Even among isogenic cells, the time to progress through the cell cycle, or the intermitotic time (IMT), is highly variable. This variability has been a topic of research for several decades and ...numerous mathematical models have been proposed to explain it. Previously, we developed a top-down, stochastic drift-diffusion+threshold (DDT) model of a cell cycle checkpoint and showed that it can accurately describe experimentally-derived IMT distributions Leander R, Allen EJ, Garbett SP, Tyson DR, Quaranta V. Derivation and experimental comparison of cell-division probability densities. J. Theor. Biol. 2014;358:129-135. Here, we use the DDT modeling approach for both descriptive and predictive data analysis. We develop a custom numerical method for the reliable maximum likelihood estimation of model parameters in the absence of a priori knowledge about the number of detectable checkpoints. We employ this method to fit different variants of the DDT model (with one, two, and three checkpoints) to IMT data from multiple cell lines under different growth conditions and drug treatments. We find that a two-checkpoint model best describes the data, consistent with the notion that the cell cycle can be broadly separated into two steps: the commitment to divide and the process of cell division. The model predicts one part of the cell cycle to be highly variable and growth factor sensitive while the other is less variable and relatively refractory to growth factor signaling. Using experimental data that separates IMT into G1 vs. S, G2, and M phases, we show that the model-predicted growth-factor-sensitive part of the cell cycle corresponds to a portion of G1, consistent with previous studies suggesting that the commitment step is the primary source of IMT variability. These results demonstrate that a simple stochastic model, with just a handful of parameters, can provide fundamental insights into the biological underpinnings of cell cycle progression.
High-throughput complementary DNA sequencing technologies have advanced our understanding of transcriptome complexity and regulation. However, these methods lose information contained in biological ...RNA because the copied reads are often short and modifications are not retained. We address these limitations using a native poly(A) RNA sequencing strategy developed by Oxford Nanopore Technologies. Our study generated 9.9 million aligned sequence reads for the human cell line GM12878, using thirty MinION flow cells at six institutions. These native RNA reads had a median length of 771 bases, and a maximum aligned length of over 21,000 bases. Mitochondrial poly(A) reads provided an internal measure of read-length quality. We combined these long nanopore reads with higher accuracy short-reads and annotated GM12878 promoter regions to identify 33,984 plausible RNA isoforms. We describe strategies for assessing 3' poly(A) tail length, base modifications and transcript haplotypes.
Whether and how landscape context and habitat traits combine to shape animal assemblages and the rate and distribution of ecological functions remains unresolved in many aquatic settings. Saltmarshes ...are one such ecosystem in which these considerations are frequently acknowledged as important, but quantitative studies of these effects are rare, especially for ecological functions. In this study, the influence of landscape configuration and habitat traits on the composition of fish assemblages and rates of predation were quantified around 30 saltmarshes in three estuaries (i.e., 10 per estuary) in eastern Australia. Fish assemblages were surveyed using unbaited underwater video cameras, and predation was quantified using videoed “Squidpop” predation assays at 10 sites at each saltmarsh. The structure of fish assemblages was best explained by the estuary in which saltmarsh was located, the proximity of sites to estuary mouth, and the area of nearby saltmarsh and mangroves. Predation was dominated (90% of total predation events) by yellowfin bream
Acanthopagrus australis
(Sparidae), and so rates of predation correlated positively with yellowfin bream abundance. Predation peaked in the lower reaches of estuaries at saltmarshes with lower vegetation cover. These findings suggest that the mouths of estuaries might function as key transition zones that concentrate prey, the products of trophic relay, and the ecological effects of predators near the estuarine-sea interface.
Human activities in coastal catchments can cause the accumulation of pollutants in seafood. We quantified the concentration of heavy metals, pesticides and PFASs in the flesh of the fisheries species ...yellowfin bream Acanthopagrus australis (n = 57) and mud crab Scylla serrata (n = 65) from 13 estuaries in southeast Queensland, Australia; a region with a variety of human land uses. Pollutants in yellowfin bream were best explained by the extent of intensive uses in the catchment. Pollutants in mud crabs were best explained by the extent of irrigated agriculture and water bodies. No samples contained detectable levels of pesticides, and only six samples contained low levels of PFASs. Metals were common in fish and crab flesh, but only mercury in yellowfin bream from the Mooloolah River breached Australian food safety standards. High pollutant presence and concentration is not the norm in seafood collected during routine surveys, even in estuaries with highly modified catchments.
•We quantified concentrations of 70 pollutants in the flesh of two fisheries species.•We sampled 13 estuaries in southeast Queensland with a variety of human land uses.•No samples contained pesticides, only six samples contained low PFAS levels.•Metals were common, but only one estuary had fish that exceeded safe mercury values.•High pollutant loads are not the norm in seafood collected during routine surveys.
Human pressures on ecosystems from landscape transformation and harvesting can result in changes to body size and functional traits of affected species. However, these effects remain very poorly ...understood in many settings. Here we examine whether and how fishing and the attributes of coastal seascapes can operate in concert to change the body size and functional traits of the giant mud crab, Scylla serrata; a prized fisheries species. We captured 65 legal sized (> 15 cm carapace width) male giant mud crabs from 13 estuaries in southeast Queensland, Australia. These estuaries span a wide range of fishing and catchment landscape transformation intensity. We made a total of 9000 external morphometric measurements in the study. There was a distinct effect of estuarine landscape context on body size, with the largest individuals captured from systems with bigger inlets and lower extent of intertidal flats. Variation in functional traits was most often associated with variation in fishing pressure and human population size in the catchment. Crabs from areas with less commercial fishing pressure and lower human populations in the catchment had the largest chelipeds. We also found effects of urbanisation (negative correlations), intertidal flats (inconsistent effects) and mangrove extent (positive correlations) on the size of some functional traits. Our results show that human pressures can have sublethal effects on animals in estuaries that alter body size and functional traits. These phenotypic responses might have consequences for the fitness and ecological roles of targeted species, and the yields of fisheries catches.
Background
Following peripheral nerve chronic constriction injury, the accumulation of the α2δ–1 auxiliary subunit of voltage-gated Ca2+ channels in primary afferent terminals contributes to the ...onset of neuropathic pain. Overexpression of α2δ–1 in Xenopus oocytes increases the opening properties of Cav1.2 L-type channels and allows Ca2+ influx at physiological membrane potentials. We therefore posited that L-type channels play a role in neurotransmitter release in the superficial dorsal horn in the chronic constriction injury model of neuropathic pain.
Results
Whole-cell recording from lamina II neurons from rats, subject to sciatic chronic constriction injury, showed that the L-type Ca2+ channel blocker, nitrendipine (2 µM) reduced the frequency of spontaneous excitatory postsynaptic currents. Nitrendipine had little or no effect on spontaneous excitatory postsynaptic current frequency in neurons from sham-operated animals. To determine whether α2δ–1 is involved in upregulating function of Cav1.2 L-type channels, we tested the effect of the α2δ–1 ligand, gabapentin (100 µM) on currents recorded from HEK293F cells expressing Cav1.2/β4/α2δ–1 channels and found a significant decrease in peak amplitude with no effect on control Cav1.2/β4/α2δ–3 expressing cells. In PC-12 cells, gabapentin also significantly reduced the endogenous dihydropyridine-sensitive calcium current. In lamina II, gabapentin reduced spontaneous excitatory postsynaptic current frequency in neurons from animals subject to chronic constriction injury but not in those from sham-operated animals. Intraperitoneal injection of 5 mg/kg nitrendipine increased paw withdrawal threshold in animals subject to chronic constriction injury.
Conclusion
We suggest that L-type channels show an increased contribution to synaptic transmission in lamina II dorsal horn following peripheral nerve injury. The effect of gabapentin on Cav1.2 via α2δ–1 may contribute to its anti-allodynic action.
Claudin 6 (Cldn6) is a tetraspanin protein expressed by barrier epithelial cells. In order to assess the effects of persistent tight junctions involving Cldn6 during lung development, a doxycycline ...(dox)-inducible conditional transgenic mouse was generated that up-regulates Cldn6 in the distal lung. Pups had unlimited access to dox from conception until sacrifice date at embryonic day (E) 18.5. Quantitative PCR, immunoblotting, and immunohistochemistry revealed significantly elevated Cldn6 expression in transgenic mice compared to non-transgenic controls. There were no differences in terms of lung size, lung weight, or whole body weight at the time of necropsy. Histological evaluations led to the discovery that E18.5 Cldn6 transgenic pups appeared to be in the early canalicular stage of development coincident with fewer, thickened respiratory airspaces. In contrast, controls appeared to have entered the saccular stage characterized by thin airspace walls and spherical architecture. Immunostaining for transcriptional regulators including TTF-1 and FoxA2 was conducted to assess cell differentiation and specific cell types were identified via staining for pro-surfactant protein C (alveolar type II epithelial cells) or Clara Cell Secretory Protein (cub or Clara cells). Lastly, cell turnover was qualitatively measured via staining for cell proliferation or apoptosis. These data suggest that Cldn6 is an important junctional protein potentially involved in the programming of epithelial cells during lung development. Furthermore, genetic down-regulation of Cldn6 as development proceeds may influence differentiation observed in the transition from the canalicular to the saccular lung.
Burst-firing in distinct subsets of thalamic relay (TR) neurons is thought to be a key requirement for the propagation of absence seizures. However, in the well-regarded Genetic Absence Epilepsy Rats ...from Strasbourg (GAERS) model as yet there has been no link described between burst-firing in TR neurons and spike-and-wave discharges (SWDs). GAERS ventrobasal (VB) neurons are a specific subset of TR neurons that do not normally display burst-firing during absence seizures in the GAERS model, and here, we assessed the underlying relationship of VB burst-firing with I
h
and T-type calcium currents between GAERS and non-epileptic control (NEC) animals. In response to 200-ms hyperpolarizing current injections, adult epileptic but not pre-epileptic GAERS VB neurons displayed suppressed burst-firing compared to NEC. In response to longer duration 1,000-ms hyperpolarizing current injections, both pre-epileptic and epileptic GAERS VB neurons required significantly more hyperpolarizing current injection to burst-fire than those of NEC animals. The current density of the Hyperpolarization and Cyclic Nucleotide-activated (HCN) current (I
h
) was found to be increased in GAERS VB neurons, and the blockade of I
h
relieved the suppressed burst-firing in both pre-epileptic P15–P20 and adult animals. In support, levels of HCN-1 and HCN-3 isoform channel proteins were increased in GAERS VB thalamic tissue. T-type calcium channel whole-cell currents were found to be decreased in P7–P9 GAERS VB neurons, and also noted was a decrease in Ca
V
3.1 mRNA and protein levels in adults. Z944, a potent T-type calcium channel blocker with anti-epileptic properties, completely abolished hyperpolarization-induced VB burst-firing in both NEC and GAERS VB neurons.
Significant changes in the intensity or distribution of human activities, like during the 2020 COVID-19 lockdowns, can cause rapid changes to the diversity, distribution and abundance of animals. ...These changes are usually viewed as positive for ecosystems. Here, we mapped the abundance and habitat associations of coastal vertebrates along approximately 50 km of ocean beaches on the Sunshine Coast in eastern Australia using baited trail cameras before (April–June 2018 and 2019) and during the April–May 2020 COVID-19 lockdown. Many Torresian crows (Corvus orru) occur in urban areas where they scavenge for human-derived food. When this food source collapsed during the COVID-19 lockdown, Torresian crows moved to beaches where we recorded a 6-fold increase in abundance. Torresian crows principally moved to beaches with greater extent of remnant vegetation and larger average tree height. Because anthropogenic food sources would be less abundant on these more natural beaches, rapid changes in the abundance of these aggressive feeders on beaches could result in Torresian crows 1) outcompeting scavengers like large raptors for naturally occurring carrion, 2) consuming insects, crustaceans and other small animals (like small mammals and reptiles) along more natural coastlines, causing potentially significant changes to animal assemblage structure along beaches, and 3) depredating eggs and hatchlings from nests of other birds. Our results highlight that the ecological effects of changes to human pressures are nuanced, depending strongly on the functional role and behaviour of species and the landscape attributes in which they interact with the broader assemblages and ecosystem.
•Changes in human activity result in changes to animal populations.•COVID-19 lockdowns provide an opportunity to test these effects.•We found six time higher crow abundance on beaches during lockdowns.•Synanthropic species like crows redistribute readily following change in conditions.•Ecological effects of human activity change depend on animal function and behaviour.
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