In silico-guided optimisation of oxygen gradients in hepatic spheroids

One of the key advantages of assessing the hepatotoxic potential of xenobiotics in spheroids rather than monolayer cell culture is the existence of a more physiologically relevant testing environment. Three-dimensional cultures support spatial gradients in nutrients such as oxygen that can be exploited to better represent in vivo gradients that exist along a fundamental sub-unit of liver microarchitecture, the liver sinusoid. The physical and physiological processes that result in the establishment of such gradients can be described mathematically. Quantification of the rates governing these processes and optimisation of cell culture conditions can be performed in silico to better inform experimental design. In this study, we take into account cell line-specific physiological properties, spheroid size and the impact of experimental equipment geometries in order to demonstrate how mathematical models can be optimised to achieve specific in vivo-like features in different scenarios. Furthermore, the sensitivity of such optimised gradients is analysed with respect to culture conditions and considerations are given to prevent the emergence of hypoxic regions in the spheroid. The methodology presented provides an enhanced understanding of the mechanisms of the system within this simulated in vitro framework such that experimental design can be more carefully calibrated when conducting experiments using hepatic spheroids. © 2019 Elsevier B.V

A novel isolator-based system promotes viability of human embryos during laboratory processing

In vitro fertilisation (IVF) and related technologies are arguably the most challenging of all cell culture applications. The starting material is a single cell from which one aims to produce an embryo capable of establishing a pregnancy eventually leading to a live birth. Laboratory processing during IVF treatment requires open manipulations of gametes and embryos, which typically involves exposure to ambient conditions. To reduce the risk of cellular stress, we have developed a totally enclosed system of interlinked isolator-based workstations designed to maintain oocytes and embryos in a physiological environment throughout the IVF process. Comparison of clinical and laboratory data before and after the introduction of the new system revealed that significantly more embryos developed to the blastocyst stage in the enclosed isolator-based system compared with conventional open-fronted laminar flow hoods. Moreover, blastocysts produced in the isolator-based system contained significantly more cells and their development was accelerated. Consistent with this, the introduction of the enclosed system was accompanied by a significant increase in the clinical pregnancy rate and in the proportion of embryos implanting following transfer to the uterus. The data indicate that protection from ambient conditions promotes improved development of human embryos. Importantly, we found that it was entirely feasible to conduct all IVF-related procedures in the isolator-based workstations

Rgs2 Mediates Pro-Angiogenic Function of Myeloid Derived Suppressor Cells in the Tumor Microenvironment via Upregulation of MCP-1

Tumor growth is intimately linked with stromal interactions. Myeloid derived suppressor cells (MDSCs) are dramatically elevated in cancer patients and tumor bearing mice. MDSCs modulate the tumor microenvironment through attenuating host immune response and increasing vascularization.In searching for molecular mediators responsible for pro-tumor functions, we found that regulator of G protein signaling-2 (Rgs2) is highly increased in tumor-derived MDSCs compared to control MDSCs. We further demonstrate that hypoxia, a common feature associated with solid tumors, upregulates the gene expression. Genetic deletion of Rgs2 in mice resulted in a significant retardation of tumor growth, and the tumors exhibit decreased vascular density and increased cell death. Interestingly, deletion of Rgs2 in MDSCs completely abolished their tumor promoting function, suggesting that Rgs2 signaling in MDSCs is responsible for the tumor promoting function. Cytokine array profiling identified that Rgs2-/- tumor MDSCs produce less MCP-1, leading to decreased angiogenesis, which could be restored with addition of recombinant MCP-1.Our data reveal Rgs2 as a critical regulator of the pro-angiogenic function of MDSCs in the tumor microenvironment, through regulating MCP-1 production

Incidence of re-amputation following partial first ray amputation associated with diabetes mellitus and peripheral sensory neuropathy: a systematic review.

Diabetes mellitus with peripheral sensory neuropathy frequently results in forefoot ulceration. Ulceration at the first ray level tends to be recalcitrant to local wound care modalities and off-loading techniques. If healing does occur, ulcer recurrence is common. When infection develops, partial first ray amputation in an effort to preserve maximum foot length is often performed. However, the survivorship of partial first ray amputations in this patient population and associated re-amputation rate remain unknown. Therefore, in an effort to determine the actual re-amputation rate following any form of partial first ray amputation in patients with diabetes mellitus and peripheral neuropathy, the authors conducted a systematic review. Only studies involving any form of partial first ray amputation associated with diabetes mellitus and peripheral sensory neuropathy but without critical limb ischemia were included. Our search yielded a total of 24 references with 5 (20.8%) meeting our inclusion criteria involving 435 partial first ray amputations. The weighted mean age of patients was 59 years and the weighted mean follow-up was 26 months. The initial amputation level included the proximal phalanx base 167 (38.4%) times; first metatarsal head resection 96 (22.1%) times; first metatarsal-phalangeal joint disarticulation 53 (12.2%) times; first metatarsal mid-shaft 39 (9%) times; hallux fillet flap 32 (7.4%) times; first metatarsal base 29 (6.7%) times; and partial hallux 19 (4.4%) times. The incidence of re-amputation was 19.8% (86/435). The end stage, most proximal level, following re-amputation was an additional digit 32 (37.2%) times; transmetatarsal 28 (32.6%) times; below-knee 25 (29.1%) times; and LisFranc 1 (1.2%) time. The results of our systematic review reveal that one out of every five patients undergoing any version of a partial first ray amputation will eventually require more proximal re-amputation. These results reveal that partial first ray amputation for patients with diabetes and peripheral sensory neuropathy may not represent a durable, functional, or predictable foot-sparing amputation and that a more proximal amputation, such as a balanced transmetatarsal amputation, as the index amputation may be more beneficial to the patient. However, this remains a matter for conjecture due to the limited data available and, therefore, additional prospective investigations are warranted

Intermediate predator naïveté and sex-skewed vulnerability predict the impact of an invasive higher predator

The spread of invasive species continues to reduce biodiversity across all regions and habitat types globally. However, invader impact prediction can be nebulous, and approaches often fail to integrate coupled direct and indirect invader effects. Here, we examine the ecological impacts of an invasive higher predator on lower trophic groups, further developing methodologies to more holistically quantify invader impact. We employ functional response (FR, resource use under different densities) and prey switching experiments to examine the trait- and density-mediated impacts of the invasive mosquitofish Gambusia affinis on an endemic intermediate predator Lovenula raynerae (Copepoda). Lovenula raynerae effectively consumed larval mosquitoes, but was naïve to mosquitofish cues, with attack rates and handling times of the intermediate predator unaffected by mosquitofish cue-treated water. Mosquitofish did not switch between male and female prey, consistently displaying a strong preference for female copepods. We thus demonstrate a lack of risk-reduction activity in the presence of invasive fish by L. raynerae and, in turn, high susceptibility of such intermediate trophic groups to invader impact. Further, we show that mosquitofish demonstrate sex-skewed predator selectivity towards intermediate predators of mosquito larvae, which may affect predator population demographics and, perversely, increase disease vector proliferations. We advocate the utility of FRs and prey switching combined to holistically quantify invasive species impact potential on native organisms at multiple trophic levels

The type VII secretion system of <i>Staphylococcus aureus</i> secretes a nuclease toxin that targets competitor bacteria

The type VII protein secretion system (T7SS) plays a critical role in the virulence of human pathogens including Mycobacterium tuberculosis and Staphylococcus aureus. Here we report that the S. aureus T7SS secretes a large nuclease toxin, EsaD. The toxic activity of EsaD is neutralised during its biosynthesis through complex formation with an antitoxin, EsaG, which binds to its C-terminal nuclease domain. The secretion of EsaD is dependent upon a further accessory protein, EsaE, that does not interact with the nuclease domain, but instead binds to the EsaD N-terminal region. EsaE has a dual cytoplasmic/membrane localization and membrane-bound EsaE interacts with the T7SS secretion ATPase, EssC, implicating EsaE in targeting the EsaDG complex to the secretion apparatus. EsaD and EsaE are co-secreted whereas EsaG is found only in the cytoplasm and may be stripped off during the secretion process. Strain variants of S. aureus that lack esaD encode at least two copies of EsaG-like proteins most likely to protect themselves from the toxic activity of EsaD secreted by esaD(+) strains. In support of this, a strain overproducing EsaD elicits significant growth inhibition against a sensitive strain. We conclude that T7SSs may play unexpected and key roles in bacterial competitiveness

Who Eats Whom in a Pool? A Comparative Study of Prey Selectivity by Predatory Aquatic Insects

Predatory aquatic insects are a diverse group comprising top predators in small fishless water bodies. Knowledge of their diet composition is fragmentary, which hinders the understanding of mechanisms maintaining their high local diversity and of their impacts on local food web structure and dynamics. We conducted multiple-choice predation experiments using nine common species of predatory aquatic insects, including adult and larval Coleoptera, adult Heteroptera and larval Odonata, and complemented them with literature survey of similar experiments. All predators in our experiments fed selectively on the seven prey species offered, and vulnerability to predation varied strongly between the prey. The predators most often preferred dipteran larvae; previous studies further reported preferences for cladocerans. Diet overlaps between all predator pairs and predator overlaps between all prey pairs were non-zero. Modularity analysis separated all primarily nectonic predator and prey species from two groups of large and small benthic predators and their prey. These results, together with limited evidence from the literature, suggest a highly interconnected food web with several modules, in which similarly sized predators from the same microhabitat are likely to compete strongly for resources in the field (observed Pianka’s diet overlap indices >0.85). Our experiments further imply that ontogenetic diet shifts are common in predatory aquatic insects, although we observed higher diet overlaps than previously reported. Hence, individuals may or may not shift between food web modules during ontogeny

Threatened reef corals of the world

10.1371/journal.pone.0034459PLoS ONE73

Human Epidermal Neural Crest Stem Cells (hEPI-NCSC)—Characterization and Directed Differentiation into Osteocytes and Melanocytes

Here we describe the isolation, characterisation and ex-vivo expansion of human epidermal neural crest stem cells (hEPI-NCSC) and we provide protocols for their directed differentiation into osteocytes and melanocytes. hEPI-NCSC are neural crest-derived multipotent stem cells that persist into adulthood in the bulge of hair follicles. Multipotency and self-renewal were determined by in vitro clonal analyses. hEPI-NCSC generate all major neural crest derivatives, including bone/cartilage cells, neurons, Schwann cells, myofibroblasts and melanocytes. Furthermore, hEPI-NCSC express additional neural crest stem cell markers and global stem cell genes. To variable degrees and in a donor-dependent manner, hEPI-NCSC express the six essential pluripotency genes C-MYC, KLF4, SOX2, LIN28, OCT-4/POU5F1 and NANOG. hEPI-NCSC can be expanded ex vivo into millions of stem cells that remain mulitpotent and continue to express stem cell genes. The novelty of hEPI-NCSC lies in the combination of their highly desirable traits. hEPI-NCSC are embryonic remnants in a postnatal location, the bulge of hair follicles. Therefore they are readily accessible in the hairy skin by minimal invasive procedure. hEPI-NCSC are multipotent somatic stem cells that can be isolated reproducibly and with high yield. By taking advantage of their migratory ability, hEPI-NCSC can be isolated as a highly pure population of stem cells. hEPI-NCSC can undergo robust ex vivo expansion and directed differentiation. As somatic stem cells, hEPI-NCSC are conducive to autologous transplantation, which avoids graft rejection. Together, these traits make hEPI-NCSC novel and attractive candidates for future cell-based therapies and regenerative medicine