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Autosomal recessive tyrosine kinase 2 (TYK2) deficiency is characterized by susceptibility to mycobacterial and viral infections. Here, we report a 4-year-old female with severe respiratory viral infections, EBV-driven Burkitt-like lymphoma, and infection with the neurotropic Jamestown Canyon virus. A novel, homozygous c.745C > T (p.R249*) variant was found in TYK2. The deleterious effects of the TYK2 lesion were confirmed by immunoblotting; by evaluating functional responses to IFN-α/β, IL-10, and IL-23; and by assessing its scaffolding effect on the cell surface expression of cytokine receptor subunits. The effects of the mutation could not be pharmacologically circumvented in vitro, suggesting that alternative modalities, such as hematopoietic stem cell transplantation or gene therapy, may be needed. We characterize the first patient from Canada with a novel homozygous mutation in TYK2., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The immune contexture of pancreatic ductal adenocarcinoma (PDAC) is generally immunosuppressive. A role for immune checkpoint inhibitors (ICIs) in PDAC has only been demonstrated for the rare and hypermutated mismatch repair (MMR) deficient (MMR-d) subtype. Homologous recombination repair (HR) deficient (HR-d) PDAC is more prevalent and may encompass up to 20% of PDAC. Its genomic instability may promote a T-cell mediated anti-tumor response with therapeutic sensitivity to ICIs. To investigate the immunogenicity of HR-d PDAC, we used multiplex immunohistochemistry (IHC) to compare the density and spatial distribution of CD8+ cytotoxic T-cells, FOXP3+ regulatory T-cells (Tregs), and CD68+ tumor-associated macrophages (TAMs) in HR-d versus HR/MMR-intact PDAC. We also evaluated the IHC positivity of programmed death-ligand 1 (PD-L1) across the subgroups. 192 tumors were evaluated and classified as HR/MMR-intact (n=166), HR-d (n=25) or MMR-d (n=1) based on germline testing and tumor molecular hallmarks. Intra-tumoral CD8+ T-cell infiltration was higher in HR-d versus HR/MMR-intact PDAC (p<0.0001), while CD8+ T-cell densities in the peri-tumoral and stromal regions were similar in both groups. HR-d PDAC also displayed increased intra-tumoral FOXP3+ Tregs (p=0.049) and had a higher CD8+:FOXP3+ ratio (p=0.023). CD68+ TAM expression was similar in HR-d and HR/MMR-intact PDAC. Finally, 6 of the 25 HR-d cases showed a PD-L1 Combined Positive Score of >=1, whereas none of the HR/MMR-intact cases met this threshold (p<0.00001). These results provide immunohistochemical evidence for intra-tumoral CD8+ T-cell enrichment and PD-L1 positivity in HR-d PDAC, suggesting that HR-d PDAC may be amenable to ICI treatment strategies., Competing Interests: P-OF has received honoraria from EMD Serono and consultation fees from Amgen, Bristol Myers Squibb, AstraZeneca Canada, Hoffmann La Roche, Merck Canada, Pfizer Canada and Roche Canada. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Golesworthy, Wang, Tanti, Pacis, Romero, Cuggia, Domecq, Bourdel, Denroche, Jang, Grant, Borgida, Grünwald, Dodd, Wilson, Bourque, O’Kane, Fischer, Kron, Fiset, Omeroglu, Foulkes, Gallinger, Guiot, Gao and Zogopoulos.)

Pathological angiogenesis is the hallmark of diseases such as cancer and retinopathies. Although tissue hypoxia and inflammation are recognized as central drivers of vessel growth, relatively little is known about the process that bridges the two. In a mouse model of ischemic retinopathy, we found that hypoxic regions of the retina showed only modest rates of apoptosis despite severely compromised metabolic supply. Using transcriptomic analysis and inducible loss-of-function genetics, we demonstrated that ischemic retinal cells instead engage the endoplasmic reticulum stress inositol-requiring enzyme 1α (IRE1α) pathway that, through its endoribonuclease activity, induces a state of senescence in which cells adopt a senescence-associated secretory phenotype (SASP). We also detected SASP-associated cytokines (plasminogen activator inhibitor 1, interleukin-6, interleukin-8, and vascular endothelial growth factor) in the vitreous humor of patients suffering from proliferative diabetic retinopathy. Therapeutic inhibition of the SASP through intravitreal delivery of metformin or interference with effectors of senescence (semaphorin 3A or IRE1α) in mice reduced destructive retinal neovascularization in vivo. We conclude that the SASP contributes to pathological vessel growth, with ischemic retinal cells becoming prematurely senescent and secreting inflammatory cytokines that drive paracrine senescence, exacerbate destructive angiogenesis, and hinder reparative vascular regeneration. Reversal of this process may be therapeutically beneficial., (Copyright © 2016, American Association for the Advancement of Science.)

Plant-root inhabiting fungi are a universal phenomenon found in all ecosystems where plants are able to grow, even in harsh environments. Interactions between fungi and plant roots can vary widely from mutualism to parasitism depending on many parameters. The role of fungal endophytes in phytoremediation of polluted sites, and characterization of the endophytic diversity and community assemblages in contaminated areas remain largely unexplored. In this study, we investigated the composition of endophytic fungal communities in the roots of two plant species growing spontaneously in petroleum-contaminated sedimentation basins of a former petro-chemical plant. The three adjacent basins showed a highly heterogeneous pattern of pollutant concentrations. We combined a culture-based isolation approach with the pyrosequencing of fungal ITS ribosomal DNA. We selected two species, Eleocharis erythropoda Steud. and Populus balsamifera L., and sampled three individuals of each species from each of three adjacent basins, each with a different concentration of petroleum hydrocarbons. We found that contamination level significantly shaped endophytic fungal diversity and community composition in E. erythropoda, with only 9.9% of these fungal Operational Taxonomic Units (OTUs) retrieved in all three basins. However, fungal community structure associated with P. balsamifera remained unaffected by the contamination level with 28.2% of fungal OTUs shared among all three basins. This could be explained by the smaller differences of pollutant concentrations in the soil around our set of P. balsamifera sampless compared to that around our set of E. erythropoda samples. Our culture-based approach allowed isolation of 11 and 30 fungal endophytic species from surface-sterilized roots of E. erythropoda and P. balsamifera, respectively. These isolates were ribotyped using ITS, and all were found in pyrosequensing datasets. Our results demonstrate that extreme levels of pollution reduce fungal diversity and shape community composition in E. erythropoda. Our findings shed light on the effect of soil petroleum contamination on fungal endophytic communities and could help to develop strategies for improving phytoremediation using fungal endophytes.

This article documents the addition of 205 microsatellite marker loci to the Molecular Ecology Resources Database. Loci were developed for the following species: Bagassa guianensis, Bulweria bulwerii, Camelus bactrianus, Chaenogobius annularis, Creontiades dilutus, Diachasmimorpha tryoni, Dioscorea alata, Euhrychiopsis lecontei, Gmelina arborea, Haliotis discus hannai, Hirtella physophora, Melanaphis sacchari, Munida isos, Thaumastocoris peregrinus and Tuberolachnus salignus. These loci were cross-tested on the following species: Halobaena caerulea, Procellaria aequinoctialis, Oceanodroma monteiroi, Camelus ferus, Creontiades pacificus, Dioscorea rotundata, Dioscorea praehensilis, Dioscorea abyssinica, Dioscorea nummularia, Dioscorea transversa, Dioscorea esculenta, Dioscorea pentaphylla, Dioscorea trifida, Hirtella bicornis, Hirtella glandulosa, Licania alba, Licania canescens, Licania membranaceae, Couepia guianensis and 7 undescribed Thaumastocoris species., (© 2010 Blackwell Publishing Ltd.)

* In the apple tree (Malus domestica), shoot architecture - the distribution of lateral bud types and growth along the parent shoot - has been extensively investigated. The distal zone of a shoot is characterized by a high proportion of vegetative or floral axillary branches mixed with latent buds and aborted laterals. The hypothesis tested here was that bud development was related to hydraulic conductance of the sap pathway to the bud, independently of an acrotonic (proximal vs distal) effect. * The distal zone of 1-yr-old shoots was studied on five cultivars for bud size and composition (number of appendages) and hydraulic conductance before bud burst. * Bud size, composition and hydraulic conductance were highly variable for all cultivars. A positive correlation was demonstrated between both the number of cataphylls and green-leaf primordia, and hydraulic conductance. Cultivar and bud size affected the intercept of these relationships more than the slope, suggesting similar scaling between these variables, but different hydraulic efficiencies. A great proportion of small buds were also characterized by null values of hydraulic conductance. * This study suggests that hydraulically mediated competition exists between adjacent buds within the same branching zone, prefiguring the variability of lateral types in the following growing season. It is hypothesized that this developmental patterning is driven by hydraulic characteristics of the whole metamer, including the subtending leaf, during bud development.

Plasma-membrane vesicles prepared from the liver of rats fed either a low-(LP) or a high-protein (HP) diet exhibited Na(+)-dependent active transport of alanine and serine. The process gave apparent kinetic parameters compatible with a single saturable component for both amino acids. Na,K-ATPase (EC 3.6.1.37), marker of the basolateral domain of the hepatocyte plasma-membrane, was chosen as reference for the expression of amino acid transport in vesicle preparations. The high-protein diet induced a significant increase in liver Na,K-ATPase activity also found in corresponding plasma-membrane preparations, in parallel with an increase in the capacity towards amino acid transport. This suggests that in rats fed the high protein diet, transcellular Na+ exchange, although increased, remains well balanced. N-Methylaminoisobutyric acid (MeAIB), due to its poor velocity, proved unsuitable to distinguish between systems A and ASC in the experimental model. Comparing Na(+)- and Li(+)-driven transport, a family of carriers with strict Na(+)-dependency (A-like) was evidenced in LP vesicles but not in HP vesicles. The sensitivity to the lowering of the pH from 7.5 to 6.5 in the external medium was similar in both type of vesicles when Na+ was the driving ion. In the HP vesicles the Li(+)-tolerant, pH-insensitive component (ASC-like) was increased in parallel with overall Na(+)-dependent transport. These functional properties suggest that the carriers involved in the stimulation of transport in HP vesicles are composite in nature. Increasing concentrations of an amino acid mixture mimicking the changes of portal aminoacidemia inhibited the transport of alanine and of serine. The degree of inhibition was correlated with the relative concentration of substrate and was independent of the nutritional treatment.

Adult rats fed proteins as a meal given during the daytime exhibit alterations of liver protein metabolism characterized by simultaneous stimulations of protein synthesis and degradation, particularly during the hours following protein ingestion. The purpose of the present work was to determine if the stimulation of liver protein breakdown could be related to biophysical alterations of the lysosomal system. There is a growing amount of evidence to suggest that the lysosomal vacuolar system is involved in the physiological regulation of overall proteolytic rate. Rats, trained to eat a protein meal 2 hrs after the onset of light, were killed 6, 9, 18, 21 and 24 hrs after protein intake. Three fractions were isolated from 0.25 M sucrose liver homogenates after differential centrifugation. The mitochondrial-lysosomal fraction was further analyzed by isopycnic centrifugation in sucrose gradients. Three specific lysosomal enzyme activities were assessed: N-acetyl-beta-D glucosaminidase (marker), cathepsin D and cathepsin C (proteolytic enzymes). Total activities remained unchanged at all time-points, but the distributions between the different fractions recovered after differential centrifugation were altered 6 and 9 hrs after protein intake. A significantly higher percentage of N-acetyl-beta-D-glucosaminidase, cathepsin D and cathepsin C activities were recovered in the M + L fraction, suggesting a shift towards lysosomal forms of lighter density. This was confirmed by density gradient analysis. Thus, even in adapted rats, acute administration of protein during the daytime quickly induced biophysical alterations in the lysosomal system. The lysosomal distribution pattern observed at 6 and 9 hrs after protein intake might be due to lysosome enlargement by active autophagy and/or by the sequestration of lighter cellular material.

The influence of protein ingestion on liver albumin synthesis and albumin content was investigated in rats fed protein as a meal (90% casein) given apart from the other dietary components provided ad libitum. In this condition, protein ingestion rapidly stimulates liver total protein synthesis. Separately fed rats were studied 6 and 20 h after the protein meal. Control rats fed mixed diets containing 13 or 80% casein were killed either during the absorptive (night) or postabsorptive (light) periods. The ratio of hepatic albumin synthesis to total protein synthesis remained fairly constant (12-15%) in all groups, indicating that albumin synthesis paralleled total protein synthesis. Liver albumin content measured in microsomes by immunonephelometry was significantly higher in separately fed rats killed 6 h postmeal than in those killed after 20 h. In rats fed 13% casein, the liver albumin content remained high regardless of the time of killing. In rats fed 80% casein, the albumin content was higher during the absorptive period than during the postabsorptive period. Immunoperoxidase staining of the hepatocyte organelles involved in albumin synthesis, especially the Golgi apparatus, was more intense for separately fed rats killed 6 h postmeal than for those killed after 20 h. Livers of rats fed 13% casein also exhibited a pattern indicative of high hepatocyte albumin content, whereas livers of rats fed 80% casein contained less. These results show that, in separate feeding, wide circadian variations of albumin synthesis run parallel to changes in liver albumin content.

Rats having a protein-free diet available ad libitum were fed a daily casein meal at the beginning of either the light- or the dark-phase of the day. A control group received a mixed-diet ad libitum. In all three groups, daily food ingestion was the same and casein corresponded to 12% of total intake. Liver activities of alanine, aspartate, ornithine and tyrosine aminotransferase, ornithine decarboxylase and serine dehydratase were assessed. In mixed-fed controls, all activities were low. Tyrosine aminotransferase and ornithine decarboxylase exhibited clear circadian rhythms of low amplitude. Feeding casein as a concentrated meal had no effect on aspartate aminotransferase. It depressed alanine aminotransferase and serine dehydratase activities. Tyrosine aminotransferase and ornithine decarboxylase exhibited rapid and strong stimulatory responses but, within 12 hours, returned to levels similar to those observed in mixed-fed controls. Ornithine aminotransferase was increased in the group receiving the casein meal during the light phase. It is concluded that the capacity for amino acid catabolism remains low in separately-fed animals, and that only tyrosine and especially ornithine, which may become limiting for urea synthesis, are actively metabolized. Thus, when high fluxes of amino acids reach the liver following the absorption of the casein meal, more amino acids are available for incorporation into newly synthesized proteins.

Experiments were designed to investigate whether the metabolic responses of pregnant females are in keeping with the known state of gestational hyperinsulinemia. Groups of female rats fed a 32% protein diet were killed on days 13, 15, 17, 19 and 21 of pregnancy, during either daytime or during night-time. Liver pyruvate kinase and glucose-6-phosphate dehydrogenase activities were increased over nonpregnant values from day 13 onward in agreement with what can be expected as a result of the gestational hyperinsulinemia. Liver malate dehydrogenase (NADP) activity was increased to lesser extent and later. Pyruvate and lactate accumulated in maternal liver from day 13 onward. The fact that this accumulation could not be related to any further increase of food intake during this time and that it correlated at day 21 with litter size was taken as indication of a probable contribution of the conceptus to maternal pyruvate and lactate accumulation in late pregnancy. Liver alanine amino-transferase activity decreased as pregnancy progressed. No change in serine dehydratase activity was found. Cytosolic aspartate aminotransferase activity remained unchanged. Mitochondrial activity increased as pregnancy progressed.

The magnitude and composition of amino acid intestinal efflux was followed over a light-dark cycle in rats ingesting the same daily amount of protein administered either in a mixed diet (12% casein) or as a separate meal (70% casein concentrate) fed 2 hours after the onset of the light period with a protein-free diet available at all times. Intestinal efflux was determined by an instant porto-aortic difference measured on pooled samples from six rats at time-points spaced every 3 hours over a light-dark cycle. During protein digestion (dark for the mixed-fed rats) and (light for the separately-fed ones), essential amino acid composition of intestinal output fell into line with that of the protein ingested (casein) while non-essential amino acid composition did not. The discrepancy bore on alanine and glycine which were released in excess and on aspartic and glutamic acids, glutamine and serine which were released in deficit from their content in casein. From the follow-up of individual amino acid release and uptake, we concluded that intestinal efflux reflects the composition of the dietary protein only with respect to the amino acids, mostly essential, that are not metabolized by the intestinal wall.

Rats fed either a low (2p. 100) or high (40 p. 100)-fat diet were exposed to 22 or 5 degrees C. The resulting hyperphagia adequately compensated energy losses as judged from body weight. The cold-induced hyperphagia was accompanied by a non-parallel increase in pancreatic hydrolases. Amylase and lipase were not increased above the adaptive levels they had respectively reached in the heat with a high-starch or high-lipid diet. Chymotrypsinogen, on the contrary, responded to increased intake of both diets. It also responded to the higher protein concentration in the high-fat diet caused by isocaloric replacement of starch by fat. Colipase varied independently of lipase and was increased additively by fat and protein intakes. Consequently, although limiting for lipase in the warm, colipase rose to a 1:1 ratio in the cold. Increased intake had a consistent pleiotropic effect evidenced by an increase of amylase with the high-fat diet and of lipase with the low-fat diet. The net effect was a significant increase in the lipid-digesting potential of the organism of lipid-fed animals upon exposure to cold, while the starch-digesting potential remained unaffected in starch-fed animals.

Hepatic tyrosine aminotransferase (EC 2.6.1.5) was induced in rats by intubation of amino acid mixtures (complete or tryptophan-free). Enzyme activity was increased 4-fold by the complete mixture and 8-fold by the tryptophan-free mixture. The enzyme was analyzed by chromatography on CM-Sephadex. Chromatographic patterns were characteristic of the type of inducer rather than of the chronology of the induction cycle: after induction by the complete amino acid mixture the three forms of the enzyme were equally increased whereas after induction by the tryptophan-free mixture Form I was preferentially increased.

The adaptative response of the exocrine pancreas to diets rich in lipids and the influence of dietary proteins are reconsidered taking into account colipase. This was particularly necessary in the rat since colipase is often limiting with respect to lipase in this species. In a first experiment, adult male rats received diets containing 10, 15, 18, 40 or 86% casein together with 2, 8 or 40% lard. Under these conditions, lipase was found to be sensitive to variations in the lipid contents of the diet and was two-fold higher with 40% than with 8% lipid, in diets containing 40% casein. Proteins exerted a permissive effect since no response was recorded with diets containing less than 18% casein. Colipase responded to protein intake, even in lipid-poor diets (2% lard), and was increased by a factor 3 when casein was raised from 18 to 40%. In a second experiment, proteins were fed as a separate meal, and the remainder of the diet (provided ad libitum) contained either 8 or 40% lard. In a diurnal study, lipase and colipase were followed every 3 hours over 24 hours. Both lipase and colipase were found to accumulate after the protein meal. Colipase was found to accumulate much faster than lipase in all cases and reach 3 times basal levels 9 hours after the protein meal. This resulted in important diurnal variations in the ratio of colipase to lipase which modulates lipolytic activity. It is concluded that colipase is particularly sensitive to protein intake, perhaps more than to lipid intake and may become and limiting factor of lipid digestion.

In agreement with what is known of pancreatic adaptation to dietary composition, a progressive lowering of amylase-to-chymotrypsinogen ratio was observed in the pancreases of rats fed mixed diets of increasing casein and decreasing carbohydrate contents. The same changes in ratio were found when casein alone was increased in otherwise isonitrogenous meals fed to rats provided ad libitum with a protein-free diet. Pancreatic accumulation of chymotrypsinogen was increased while that of amylase was decreased in spite of low-protein and high-carbohydrate intakes. Lowering the lipid content of the protein-free diet reduced the effectiveness of casein meals in lowering the amylase-to-chymotrypsinogen ratio. It is concluded that 1) the capacity of protein-rich diets to induce adaptative changes in pancreatic secretion does not depend on the absolute amount of proteins passing through the duodenum but rather on the increased protein concentration in the alimentary bolus, 2) protein concentration affects both amylase and chymotrypsinogen, and 3) lipids have a cooperative effect.