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Powdery mildew, caused by the ascomycete Podosphaera leucotricha, is an endemic disease found wherever apples are grown that reduces both tree vigor and fresh market yield. In the absence of durable host resistance, chemical management is the primary means of disease control. Demethylation inhibitor (DMI) fungicides are widely used to manage apple powdery mildew, but members within this fungicide class have been observed to differ in efficacy with respect to disease control. Moreover, debate exists as to the optimal timing of DMI fungicide applications for management of apple powdery mildew. In this regard, the goal of this study was to determine the best-use practices for DMI fungicides to manage apple powdery mildew in New York State. Multiyear trials were conducted to evaluate the potential differential efficacy performance of four common DMI fungicides, and additional trials were conducted to assess optimal application timing. In all years, we observed that treatments of flutriafol and myclobutanil consistently had the lowest incidences of powdery mildew compared with difenoconazole and fenbuconazole. In the 2018 and 2021 trials, the newly registered mefentrifluconazole was more comparable to the difenoconazole program with respect to powdery mildew disease incidence. We hypothesize that differences in DMI efficacy may result from each fungicide’s water solubility and lipophilicity characteristics and thus their ability to move systemically in the host or more easily penetrate the surface of germinating conidia. Applications timed between petal fall and first cover resulted in the lowest incidence of powdery mildew on terminal leaves of apple shoots compared with applications timed before petal fall. These observations are contrary to previous studies conducted in regions with differing climates. We also found that the incidence of secondary powdery mildew observed 2 weeks after petal fall was influenced by applications of DMI fungicides during the previous season. For example, management programs consisting of applications of flutriafol or myclobutanil in the previous season tended to have lower incidence of apple powdery in the next spring, presumably because of reductions in overwintering inoculum. Despite reports of DMI resistance in other apple pathosystems, the DMI fungicide class is still relevant for the successful management of apple powdery mildew in New York State.

The well-cataloged marine fish fauna of the Galapagos Islands includes eight of the 12 species of snappers (Lutjanidae) found in the Tropical Eastern Pacific. A recent recreational scuba dive in the Galapagos produced photographs of an additional snapper species, Lutjanus inermis (Peters, 1869), which was sufficiently common as to likely have a recently established resident population.

Piezo1 is a mechanosensitive ion channel with essential roles in cardiovascular, lung, urinary, and immune functions. Piezo1 is widely distributed in different tissues in the human body and its specific roles have been identified following a decade of research; however, not all are well understood. Many structural and functional characteristics of Piezo1 have been discovered and are known to differ greatly from the characteristics of other mechanosensitive ion channels. Understanding the mechanisms by which this ion channel functions may be useful in determining its physiological roles in various organ systems. This review provides insight into the signalling pathways activated by mechanical stimulation of Piezo1 in various organ systems and cell types. We discuss downstream targets of Piezo1 and the overall effects resulting from Piezo1 activation, which may provide insights into potential treatment targets for diseases involving this ion channel.

Apple scab is one of the most economically important diseases of apple in temperate production regions. In the absence of durable host resistance in commercially preferred cultivars, considerable applications of fungicides are needed to manage this disease. With the sequential development of resistance to nearly all classes of single-site fungicides in the apple scab pathogen Venturia inaequalis, synthetic multisite fungicides, such as mancozeb and captan, often comprise the core of chemical management programs for apple scab. Although these fungicides have demonstrable benefits for both disease and fungicide resistance management, the sustainability movement within agriculture aims to reduce reliance on such fungicides because of their broader environmental impacts. In this study, we establish a framework to enhance the feasibility of chemical management programs that do not rely on use of synthetic multisite protectant fungicides to manage apple scab. Specifically, we wish to evaluate chemical programs that integrate the biopesticide Bacillus subtilis QST 713 (Serenade Opti) in rotation with benzovindiflupyr (Aprovia), a single-site fungicide belonging to the class of succinate dehydrogenase inhibitors (SDHI), to circumvent the need for applications of synthetic multisite fungicides. During implementation of these programs, disease incidence data were taken at biweekly intervals. Regardless of the seasonal challenges presented in the 2 years of this study, when Bacillus subtilis QST 713 was used in place of captan and mancozeb mixtures, we did not observe any significant differences (P > 0.05) in development of apple scab symptoms between any of the management programs for the vertical axis or super spindle orchards in either year. This potential for substituting synthetic multisite fungicides with biopesticides is best realized when the programs are used with a decision support system in a super spindle planting system, where trees have reduced canopy densities. This 2-year study shows the potential to achieve adequate disease control using the integration of SDHI fungicides and biological controls without the use of synthetic multisite fungicides.

Fire blight, caused by the bacterium Erwinia amylovora, is one of the most important diseases of apple. The antibiotic streptomycin is routinely used in the commercial apple industries of New York (NY) and New England to manage the disease. In 2002 and again, from 2011 to 2014, outbreaks of streptomycin resistance (SmR) were reported and investigated in NY. Motivated by new grower reports of control failures, we conducted a follow-up investigation of the distribution of SmR and E. amylovora strains for major apple production regions of NY over the last 6 years (2015 to 2020). Characterization of clustered regularly interspaced short palindromic repeat (CRISPR) profiles revealed that a few “cosmopolitan” strains were widely prevalent across regions, whereas many other “resident” strains were confined to one location. In addition, we uncovered novel CRISPR profile diversity in all investigated regions. SmR E. amylovora was detected only in a small area spanning two counties from 2017 to 2020 and was always associated with one CRISPR profile (41:23:38), which matched the profile of SmR E. amylovora, discovered in 2002. This suggests the original SmR E. amylovora was never fully eradicated and went undetected because of several seasons of low disease pressure in this region. Investigation of several representative isolates under controlled greenhouse conditions indicated significant differences in aggressiveness on ‘Gala’ apples. Potential implications of strain differences include the propensity of strains to become distributed across wide geographic regions and associated resistance management practices. Results from this work will directly influence sustainable fire blight management recommendations for commercial apple industries in NY state and other regions.

Athletes abuse recombinant human erythropoietin (rhEPO) and erythropoiesis stimulating agents to increase hemoglobin mass and improve performance. To evade detection, athletes have developed sophisticated blood doping regimens, which often include rhEPO micro-dosing. Detection of these methods requires biomarkers with increased sensitivity and a sample matrix that is more amenable to frequent testing in the field. We have developed a method to measure two immature reticulocyte proteins, CD71 and ferrochelatase (FECH), and one total erythrocyte protein, Band 3, in dried blood spots (DBS). This method was tested in response to rhEPO administration after low doses, 40 IU/kg, micro-doses, 900 IU, or saline injection in 20 healthy subjects. During administration of low-dose rhEPO, the mean CD71/Band 3 and FECH/Band 3 ratio increased by 412 ± 197% and 250 ± 44%, respectively. The mean response for the current biomarker, RET%, increased by 195 ± 35%. During administration of rhEPO micro-doses, CD71/Band 3 increased to 127 ± 25% on day 35 and 139 ± 36% on day 39, while no increase was observed in RET%. After rhEPO administration, during the washout phase, mean values decreased to a minimum of 64 ± 4% and 64 ± 11% for CD71/Band 3 and RET%, respectively. However, CD71/Band 3 remained below 75% of baseline for at least 4 weeks after rhEPO injection, while RET% returned to baseline levels. The results demonstrate that immature reticulocyte proteins have a larger response to rhEPO administration than the current biomarker, RET%, and can be monitored in the DBS matrix.

Haemophilus influenzae is a leading cause of meningitis disease and mortality, particularly in young children. Since the introduction of a licensed conjugate vaccine (targeting the outer capsular polysaccharide) against the most prevalent serotype, Haemophilus influenzae serotype b, the epidemiology of the disease has changed and Haemophilus influenzae serotype a is on the rise, especially in Indigenous North American populations. Here we apply molecular modeling to explore the preferred conformations of the serotype a and b capsular polysaccharides as well as a modified hydrolysis resistant serotype b polysaccharide. Although both serotype b and the modified serotype b have similar random coil behavior, our simulations reveal some differences in the polysaccharide conformations and surfaces which may impact antibody cross-reactivity between these two antigens. Importantly, we find significant conformational differences between the serotype a and b polysaccharides, indicating a potential lack of cross-reactivity that is corroborated by immunological data showing little recognition or killing between heterologous serotypes. These findings support the current development of a serotype a conjugate vaccine.

We and others previously reported increased signaling through the Prostaglandin E3 Receptor (EP3), a G protein-coupled receptor (GPCR) for the arachidonic acid metabolite, prostaglandin E2 (PGE2), is associated with {beta}-cell dysfunction of type 2 diabetes (T2D). Yet, the relationship between PGE2 production and signaling and {beta}-cell function during the progression to T2D remains unclear. In this work, we assessed gene expression from a panel of cadaveric human islets from 40 non-diabetic donors with BMI values spanning the spectrum from lean to high-risk obesity. Interleukin-6 (gene symbol: IL6) and cyclooxygenase-2 (COX-2) (gene symbol: PTGS2) mRNA levels were positively correlated with donor body mass index (BMI), while EP3 (gene symbol: PTGER3) was not. IL6 was itself strongly correlated with PTGS2 and all but one of the other PGE2 synthetic pathway genes tested. About half of the islet preparations were used in glucose-stimulated- and incretin-potentiated insulin secretion assays using an EP3-specific antagonist, confirming functionally-relevant up-regulation of PGE2 production. Islets from obese donors showed no inherent {beta}-cell dysfunction and were at least equally as glucose- and incretin-responsive as islets from non-obese donors. Furthermore, insulin content, a marker of islet size known to be associated with donor BMI, was also significantly and positively correlated with islet PTGS2 expression. We conclude up-regulated islet PGE2 production and signaling may be a necessary part of the {beta}-cell adaption response, compensating for obesity and insulin resistance. Analysis of plasma PGE2 metabolite levels from a clinical cohort reveal these findings are not in conflict with the concept of further elevations in PGE2 production contributing to T2D-related {beta}-cell dysfunction where islet EP3 expression has also been up-regulated. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=126 HEIGHT=200 SRC="FIGDIR/small/429205v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@14056a7org.highwire.dtl.DTLVardef@f8fcf1org.highwire.dtl.DTLVardef@a66915org.highwire.dtl.DTLVardef@13ce208_HPS_FORMAT_FIGEXP M_FIG C_FIG

Background: Diarrhoea is a major cause of morbidity in Cape Town, South Africa, and mortality is attributed to a failure to recognize the severity of the condition. Cryptosporidium and Giardia are increasingly recognized as important causes of diarrhoea in Africa however, suboptimal diagnostic techniques may lead to underappreciation of their significance. Our objectives are to compare the diagnostic yield of direct immunofluorescent antigen (DFA) microscopy on concentrated stool samples for Cryptosporidium and Giardia, with the current approach of wet mount microscopy for Giardia and auramine fluorescent stain for Cryptosporidium on unconcentrated stool.Methodology: Stool specimens (n=104) received at our hospital laboratory for routine microbiological investigations were used for the study. Direct wet-mount auramine-phenol fluorescent microscopy (auramine) detection of Cryptosporidium oocysts and wet mount iodine microscopy for Giardia detection, were performed on unconcentrated stool samples, while DFA stain for simultaneous detection of Cryptosporidium and Giardia was performed on sodium-acetate formalin concentrated stool samples. The diagnostic yields of the tests were compared using the MEDCALC® version 18.0Results: Of the 104 stool specimens received for microbiological analysis, only 66 (63.5%) had specific Cryptosporidium requests while 38 (36.5%) had no Cryptosporidium specific requests. Of the 66 specimens, 9 (13.6%) were positive for Cryptosporidium oocysts with DFA while only 1 (1.5%) was positive with auramine staining (p=0.013). The one auramine-positive specimen was also positive by DFA. Auramine stain microscopy gave a sensitivity of 11.1% (95%CI: 0.28-48.25%) and specificity of 100% (95%CI: 93.7%-100%) when compared to DFA. Of the 38 stool specimens without specific Cryptosporidium request, DFA yielded 5 (13.2%) additional positive results. Taken together, Cryptosporidium was detected in 14/104 (13.5%; 95%CI: 8.36–21.7%) specimens and only 1 of 14 (7.1%) specimens with the current routine laboratory testing approach. Giardia was detected by DFA in 3/104 (0.9%) specimens, while direct iodine wet mount microscopy did not yield any positive results (0%). All 3 Giardia-positive specimens had Cryptosporidium oocysts detected by DFA.Conclusion: These data suggest that a large proportion of Cryptosporidium cases remain undetected by the laboratory due to suboptimal testing methods, and failure by clinicians to specifically request for Cryptosporidium detection. There is need to periodically assess the effectiveness of diagnostic microbiology laboratory approaches to diarrhoea, and access to improved diagnostic laboratory techniques will contribute to more accurate differential diagnosis and a broadened understanding of local aetiology of diarrhoea diseases in Africa. Keywords: Cryptosporidium, Giardia, diarrhoea, stool concentration, DFA, microscopy French Title: Amélioration des découvertes de cas de Cryptosporidium à l'aide de la microscopie immunofluorescente sur des selles concentrées Contexte: La diarrhée est une cause majeure de morbidité au Cap, en Afrique du Sud, et la mortalité est attribuée à l'incapacité de reconnaître la gravité de la maladie. Cryptosporidium et Giardia sont de plus en plus reconnus comme des causes importantes de diarrhée en Afrique, cependant, des techniques de diagnostic sous-optimales peuvent conduire à une sous-estimation de leur importance. Nos objectifs sont de comparer le rendement diagnostique de la microscopie à antigène immunofluorescent direct (DFA) sur des échantillons de selles concentrées pour Cryptosporidium et Giardia, avec l'approche actuelle de la microscopie à montage humide pour Giardia et la coloration fluorescente auramine pour Cryptosporidium sur des selles non concentrées.Méthodologie: Des échantillons de selles (n=104) reçus au laboratoire de notre hôpital pour des examens microbiologiques de routine ont été utilisés pour l'étude. La détection directe par microscopie fluorescente auramine-phénol à montage humide (auramine) des oocystes de Cryptosporidium et la microscopie à l'iode à montage humide pour la détection de Giardia, ont été effectuées sur des échantillons de selles non concentrées, tandis que la coloration DFA pour la détection simultanée de Cryptosporidium et de Giardia a été réalisée sur de l'acétate de sodium formaline concentré échantillons de selles. Les rendements diagnostiques des tests ont étécomparés à l'aide de MEDCALC® version 18.0Résultats: Sur les 104 échantillons de selles reçus pour l'analyse microbiologique, seuls 66 (63,5%) avaient des demandes spécifiques de Cryptosporidium tandis que 38 (36,5%) n'avaient pas de demandes spécifiques de Cryptosporidium. Sur les 66 échantillons, 9 (13,6%) étaient positifs pour les oocystes de Cryptosporidium avec DFA tandis que seulement 1 (1,5%) était positif avec coloration à l'auramine (p=0,013). Le seul échantillon positif à l'auramine était également positif au DFA. La microscopie à l'auramine a donné une sensibilité de 11,1% (IC 95%: 0,28-48,25%) et une spécificité de 100% (IC 95%: 93,7% -100%) par rapport au DFA. Sur les 38 échantillons de selles sans demande spécifique de Cryptosporidium, le DFA a donné 5 (13,2%) résultats positifs supplémentaires. Pris ensemble, Cryptosporidium a été détecté dans 14/104 (13,5%; IC à 95%: 8,36–21,7%) et seulement 1 des 14 échantillons (7,1%) avec l'approche actuelle des tests de routine en laboratoire. Giardia a été détecté par DFA dans 3/104 (0,9%) échantillons, tandis que la microscopie directe à l'iode sur monture humide n'a donné aucun résultat positif (0%). Les 3 échantillons positifs à Giardia avaient des oocystes deCryptosporidium détectés par DFA.Conclusion: Ces données suggèrent qu'une grande proportion des cas de Cryptosporidium ne sont pas détectés par le laboratoire en raison de méthodes de test sous-optimales et de l'échec des cliniciens à demander spécifiquement la détection de Cryptosporidium. Il est nécessaire d'évaluer périodiquement l'efficacité des approches de laboratoire de microbiologie diagnostique pour la diarrhée, et l'accès à des techniques de laboratoire de diagnostic améliorées contribuera à un diagnostic différentiel plus précis et à une compréhension élargie de l'étiologie locale des maladies diarrhéiques en Afrique. Mots clés: Cryptosporidium, Giardia, diarrhée, concentration des selles, DFA, microscopie

The role of insects in dissemination of Erwinia amylovora has been studied for over 100 years. Pollinating bees do not feed on bacterial ooze but are suggested to transmit between flowers. It has been suggested that various hemipteran species walk on bacterial ooze and subsequently shed acquired bacteria into their own feeding wounds. Dipterans have been observed readily feeding on ooze, but their importance has been understudied. The goal of this study was to advance understanding of the ecology of insect-mediated transmission of E. amylovora through field collections and observations conducted in a research apple orchard with actively oozing fire blight symptoms. We found that field-collected pollinating bees did not test positive for the bacterium, suggesting that their role in blossom blight dissemination may be overstated. Flies were prominent flower visitors, underscoring the need for further research into their role in bloom time bacterial dissemination. Flies were observed feeding on ooze droplets in the late spring and early summer and the insects retained bacteria for at least 7 days. Flies shed transmissible amounts of E. amylovora for the duration of the experiment. The role of hemipterans was not clarified in this study but it is possible that their role is indirect through interactions with other insects. Collectively, this research outlines the ecological role of different insects in disease transmission and underscores the underappreciated potential importance of flies, providing a roadmap toward a better understanding of the complex dynamics at play. [Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

The adoption of mechanical thinning and pruning in commercial apple orchards has been limited largely by the risk of development and spread of fire blight. This devastating disease, caused by the bacterial pathogen Erwinia amylovora, may be transmitted by mechanical injury such as pruning, especially under warm, moist conditions conducive to bacterial growth, infection, and disease development. However, risk may be mitigated by avoiding highest-risk times and applying a bactericide, such as streptomycin, after mechanical thinning or pruning. In ‘Gala’ and ‘Idared’ orchards, we evaluated the risk of fire blight development and spread after mechanical thinning early in bloom (20% bloom), when seasonal temperatures are cooler and there are few open flowers available for infection. In both orchards, we also evaluated the spread and development of fire blight by mechanical pruning in July and in August, before and after terminal bud set, when shoot growth is slowed and the tree is less susceptible to infection. We also assessed the potential efficacy of a streptomycin or Bacillus subtilis biopesticide application after mechanical thinning and pruning to mitigate the spread of fire blight. In the ‘Gala’ orchard, disease never developed beyond the inoculated tree after thinning or pruning, which was unexpected for this highly susceptible cultivar. In the ‘Idared’ orchard, incidence of blossom or shoot blight from the point source, represented as relative area under the disease progress curve, was rarely different for trees that underwent mechanical thinning or mechanical pruning compared with untreated trees, and it was often reduced or eliminated when the antibiotic streptomycin or the B. subtilis biopesticide was applied within 24 h of mechanical thinning or pruning. For both thinning and pruning, incidence of fire blight dropped off quickly beyond the inoculated tree in the ‘Idared’ orchard and generally was not observed in trees beyond 10 to 15 m from the inoculated point source or predicted beyond 10 m by exponential and power law models fit to the disease progress curves. The results of this work demonstrate the low risk for fire blight development and spread by mechanical thinning and pruning when practiced under low-risk conditions—early in bloom for mechanical thinning and after terminal bud set (in August) for mechanical pruning—especially when paired with a subsequent bactericide application. This study demonstrates the safe use of mechanical thinning and pruning in commercial apple production, corroborated by anecdotal evidence from apple growers in western New York State.

A novel series of histone deacetylase (HDAC) inhibitors lacking a zinc-binding moiety has been developed and described herein. HDAC isozyme profiling and kinetic studies indicate that these inhibitors display a selectivity preference for HDACs 1, 2, 3, 10, and 11 via a rapid equilibrium mechanism, and crystal structures with HDAC2 confirm that these inhibitors do not interact with the catalytic zinc. The compounds are nonmutagenic and devoid of electrophilic and mutagenic structural elements and exhibit off-target profiles that are promising for further optimization. The efficacy of this new class in biochemical and cell-based assays is comparable to the marketed HDAC inhibitors belinostat and vorinostat. These results demonstrate that the long-standing pharmacophore model of HDAC inhibitors requiring a metal binding motif should be revised and offers a distinct class of HDAC inhibitors.

Within is a comprehensive review of current apple powdery mildew literature, with the taxonomy, morphology, and life cycle of the causal phytopathogen Podosphaera leucotricha described. A discussion of economic impact, environmental aspects contributing to apple powdery mildew epidemiology, options for cultural, chemical, and biopesticide disease control, and fungicide resistance in P. leucotricha is also detailed. The present status of breeding for host resistance and apple cultivar susceptibility is discussed. Finally, emerging research into the biology and management of apple powdery mildew is discussed along with how they may aid future research endeavors.

Regeneration is a complex chain of events that restores a tissue to its original size and shape. The tissue-wide coordination of cellular dynamics that is needed for proper morphogenesis is challenged by the large dimensions of regenerating body parts. Feedback mechanisms in biochemical pathways can provide effective communication across great distances1–5, but how they might regulate growth during tissue regeneration is unresolved6,7. Here we report that rhythmic travelling waves of Erk activity control the growth of bone in time and space in regenerating zebrafish scales, millimetre-sized discs of protective body armour. We find that waves of Erk activity travel across the osteoblast population as expanding concentric rings that are broadcast from a central source, inducing ring-like patterns of tissue growth. Using a combination of theoretical and experimental analyses, we show that Erk activity propagates as excitable trigger waves that are able to traverse the entire scale in approximately two days and that the frequency of wave generation controls the rate of scale regeneration. Furthermore, the periodic induction of synchronous, tissue-wide activation of Erk in place of travelling waves impairs tissue growth, which indicates that wave-distributed Erk activation is key to regeneration. Our findings reveal trigger waves as a regulatory strategy to coordinate cell behaviour and instruct tissue form during regeneration. The rate of scale regeneration in zebrafish is controlled by the frequency of rhythmic travelling waves of Erk activity, which are broadcast from a central source to induce ring-like patterns of osteoblast tissue growth.

Erwinia amylovora is a bacterial pathogen of rosaceous plants that can be devastating to commercial apple and pear production worldwide. Exopolysaccharide (EPS) production is essential for pathogenicity, aiding in biofilm production, and plant defense protection. EPS also plays an epidemiological role in the form of bacterial ooze, which is generated when E. amylovora builds to such high levels within the parenchyma that it ruptures the plant epidermis and a mixture of bacteria encased in EPS exudes from the injury. Insects such as Delia platura feed on the ooze and become potential vectors in the process. The goal of this study was to investigate interactions between D. platura and E. amylovora to better understand how insects facilitate shoot blight. We demonstrate for the first time that D. platura can successfully transmit pathogen cells that initiate new infections in mechanically damaged apple shoots and that EPS aids in adherence of E. amylovora to the insect surface. We show that flies can carry the bacteria externally for at least 5 days and that they shed a constant daily rate. We also show that E. amylovora strains differing in virulence do not behave differently when associated with D. platura and that consumption of E. amylovora has no effect on insect survival. Our data demonstrate that flies can be efficient facilitators of new shoot blight infections, but the field conditions that make this type of transmission possible require further investigation. [Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY 4.0 International license .

In apple fruit, streptomycin is routinely used to manage fire blight, a devastating disease that greatly affects commercial apple production. Applications of streptomycin may affect the endophytic bacterial communities, which are increasingly recognized for their valuable roles in plant hosts. Altering these communities may have implications for plant health and selection for streptomycin resistance in off-target bacteria. In a previous study, we found rapid selection of streptomycin-resistant epiphytes following season-long streptomycin use, and now sought to investigate the season-long impact of streptomycin applications (one, three, or nine postbloom applications) on endophytic bacterial communities. Studies were conducted in two orchards, and both culture-dependent and independent methods were used to assess the bacterial endophytic community. Streptomycin programs were compared with untreated trees and two programs approved for organic production. Treatments minimally affected abundance of culturable endophytic bacteria as determined by culture-dependent methods. Similarly, there were no significant differences in relative bacterial abundance or community composition between treatments as determined by 16S ribosomal RNA high-throughput sequencing (culture independent). There were greater differences between orchards than between treatments, suggesting a stronger influence of locality. Communities were dominated by the families Pseudomonadaceae, Enterobacteriaceae, Desulfovibrionaceae, Bacillaceae, and Burkholderiaceae, which are commonly described in other investigations of microbial communities of apple orchards. In addition, we found a high abundance of Amoebophiliaceae, a family dominated by arthropod parasites. Our results provide evidence that season-long applications of streptomycin in apple orchards have minimal impact on endophytic bacterial communities, which is in contrast to some reports on epiphytic communities.

Fire blight, caused by the bacteria Erwinia amylovora, is an incredibly destructive disease of apples, capable of spreading rapidly through an orchard block. The pathogen is endemic to many apple production regions worldwide, but it is often introduced into newly planted sites on infested host material, while locally it is typically vectored by wind-driven rain, hail, and insects. Here we presented two case studies of orchard blocks infected with fire blight at the Cornell AgriTech Research orchards in which CRISPR pattern characterization was used to identify strains present in the blocks and spatial analyses were used to describe the distribution and spread of the disease over the course of 2 years. Results indicated two very different sources of introduction (a nearby block or planting material) and patterns of spread (from a corner of the field or from focal points within the block). Describing the distribution and spread of fire blight within and between orchard blocks has the potential to improve our understanding of the disease movement, inform appropriate management recommendations, and facilitate traceback efforts.

Huisache (Vachellia farnesiana [L.] Wight & Arn.) is a woody species native to the western hemisphere that can invade a variety of native rangeland habitats around the world. In South Texas, it is native yet increasing in density and range while displacing more desirable forage plants, and because it resprouts prolifically it has been difficult to control. We therefore attempted to characterize optimal timing and environmental conditions for herbicide control. We collected weather data in four huisache-invaded pastures along the Texas Coastal Bend and recorded the average huisache phenological stage at those pastures each month. We also analyzed root crown total nonstructural carbohydrate (TNC) and huisache mortality resulting from herbicide treatment each month between April 2012 and November 2014. Huisache mortality and TNC data were analyzed using a randomized complete block analysis of variance, with post hoc tests to separate means; Akaike's information criterion determined best-fit models for mortality data. Root crown TNC increased during May, August, and December, indicating downward translocation and the potential for increased mortality of treated plants. Actual mortality was greatest during the months of May, September, October, and November. The best fit model for mortality was a sixth-order polynomial function of mortality versus month; when month was removed as a model factor, the best fit model was a quadratic function of soil temperature (peaking at ≈24.5°C), combined with a quadratic function of phenology (highest at full canopy stage). These results indicate that huisache is best treated with chemicals when soil temperature is near 24.5°C and during the full canopy stage. In the coastal plains of South Texas these variables coincide with two windows of optimal mortality for huisache treatment: May and September−November.

Fusobacterium nucleatum is becoming increasingly recognised as an emerging pathogen, gaining attention as a potential factor for exacerbating colorectal cancer and is strongly linked with pregnancy complications including pre-term and still births. Little is known about the virulence factors of this organism; thus, we have initiated studies to examine the bacterium’s surface glycochemistry. In an effort to characterise the surface carbohydrates of F. nucleatum, the aims of this study were to investigate the structure of the lipopolysaccharide (LPS) O-antigen of the cancer-associated isolate F. nucleatum strain CC 7/3 JVN3 C1 (hereafter C1) and to develop monoclonal antibodies (mAbs) to the LPS O-antigen that may be beneficial to the growing field of F. nucleatum research. In this study, we combined several technologies, including nuclear magnetic resonance (NMR) spectroscopy, to elucidate the structure of the LPS O-antigen repeat unit as -[-4-β-Gal-3-α-FucNAc4N-4-α-NeuNAc-]-. We have previously identified this structure as the LPS O-antigen repeat unit from strain 10953. In this present study, we developed a mAb to the C1 LPS O-antigen and confirmed the mAbs cross-reactivity to the 10953 strain, thus confirming the structural identity.

In Texas, mesquite and yellow-bluestem invasions are widespread. Identifying and monitoring juvenile and adult plants using high-resolution imagery from airborne sensors while they colonize new areas across the landscape can help land managers prioritize locations for treatment and eradication. In this study, we evaluated how data collection design using an unmanned aerial system (UAS) can affect plant detection and mapping. We used a Phantom 3 Professional unmanned aerial vehicle with a Parrot Sequoia multispectral camera for detecting and mapping native honey mesquite (Prosopis glandulosa) and non-native yellow bluestem (Bothriochloa ischaemum) at a rangeland site in northwest Texas. Flights were conducted seasonally during the period from summer 2017 to fall 2018 to test the seasonal impact of detecting plant species. Flights were conducted at altitudes of 30, 60, and 100 m, and four image classification techniques were tested to determine their viability of detecting distinct plant species. Results suggest that flights at 100-m aircraft altitude during the spring season are more effective (>80% user accuracies) for mapping mesquite canopies based on reflectance values and image segmentation information. Yellow bluestem mapping accuracies were low (

Two known Clostridiodes (Clostridium) difficile surface antigens, a lipoteichoic acid (LTA) and a polysaccharide (PS-II) were isolated and purified in order to prepare glycoconjugate vaccines to the carrier protein human serum albumin utilising a reductive amination strategy. Mice and rabbits were immunized with a prime and two boost strategy and the resulting sera were examined for their ability to recognise the purified homologous antigens and subsequently killed whole cells of C. difficile strains and other Clostridia species. Immunisation derived antisera from rabbits and mice, recognised all strains of C. difficile vegetative cells examined, with generally similar titers from animals that received the LTA or the PS-II conjugates. Sera raised to the LTA conjugates were able to recognise other Clostridia species C. butyricum, C. bifermentans and C. subterminale whereas sera raised to the PS-II conjugates were not. These LTA and PS-II sera recognised live cells in an immunofluorescence assay and were also able to recognise the spore form of the bacterium. This study has confirmed that the LTA and PS-II polysaccharides are both highly conserved surface polymers of C. difficile that are easily accessible to the immune system and as such may have potential as vaccine antigens or as targets for therapeutics to combat C. difficile infection.

We describe the structural characterization of the capsular polysaccharides (CPSs) of Pasteurella multocida serotypes B and E. CPS was isolated following organic solvent precipitation of the supernatant from flask grown cells. Structural analysis utilizing nuclear magnetic resonance spectroscopy enabled the determination of the CPS structures and revealed significant structural similarities between the two serotypes, but also provided an explanation for the serological distinction. This observation was extended by the development of polyclonal sera to the glycoconjugate of serotype B CPS that corroborated the structural likenesses and differences. Finally, identification of these structures enabled a more comprehensive interrogation of the genetic loci and prediction of roles for some of the encoded proteins in repeat unit biosynthesis.

Fire blight, a bacterial disease of rosaceous plants caused by Erwinia amylovora, is one of the most important diseases affecting commercial apple production worldwide. Antibiotics, applied at bloom to protect against blossom infection, are the most effective means of management but raise concern due to the potential for antibiotic resistance in both the pathogen population and nontarget organisms. In addition, most fire blight outbreaks in New York State often emerge in late June to July as shoot blight, calling into question the role of blossom infections and the antibiotic applications made to manage them. Prohexadione-calcium (PhCa) is a gibberellic acid inhibitor used post-bloom to control shoot vigor and to manage shoot blight. However, the magnitude of shoot blight management is directly related to the suppression of shoot growth, which is undesirable, especially in young orchards during establishment years. PhCa is believed to control shoot blight by thickening cell walls in cortical parenchyma, preventing invasion of host tissues by E. amylovora. We hypothesize that PhCa applied pre-bloom could similarly prevent invasion of blossom pedicels following infection, leading to reduced disease incidence. We evaluated novel pre-bloom PhCa programs for their effects on disease management (blossom and shoot blight) as well as their impact on shoot growth for three years in a mature ‘Gala’ orchard in New York. In all three years of the study, all PhCa programs resulted in less than 27% incidence (71% control) of blossom blight and less than 13% incidence (77% control) of shoot blight with minimal effect on tree growth. Inclusion of a biopesticide during bloom further reduced the incidence of blossom blight in one year of three. Using light microscopy, we found that cell walls in the cortical parenchyma of fruitlet pedicels on trees receiving pre-bloom PhCa applications were significantly thicker than those of untreated trees 40 days after full bloom and inoculation. Overall, we found that pre-bloom applications of PhCa had utility in reducing blossom blight and shoot blight with minimal impacts on tree growth. These pre-bloom programs would fit with standard production practices and may contribute toward the development of fire blight management programs without the use of antibiotics.

The small GTPase ARF family member ARL15 gene locus is associated in population studies with increased risk of type 2 diabetes, lower adiponectin and higher fasting insulin levels. Previously, loss of ARL15 was shown to reduce insulin secretion in a human β-cell line and loss-of-function mutations are found in some lipodystrophy patients. We set out to understand the role of ARL15 in adipogenesis and showed that endogenous ARL15 palmitoylated and localised in the Golgi of mouse liver. Adipocyte overexpression of palmitoylation-deficient ARL15 resulted in redistribution to the cytoplasm and a mild reduction in expression of some adipogenesis-related genes. Further investigation of the localisation of ARL15 during differentiation of a human white adipocyte cell line showed that ARL15 was predominantly co-localised with a marker of the cis face of Golgi at the preadipocyte stage and then translocated to other Golgi compartments after differentiation was induced. Finally, co-immunoprecipitation and mass spectrometry identified potential interacting partners of ARL15, including the ER-localised protein ARL6IP5. Together, these results suggest a palmitoylation dependent trafficking-related role of ARL15 as a regulator of adipocyte differentiation via ARL6IP5 interaction. This article has an associated First Person interview with the first author of the paper., Summary: ARL15 (GTPase ARF family) is associated with adipose traits. ARL15 is palmitoylated, localised to Golgi in preadipocytes and translocated to other Golgi compartments during differentiation. ARL15 interacts with ER-localised ARL6IP5.

ECT2 is an activator of RHO GTPases that is essential for cytokinesis. In addition, ECT2 was identified as an oncoprotein when expressed ectopically in NIH/3T3 fibroblasts. However, oncogenic activation of ECT2 resulted from N-terminal truncation, and such truncated ECT2 proteins have not been found in patients with cancer. In this study, we observed elevated expression of full-length ECT2 protein in preneoplastic colon adenomas, driven by increased ECT2 mRNA abundance and associated with APC tumor-suppressor loss. Elevated ECT2 levels were detected in the cytoplasm and nucleus of colorectal cancer tissue, suggesting cytoplasmic mislocalization as one mechanism of early oncogenic ECT2 activation. Importantly, elevated nuclear ECT2 correlated with poorly differentiated tumors, and a low cytoplasmic:nuclear ratio of ECT2 protein correlated with poor patient survival, suggesting that nuclear and cytoplasmic ECT2 play distinct roles in colorectal cancer. Depletion of ECT2 reduced anchorage-independent cancer cell growth and invasion independent of its function in cytokinesis, and loss of Ect2 extended survival in a KrasG12D Apc-null colon cancer mouse model. Expression of ECT2 variants with impaired nuclear localization or guanine nucleotide exchange catalytic activity failed to restore cancer cell growth or invasion, indicating that active, nuclear ECT2 is required to support tumor progression. Nuclear ECT2 promoted ribosomal DNA transcription and ribosome biogenesis in colorectal cancer. These results support a driver role for both cytoplasmic and nuclear ECT2 overexpression in colorectal cancer and emphasize the critical role of precise subcellular localization in dictating ECT2 function in neoplastic cells. Significance: ECT2 overexpression and mislocalization support its role as a driver in colon cancer that is independent from its function in normal cell cytokinesis.

Clostridiodes (Clostridium) difficile is an anaerobic Gram-positive, spore-forming nosocomial, gastrointestinal pathogen causing C. difficile-associated disease with symptoms ranging from mild cases of antibiotic-associated diarrhea to fatal pseudomembranous colitis. We developed murine monoclonal antibodies (mAbs) specific for a conserved cell surface antigen, lipoteichoic acid (LTA)of C. difficile. The mAbs were characterized in terms of their thermal stability, solubility, and their binding to LTA by surface plasmon resonance and competitive ELISA. Synthetic LTA molecules were prepared in order to better define the minimum epitope required to mimic the natural antigen, and three repeat units of the polymer were required for optimal recognition. One of the murine mAbs was chimerized with human constant region domains and was found to recognize the target antigen identically to the mouse version. These mAbs may be useful as therapeutics (standalone, in conjunction with known antitoxin approaches, or as delivery vehicles for antibody drug conjugates targeting the bacterium), as diagnostic agents, and in infection control applications.

The proprotein convertase subtilisin/Kexin type 1 (PCSK1/PC1) protein processes inactive pro-hormone precursors into biologically active hormones in a number of neuroendocrine and endocrine cell types. Patients with recessive mutations in PCSK1 exhibit a complex spectrum of traits including obesity, diarrhoea and endocrine disorders. We describe here a new mouse model with a point mutation in the Pcsk1 gene that exhibits obesity, hyperphagia, transient diarrhoea and hyperproinsulinaemia, phenotypes consistent with human patient traits. The mutation results in a pV96L amino acid substitution and changes the first nucleotide of mouse exon 3 leading to skipping of that exon and in homozygotes very little full-length transcript. Overexpression of the exon 3 deleted protein or the 96L protein results in ER retention in Neuro2a cells. This is the second Pcsk1 mouse model to display obesity phenotypes, contrasting knockout mouse alleles. This model will be useful in investigating the basis of endocrine disease resulting from prohormone processing defects.

Allele-specific signaling by different KRAS alleles remains poorly understood. The KRASG12R mutation displays uneven prevalence among cancers that harbor the highest occurrence of KRAS mutations: It is rare (∼1%) in lung and colorectal cancers, yet relatively common (∼20%) in pancreatic ductal adenocarcinoma (PDAC), suggesting context-specific properties. We evaluated whether KRASG12R is functionally distinct from the more common KRASG12D- or KRASG12V-mutant proteins (KRASG12D/V). We found that KRASG12D/V but not KRASG12R drives macropinocytosis and that MYC is essential for macropinocytosis in KRASG12D/V- but not KRASG12R-mutant PDAC. Surprisingly, we found that KRASG12R is defective for interaction with a key effector, p110α PI3K (PI3Kα), due to structural perturbations in switch II. Instead, upregulated KRAS-independent PI3Kγ activity was able to support macropinocytosis in KRASG12R-mutant PDAC. Finally, we determined that KRASG12R-mutant PDAC displayed a distinct drug sensitivity profile compared with KRASG12D-mutant PDAC but is still responsive to the combined inhibition of ERK and autophagy.Significance:We determined that KRASG12R is impaired in activating a key effector, p110α PI3K. As such, KRASG12R is impaired in driving macropinocytosis. However, overexpression of PI3Kγ in PDAC compensates for this deficiency, providing one basis for the prevalence of this otherwise rare KRAS mutant in pancreatic cancer but not other cancers.See related commentary by Falcomatà et al., p. 23.This article is highlighted in the In This Issue feature, p. 1

The cytosine (C)-rich sequences that can fold into tetraplex structures known as i-motif are prevalent in genomic DNA. Recent studies of i-motif–forming sequences have shown increasing evidence of their roles in gene regulation. However, most of these studies have been performed in short single-stranded oligonucleotides, far from the intracellular environment. In cells, i-motif–forming sequences are flanked by DNA duplexes and packed in the genome. Therefore, exploring the conformational dynamics and kinetics of i-motif under such topologically constrained environments is highly relevant in predicting their biological roles. Using single-molecule fluorescence analysis of self-assembled DNA duplexes and nanocircles, we show that the topological environments play a key role on i-motif stability and dynamics. While the human telomere sequence (C3TAA)3C3 assumes i-motif structure at pH 5.5 regardless of topological constraint, it undergoes conformational dynamics among unfolded, partially folded and fully folded states at pH 6.5. The lifetimes of i-motif and the partially folded state at pH 6.5 were determined to be 6 ± 2 and 31 ± 11 s, respectively. Consistent with the partially folded state observed in fluorescence analysis, interrogation of current versus time traces obtained from nanopore analysis at pH 6.5 shows long-lived shallow blockades with a mean lifetime of 25 ± 6 s. Such lifetimes are sufficient for the i-motif and partially folded states to interact with proteins to modulate cellular processes.

Succinate dehydrogenase inhibitors (SDHI) are an important class of fungicides for management of apple scab, especially as resistance to other classes of fungicides has become prevalent in the northeastern United States. Considering their single-site mode of action, there is high risk of resistance development to SDHI fungicides. Such risk mandates the need for proper monitoring of shifts in population sensitivity. This study aims to provide a means for phenotypic and genotypic characterization of SDHI fungicide resistance for Venturia inaequalis, the causal agent of apple scab. To complement the published sequence of VisdhB, target genes VisdhC and VisdhD were identified using sequences of homologous genes in other fungal organisms and a draft genome of V. inaequalis. Using mycelial growth and conidial germination assays, baseline sensitivities and cross sensitivities of V. inaequalis were determined for several SDHI fungicides. Mean baseline EC50 values for conidial germination of benzovindiflupyr, fluxapyroxad, pydiflumetofen, and inpyrfluxam were found to be 0.0021, 0.0284, 0.014, and 0.0137 μg ml−1, respectively. Mean baseline EC50 values for mycelial growth of benzovindiflupyr, fluxapyroxad, pydiflumetofen, and inpyrfluxam were found to be 0.0575, 0.228, 0.062, and 0.0291 μg ml−1, respectively. A significant and positive correlation in sensitivity was found between benzovindiflupyr, fluxapyroxad, pydiflumetofen, and inpyrfluxam as well as penthiopyrad and fluopyram, with the highest correlation between benzovindiflupyr and penthiopyrad for mycelial inhibition of V. inaequalis (r = 0.950, P < 0.001). For inhibition of conidial germination, the highest correlation was observed between penthiopyrad and fluopyram (r = 0.775, P < 0.001). Furthermore, the sequences of the VisdhC and VisdhD genes were identified and characterized for baseline isolates of V. inaequalis. Residues of similar position to mutations found in other systems that confer resistance to SDHI fungicides were identified in baseline isolates, but no mutations were identified in baseline isolates or those previously exposed to SDHI fungicides. This study will serve as a reference for future monitoring of resistance to SDHI fungicides in V. inaequalis at both a phenotypic and genotypic level.

Missense mutations in the gene that encodes for the mechanically-gated ion channel Piezo1 have been linked to a number of diseases. Gain-of-function variants are linked to a hereditary anaemia and loss-of-function variants have been linked to generalized lymphatic dysplasia and bicuspid aortic valve. Two previously characterized mutations, S217L and G2029R, both exhibit reduced plasma membrane trafficking. Here we show that both mutations also display reduced stability and higher turnover rates than wild-type Piezo1 channels. This occurs through increased ubiquitination and subsequent proteasomal degradation. Congruent with this, proteasome inhibition using N-acetyl-l-leucyl-l-leucyl-l-norleucinal (ALLN) reduced the degradation of both mutant proteins. While ALLN treatment could not rescue the function of S217L we show via multiple complementary methodologies that proteasome inhibition via ALLN treatment can not only prevent G2029R turnover but increase the membrane localized pool of this variant and the functional Piezo1 mechanosensitive currents. This data in combination with a precision medicine approach provides a new potential therapeutic avenue for the treatment of Piezo1 mediated channelopathies.

The obesity-linked FTO regulatory circuitry shows cross-species molecular, cellular, metabolic, and organismal conservation., Variants in FTO have the strongest association with obesity; however, it is still unclear how those noncoding variants mechanistically affect whole-body physiology. We engineered a deletion of the rs1421085 conserved cis-regulatory module (CRM) in mice and confirmed in vivo that the CRM modulates Irx3 and Irx5 gene expression and mitochondrial function in adipocytes. The CRM affects molecular and cellular phenotypes in an adipose depot–dependent manner and affects organismal phenotypes that are relevant for obesity, including decreased high-fat diet–induced weight gain, decreased whole-body fat mass, and decreased skin fat thickness. Last, we connected the CRM to a genetically determined effect on steroid patterns in males that was dependent on nutritional challenge and conserved across mice and humans. Together, our data establish cross-species conservation of the rs1421085 regulatory circuitry at the molecular, cellular, metabolic, and organismal level, revealing previously unknown contextual dependence of the variant’s action.

Xanthomonas axonopodis infects common bean (Phaseolus vulgaris L.) causing the disease common bacterial blight (CBB). The aim of this study was to investigate the molecular and metabolic mechanisms underlying CBB resistance in P. vulgaris. Trifoliate leaves of plants of a CBB-resistant P. vulgaris recombinant inbred line (RIL) and a CBB-susceptible RIL were inoculated with X. axonopodis or water (mock treatment). Leaves sampled at defined intervals over a 48-h post-inoculation (PI) period were monitored for alterations in global transcript profiles. A total of 800 genes were differentially expressed between pathogen and mock treatments across both RILs, approximately half were differentially expressed in the CBB-resistant RIL at 48 h PI. Notably, there was a 4- to 32-fold increased transcript abundance for isoflavone biosynthesis genes, including several isoflavone synthases, isoflavone 2′-hydroxylases and isoflavone reductases. Ultra-high performance liquid chromatography-tandem mass spectrometry assessed leaf metabolite levels as a function of the PI period. The concentrations of the isoflavones daidzein and genistein and related metabolites coumestrol and phaseollinisoflavan were increased in CBB-resistant RIL plant leaves after exposure to the pathogen. Isoflavone pathway transcripts and metabolite profiles were unaffected in the CBB-susceptible RIL. Thus, induction of the isoflavone pathway is associated with CBB-resistance in P. vulgaris.

Pathological left ventricular hypertrophy (LVH) occurs in response to pressure overload and remains the single most important clinical predictor of cardiac mortality. The molecular pathways in the induction of pressure overload LVH are potential targets for therapeutic intervention. Current treatments aim to remove the pressure overload stimulus for LVH, but do not completely reverse adverse cardiac remodelling. Although numerous molecular signalling steps in the induction of LVH have been identified, the initial step by which mechanical stretch associated with cardiac pressure overload is converted into a chemical signal that initiates hypertrophic signalling remains unresolved. In this study, we show that selective deletion of transient receptor potential melastatin 4 (TRPM4) channels in mouse cardiomyocytes results in an approximately 50% reduction in the LVH induced by transverse aortic constriction. Our results suggest that TRPM4 channel is an important component of the mechanosensory signalling pathway that induces LVH in response to pressure overload and represents a potential novel therapeutic target for the prevention of pathological LVH. ispartof: ELIFE vol:10 ispartof: location:England status: published

White adipose tissue (WAT) plays a central role in metabolism, with multiple diseases and genetic mutations causing its remodeling. Quantitative analysis of white adipocyte size is of great interest to understand physiology and disease, but previous studies of H&E histology have been limited to a subsample of whole depot cross-sections. In this paper, we present the deep learning pipeline DeepCytometer, that can segment mouse and human whole slides (≃40,000 cells per mouse slide on average) using an adaptive tiling method, correct for cell overlap and reject non-white adipocytes from the segmentation. Using quantile colour maps we show intra- and inter-depot cell size heterogeneity with local correlation; quantile estimates also suggest significant differences in population estimates from 75 whole slides compared to smaller data sets. We propose three linked levels (body weight BW, depot weight DW and cell area quartiles) for exploratory analysis of mouse Klf14 phenotypes in gonadal and subcutaneous depots. We find a rich set of phenotypes when stratifying by sex, depot and three genotype strata: (1) WTs/Hets with a Het father (Controls), (2) WTs with a Het mother, and (3) Hets with a Het mother (functional KOs or FKOs). Namely, at BW level, mean difference testing suggests that female FKOs are similar to Controls, but WTs with a Het mother are significantly larger. At DW and cell levels, linear models with interaction terms and BW or DW covariates, respectively, reveal phenotypes not shown by difference of means tests. For example, at DW level, gonadal and subcutaneous models are similar, and female FKOs have lower fat percentage than Controls due to both an offset and the DW/BW slope in the linear model. Meanwhile, female WTs with a Het mother have on average similar fat percentage to Controls, but as their slopes are close to zero, their DWs are uncorrelated to BW, suggesting that larger female WTs with a Het mother have lower fat percentage than smaller ones. In contrast to depot level, at cell level female gonadal phenotypes diverge from subcutaneous ones. Furthermore, male Controls and FKOs have similar average area values in subcutaneous depots, but area~DW slope flattening in FKOs suggests that larger DWs could be caused by cell size increase in Controls and by cell count increase in FKOs. Thus, DeepCytometer and associated exploratory analysis reveal new insights into adipocyte heterogeneity and phenotyping.

Synaptic disturbances in excitatory to inhibitory (E/I) balance in forebrain circuits are thought to contribute to the progression of Alzheimer’s disease (AD) and dementia, although direct evidence for such imbalance in humans is lacking. We assessed anatomical and electrophysiological synaptic E/I ratios in post-mortem parietal cortex samples from middle-aged individuals with AD (early-onset) or Down syndrome (DS) by fluorescence deconvolution tomography and microtransplantation of synaptic membranes. Both approaches revealed significantly elevated E/I ratios for AD, but not DS, versus controls. Gene expression studies in an independent AD cohort also demonstrated elevated E/I ratios in individuals with AD as compared to controls. These findings provide evidence of a marked pro-excitatory perturbation of synaptic E/I balance in AD parietal cortex, a region within the default mode network that is overly active in the disorder, and support the hypothesis that E/I imbalances disrupt cognition-related shifts in cortical activity which contribute to the intellectual decline in AD., Synaptic loss may disturb the excitatory to inhibitory balance (E/I ratio) in circuits vulnerable in Alzheimer’s disease (AD). The authors find reduced synaptic levels of PSD-95 and gephyrin and show that individuals with AD exhibit a pro-excitatory shift of postsynaptic densities and the electrophysiological synaptic E/I ratio in the parietal cortex.

Oncogenic KRAS drives cancer growth by activating diverse signaling networks, not all of which have been fully delineated. We set out to establish a system-wide profile of the KRAS-regulated kinase signaling network (kinome) in KRAS-mutant pancreatic ductal adenocarcinoma (PDAC). We knocked down KRAS expression in a panel of six cell lines, and then applied Multiplexed Inhibitor Bead/Mass Spectrometry (MIB/MS) chemical proteomics to monitor changes in kinase activity and/or expression. We hypothesized that depletion of KRAS would result in downregulation of kinases required for KRAS-mediated transforming activities, and in upregulation of other kinases that could potentially compensate for the deleterious consequences of the loss of KRAS. We identified 15 upregulated and 13 downregulated kinases in common across the panel. In agreement with our hypothesis, all 15 of the upregulated kinases have established roles as cancer drivers (e.g., SRC, TGFBR1, ILK), and pharmacologic inhibition of the upregulated kinase, DDR1, suppressed PDAC growth. Interestingly, 11 of the 13 downregulated kinases have established driver roles in cell cycle progression, particularly in mitosis (e.g., WEE1, Aurora A, PLK1). Consistent with a crucial role for the downregulated kinases in promoting KRAS-driven proliferation, we found that pharmacologic inhibition of WEE1 also suppressed PDAC growth. The unexpected paradoxical activation of ERK upon WEE1 inhibition led us to inhibit both WEE1 and ERK concurrently, which caused further potent growth suppression and enhanced apoptotic death than WEE1 inhibition alone. We conclude that system-wide delineation of the KRAS-regulated kinome can identify potential therapeutic targets for KRAS-mutant pancreatic cancer.

Using model organisms to identify novel therapeutic targets is frequently constrained by pre-existing genetic toolkits. To expedite positive selection for identification of novel downstream effectors, we engineered conditional expression of activated CED-10/Rac to disrupt Caenorhabditis elegans embryonic morphogenesis, titrated to 100% lethality. The strategy of engineering thresholds for positive selection using experimental animals was validated with pharmacological and genetic suppression and is generalizable to diverse molecular processes and experimental systems.

Why layers II/III of entorhinal cortex (EC) deteriorate in advance of other regions during the earliest stages of Alzheimer's disease is poorly understood. Failure of retrograde trophic support from synapses to cell bodies is a common cause of neuronal atrophy, and we accordingly tested for early-life deterioration in projections of rodent layer II EC neurons. Using electrophysiology and quantitative imaging, changes in EC terminals during young adulthood were evaluated in male rats and mice. Field excitatory postsynaptic potentials, input/output curves, and frequency following capacity by lateral perforant path (LPP) projections from lateral EC to dentate gyrus were unchanged from 3 to 8–10 months of age. In contrast, the unusual presynaptic form of long-term potentiation (LTP) expressed by the LPP was profoundly impaired by 8 months in rats and mice. This impairment was accompanied by a reduction in the spine to terminal endocannabinoid signaling needed for LPP-LTP induction and was offset by an agent that enhances signaling. There was a pronounced age-related increase in synaptophysin within LPP terminals, an effect suggestive of incipient pathology. Relatedly, presynaptic levels of TrkB—receptors mediating retrograde trophic signaling—were reduced in the LPP terminal field. LTP and TrkB content were also reduced in the medial perforant path of 8- to 10-month-old rats. As predicted, performance on an LPP-dependent episodic memory task declined by late adulthood. We propose that memory-related synaptic plasticity in EC projections is unusually sensitive to aging, which predisposes EC neurons to pathogenesis later in life.SIGNIFICANCE STATEMENTNeurons within human superficial entorhinal cortex are particularly vulnerable to effects of aging and Alzheimer's disease, although why this is the case is not understood. Here we report that perforant path projections from layer II entorhinal cortex to the dentate gyrus exhibit rapid aging in rodents, including reduced synaptic plasticity and abnormal protein content by 8–10 months of age. Moreover, there was a substantial decline in the performance of an episodic memory task that depends on entorhinal cortical projections at the same ages. Overall, the results suggest that the loss of plasticity and related trophic signaling predispose the entorhinal neurons to functional decline in relatively young adulthood.

BackgroundSteroid 5β-reductase (AKR1D1) plays important roles in hepatic glucocorticoid clearance and bile acid synthesis. Glucocorticoids and bile acids are potent metabolic regulators, but whether AKR1D1 controls metabolic phenotype in vivo is unknown.MethodsAkr1d1-/-mice were generated on a C57BL/6 background. Liquid chromatography / mass spectrometry, metabolomic and transcriptomic approaches were used to determine effects on glucocorticoid and bile acid homeostasis. Metabolic phenotypes including body weight and composition, lipid homeostasis, glucose tolerance and insulin sensitivity were evaluated. Molecular changes were assessed by RNASeq and western blotting. Male Akr1d1-/-mice were challenged with a 60% high fat diet.ResultsAkr1d1-/-mice had a sex specific metabolic phenotype. At 30-weeks of age male, but not female, Akr1d1-/-mice were more insulin sensitive and had reduced lipid accumulation in the liver and adipose tissue, concomitant with hypertriglyceridemia and increased intramuscular triacylglycerol. This phenotype was underpinned by sexually dimorphic changes in bile acid metabolism and composition, but without overt effects on glucocorticoid action. Male Akr1d1-/-mice were not protected against diet induced obesity and insulin resistance.ConclusionThis study shows that AKR1D1 controls bile acid homeostasis in vivo and that altering its activity can affect insulin sensitivity and lipid homeostasis in a sex dependent manner.

The transition from &beta, cell compensation to &beta, cell failure is not well understood. Previous works by our group and others have demonstrated a role for Prostaglandin EP3 receptor (EP3), encoded by the Ptger3 gene, in the loss of functional &beta, cell mass in Type 2 diabetes (T2D). The primary endogenous EP3 ligand is the arachidonic acid metabolite prostaglandin E2 (PGE2). Expression of the pancreatic islet EP3 and PGE2 synthetic enzymes and/or PGE2 excretion itself have all been shown to be upregulated in primary mouse and human islets isolated from animals or human organ donors with established T2D compared to nondiabetic controls. In this study, we took advantage of a rare and fleeting phenotype in which a subset of Black and Tan BRachyury (BTBR) mice homozygous for the Leptinob/ob mutation&mdash, a strong genetic model of T2D&mdash, were entirely protected from fasting hyperglycemia even with equal obesity and insulin resistance as their hyperglycemic littermates. Utilizing this model, we found numerous alterations in full-body metabolic parameters in T2D-protected mice (e.g., gut microbiome composition, circulating pancreatic and incretin hormones, and markers of systemic inflammation) that correlate with improvements in EP3-mediated &beta, cell dysfunction.

Summary Skeletal and glycemic traits have shared etiology, but the underlying genetic factors remain largely unknown. To identify genetic loci that may have pleiotropic effects, we studied Genome-wide association studies (GWASs) for bone mineral density and glycemic traits and identified a bivariate risk locus at 3q21. Using sequence and epigenetic modeling, we prioritized an adenylate cyclase 5 (ADCY5) intronic causal variant, rs56371916. This SNP changes the binding affinity of SREBP1 and leads to differential ADCY5 gene expression, altering the chromatin landscape from poised to repressed. These alterations result in bone- and type 2 diabetes-relevant cell-autonomous changes in lipid metabolism in osteoblasts and adipocytes. We validated our findings by directly manipulating the regulator SREBP1, the target gene ADCY5, and the variant rs56371916, which together imply a novel link between fatty acid oxidation and osteoblast differentiation. Our work, by systematic functional dissection of pleiotropic GWAS loci, represents a framework to uncover biological mechanisms affecting pleiotropic traits., Graphical abstract, Highlights • 3q21 variants are associated with type 2 diabetes and increased bone mineral density • 3q21 variants affect chromatin accessibility in mesenchymal cells • rs56371916 is a 3q21 causal variant and ADCY5 its target in adipocytes and osteoblasts • ADCY5 and rs56371916 affect lipid oxidation processes in adipocytes and osteoblasts, Nasa Sinnott-Armstrong and colleagues identify a pleiotropic risk locus at 3q21 that is associated with type 2 diabetes (T2D) and greater bone mineral density (BMD) and its associated cell-autonomous mechanisms in adipocytes and osteoblasts. Together, these findings provide a possible explanation for the perplexing finding that individuals with T2D have higher BMD but greater susceptibility to bone fracture.

Fusobacterium nucleatum is a gram-negative bacterium, part of the normal human microflora. It is associated with various health complications, including periodontitis and colorectal cancer. Its surface is covered with lipopolysaccharide, which interacts with the immune system and can be involved in various processes in health and disease conditions. Here we present the results of structural analysis of core oligosaccharides from the lipopolysaccharides of several strains of F. nucleatum. Pure compounds were isolated using mild acid hydrolysis or alkaline deacylation of the lipopolysaccharides and analyzed by NMR spectroscopy, mass-spectrometry and chemical methods. All cores analyzed had a common octasaccharide region, including five heptose residues and a non-phosphorylated 3-deoxy-d-manno-oct-2-ulosonic acid residue. The common region is substituted with different additional components specific for each strain. By structure type the F. nucleatum core is similar to that produced by Aeromonas.

Oncogenic mutations in the KRAS gene are well-established drivers of cancer. While the recently developed KRASG12C inhibitors offer a targeted KRAS therapy and have shown success in the clinic, KRASG12C represents only 11% of all KRAS mutations. Current therapeutic approaches for all other KRAS mutations are both indirect and non-mutant-selective, largely focusing on inhibition of downstream KRAS effectors such as MAP kinases. Inhibition of KRAS downstream signaling results in a system-wide down-modulation of the respective targets, raising concerns about systemic cell toxicity. Here, we describe a custom short interfering RNA (siRNA) oligonucleotide (EFTX-D1) designed to preferentially bind mRNA of the most commonly occurring KRAS missense mutations in codons 12 and 13. We determined that EFTX-D1 preferentially reduced the mutant KRAS sequence versus wild-type at the levels of both transcription and translation, and reversed oncogenic KRAS-induced morphologic and growth transformation. Furthermore, EFTX-D1 significantly impaired the proliferation of several KRAS mutant cancer cell lines in 2-D as well as 3-D assays. Taken together, our data indicate a novel use of RNA interference (RNAi) to target oncogenic KRAS-driven cancers specifically.

[Image: see text] Oncogenic mutations in the KRAS gene are well-established drivers of cancer. While the recently developed KRAS(G12C) inhibitors offer a targeted KRAS therapy and have shown success in the clinic, KRAS(G12C) represents only 11% of all KRAS mutations. Current therapeutic approaches for all other KRAS mutations are both indirect and nonmutant-selective, largely focusing on inhibition of downstream KRAS effectors such as MAP kinases. Inhibition of KRAS downstream signaling results in a system-wide down-modulation of the respective targets, raising concerns about systemic cell toxicity. Here, we describe a custom short interfering RNA oligonucleotide (EFTX-D1) designed to preferentially bind mRNA of the most commonly occurring KRAS missense mutations in codons 12 and 13. We determined that EFTX-D1 preferentially reduced the mutant KRAS sequence versus wild-type at the levels of both transcription and translation and reversed oncogenic KRAS-induced morphologic and growth transformation. Furthermore, EFTX-D1 significantly impaired the proliferation of several KRAS mutant cancer cell lines in 2-D as well as 3-D assays. Taken together, our data indicate a novel use of RNA interference to target oncogenic KRAS-driven cancers specifically.

Background Blue mold is a globally important and economically impactful postharvest disease of apples caused by multiple Penicillium spp. There are currently four postharvest fungicides registered for blue mold control, and some isolates have developed resistance manifesting in decay on fungicide-treated fruit during storage. To date, mechanisms of fungicide resistance have not been explored in this fungus using a transcriptomic approach. Results We have conducted a comparative transcriptomic study by exposing naturally-occurring difenoconazole (DIF) resistant (G10) and sensitive (P11) blue mold isolates to technical grade difenoconazole, an azole fungicide in the commercial postharvest product Academy (Syngenta Crop Protection, LLC). Dynamic changes in gene expression patterns were observed encompassing candidates involved in active efflux and transcriptional regulators between the resistant and sensitive isolates. Unlike other systems, 3 isoforms of cytochrome P450 monoxygenase (CYP51A-C) were discovered and expressed in both sensitive and resistant strains upon difenoconazole treatment. Active efflux pumps were coordinately regulated in the resistant isolate and were shown to mediate the global resistance response as their inhibition reversed the difenoconazole-resistant phenotype in vitro. Conclusions Our data support the observation that global transcriptional changes modulate difenoconazole resistance in Penicillium spp. While the dogma of CYP51 overexpression is supported in the resistant isolate, our studies shed light on additional new mechanisms of difenoconazole resistance on a global scale in Penicillium spp. These new findings broaden our fundamental understanding of azole fungicide resistance in fungi, which has identified multiple genetic targets, that can be used for the detection, management, and abatement of difenoconazole-resistant blue mold isolates during long-term storage of apples.

Understanding how fungicide application practices affect selection for fungicide resistance is imperative for continued sustainable agriculture. Here, we examined the effect of field applications of the succinate dehydrogenase inhibitor (SDHI) fluxapyroxad at different doses and mixtures on the SDHI sensitivity of Venturia inaequalis, the apple scab pathogen. Fungicide applications were part of selection programs involving different doses (high or low) and mixtures (with a second single-site fungicide or a multisite fungicide). These programs were tested in two apple orchards over 4 years to determine potential cumulative selection effects on resistance. Each year after program applications, apple scab lesions were collected, and relative growth assays were conducted to understand shifts in fluxapyroxad sensitivity. After 4 years, there was a trend toward a reduction in sensitivity to fluxapyroxad for most selection programs in comparison to that in the non-selective-pressure control. In most years, the selection program plots treated with low-dose fluxapyroxad applications resulted in a larger number of isolates with reduced sensitivity, supporting the use of higher doses for disease management. Few significant differences (P

The Vector Manipulation Hypothesis (VMH) posits that phytopathogens develop strategies to enhance dissemination by mediating behavior change in insect vectors. The VMH is poorly studied in phytopathogenic bacteria, especially in systems with numerous, occasional vectors. Erwinia amylovora is a bacterial pathogen of pome fruit that produces a bacterial ooze and is mechanically vectored by insects after they feed on ooze. The blossom blight phase of the disease exhibits manipulation of honeybees, leading to enhanced transmission, but whether the same occurs during the shoot blight phase of the disease is unknown. The goal of this study was to evaluate the effect of E. amylovora on the behavior of Delia platura, a fly with a worldwide endemic presence that may transmit E. amylovora. We show that D. platura prefer infected, oozing fruit to uninfected fruit in choice tests and that preference subsides when bacterial ooze is removed from the infected fruit. Flies did not exhibit a preference between infected saplings and uninfected saplings. The volatiles of infected fruit did not attract D. platura, indicating that diseased fruit odor is not responsible for the observed preference for infected fruit. Flies did not differentiate between sapling odors until infected trees had died, at which point they preferred uninfected tree odors. This study supports previous hypotheses suggesting that E. amylovora takes advantage of existing plant–insect interactions, though it is not fully understood how significantly behavioral changes affect transmission. Additional pathosystems with occasional, nonspecific vectors should be studied to further understanding of the VMH.