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

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

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

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

Threatened reef corals of the world

10.1371/journal.pone.0034459PLoS ONE73

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

Safety of induced sputum collection in children hospitalized with severe or very severe pneumonia

Background Induced sputum (IS) may provide diagnostic information about the etiology of pneumonia. The safety of this procedure across a heterogeneous population with severe pneumonia in low- and middle-income countries has not been described. Methods IS specimens were obtained as part a 7-country study of the etiology of severe and very severe pneumonia in hospitalized children <5 years of age. Rigorous clinical monitoring was done before, during, and after the procedure to record oxygen requirement, oxygen saturation, respiratory rate, consciousness level, and other evidence of clinical deterioration. Criteria for IS contraindications were predefined and serious adverse events (SAEs) were reported to ethics committees and a central safety monitor. Results A total of 4653 IS procedures were done among 3802 children. Thirteen SAEs were reported in relation to collection of IS, or 0.34% of children with at least 1 IS specimen collected (95% confidence interval, 0.15%–0.53%). A drop in oxygen saturation that required supplemental oxygen was the most common SAE. One child died after feeding was reinitiated 2 hours after undergoing sputum induction; this death was categorized as “possibly related” to the procedure. Conclusions The overall frequency of SAEs was very low, and the nature of most SAEs was manageable, demonstrating a lowrisk safety profile for IS collection even among severely ill children in low-income-country settings. Healthcare providers should monitor oxygen saturation and requirements during and after IS collection, and assess patients prior to reinitiating feeding after the IS procedure, to ensure patient safety

Pertussis-associated pneumonia in infants and children from low- and middle-income countries participating in the PERCH study

Background. Few data exist describing pertussis epidemiology among infants and children in low-and middle-income countries to guide preventive strategies. Methods. Children 1-59 months of age hospitalized withWorld Health Organization-defined severe or very severe pneumonia in 7 African and Asian countries and similarly aged community controls were enrolled in the Pneumonia Etiology Research for Child Health study. They underwent a standardized clinical evaluation and provided nasopharyngeal and oropharyngeal swabs and induced sputum (cases only) for Bordetella pertussis polymerase chain reaction. Risk factors and pertussis-associated clinical findings were identified. Results. Bordetella pertussis was detected in 53 of 4200 (1.3%) cases and 11 of 5196 (0.2%) controls. In the age stratum1-5months, 40 (2.3% of 1721) cases were positive, all from African sites, as were 8 (0.5% of 1617) controls. Pertussis-positive African cases 1-5 months old, compared to controls, were more often human immunodeficiency virus (HIV) uninfected-exposed (adjusted odds ratio [aOR], 2.2), unvaccinated (aOR, 3.7), underweight (aOR, 6.3), and too young to be immunized (aOR, 16.1) (all P ? .05). Compared with pertussisnegative African cases in this age group, pertussis-positive cases were younger, more likely to vomit (aOR, 2.6), to cough ?14 days (aOR, 6.3), to have leukocyte counts > 20 000 cells/?L (aOR, 4.6), and to have lymphocyte counts > 10 000 cells/?L (aOR, 7.2) (all P ? .05). The case fatality ratio of pertussis-infected pneumonia cases 1-5 months of age was 12.5% (95% confidence interval, 4.2%-26.8%; 5/40); pertussis was identified in 3.7% of 137 in-hospital deaths among African cases in this age group. Conclusions. In the postneonatal period, pertussis causes a small fraction of hospitalized pneumonia cases and deaths; however, case fatality is substantial. The propensity to infect unvaccinated infants and those at risk for insufficient immunity (too young to be vaccinated, premature, HIV-infected/exposed) suggests that the role for maternal vaccination should be considered along with efforts to reduce exposure to risk factors and to optimize childhood pertussis vaccination coverage

Microscopic analysis and quality assessment of induced sputum from children with pneumonia in the PERCH study

Background It is standard practice for laboratories to assess the cellular quality of expectorated sputum specimens to check that they originated from the lower respiratory tract. The presence of low numbers of squamous epithelial cells (SECs) and high numbers of polymorphonuclear (PMN) cells are regarded as indicative of a lower respiratory tract specimen. However, these quality ratings have never been evaluated for induced sputum specimens from children with suspected pneumonia. Methods We evaluated induced sputum Gram stain smears and cultures from hospitalized children aged 1–59 months enrolled in a large study of community-acquired pneumonia. We hypothesized that a specimen representative of the lower respiratory tract will contain smaller quantities of oropharyngeal flora and be more likely to have a predominance of potential pathogens compared to a specimen containing mainly saliva. The prevalence of potential pathogens cultured from induced sputum specimens and quantity of oropharyngeal flora were compared for different quantities of SECs and PMNs. Results Of 3772 induced sputum specimens, 2608 (69%) had 25 PMNs per LPF, measures traditionally associated with specimens from the lower respiratory tract in adults. Using isolation of low quantities of oropharyngeal flora and higher prevalence of potential pathogens as markers of higher quality, 25 PMNs per LPF) was the microscopic variable most associated with high quality of induced sputum. Conclusions Quantity of SECs may be a useful quality measure of induced sputum from young children with pneumonia