Sarcocornia, and Suaeda. Of the 29 susceptible species, 10 native or commercially important species in N. America were identified as needing additional tests to determine the extent of any damage caused by infection.     

Gene flow and rapid differentiation characterize a rapid insular radiation in the southwest Pacific (Aves: Zosterops)

As a dispersive lineage expands its distribution across a heterogeneous landscape, it leaves behind allopatric populations with varying degrees of geographic isolation that often differentiate rapidly. In the case of oceanic islands, even narrowly separated populations often differentiate, which seems contrary to the highly dispersive nature of the founding lineage. This pattern of highly dispersive lineages differentiating across narrow sea barriers has perplexed biologists for more than a century. We used two reduced-representation genomic datasets to examine the diversification of a recent, rapid geographic radiation, the white-eyes (Aves: Zosterops) of the Solomon Islands. We incorporated methods that targeted phylogenetic structure, population structure, and explicit tests for gene flow. Both datasets showed evidence of gene flow among species, but not involving the closely spaced islands in the New Georgia Group. Instead, gene flow has occurred among the larger islands in the archipelago, including those recently connected by land bridges as well as those isolated by large expanses of deep ocean. Populations separated by shallow seas, and connected by land bridges during glacial cycles, ranged from no differentiation to both phenotypic and genomic differentiation. These complex patterns of gene flow and divergence support a model of rapid geographic radiation in which lineages differentially evolve dispersal disparity and phenotypic differences.     

Distribution of Competition Potential Between Native Ungulates and Free-Roaming Equids on Western Rangelands

Free-roaming equids (i.e., feral horses [Equus caballus] and burros [Equus asinus]) are widely distributed and locally abundant across the rangelands of the western United States. The 1971 Wild Free Roaming Horse and Burro Act (WFRHBA) gave the Bureau of Land Management (BLM) and United States Forest Service (USFS) the legal authority to manage these animals on designated public lands. To fulfill this responsibility, federal agencies established an Appropriate Management Level (AML), defined as the number of horses or burros that can be sustained on a given management unit under prevailing environmental conditions and land uses. Although the WFRHBA specifies that feral equids must be managed in ecological balance with other land uses, including conservation of native wildlife, population control measures such as gathers, contraception, and adoptions have failed to keep pace with intrinsic growth rates. Over 80% of federally managed herds currently exceed prescribed population levels, making the potential for competition between native ungulates and feral equids a growing concern among state wildlife agencies. Mule deer (Odocoileus hemionus), pronghorn (Antilocapra americana), elk (Cervus canadensis), and bighorn sheep (Ovis canadensis) are of ecological and economic value to the states where they occur, and all exhibit some degree of distributional, habitat, or dietary overlap with horses or burros. Notwithstanding the scale of the problem, to date there have been no range-wide assessments of competition potential among native and feral ungulates for space, forage, or water. To address this need, we compiled demographic, jurisdictional, and species occurrence data collected from 2010–2019 by federal and state agencies. We used these data to map the distributions of 4 native ungulate species across federal equid management units (FEMUS) in 10 western states (n = 174). We then made within-state rankings of the 50 units that were ≥2 times over AML and encompassed ≥3 native ungulates. Collectively, FEMUs covered approximately 225,000 km², representing 18% of all BLM and USFS lands in affected states. Each FEMU supported ≥1 native ungulate and 14% contained all 4. The degree of overlap between native and feral species varied by state, ranging from <1% for mule deer in Montana, to 40% for bighorn sheep in Nevada. Oregon had the largest proportion of units that supported all 4 native ungulates (58%), whereas Montana and New Mexico had the fewest equids, but all populations were over target densities. Despite the perception that the problem of equid abundance is limited to the Great Basin states, high intrinsic growth rates and social constraints on management practices suggest all affected states should monitor range conditions and native ungulate demography in areas where forage and water resources are limited and expanding equid populations are a concern. © 2021 The Wildlife Society.     

Trefoil factor 2 secreted from damaged hepatocytes activates hepatic stellate cells to induce fibrogenesis

Liver fibrosis is a common characteristic of chronic liver diseases. The activation of hepatic stellate cells (HSCs) plays a key role in fibrogenesis in response to liver injury, yet the mechanism by which damaged hepatocytes modulate the activation of HSCs is poorly understood. Our previous studies have established that liver-specific deletion of O-GlcNAc transferase (OGT)leads to hepatocyte necroptosis and spontaneous fibrosis. Here, we report that OGT-deficient hepatocytes secrete trefoil factor 2 (TFF2) that activates HSCs and contributes to the fibrogenic process. The expression and secretion of TFF2 are induced in OGT-deficient hepatocytes but not in WT hepatocytes. TFF2 activates the platelet-derived growth factor receptor beta signaling pathway that promotes the proliferation and migration of primary HSCs. TFF2 protein expression is elevated in mice with carbon tetrachloride-induced liver injury. These findings identify TFF2 as a novel factor that mediates intercellular signaling between hepatocytes and HSCs and suggest a role of the hepatic OGT–TFF2 axis in the process of fibrogenesis.     

Targeting of T7 RNA polymerase to tobacco nuclei mediated by an SV40 nuclear location signal

We have expressed two T7 RNA polymerase genes by electroporation into tobacco protoplasts. One of the genes was modified by inserting nucleotides encoding a viral nuclear localization signal (NLS) from the large T antigen of SV40. Both T7 RNA polymerase genes directed synthesis of a ca. 100 kDa protein in the electroporated protoplasts. T7 RNA polymerase activity was detected in extracts of protoplasts electroporated with both genes. Immunofluorescence analysis of these protoplasts indicated that only the polymerase carrying the NLS accumulated in the cell nucleus. These experiments suggest that mechanisms involved in the transport from the cytoplasm to the nucleus are similar in plant and animal cells. This system demonstrates the feasibility of T7 RNA polymerase-based approaches for the high-level expression of introduced genes in plant cells.     

Models of seasonal growth of the equatorial carp Labeo dussumieri in response to the river flood cycle

Synopsis The phenology of Labeo dussumieri, an omnivorous carp common to South Asia, was investigated in a population inhabiting a flood plain anabranch of the Mahaweli Ganga, Sri Lanka. The Mahaweli Ganga exhibited a bimodal discharge pattern typical of many equatorial rivers, with a minor peak during the S.W. monsoon and a major peak during the N.E. monsoon. Seasonal changes in several lotic variables were measured in an attempt to correlate changes in environmental conditions to reproduction and growth in L. dussumieri. The onset of gonad recrudescence and spawning were synchronized with the increased river discharge during the S.W. and N.E. monsoons: gonad development followed one monsoonal discharge peak and spawning took place at the beginning of the other. Most fish spawned at the beginning of the major discharge peak in October and November, following the September dry season. Increased discharge was concomitant with a fall in temperature, light intensity, pH and conductivity. Growth was shown to be seasonal, exhibiting an annual bimodal pattern with peaks coincident with S.W. and N.E. monsoonal rains. Seasonal changes in growth were expressed by two models in terms of: (a) change of somatic weight or fork length with time, (b) change of specific growth rate in response to river discharge, modified by somatic weight. Gonad recrudescence and spawning stress did not appear to influence growth rate.