Natural spider silk is one of the world's toughest proteinaceous materials, yet a truly biomimetic spider silk is elusive even after several decades of intense focus. In this study, Next-Generation Sequencing was utilised to produce transcriptomes of the major ampullate gland of two Australian golden orb-weavers, Nephila plumipes and Nephila pilipes, in order to identify highly expressed predicted proteins that may co-factor in the construction of the final polymer. Furthermore, proteomics was performed by liquid chromatography tandem-mass spectroscopy to analyse the natural solid silk fibre of each species to confirm highly expressed predicted proteins within the silk gland are present in the final silk product. We assembled the silk gland transcriptomes of N. plumipes and N. pilipes into 69,812 and 70,123 contigs, respectively. Gene expression analysis revealed that silk gene sequences were among the most highly expressed and we were able to procure silk sequences from both species in excess of 1,300 amino acids. However, some of the genes with the highest expression values were not able to be identified from our proteomic analysis. Proteome analysis of "reeled" silk fibres of N. plumipes and N. pilipes revealed 29 and 18 proteins, respectively, most of which were identified as silk fibre proteins. This study is the first silk gland specific transcriptome and proteome analysis for these species and will assist in the future development of a biomimetic spider silk.

This study explores the effects of maternal separation as a chronic early life stress (ELS) on pancreatic islets insulin content and secretion, and their potential relationship with the hippocampus insulin content and spatial memory in young adulthood. Male rat offspring were divided into two groups: stress (STR) and non-stress (non-STR) groups. The animals of the STR group were separated from their mothers during postnatal days (PND) 1 to 21. During the weaning time, that is, PND-0 to PND-21, the body weight and length of the pups were measured. Blood samples were collected on PND-1, 21, 29 and 34 and during young adulthood (53±2 days) to determine plasma corticosterone and insulin levels. The young adult animals were also tested for spatial memory. One day after the memory test, the animals were decapitated and their pancreases were removed to measure the islets insulin content and secretion. Finally, the animals' hippocampi were isolated to determine their insulin content and insulin receptor protein amounts. During the period of weaning, the body weight and length of pups belonging to the STR group were significantly lower as compared to those in the non-STR group. Maternal separation did not change the plasma levels of insulin but increased plasma corticosterone levels from PND-21 to young adulthood and also reduced the islets insulin content but did not affect insulin secretion and the hippocampus insulin content and insulin receptor protein amount. Although, at the end of the memory tests, rats of the STR group reached the escape box at almost the same time and distance and with the same errors as rats of the non-STR group, the distance traveled to reach the escape box showed a steep reduction in the non-STR group as compared to the STR group after the first trial. Moreover, as compared to the STR group, the non-STR group showed an increasing trend for direct strategy to find the escape box. The islets insulin content and secretion, and the plasma insulin concentration were not significantly correlated with the hippocampus insulin content. From the results of the present study, it appears that the main behavioral effect of the maternal separation stress in the spatial memory task was to impair the strategy used by the animals to reach the escape box. This may indicate that maternal separation stress affects brain regions other than the hippocampus. Moreover, due to the reduction of the body weight and length of offspring belonging to the STR group, it should be further considered that both maternal separation and early life malnutrition are directly (and mechanistically) linked to cognitive alterations later in life in ways that are not dependent on peripheral and hippocampal insulin content.

Physical activity is associated with a lower risk of disease recurrence among colon cancer patients. Circulating tumor cells (CTC) are prognostic of disease recurrence among stage I-III colon cancer patients. The pathways through which physical activity may alter disease outcomes are unknown, but may be mediated by changes in CTCs.

Diabetes mellitus causes secondary osteoporosis and muscle atrophy. The ability of alfacalcidol (ALF) and exercise (Exe) to inhibit osteoporosis and muscle atrophy in type 2 diabetes mellitus (T2DM) model rats was examined. Twenty-week-old Otsuka Long-Evans Tokushima Fatty rats were randomized to ALF (orally 0.1 μg/kg/day), Exe (treadmill exercise at 10 m/min, 60 min/day, 5 days/week), Comb (ALF and Exe), and Cont (T2DM control treated with vehicle and no exercise) groups (n = 8-10 per group). Sedentary Long-Evans Tokushima Otsuka rats were used as a non-hyperphagic control. After treatment for 2 or 6 weeks, blood glucose (BG) levels, cross-sectional area (CSA) of tibialis anterior muscle fibers, femoral bone mineral density (BMD), and relative quantities of muscle anabolic markers (Pax7, MyoD, and myogenin) and catabolic markers (Atrogin-1, MuRF1, and REDD1) of the soleus muscle assessed by real-time polymerase chain reaction assays were measured. Exe and Comb treatments for 6 weeks decreased BG levels compared with those of the Cont group. ALF, Exe, and Comb treatments for 2 and 6 weeks recovered the CSA compared with that of the Cont group. ALF and Comb treatments for 6 weeks increased femoral BMDs compared with those of the Cont group. After 2 weeks of treatment, Comb treatment increased MyoD expression and decreased MuRF1 expression. ALF or Exe monotherapy significantly decreased Atrogin-1 or MuRF1 expression after 2 weeks of treatment, respectively. After 6 weeks of treatment, ALF and Comb treatments decreased Atrogin-1 and REDD1. These results demonstrate that a combination of ALF and Exe improved CSA from the early phase of treatment by stimulating skeletal muscle differentiation and suppressing muscle catabolic genes. Improvements in BG, BMD, and CSA were observed as long-term effects of the combination therapy. Continued suppression of muscle catabolic genes was observed as a background to these effects.

Modeling of neurodegenerative diseases in vitro holds great promise for biomedical research. Human cell lines harboring a mutations in disease-causing genes are thought to recapitulate early stages of the development an inherited disease. Modern genome-editing tools allow researchers to create isogenic cell clones with an identical genetic background providing an adequate "healthy" control for biomedical and pharmacological experiments. Here, we generated isogenic mutant cell clones with 150 CAG repeats in the first exon of the huntingtin (HTT) gene using the CRISPR/Cas9 system and performed ultrastructural and morphometric analyses of the internal organization of the mutant cells. Electron microscopy showed that deletion of three CAG triplets or an HTT gene knockout had no significant influence on the cell structure. The insertion of 150 CAG repeats led to substantial changes in quantitative and morphological parameters of mitochondria and increased the association of mitochondria with the smooth and rough endoplasmic reticulum while causing accumulation of small autolysosomes in the cytoplasm. Our data indicate for the first time that expansion of the CAG repeat tract in HTT introduced via the CRISPR/Cas9 technology into a human cell line initiates numerous ultrastructural defects that are typical for Huntington's disease.

Reproductive seasonality in Neotropical bats has been assessed to the better understand their reproductive behavior. This knowledge is especially important for the control of Desmodus rotundus population as it is a transmitter of rabies virus. Therefore, we aimed to evaluate the functional activity of testis and epididymis of D. rotundus in dry and rainy seasons under a morphological approach. We observed an increase in tubular diameter and epithelial height of the seminiferous tubules during the rainy season. In the latter, additionally, stereological analysis of the testis showed increased proportion of seminiferous epithelium and reduced percentage of lumen. The sperm number in caput/corpus epididymis increased in rainy season, whereas sperm count and transit time were reduced in cauda region. These alterations were probably related to the recovery of epithelium activities after mating season in dry season. Despite altered nuclear and cytoplasm parameters of Leydig cells between seasons, the volume and number of these cells were constant. Moreover, no change in serum testosterone levels, daily sperm production, and apoptotic index were observed, which indicates that the reproductive pattern in D. rotundus does not change between seasons. Our study offers a baseline for the management of vampire bat population as an attempt to control rabies disease.

Student engagement during classes includes behavioural, cognitive and emotional components, and is a pre-requisite for successful active learning environments. A novel approach to measuring student engagement was developed, involving triangulation of real-time student-self report, observation by trained observers and heart rate measurement. The self-report instrument was evaluated in four separate cohorts (n = 123) at Monash University and the University of North Carolina. The six item self-report demonstrated good reliability (Cronbach's alpha values ranged from 0.7-0.81). The self-report showed predictive validity in that small group activities were rated as significantly more engaging than didactic lecturing. Additionally, there was significant inter-instructor variability and within-class variability, indicating good discrimination between classroom activities. This self-report may prove useful to academic teaching staff in evaluating and refining their active learning activities. Independent observation was not found to correlate with student self-report, due in part to students who were pretending to engage being rated as engaged by an observer. Strikingly, students reported that they were pretending to engage for 23% of class time, even for highly regarded instructors. Individual participants were rated as engaged for 42 of the 46 intervals for which they reported that they had "pretended to engage", indicating that the two observers were unable to detect disengagement during periods in which students pretended to engage. Instructors should be aware that student cues such as eye contact and nodding may indicate pretending to engage. One particular self-report item; "I tried a new approach or way of thinking about the content", correlated positively with heart rates, and a controlled study reproduced this finding during two activities that required students to try a new approach to understanding a concept. Agreement with this item also correlated with superior performance on two in-class written assessment tasks (n = 101, p<0.01). Further use of this tool and related educational research may be useful to identify in-class activities that are engaging and likely to lead to improved student attainment of learning outcomes.

Undernutrition resulting from inadequate access to high-quality, nutritious food is a widespread issue in sub-Saharan Africa impacting the health and survival of mothers and their children. Inadequate dietary intake leads to a deficiency in nutrients including calcium, required for growth and physiological functioning. This study investigated the potential of increasing dietary calcium intake by the addition of heat-treated ground eggshell to locally prepared food. A mixed methods approach of literature review, Delphi expert survey and focus group discussions with women of childbearing age in rural Tanzania, were used to assess the practicality, safety, and acceptability of consumption of ground eggshell. Chicken eggshell has high calcium content (380 mg of calcium/gram) and bioavailability comparable to calcium carbonate (~39%) with 1 g sufficient to provide one half of a sub-Saharan African adult female's dietary calcium needs. Salmonella was indicated as the most likely threat to human health through eggshell consumption. Experts agreed that eggshells boiled for 10 min when preparing hard-boiled eggs with a further 20 min cooking of crushed eggshell in staple foods would eliminate identified egg-associated pathogens. Five focus groups (n = 46) indicated eggshells were perceived as waste. However, there was an indication of general acceptance of the approach and a willingness to consider the incorporation of ground eggshells into their diets. Development of suitable communication methods are required to convey benefits and safe preparation methods. Ground eggshell could be a highly equitable method of increasing calcium intakes across rural sub-Saharan Africa where calcium intake is low and village poultry ownership common.

During 2018, an unusual increase in Lassa fever cases occurred in Nigeria, raising concern among national and international public health agencies. We analyzed 220 Lassa virus genomes from infected patients, including 129 from the 2017-2018 transmission season, to understand the viral populations underpinning the increase. A total of 14 initial genomes from 2018 samples were generated at Redeemer's University in Nigeria, and the findings were shared with the Nigerian Center for Disease Control in real time. We found that the increase in cases was not attributable to a particular Lassa virus strain or sustained by human-to-human transmission. Instead, the data were consistent with ongoing cross-species transmission from local rodent populations. Phylogenetic analysis also revealed extensive viral diversity that was structured according to geography, with major rivers appearing to act as barriers to migration of the rodent reservoir.

Metastatic disease is a major cause of mortality in cancer patients. While many drug delivery strategies for anticancer therapeutics have been developed in preclinical studies of primary tumors, the drug delivery properties of metastatic tumors have not been sufficiently investigated. Therapeutic efficacy hinges on efficient drug permeation into the tumor microenvironment, which is known to be heterogeneous thus potentially making drug permeation heterogeneous, also. In this study, we have identified that 4 T1 liver metastases, treated with pegylated liposomal doxorubicin, have unfavorable and heterogeneous transport of doxorubicin. Our drug extravasation results differ greatly from analogous studies with 4 T1 tumors growing in the primary site. A probabilistic tumor population model was developed to estimate drug permeation efficiency and drug kinetics of liver metastases by integrating the transport and structural properties of tumors and delivered drugs. The results demonstrate significant heterogeneity in metastases with regard to transport properties of doxorubicin within the same animal model, and even within the same organ. These results also suggest that the degree of heterogeneity depends on the stage of tumor progression and that differences in transport properties can define transport-based tumor phenotypes. These findings may have valuable clinical implications by illustrating that therapeutic agents can permeate and eliminate metastases of "less resistant" transport phenotypes, while sparing tumors with more "resistant" transport properties. We anticipate that these results could challenge the current paradigm of drug delivery into metastases, highlight potential caveats for therapies that may alter tumor perfusion, and deepen our understanding of the emergence of drug transport-based therapeutic resistance.

Macrophages polarize into distinct phenotypes in response to complex environmental cues. We found that the nuclear receptor PPARγ drove robust phenotypic changes in macrophages upon repeated stimulation with interleukin (IL)-4. The functions of PPARγ on macrophage polarization in this setting were independent of ligand binding. Ligand-insensitive PPARγ bound DNA and recruited the coactivator P300 and the architectural protein RAD21. This established a permissive chromatin environment that conferred transcriptional memory by facilitating the binding of the transcriptional regulator STAT6 and RNA polymerase II, leading to robust production of enhancer and mRNAs upon IL-4 re-stimulation. Ligand-insensitive PPARγ binding controlled the expression of an extracellular matrix remodeling-related gene network in macrophages. Expression of these genes increased during muscle regeneration in a mouse model of injury, and this increase coincided with the detection of IL-4 and PPARγ in the affected tissue. Thus, a predominantly ligand-insensitive PPARγ:RXR cistrome regulates progressive and/or reinforcing macrophage polarization.

Naegleria gruberi is a free-living non-pathogenic amoeboflagellate and relative of Naegleria fowleri, a deadly pathogen causing primary amoebic meningoencephalitis (PAM). A genomic analysis of N. gruberi exists, but physiological evidence for its core energy metabolism or in vivo growth substrates is lacking. Here, we show that N. gruberi trophozoites need oxygen for normal functioning and growth and that they shun both glucose and amino acids as growth substrates. Trophozoite growth depends mainly upon lipid oxidation via a mitochondrial branched respiratory chain, both ends of which require oxygen as final electron acceptor. Growing N. gruberi trophozoites thus have a strictly aerobic energy metabolism with a marked substrate preference for the oxidation of fatty acids. Analyses of N. fowleri genome data and comparison with those of N. gruberi indicate that N. fowleri has the same type of metabolism. Specialization to oxygen-dependent lipid breakdown represents an additional metabolic strategy in protists.

Neural network remodeling underpins the ability to remember life experiences, but little is known about the long-term plasticity of neural populations. To study how the brain encodes episodic events, we used time-lapse two-photon microscopy and a fluorescent reporter of neural plasticity based on an enhanced form of the synaptic activity-responsive element (E-SARE) within the Arc promoter to track thousands of CA1 hippocampal pyramidal cells over weeks in mice that repeatedly encountered different environments. Each environment evokes characteristic patterns of ensemble neural plasticity, but with each encounter, the set of activated cells gradually evolves. After repeated exposures, the plasticity patterns evoked by an individual environment progressively stabilize. Compared with young adults, plasticity patterns in aged mice are less specific to individual environments and less stable across repeat experiences. Long-term consolidation of hippocampal plasticity patterns may support long-term memory formation, whereas weaker consolidation in aged subjects might reflect declining memory function.

A hallmark of aging is a decline in metabolic homeostasis, which is attenuated by dietary restriction (DR). However, the interaction of aging and DR with the metabolome is not well understood. We report that DR is a stronger modulator of the rat metabolome than age in plasma and tissues. A comparative metabolomic screen in rodents and humans identified circulating sarcosine as being similarly reduced with aging and increased by DR, while sarcosine is also elevated in long-lived Ames dwarf mice. Pathway analysis in aged sarcosine-replete rats identify this biogenic amine as an integral node in the metabolome network. Finally, we show that sarcosine can activate autophagy in cultured cells and enhances autophagic flux in vivo, suggesting a potential role in autophagy induction by DR. Thus, these data identify circulating sarcosine as a biomarker of aging and DR in mammalians and may contribute to age-related alterations in the metabolome and in proteostasis.

CDH1 and PIK3CA are the two most frequently mutated genes in invasive lobular carcinoma (ILC) of the breast. Transcription profiling has identified molecular subtypes for ILC, one of which, immune-related (IR), is associated with gene expression linked to lymphocyte and macrophage infiltration. Here, we report that deletion of Cdh1, together with activation of Pik3ca in mammary epithelium of genetically modified mice, leads to formation of IR-ILC-like tumors with immune cell infiltration, as well as gene expression linked to T-regulatory (Treg) cell signaling and activation of targetable immune checkpoint pathways. Interestingly, these tumors show enhanced Rac1- and Yap-dependent transcription and signaling, as well as sensitivity to PI3K, Rac1, and Yap inhibitors in culture. Finally, high-dimensional immunophenotyping in control mouse mammary gland and IR-ILC tumors by mass cytometry shows dramatic alterations in myeloid and lymphoid populations associated with immune suppression and exhaustion, highlighting the potential for therapeutic intervention via immune checkpoint regulators.

Despite advances made in understanding the effects of promoter structure on transcriptional activity, limited knowledge exists regarding the role played by chromatin architecture in transcription. Previous work hypothesized that transcription from the bidirectional GAL1/GAL10 promoter is controlled through looping of its UAS region around a nonstandard nucleosome. Here, by editing the GAL1/GAL10 promoter at high resolution, we provide insights into bidirectional expression control. We demonstrate that the first and fourth Gal4 binding sites within the UAS do not functionally contribute to promoter activation. Instead, these sites, along with nearby regulatory regions, contribute to the directional regulation of gene expression. Furthermore, Gal4 binding to the third binding site is critical for gene expression, while binding to the other three sites is not sufficient for transcriptional activation. Because the GAL1/GAL10 UAS can activate gene expression in many eukaryotes, the regulatory mechanism presented is expected to operate broadly across the eukaryotic clade.

Deviating from the normal karyotype dramatically changes gene dosage, in turn decreasing the robustness of biological networks. Consequently, aneuploidy is poorly tolerated by normal somatic cells and acts as a barrier to transformation. Paradoxically, however, karyotype heterogeneity drives tumor evolution and the emergence of therapeutic drug resistance. To better understand how cancer cells tolerate aneuploidy, we focused on the p38 stress response kinase. We show here that p38-deficient cells upregulate glycolysis and avoid post-mitotic apoptosis, leading to the emergence of aneuploid subclones. We also show that p38 deficiency upregulates the hypoxia-inducible transcription factor Hif-1α and that inhibiting Hif-1α restores apoptosis in p38-deficent cells. Because hypoxia and aneuploidy are both barriers to tumor progression, the ability of Hif-1α to promote cell survival following chromosome missegregation raises the possibility that aneuploidy tolerance coevolves with adaptation to hypoxia.

Paxillin is a scaffold protein that participates in focal adhesion signaling in mammalian cells. Fission yeast paxillin ortholog, Pxl1, is required for contractile actomyosin ring (CAR) integrity and collaborates with the β-glucan synthase Bgs1 in septum formation. We show here that Pxl1's main function is to recruit calcineurin (CN) phosphatase to the actomyosin ring; and thus the absence of either Pxl1 or calcineurin causes similar cytokinesis defects. In turn, CN participates in the dephosphorylation of the Cdc15 F-BAR protein, which recruits and concentrates Pxl1 at the CAR. Our findings suggest the existence of a positive feedback loop between Pxl1 and CN and establish that Pxl1 is a crucial component of the CN signaling pathway during cytokinesis.

The immune system is highly diverse, but characterization of its genetic architecture has lagged behind the vast progress made by genome-wide association studies (GWASs) of emergent diseases. Our GWAS for 54 functionally relevant phenotypes of the adaptive immune system in 489 healthy individuals identifies eight genome-wide significant associations explaining 6%-20% of variance. Coding and splicing variants in PTPRC and COMMD10 are involved in memory T cell differentiation. Genetic variation controlling disease-relevant T helper cell subsets includes RICTOR and STON2 associated with Th2 and Th17, respectively, and the interferon-lambda locus controlling regulatory T cell proliferation. Early and memory B cell differentiation stages are associated with variation in LARP1B and SP4. Finally, the latrophilin family member ADGRL2 correlates with baseline pro-inflammatory interleukin-6 levels. Suggestive associations reveal mechanisms of autoimmune disease associations, in particular related to pro-inflammatory cytokine production. Pinpointing these key human immune regulators offers attractive therapeutic perspectives.

Esthesioneuroblastoma (ENB) is a rare cancer of the olfactory mucosa, with no established molecular stratification to date. We report similarities of ENB with tumors arising in the neural crest and perform integrative analysis of these tumors. We propose a molecular-based subtype classification of ENB as basal or neural, both of which have distinct pathological, transcriptomic, proteomic, and immune features. Among the basal subtype, we uncovered an IDH2 R172 mutant-enriched subgroup (∼35%) harboring a CpG island methylator phenotype reminiscent of IDH2 mutant gliomas. Compared with the basal ENB methylome, the neural ENB methylome shows genome-wide reprogramming with loss of DNA methylation at the enhancers of axonal guidance genes. Our study reveals insights into the molecular pathogenesis of ENB and provides classification information of potential therapeutic relevance.