Pax6 limits the competence of developing cerebral cortical cells to respond to inductive intercellular signals.

The development of stable specialized cell types in multicellular organisms relies on mechanisms controlling inductive intercellular signals and the competence of cells to respond to such signals. In developing cerebral cortex, progenitors generate only glutamatergic excitatory neurons despite being exposed to signals with the potential to initiate the production of other neuronal types, suggesting that their competence is limited. Here, we tested the hypothesis that this limitation is due to their expression of transcription factor Pax6. We used bulk and single-cell RNAseq to show that conditional cortex-specific Pax6 deletion from the onset of cortical neurogenesis allowed some progenitors to generate abnormal lineages resembling those normally found outside the cortex. Analysis of selected gene expression showed that the changes occurred in specific spatiotemporal patterns. We then compared the responses of control and Pax6-deleted cortical cells to in vivo and in vitro manipulations of extracellular signals. We found that Pax6 loss increased cortical progenitors' competence to generate inappropriate lineages in response to extracellular factors normally present in developing cortex, including the morphogens Shh and Bmp4. Regional variation in the levels of these factors could explain spatiotemporal patterns of fate change following Pax6 deletion in vivo. We propose that Pax6's main role in developing cortical cells is to minimize the risk of their development being derailed by the potential side effects of morphogens engaged contemporaneously in other essential functions.

Glutamine deprivation alters the origin and function of cancer cell exosomes.

Exosomes are secreted extracellular vesicles carrying diverse molecular cargos, which can modulate recipient cell behaviour. They are thought to derive from intraluminal vesicles formed in late endosomal multivesicular bodies (MVBs). An alternate exosome formation mechanism, which is conserved from fly to human, is described here, with exosomes carrying unique cargos, including the GTPase Rab11, generated in Rab11-positive recycling endosomal MVBs. Release of Rab11-positive exosomes from cancer cells is increased relative to late endosomal exosomes by reducing growth regulatory Akt/mechanistic Target of Rapamycin Complex 1 (mTORC1) signalling or depleting the key metabolic substrate glutamine, which diverts membrane flux through recycling endosomes. Vesicles produced under these conditions promote tumour cell proliferation and turnover and modulate blood vessel networks in xenograft mouse models in vivo. Their growth-promoting activity, which is also observed in vitro, is Rab11a-dependent, involves ERK-MAPK-signalling and is inhibited by antibodies against amphiregulin, an EGFR ligand concentrated on these vesicles. Therefore, glutamine depletion or mTORC1 inhibition stimulates release from Rab11a compartments of exosomes with pro-tumorigenic functions, which we propose promote stress-induced tumour adaptation.

Modified fructan accumulation through overexpression of wheat fructan biosynthesis pathway fusion genes Ta1SST:Ta6SFT.

BACKGROUND: Fructans are water-soluble carbohydrates that accumulate in wheat and are thought to contribute to a pool of stored carbon reserves used in grain filling and tolerance to abiotic stress. RESULTS: In this study, transgenic wheat plants were engineered to overexpress a fusion of two fructan biosynthesis pathway genes, wheat sucrose: sucrose 1-fructosyltransferase (Ta1SST) and wheat sucrose: fructan 6-fructosyltransferase (Ta6SFT), regulated by a wheat ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit (TaRbcS) gene promoter. We have shown that T4 generation transgene-homozygous single-copy events accumulated more fructan polymers in leaf, stem and grain when compared in the same tissues from transgene null lines. Under water-deficit (WD) conditions, transgenic wheat plants showed an increased accumulation of fructan polymers with a high degree of polymerisation (DP) when compared to non-transgenic plants. In wheat grain of a transgenic event, increased deposition of particular fructan polymers such as, DP4 was observed. CONCLUSIONS: This study demonstrated that the tissue-regulated expression of a gene fusion between Ta1SST and Ta6SFT resulted in modified fructan accumulation in transgenic wheat plants and was influenced by water-deficit stress conditions.

Electrographic screening for infantile epileptic spasms syndrome in a single sleep-wake cycle.

OBJECTIVE: Infantile epileptic spasms syndrome (IESS) is a common and urgent diagnosis with seizure and nonseizure mimics. Evaluation with prolonged video-electroencephalography (EEG) can be time-consuming and costly. This study investigated the use of EEG review of a single sleep-wake cycle to exclude IESS. METHODS: We retrospectively reviewed video-EEG studies to rule out IESS in children between the ages of 2 months and 2 years in the period from January 2019 through June 2020. EEG studies were reviewed from the start of the recording through the first sleep-wake cycle and scored as "normal," "consistent with IESS," or "abnormal but not diagnostic of IESS." Scores were compared to the clinical report created by analysis of the entire video-EEG. RESULTS: Inclusion criteria were met in 238 EEG studies. The mean patient age was 7.6 months. The median duration of the full study was 908 min, compared to 107.5 min for the first sleep-wake cycle only. The median difference in recording time was 801 min, p-value < .01. Scored outcomes were similar. Sixty-eight percent of EEG studies were scored as "normal" on first sleep-wake cycle review as compared to 63% on full study review, 13% scored as "consistent with IESS" compared to 16% and 19% scored as "abnormal but not diagnostic of IESS" compared to 21%. Sensitivity and specificity of the first sleep-wake cycle review for studies "consistent with IESS" was 84% and 100%, respectively. No cases of IESS were scored as normal on first sleep-wake cycle review. SIGNIFICANCE: A single sleep-wake cycle captured on EEG can triage studies when IESS is suspected. A normal first sleep-wake cycle did not miss cases of IESS and could result in reduced EEG recording time. Because most of these cases presented to an emergency department, a normal first sleep-wake cycle may help providers determine the acuity, or necessity, of further testing.

The transcription factor E2A drives neural differentiation in pluripotent cells.

The intrinsic mechanisms that link extracellular signalling to the onset of neural differentiation are not well understood. In pluripotent mouse cells, BMP blocks entry into the neural lineage via transcriptional upregulation of inhibitor of differentiation (Id) factors. We have previously identified the major binding partner of Id proteins in pluripotent cells as the basic helix-loop-helix (bHLH) transcription factor (TF) E2A. Id1 can prevent E2A from forming heterodimers with bHLH TFs or from forming homodimers. Here, we show that overexpression of a forced E2A homodimer is sufficient to drive robust neural commitment in pluripotent cells, even under non-permissive conditions. Conversely, we find that E2A null cells display a defect in their neural differentiation capacity. E2A acts as an upstream activator of neural lineage genes, including Sox1 and Foxd4, and as a repressor of Nodal signalling. Our results suggest a crucial role for E2A in establishing neural lineage commitment in pluripotent cells.

Understanding the Mechanism for Adsorption of Pb(II) Ions by Cu-BTC Metal-Organic Frameworks.

With the growing population, industrialization, and agriculture, water contamination not only affects people but entire ecosystems. Metal-organic frameworks (MOFs), because of their large surface area and porosity, show great potential as adsorbents for removing pollutants, such as heavy metals, from contaminated water. The current research aims at examining copper (II) benzene-1,3,5-tricarboxylate (Cu-BTC) MOFs and understanding the mechanism for their adsorption of Pb(II) from aqueous solution. The Cu-BTC samples were characterized using FTIR and XRD, and their surface area and porosity were determined based on N2 adsorption isotherms. The concentration of Pb(II) in the solutions was measured using atomic absorption spectroscopy (AAS). Both kinetic and equilibrium adsorption data were collected and then analyzed using numerical models. The analyses led to the findings that the limiting steps in the adsorption of Pb(II) on Cu-BTC are (a) pore diffusion of Pb(II) and (b) the availability of the active sites on Cu-BTC MOFs. It was further revealed that the former step is more dominant in the adsorption of Pb(II) when the lead concentration is low. The latter step, which is directly proportional to the surface areas of the MOFs, affects the adsorption to a greater extent when the lead concentration is high. The results also show that adsorption of Pb(II) ions on Cu-BTC is mainly a multi-layer heterogeneous process.

Mating induces switch from hormone-dependent to hormone-independent steroid receptor-mediated growth in Drosophila secondary cells.

Male reproductive glands like the mammalian prostate and the paired Drosophila melanogaster accessory glands secrete seminal fluid components that enhance fecundity. In humans, the prostate, stimulated by environmentally regulated endocrine and local androgens, grows throughout adult life. We previously showed that in fly accessory glands, secondary cells (SCs) and their nuclei also grow in adults, a process enhanced by mating and controlled by bone morphogenetic protein (BMP) signalling. Here, we demonstrate that BMP-mediated SC growth is dependent on the receptor for the developmental steroid ecdysone, whose concentration is reported to reflect sociosexual experience in adults. BMP signalling appears to regulate ecdysone receptor (EcR) levels via one or more mechanisms involving the EcR's N terminus or the RNA sequence that encodes it. Nuclear growth in virgin males is dependent on ecdysone, some of which is synthesised in SCs. However, mating induces additional BMP-mediated nuclear growth via a cell type-specific form of hormone-independent EcR signalling, which drives genome endoreplication in a subset of adult SCs. Switching to hormone-independent endoreplication after mating allows growth and secretion to be hyperactivated independently of ecdysone levels in SCs, permitting more rapid replenishment of the accessory gland luminal contents. Our data suggest mechanistic parallels between this physiological, behaviour-induced signalling switch and altered pathological signalling associated with prostate cancer progression.

Can surgical skills be taught using technological advances online? A comparative study of online and face-to-face surgical skills training.

INTRODUCTION: Online teaching has rapidly emerged as a viable alternative to traditional face-to-face education. How to teach surgical skills in the online environment, however, has not yet been fully established nor evaluated. METHODS: An international 1-day online surgical skills course consisting of lectures, pre-recorded virtual workshops, live demonstrations and along with surgical skills teaching in breakout rooms was organised. Based on existing learning theories, new methods were developed to deliver skills teaching online. Simultaneously, traditional in-person surgical skills teaching was also conducted and used as a benchmark. Skills development was assessed by trained demonstrators and self-reported competency scores were compared between the online and face-to-face event. RESULTS: 553 delegates from 20 different countries attended the online course. Of these, 64 were trained in breakout rooms with a 1:5 demonstrator-to-delegate ratio whilst the remaining 489 delegates participated in didactic skills development sessions. In a separate face-to-face course, 20 delegates were trained with traditional methods. Demonstrators rated the competency of delegates for suturing, tendon repair and vascular anastomosis. There was no significant difference in the competency ratings of delegates receiving online teaching or face-to-face teaching (p = 0.253, p = 0.084, p = 1.00, respectively). The development of the same skills to "articulation" were not different between formats (p = 0.841, p = 0.792, p = 1.00, respectively). Post course self-rated competency scores improved for all technical skills (p < 0.001). Small group sessions, both online and face-to-face, received higher satisfaction ratings compared to large group sessions in terms of clarity of instructions, answers to questions and demonstrator feedback. Overall feedback on teaching quality, however, was equivalent across both groups. DISCUSSION: Online teaching of surgical skills for early training years is an appropriate alternative to face-to-face teaching.

Visual outcomes of macula-involving rhegmatogenous retinal detachment in patients with and without age-related macular degeneration.

BACKGROUND: The prognosis for patients with macula-off rhegmatogenous retinal detachment (RRD) and concomitant age-related macular degeneration (AMD) is not well known. The purpose of this study is to compare visual outcomes in macula-off RRD in eyes with AMD versus a group of comparison eyes without AMD. METHODS: This was a retrospective chart review of 1149 patients. A total of 191 eyes met study criteria, 162 non-AMD eyes (controls), and 29 AMD eyes. The main outcome measure was postoperative visual acuity following pars plana vitrectomy (PPV), scleral buckle (SB), or combined PPV/SB in control eyes versus AMD eyes. This was compared using Fisher's exact test. RESULTS: There was a statistically significant difference in postoperative visual acuity by AMD status, with those without AMD having a worse visual outcome overall (p = 0.0048). A similar percentage of AMD versus non-AMD eyes achieved vision better than 20/40. More patients in the non-AMD group achieved a final visual acuity between 20/40 and 20/200. Of patients with AMD, more had vision worse than 20/200 though 58% maintained functional vision (better than 20/200). Those without AMD had a higher frequency of Count Fingers (CF), Hand Motion (HM), Light Perception (LP), or No Light Perception (NLP) vision (p = 0.023). CONCLUSIONS: Though postoperative visual acuity was worse overall in the non-AMD group with a higher frequency of patients having final vision of CF, HM, LP, or NLP, this is likely a function of the difference in sample size and composition between the two groups. Importantly, this study suggests AMD patients can expect similar outcomes to non-AMD patients after RRD repair. We conclude that AMD patients can achieve functional vision after RRD surgery, similar to those without AMD.

The Effect of Blood Flow Restriction Training on Muscle Atrophy Following Meniscal Repair or Chondral Restoration Surgery in Active Duty Military: A Randomized Controlled Trial.

CONTEXT: Recently, blood flow restriction (BFR) training has gained popularity as an alternative to high-load resistance training for improving muscle strength and hypertrophy. Previous BFR studies have reported positive treatment effects; however, clinical benefits to using BFR following meniscal repair or chondral surgery are unknown. The purpose of this study was to determine the effect of resistance exercises with BFR training versus exercises alone on self-reported knee function, thigh circumference, and knee flexor/extensor strength postmeniscal or cartilage surgery. DESIGN: Single-blinded randomized controlled trial in an outpatient military hospital setting. Twenty participants were randomized into 2 groups: BFR group (n = 11) and control group (n = 9). METHODS: Participants completed 12 weeks of postoperative thigh strengthening. The BFR group performed each exercise with the addition of BFR. Both groups continued with the prescribed exercises without BFR from 12 weeks until discharged from therapy. Thigh circumference and self-reported knee function were measured at 1, 6, 12, and 24 weeks postoperatively along with knee extensor and flexor strength at 12 and 24 weeks. Change scores between time points were calculated for knee function. Limb symmetry indices (LSI) were computed for thigh circumference and knee strength variables. RESULTS: Seventeen participants were included in the final analyses (BFR = 8 and control = 9) due to COVID-19 restrictions. There were no interactions or main effects for group. Time main effects were established for change in knee function scores, thigh circumference LSI, and knee extensor strength LSI. However, knee flexor strength LSI had no main effect for time. CONCLUSION: The outcomes of this trial suggest that resistance exercises with and without BFR training may result in similar changes to function, thigh atrophy, and knee extensor strength postmeniscus repair/chondral restoration, though further study with larger sample sizes is needed.

Impact of donor extracellular vesicle release on recipient cell "cross-dressing" following clinical liver and kidney transplantation.

In several murine models of transplantation, the "cross-dressing" of recipient antigen presenting cells (APCs) with intact donor major histocompatibility complex (MHC) derived from allograft-released small extracellular vesicles (sEVs) has been recently described as a key mechanism in eliciting and sustaining alloimmune responses. Investigation of these processes in clinical organ transplantation has, however, been hampered by the lack of sensitivity of conventional instruments and assays. We have employed advanced imaging flow cytometry (iFCM) to explore the kinetics of allograft sEV release and the extent to which donor sEVs might induce cross-dressing following liver and kidney transplantation. We report for the first time that recipient APC cross-dressing can be transiently detected in the circulation shortly after liver, but not kidney, transplantation in association with the release of HLA-bearing allograft-derived sEVs. In liver transplant recipients the majority of circulating cells exhibiting donor HLA are indeed cross-dressed cells and not passenger leukocytes. In keeping with experimental animal data, the downstream functional consequences of the transfer of circulating sEVs harvested from human transplant recipients varies depending on the type of transplant and time posttransplant. sEVs released shortly after liver, but not kidney, transplantation exhibit immunoinhibitory effects that could influence liver allograft immunogenicity.

Gli3 controls the onset of cortical neurogenesis by regulating the radial glial cell cycle through Cdk6 expression.

The cerebral cortex contains an enormous number of neurons, allowing it to perform highly complex neural tasks. Understanding how these neurons develop at the correct time and place and in accurate numbers constitutes a major challenge. Here, we demonstrate a novel role for Gli3, a key regulator of cortical development, in cortical neurogenesis. We show that the onset of neuron formation is delayed in Gli3 conditional mouse mutants. Gene expression profiling and cell cycle measurements indicate that shortening of the G1 and S phases in radial glial cells precedes this delay. Reduced G1 length correlates with an upregulation of the cyclin-dependent kinase gene Cdk6, which is directly regulated by Gli3. Moreover, pharmacological interference with Cdk6 function rescues the delayed neurogenesis in Gli3 mutant embryos. Overall, our data indicate that Gli3 controls the onset of cortical neurogenesis by determining the levels of Cdk6 expression, thereby regulating neuronal output and cortical size.

Digital PCR (dPCR) and qPCR mediated determination of transgene copy number in the forage legume white clover (Trifolium repens).

Obtaining data on transgene copy number is an integral step in the generation of transgenic plants. Techniques such as Southern blot, segregation analysis, and quantitative PCR (qPCR) have routinely been used for this task, in a range of species. More recently, use of Digital PCR (dPCR) has become prevalent, with a measurement accuracy higher than qPCR reported. Here, the relative merits of qPCR and dPCR for transgene copy number estimation in white clover were investigated. Furthermore, given that single copy reference genes are desirable for estimating gene copy number by relative quantification, and that no single-copy genes have been reported in this species, a search and evaluation of suitable reference genes in white clover was undertaken. Results demonstrated a higher accuracy of dPCR relative to qPCR for copy number estimation in white clover. Two genes, Pyruvate dehydrogenase (PDH), and an ATP-dependent protease, identified as single-copy genes, were used as references for copy number estimation by relative quantification. Identification of single-copy genes in white clover will enable the application of relative quantification for copy number estimation of other genes or transgenes in the species. The results generated here validate the use of dPCR as a reliable strategy for transgene copy number estimation in white clover, and provide resources for future copy number studies in this species.

Heparan Sulfate Sulfation by Hs2st Restricts Astroglial Precursor Somal Translocation in Developing Mouse Forebrain by a Non-Cell-Autonomous Mechanism.

Heparan sulfate (HS) is a cell surface and extracellular matrix carbohydrate extensively modified by differential sulfation. HS interacts physically with canonical fibroblast growth factor (FGF) proteins that signal through the extracellular signal regulated kinase (ERK)/mitogen activated protein kinase (MAPK) pathway. At the embryonic mouse telencephalic midline, FGF/ERK signaling drives astroglial precursor somal translocation from the ventricular zone of the corticoseptal boundary (CSB) to the induseum griseum (IG), producing a focus of Slit2-expressing astroglial guidepost cells essential for interhemispheric corpus callosum (CC) axon navigation. Here, we investigated the cell and molecular function of a specific form of HS sulfation, 2-O HS sulfation catalyzed by the enzyme Hs2st, in midline astroglial development and in regulating FGF protein levels and interaction with HS. Hs2st-/- embryos of either sex exhibit a grossly enlarged IG due to precocious astroglial translocation and conditional Hs2st mutagenesis and ex vivo culture experiments show that Hs2st is not required cell autonomously by CC axons or by the IG astroglial cell lineage, but rather acts non-cell autonomously to suppress the transmission of translocation signals to astroglial precursors. Rescue of the Hs2st-/- astroglial translocation phenotype by pharmacologically inhibiting FGF signaling shows that the normal role of Hs2st is to suppress FGF-mediated astroglial translocation. We demonstrate a selective action of Hs2st on FGF protein by showing that Hs2st (but not Hs6st1) normally suppresses the levels of Fgf17 protein in the CSB region in vivo and use a biochemical assay to show that Hs2st (but not Hs6st1) facilitates a physical interaction between the Fgf17 protein and HS.SIGNIFICANCE STATEMENT We report a novel non-cell-autonomous mechanism regulating cell signaling in developing brain. Using the developing mouse telencephalic midline as an exemplar, we show that the specific sulfation modification of the cell surface and extracellular carbohydrate heparan sulfate (HS) performed by Hs2st suppresses the supply of translocation signals to astroglial precursors by a non-cell-autonomous mechanism. We further show that Hs2st modification selectively facilitates a physical interaction between Fgf17 and HS and suppresses Fgf17 protein levels in vivo, strongly suggesting that Hs2st acts selectively on Fgf17 signaling. HS interacts with many signaling proteins potentially encoding numerous selective interactions important in development and disease, so this class of mechanism may apply more broadly to other biological systems.

Radiographic and Clinical Outcomes of Modular Tapered Fluted Stems for Femoral Revision for Paprosky III and IV Femoral Defects or Vancouver B2 and B3 Femoral Fractures.

BACKGROUND: Extensive femoral bone loss poses a challenge in revision total hip arthroplasty (rTHA). Many techniques have been developed to address this problem including fully porous cylindrical stems, impaction bone grafting, and cementation of long stems, which have had varied success. Modular tapered fluted femoral stems (MTFS) show favorable results. We sought to determine the minimum 2-year radiographic and clinical performance of MTFS in rTHA in a population with extensive proximal femoral bone loss. METHODS: Our clinical database was queried retrospectively for all patients who underwent rTHA with an MTFS. We included patients with Paprosky 3 and 4 femoral bone loss and patients with Vancouver B2 and B3 periprosthetic femur fractures. Patients without 2-year follow-up were invited to return to clinic for X-ray evaluation and to complete clinical questionnaires. We assessed distance of stem subsidence and presence of stem fixation on final X-ray. We recorded all-cause revision and survival of the stem at final follow-up. RESULTS: One hundred twenty-nine patients were available for follow-up. Average follow-up time was 3.75 years. One hundred twenty-two stems (95%) remained in place at final follow-up. Median subsidence was 1.4 mm (range 0-21). All-cause revision rate was 16.3% (21 patients). Of the hips revised, 10 were for instability, 6 for infection, 1 for aseptic loosening, and 1 for periprosthetic femur fracture. Three were revised for other reasons. The stem was revised in 7 patients (5.4%), and the most common reason for stem revision was infection (5 patients). The other 2 stems were revised for aseptic loosening in a Paprosky 4 femur and periprosthetic femur fracture. Survival of tapered modular fluted stems with aseptic failure as an endpoint was 98.4%. The mean Hip disability and Osteoarthritis Outcome Score, Joint Replacement score at final follow-up was 73, and mean Veterans Rand 12 item health survey physical and mental scores were 32.8 and 52.2, respectively. CONCLUSION: In patients with Paprosky 3, 4 femoral defects or Vancouver type B2, B3 fractures, modular tapered fluted stems for femoral revision show excellent outcomes at minimum 2-year follow-up.

Carbon dynamics, net primary productivity and human-appropriated net primary productivity across a forest-cocoa farm landscape in West Africa.

Terrestrial net primary productivity (NPP) is an important metric of ecosystem functioning; however, there are little empirical data on the NPP of human-modified ecosystems, particularly smallholder, perennial crops like cocoa (Theobroma cacao), which are extensive across the tropics. Human-appropriated NPP (HANPP) is a measure of the proportion of a natural system's NPP that has either been reduced through land-use change or harvested directly and, previously, has been calculated to estimate the scale of the human impact on the biosphere. Additionally, human modification can create shifts in NPP allocation and decomposition, with concomitant impacts on the carbon cycle. This study presents the results of 3 years of intensive monitoring of forest and smallholder cocoa farms across disturbance, management intensity, distance from forest and farm age gradients. We measured among the highest reported NPP values in tropical forest, 17.57 ± 2.1 and 17.7 ± 1.6 Mg C ha-1  year-1 for intact and logged forest, respectively; however, the average NPP of cocoa farms was still higher, 18.8 ± 2.5 Mg C ha-1  year-1 , which we found was driven by cocoa pod production. We found a dramatic shift in litterfall residence times, where cocoa leaves decomposed more slowly than forest leaves and shade tree litterfall decomposed considerably faster, indicating significant changes in rates of nutrient cycling. The average HANPP value for all cocoa farms was 2.1 ± 1.1 Mg C ha-1  year-1 ; however, depending on the density of shade trees, it ranged from -4.6 to 5.2 Mg C ha-1  year-1 . Therefore, rather than being related to cocoa yield, HANPP was reduced by maintaining higher shade levels. Across our monitored farms, 18.9% of farm NPP was harvested (i.e., whole cocoa pods) and only 1.1% (i.e., cocoa beans) was removed from the system, suggesting that the scale of HANPP in smallholder cocoa agroforestry systems is relatively small.

An energetic reformulation of kinetic rate laws enables scalable parameter estimation for biochemical networks.

The technology for building functionally complete or 'whole-cell' biological simulations is rapidly developing. However, the predictive capabilities of these simulations are hindered by the availability of parameter values, which are often difficult or even impossible to obtain experimentally and must therefore be estimated. Using E. coli's glycolytic network as a model system, we describe and apply a new method which can estimate the values of all the system's 102 parameters - fit to observations from studies of proteomics, metabolomics, enzyme kinetics and chemical energetics - and find that the resulting metabolic models are not only well-fit, but also dynamically stable. An analysis of how well parameter values in the network were determined by the training data revealed that over 80% of the parameter values were not well-specified. Moreover, the distribution of well-determined values was biased to a specific part of the network and against certain types of experimental data. Our results also suggest that perturbing the functional, energetic space of parameters (rather than traditional metabolic parameters) is a superior strategy for exploring the space of biological dynamics. The estimated parameter values matched both training data and previously withheld validation data within an order of magnitude for over 85% of the data points; notably, the area of greatest frustration in the network was also the most fully determined. Finally, our estimation method showed that fidelity to physiological observations such as network response time is enforced at the cost of fit to molecular parameter values. In summary, our reformulation enables estimation of accurate, biologically relevant parameters, generates insight into the biology of the simulated network, and appears generalizable to any biochemical network - potentially including whole-cell models.

Somatic POLE exonuclease domain mutations are early events in sporadic endometrial and colorectal carcinogenesis, determining driver mutational landscape, clonal neoantigen burden and immune response.

Genomic instability, which is a hallmark of cancer, is generally thought to occur in the middle to late stages of tumourigenesis, following the acquisition of permissive molecular aberrations such as TP53 mutation or whole genome doubling. Tumours with somatic POLE exonuclease domain mutations are notable for their extreme genomic instability (their mutation burden is among the highest in human cancer), distinct mutational signature, lymphocytic infiltrate, and excellent prognosis. To what extent these characteristics are determined by the timing of POLE mutations in oncogenesis is unknown. Here, we have shown that pathogenic POLE mutations are detectable in non-malignant precursors of endometrial and colorectal cancer. Using genome and exome sequencing, we found that multiple driver mutations in POLE-mutant cancers show the characteristic POLE mutational signature, including those in genes conventionally regarded as initiators of tumourigenesis. In POLE-mutant cancers, the proportion of monoclonal predicted neoantigens was similar to that in other cancers, but the absolute number was much greater. We also found that the prominent CD8+ T-cell infiltrate present in POLE-mutant cancers was evident in their precursor lesions. Collectively, these data indicate that somatic POLE mutations are early, quite possibly initiating, events in the endometrial and colorectal cancers in which they occur. The resulting early onset of genomic instability may account for the striking immune response and excellent prognosis of these tumours, as well as their early presentation. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Zinc finger nuclease-mediated precision genome editing of an endogenous gene in hexaploid bread wheat (Triticum aestivum) using a DNA repair template.

Sequence-specific nucleases have been used to engineer targeted genome modifications in various plants. While targeted gene knockouts resulting in loss of function have been reported with relatively high rates of success, targeted gene editing using an exogenously supplied DNA repair template and site-specific transgene integration has been more challenging. Here, we report the first application of zinc finger nuclease (ZFN)-mediated, nonhomologous end-joining (NHEJ)-directed editing of a native gene in allohexaploid bread wheat to introduce, via a supplied DNA repair template, a specific single amino acid change into the coding sequence of acetohydroxyacid synthase (AHAS) to confer resistance to imidazolinone herbicides. We recovered edited wheat plants having the targeted amino acid modification in one or more AHAS homoalleles via direct selection for resistance to imazamox, an AHAS-inhibiting imidazolinone herbicide. Using a cotransformation strategy based on chemical selection for an exogenous marker, we achieved a 1.2% recovery rate of edited plants having the desired amino acid change and a 2.9% recovery of plants with targeted mutations at the AHAS locus resulting in a loss-of-function gene knockout. The latter results demonstrate a broadly applicable approach to introduce targeted modifications into native genes for nonselectable traits. All ZFN-mediated changes were faithfully transmitted to the next generation.

Standardizing the evaluation of community-based conservation success.

Community-based conservation, which strives to simultaneously improve nature conservation and alleviate poverty, must provide biological and socioeconomic benefits that are linked through effective resilience mechanisms. To date, few community-based conservation initiatives have published comprehensive assessments that track performance in these elements of success. With 45% of the world's protected areas in comanagement with local communities, standardized measures to effectively evaluate the dual goals of community-based conservation are needed. We here introduce SPECCS, a user-friendly Standardized Protocol for Evaluating Community Conservation Success that incorporates an appraisal of data quality to responsibly assess progress over time or to compare effectiveness among different initiatives. We illustrate SPECCS's use by evaluating the Wechiau Community Hippo Sanctuary (WCHS) of northern Ghana 10 and 20 yr after its inception. The WCHS has the dual objective of protecting one of Ghana's few remaining hippopotamus populations while alleviating poverty in the surrounding communities through the creation of economic opportunity and infrastructure development. Results suggest stable project performance in the 10-yr (76%) and 20-yr (76%) evaluation, with an improvement in evaluation quality from 30% to 34%. The project is currently stronger in socioeconomic (performance 86%; quality 30%) than biological (60%; 32%) outcomes and in benefits (83%, 42%) than resilience (63%, 21%). Biological resilience is challenged by poor connectivity and limited project control over threats, whereas socioeconomic resilience is affected by a decision balance that continues to favor external stakeholders. SPECCS helps pinpoint strengths and weaknesses for timely adaptive management, strategic investments, and evidence-based recognition of community-based conservation successes.