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Resisting and tolerating microbes are alternative strategies to survive infection, but little is known about the evolutionary mechanisms controlling this balance. Here genomic analyses of anatomically modern humans, extinct Denisovan hominins and mice revealed a TNFAIP3 allelic series with alterations in the encoded immune response inhibitor A20. Each TNFAIP3 allele encoded substitutions at non-catalytic residues of the ubiquitin protease OTU domain that diminished IκB kinase-dependent phosphorylation and activation of A20. Two TNFAIP3 alleles encoding A20 proteins with partial phosphorylation deficits seemed to be beneficial by increasing immunity without causing spontaneous inflammatory disease: A20 T108A;I207L, originating in Denisovans and introgressed in modern humans throughout Oceania, and A20 I325N, from an N-ethyl-N-nitrosourea (ENU)-mutagenized mouse strain. By contrast, a rare human TNFAIP3 allele encoding an A20 protein with 95% loss of phosphorylation, C243Y, caused spontaneous inflammatory disease in humans and mice. Analysis of the partial-phosphorylation A20 I325N allele in mice revealed diminished tolerance of bacterial lipopolysaccharide and poxvirus inoculation as tradeoffs for enhanced immunity.

The extracellular matrix (ECM) is a major component of the ovarian stroma. Collagen and hyaluronan (HA) are critical ovarian stromal ECM molecules that undergo age-dependent changes in the mouse and human. How these matrix components are regulated and organized in other mammalian species with reproductive characteristics similar to women such as cows and pigs, has not been systematically investigated. Therefore, we performed histological, molecular, and biochemical analyses to characterize collagen and HA in these animals. Bovine ovaries had more collagen than porcine ovaries when assessed biochemically, and this was associated with species-specific differences in collagen gene transcripts: Col3a1 was predominant in cow ovaries while Col1a1 was predominant in pig ovaries. We also observed more HA in the porcine vs. bovine ovary. HA was distributed across three molecular weight ranges (&lt, 100 kDa, 100–300 kDa, and &gt, 300 kDa) in ovarian tissue and follicular fluid, with tissue having more &gt, 300 kDa HA than the other two ranges. Transcripts for HA synthesis and degradation enzymes, Has3 and Hyal2, respectively, were predominant in cow ovaries, whereas Has2, Kiaa1199, and Tmem2 tended to be predominant in pig ovaries. Together, our findings have implications for the composition, organization, and regulation of the ovarian ECM in large mammalian species, including humans.

Fibrosis is a hallmark of aging tissues which often leads to altered architecture and function. The ovary is the first organ to show overt signs of aging, including increased fibrosis in the ovarian stroma. How this fibrosis affects ovarian biomechanics and the underlying mechanisms are unknown. Using instrumental indentation, we demonstrated a quantitative increase in ovarian stiffness, as evidenced by an increase in Young's modulus, when comparing ovaries from reproductively young (6–12 weeks) and old (14–17 months) mice. This ovarian stiffness was dependent on collagen because ex vivo enzyme‐mediated collagen depletion in ovaries from reproductively old mice restored their collagen content and biomechanical properties to those of young controls. In addition to collagen, we also investigated the role of hyaluronan (HA) in regulating ovarian stiffness. HA is an extracellular matrix glycosaminoglycan that maintains tissue homeostasis, and its loss can change the biomechanical properties of tissues. The total HA content in the ovarian stroma decreased with age, and this was associated with increased hyaluronidase (Hyal1) and decreased hyaluronan synthase (Has3) expression. These gene expression differences were not accompanied by changes in ovarian HA molecular mass distribution. Furthermore, ovaries from mice deficient in HAS3 were stiffer compared to age‐matched WT mice. Our results demonstrate that the ovary becomes stiffer with age and that both collagen and HA matrices are contributing mechanisms regulating ovarian biomechanics. Importantly, the age‐associated increase in collagen and decrease in HA are conserved in the human ovary and may impact follicle development and oocyte quality., Advanced reproductive age is associated with a quantitative increase in ovarian tissue stiffness. Both collagen and hyaluronan (HA) matrices regulate the biomechanical properties of the ovary, and with age, there is an increase in ovarian collagen and a decrease in HA (without a change in HA polydispersity). These age‐dependent changes in extracellular matrix molecules are conserved in mouse and human. The increased stiffness of the aging ovary likely influences gamete quantity and quality and may be an important therapeutic target to extend reproductive longevity.

Bacterial type III secretion systems (T3SS) are used to inject proteins into mammalian cells to subvert cellular functions. The Shigella T3SS apparatus (T3SA) is comprised of a basal body, cytoplasmic sorting platform and exposed needle with needle "tip complex" (TC). TC maturation occurs when the translocator protein IpaB is recruited to the needle tip where both IpaD and IpaB control secretion induction. IpaB insertion into the host membrane is the first step of translocon pore formation and secretion induction. We employed disruptive insertional mutagenesis, using bacteriophage T4 lysozyme (T4L), within predicted IpaB loops to show how topological features affect TC functions (secretion control, translocon formation and effector secretion). Insertions within the N-terminal half of IpaB were most likely to result in a loss of steady-state secretion control, however, all but the two that were not recognized by the T3SA retained nearly wild-type hemolysis (translocon formation) and invasiveness levels (effector secretion). In contrast, all but one insertion in the C-terminal half of IpaB maintained secretion control but were impaired for hemolysis and invasion. These nature of the data suggest the latter mutants are defective in a post-secretion event, most likely due to impaired interactions with the second translocator protein IpaC. Intriguingly, only two insertion mutants displayed readily detectable T4L on the bacterial surface. The data create a picture in which the makeup and structure of a functional T3SA TC is highly amenable to physical perturbation, indicating that the tertiary structure of IpaB within the TC is more plastic than previously realized.

Summary The advent of large-scale in vitro differentiation of human stem cell-derived insulin-producing cells (SCIPC) has brought us closer to treating diabetes using stem cell technology. However, decades of experiences from islet transplantation show that ischemia-induced islet cell death after transplant severely limits the efficacy of the therapy. It is unclear to what extent human SCIPC are susceptible to ischemia. In this study, we show that more than half of SCIPC die shortly after transplantation. Nutrient deprivation and hypoxia acted synergistically to kill SCIPC in vitro. Amino acid supplementation rescued SCIPC from nutrient deprivation, likely by providing cellular energy. Generating SCIPC under physiological oxygen tension of 5% conferred hypoxia resistance without affecting their differentiation or function. A two-pronged strategy of physiological oxygen acclimatization during differentiation and amino acid supplementation during transplantation significantly improved SCIPC survival after transplant., Graphical Abstract, Highlights • Stem cell-derived insulin-producing cells (SCIPC) are susceptible to ischemic injury • Amino acid supplementation prevents nutrient-deprivation-induced SCIPC death • Generation of SCIPC at physiological oxygen levels protects them against hypoxia • Both strategies combined preserve SCIPC graft viability in vivo upon transplant, Cell death after transplant due to ischemic injury is a major obstacle to successful β cell replacement therapy for diabetes. In this issue of Stem Cell Reports, Faleo, Russ et al. analyzed the effects of hypoxia and nutrient deprivation on human stem cell-derived insulin-producing cells and developed effective strategies to promote their survival after transplant.

Objetive It has been claimed that micro-pulse vibration can accelerate the rate of tooth movement during orthodontic treatment; however, the underlying cellular mechanism has yet to be elucidated. The purpose of this study was to understand the mechanisms underlying tooth movement acceleration by measuring alterations in a panel of intercellular signalling molecules and markers of osteoblast/osteoclast function following micro-pulse vibration for 20 min at 30 Hz. Design Primary BALB/c mouse calvarial osteoblasts were cultivatedin vitro and subjected to micro-pulse vibration (0.25 N; 30 Hz) with the AcceleDent® Aura appliance for 20 min and assayed for IL-4, IL-13, IL-17, OPG, soluble RANKL and TGF-β protein by ELISA; for PCNA in osteoblasts and caspase 3/7 in osteoclasts by immunohistochemistry; for IL-4, IL-13, and Il-17 in osteoclasts by ELISA; and for cathepsin K by flow cytometry. Results After micro-pulse vibration, the murine osteoblast culture supernatant showed increased IL-4, IL-13, IL-17, OPG and TGF-β levels and decreased RANKL levels; PCNA in osteoblasts and caspase 3/7 in osteoclasts were also upregulated. The osteoclast culture supernatant had increased levels of IL-4, IL-13 and IL-17, and cathepsin K was upregulated in the treatment group compared with the control group. Conclusions Micro-pulse vibration promotes the production of soluble factors that inhibit osteoclasts, promote apoptosis and activate osteoblasts in vitro, which could increase bone mineral density. Further studies should be conducted in order to understand the biological mechanism of how micro-vibration might influence tooth movement during orthodontic treatment.

Resisting or tolerating microbes are alternative strategies to survive infection, but little is known about the evolutionary mechanisms controlling this balance. Here, genomic analyses of anatomically modern humans, extinct Denisovan hominins, and mice revealed a series of missense variants in the immune response inhibitor A20 (encoded byTNFAIP3), substituting non-catalytic residues of the ubiquitin protease domain to diminish IκB-dependent phosphorylation and activation of A20. Two A20 variants with partial phosphorylation deficits appeared beneficial: one originating in Denisovans and introgressed in modern humans throughout Oceania, and another in a mouse strain resistant to Coxsackievirus. By contrast, a variant with 95% loss of phosphorylation caused spontaneous inflammatory disease in humans and mice. Analysis of the partial phosphorylation variant in mice revealed diminished tolerance of bacterial lipopolysaccharide or to poxvirus inoculation as trade-offs for enhanced immunity.One Sentence SummaryModern and ancient variants reveal a genetically tunable element for balancing immunity and microbial tolerance.

Administration of anti-CD40 ligand (CD40L) antibodies has been reported to allow long-term islet allograft survival in non-human primates without the need for exogenous immunosuppression. However, the use of anti-CD40L antibodies was associated with thromboembolic complications. Targeting downstream intracellular components shared between CD40 and other TNF family co-stimulatory molecules could bypass these complications. TNF receptor associated factor 2 (TRAF2) integrates multiple TNF receptor family signalling pathways that are critical for T cell activation and may be a central node of alloimmune responses.T cell-specific Traf2-deficient mice (Traf2TKO) were generated to define the role of TRAF2 in CD4Traf2TKO mice exhibited permanent survival of full MHC-mismatched pancreatic islet allografts without exogenous immunosuppression. Traf2TKO CD4Targeting TRAF2 may be useful as a therapeutic approach for immunosuppression-free islet allograft survival that avoids the thromboembolic complications associated with the use of anti-CD40L antibodies.

Among the pectic enzymes present in fruits and vegetables, pectin methylesterase (PME) is usually related to the loss of quality and it causes adverse effects on finished products. In this research, the kinetic of ultrasound and thermal treatments are evaluated in the PME activity in papaya juice. The results showed that the ultrasound treatment caused an increase in the catalytic activity up to 52%. After a while, the catalytic activity decreased in 27% indicating that the ultrasound was not effective in the enzymatic inactivation, whereas the thermal treatment inactivated 71% of the PME. However, these results open perspectives to evaluate the effect of ultrasound and enhance the catalytic activity of enzymes of industrial interest.

Objetivos . Evaluar el efecto del endospermo de semilla de tara (EST) y polvo de hojas del Agave americana (HAA) sobre el peso corporal y velocidad de transito intestinal en ratas Holtzman. Materiales y metodos . Veinticinco ratas machos Holtzman distribuidas en cinco grupos y alojadas en jaulas individuales, fueron alimentadas durante 21 dias con uno de los siguientes tratamientos: T1, dieta con 6% de alfa celulosa (control); T2, dieta con 6% de EST; T3, dieta con 6% de HAA; T4, dieta con 10% de EST y T5, dieta con 10% de HAA. Se registraron el consumo de alimento, ganancia de peso corporal, digestibilidad aparente de la grasa, caracteristicas de las heces (contenido de grasa, peso, humedad, volumen y pH) y tiempo de transito intestinal. Se realizaron analisis de varianza (ANOVA) de una via y a traves de la comparacion multiple de medias de Tukey. Resultados . Dietas con 6% y 10% del EST exhibieron una reduccion en el consumo de alimento, digestibilidad aparente de la grasa y pH fecal, cuyos resultados tuvieron efectos en la reduccion de la ganancia del peso corporal de 37,0% (p=0,008) y 50,9% (p=0,001) comparados con la dieta control. Dieta con 10% del polvo de HAA redujo el tiempo de transito intestinal de 642 min (control) a 532 min (p=0,242). Conclusiones. Dietas que contienen EST regulan la ganancia del peso corporal; en cambio, dieta con polvo de HAA, no tuvo efectos sobre la velocidad de transito intestinal en ratas.

Pathological obesity can result from genetic predisposition, obesogenic diet, and circadian rhythm disruption. Obesity compromises function of muscle, which accounts for a majority of body mass. Behavioral intervention that can counteract obesity arising from genetic, diet or circadian disruption and can improve muscle function holds untapped potential to combat the obesity epidemic. Here we show that Drosophila melanogaster (fruit fly) subject to obesogenic challenges exhibits metabolic disease phenotypes in skeletal muscle; sarcomere disorganization, mitochondrial deformation, upregulation of Phospho-AKT level, aberrant intramuscular lipid infiltration, and insulin resistance. Imposing time-restricted feeding (TRF) paradigm in which flies were fed for 12 h during the day counteracts obesity-induced dysmetabolism and improves muscle performance by suppressing intramuscular fat deposits, Phospho-AKT level, mitochondrial aberrations, and markers of insulin resistance. Importantly, TRF was effective even in an irregular lighting schedule mimicking shiftwork. Hence, TRF is an effective dietary intervention for combating metabolic dysfunction arising from multiple causes., Time-restricted feeding (TRF) has beneficial metabolic effects. Here the authors examine how TRF impacts muscle physiology using fly models of metabolically adverse conditions, including diet and genetic models of obesity as well as circadian rhythm disruption, and find that TRF ameliorates skeletal muscle dysfunction.

Malle et al. identify a role for nuclear factor inducing κB (NIK) in pancreatic β cell failure. NIK activation disrupts glucose homeostasis in zebrafish in vivo and impairs glucose-stimulated insulin secretion in mouse and human islets in vitro. NIK activation also perturbs β cell insulin secretion in a diet-induced obesity mouse model. These studies reveal that NIK contributes a central mechanism for β cell failure in obesity., The nuclear factor κB (NF-κB) pathway is a master regulator of inflammatory processes and is implicated in insulin resistance and pancreatic β cell dysfunction in the metabolic syndrome. Whereas canonical NF-κB signaling is well studied, there is little information on the divergent noncanonical NF-κB pathway in the context of pancreatic islet dysfunction. Here, we demonstrate that pharmacological activation of the noncanonical NF-κB–inducing kinase (NIK) disrupts glucose homeostasis in zebrafish in vivo. We identify NIK as a critical negative regulator of β cell function, as pharmacological NIK activation results in impaired glucose-stimulated insulin secretion in mouse and human islets. NIK levels are elevated in pancreatic islets isolated from diet-induced obese (DIO) mice, which exhibit increased processing of noncanonical NF-κB components p100 to p52, and accumulation of RelB. TNF and receptor activator of NF-κB ligand (RANKL), two ligands associated with diabetes, induce NIK in islets. Mice with constitutive β cell–intrinsic NIK activation present impaired insulin secretion with DIO. NIK activation triggers the noncanonical NF-κB transcriptional network to induce genes identified in human type 2 diabetes genome-wide association studies linked to β cell failure. These studies reveal that NIK contributes a central mechanism for β cell failure in diet-induced obesity.

In this study, a critical and novel role for TNF receptor (TNFR) associated factor 2 (TRAF2) is elucidated for peripheral CD8(+) T-cell and NKT-cell homeostasis. Mice deficient in TRAF2 only in their T cells (TRAF2TKO) show ∼40% reduction in effector memory and ∼50% reduction in naive CD8(+) T-cell subsets. IL-15-dependent populations were reduced further, as TRAF2TKO mice displayed a marked ∼70% reduction in central memory CD8(+) CD44(hi) CD122(+) T cells and ∼80% decrease in NKT cells. TRAF2TKO CD8(+) CD44(hi) T cells exhibited impaired dose-dependent proliferation to exogenous IL-15. In contrast, TRAF2TKO CD8(+) T cells proliferated normally to anti-CD3 and TRAF2TKO CD8(+) CD44(hi) T cells exhibited normal proliferation to exogenous IL-2. TRAF2TKO CD8(+) T cells expressed normal levels of IL-15-associated receptors and possessed functional IL-15-mediated STAT5 phosphorylation, however TRAF2 deletion caused increased AKT activation. Loss of CD8(+) CD44(hi) CD122(+) and NKT cells was mechanistically linked to an inability to respond to IL-15. The reduced CD8(+) CD44(hi) CD122(+) T-cell and NKT-cell populations in TRAF2TKO mice were rescued in the presence of high dose IL-15 by IL-15/IL-15Rα complex administration. These studies demonstrate a critical role for TRAF2 in the maintenance of peripheral CD8(+) CD44(hi) CD122(+) T-cell and NKT-cell homeostasis by modulating sensitivity to T-cell intrinsic growth factors such as IL-15.

It is well known that vitamin C is an important enhancer of nonheme iron bioavailability due to its high reducing capacity. Camu-camu, a fruit that grows in the jungle of Peru, contains high amount of vitamin C (2,780 mg per 100 g). In this study, we investigated the effect of camu-camu on nonheme iron bioavailability from two different meals (rice with lentils and wheat flour porridge) using an in vitro Caco-2 cell model. These two meals were treated with three different camu-camu juice concentrations (C 0 = 0 g, C 1 = typical consumption, and C 2 = 3X typical consumption). The results showed that camu-camu reduced rather than enhanced nonheme iron bioavailability. The inhibiting trend was significant (p

Targeting the BAFF/APRIL system has shown to be effective in preventing T-cell dependent autoimmune disease in the NOD mouse, a spontaneous model of type 1 diabetes. In this study we generated BAFF-deficient NOD mice to examine how BAFF availability would influence T-cell responses in vivo and the development of spontaneous diabetes. BAFF-deficient NOD mice which lack mature B cells, were protected from diabetes and showed delayed rejection of an allogeneic islet graft. Diabetes protection correlated with a failure to expand pathogenic IGRP-reactive CD8(+) T cells, which were maintained in the periphery at correspondingly low levels. Adoptive transfer of IGRP-reactive CD8(+) T cells with B cells into BAFF-deficient NOD mice enhanced IGRP-reactive CD8(+) T-cell expansion. Furthermore, when provoked with cyclophosphamide, or transferred to a secondary lymphopenic host, the latent pool of self-reactive T cells resident in BAFF-deficient NOD mice could elicit beta cell destruction. We conclude that lack of BAFF prevents the procurement of B-cell-dependent help necessary for the emergence of destructive diabetes. Indeed, treatment of NOD mice with the BAFF-blocking compound, BR3-Fc, resulted in a delayed onset and reduced incidence of diabetes.

Islet grafts can contribute to their own destruction via the elaboration of proinflammatory genes, many of which are transcriptionally regulated by nuclear factor κ-light-chain-enhancer of activated B-cells (NF-κB). Thus, NF-κB constitutes an enticing gene therapy candidate to improve the success of islet transplantation. To test this hypothesis in vivo, we blocked NF-κB in BALB/c (H2d) to C57/BL6 (H2b) mouse islet allografts by genetically engineering islets to express the NF-κB superrepressor, IκBα. Here we show by microarray and RTqPCR that islets exhibit an intrinsic early immediate proinflammatory response, with the most highly upregulated proinflammatory genes comprising the chemokines Cxcl1, Cxcl2, Cxcl10, and Ccl2; the cytokines Tnf-α and Il-6; and the adhesion molecule Icam1. Overexpression of IκBα inhibited the expression of these genes by 50–95% in islets and MIN6 β-cells in vitro, by inhibiting NF-κB-dependent gene transcription. Histological and RTqPCR analysis at postoperative day (POD) 10 revealed that IκBα-transduced islet allografts exhibited improved islet architecture and strong insulin-labeling with decreased Ccl2 and Il-6 mRNA levels compared to the GFP-transduced control grafts. Despite these protective effects, NF-κB-blocked islet allografts were promptly rejected in our MHC-mismatched mouse model. However, IκBα-expressing grafts did harbor localized “pockets” of Foxp3+ mononuclear cells not evident in the control grafts. This result suggested that the effect of the NF-κB blockade might synergize with regulatory T-cell-sparing rapamycin. Indeed, combining intragraft IκBα expression with low-dose rapamycin increased the mean survival time of islet allografts from 20 to 81 days, with 20% of the grafts surviving for greater than 100 days. In conclusion, rapamycin unmasks the protective potential of intragraft NF-κB blockade, which can, in some cases, permit permanent allograft survival without continuous systemic immunosuppression.

Summary Turner syndrome (TS) is one of the most common sexual chromosome abnormalities and is clearly associated with an increased risk of autoimmune diseases, particularly thyroid disease and coeliac disease (CD). Single-nucleotide polymorphism analyses have been shown to provide correlative evidence that specific genes are associated with autoimmune disease. Our aim was to study the functional polymorphic variants of PTPN22 and ZFAT in relation to thyroid disease and those of MYO9B in relation to CD. A cross-sectional comparative analysis was performed on Mexican mestizo patients with TS and age-matched healthy females. Our data showed that PTPN22 C1858T (considered a risk variant) is not associated with TS (X2 = 3.50, p = .61, and OR = 0.33 [95% CI = 0.10–1.10]). Also, ZFAT was not associated with TS (X2 = 1.2, p = .28, and OR = 1.22 [95% CI = 0.84–1.79]). However, for the first time, rs2305767 MYO9B was revealed to have a strong association with TS (X2 = 58.6, p = .0001, and OR = 10.44 [95% C = 5.51–19.80]), supporting a high level of predisposition to CD among TS patients. This report addresses additional data regarding the polymorphic variants associated with autoimmune disease, one of the most common complications in TS.

Current approaches in tissue engineering are geared toward generating tissue-specific stem cells. Given the complexity and heterogeneity of tissues, this approach has its limitations. An alternate approach is to induce terminally differentiated cells to dedifferentiate into multipotent proliferative cells with the capacity to regenerate all components of a damaged tissue, a phenomenon used by salamanders to regenerate limbs. 5-Azacytidine (AZA) is a nucleoside analog that is used to treat preleukemic and leukemic blood disorders. AZA is also known to induce cell plasticity. We hypothesized that AZA-induced cell plasticity occurs via a transient multipotent cell state and that concomitant exposure to a receptive growth factor might result in the expansion of a plastic and proliferative population of cells. To this end, we treated lineage-committed cells with AZA and screened a number of different growth factors with known activity in mesenchyme-derived tissues. Here, we report that transient treatment with AZA in combination with platelet-derived growth factor–AB converts primary somatic cells into tissue-regenerative multipotent stem (iMS) cells. iMS cells possess a distinct transcriptome, are immunosuppressive, and demonstrate long-term self-renewal, serial clonogenicity, and multigerm layer differentiation potential. Importantly, unlike mesenchymal stem cells, iMS cells contribute directly to in vivo tissue regeneration in a context-dependent manner and, unlike embryonic or pluripotent stem cells, do not form teratomas. Taken together, this vector-free method of generating iMS cells from primary terminally differentiated cells has significant scope for application in tissue regeneration.

The authors had validated a proprietary method, Transcriptome-To-Metabolome™ (TTM™) Biosimulation, for using the transcriptome to determine parameters for kinetic biosimulation of 16 core metabolic pathways. In vivo and in silico evidence confirmed that hippocampal cholesterol metabolism decreases with aging and increases with Alzheimer’s disease (AD). The molecular studies on aging primate and human hippocampus, including AD samples, provided internal validations on the biosimulations, while evidence from the literature, bibliome, provided external validations. This study extends the investigations with the TTM™ Biosimulations into the changes in these 16 metabolic pathways in aging male human hippocampus and for stages of AD. The authors report robust hippocampal hypometabolism in the fifth to tenth decade of life involving glucose and lipid metabolism in male humans. These findings are validated externally from the bibliome. Several changes in AD are demonstrated to be exaggerations or deviations of very late stage changes of normal aging among these pathways.

With the purpose to evaluate the effect in the flowering and growth of the plant of chrysanthemum var. Polaris White several concentrations of salicylic acid (SA) 10-6, 10-8 y 10-10 M and dimethyl sulphoxide (DMSO) 10-4 M were sprinkled on chrysanthemum foliage. The salicylic acid applications to the foliage were made 16 days after the transplant. Four applications were made by dripping with an interval of seven days between each application. A completely randomized design with five treatments and five repetitions was used. The DMSO sprinkled plants grew more (83.6 cm) than the plants sprinkled with 10-6 M (81.0 cm) of AS and surpassed the control plant. The stem diameter of the AS and DMSO sprinkled plants was greater in comparison with the control plant, and the 10-8 M treatment obtained the greatest values (8.9 mm). The salicylic acid (10-6, 10-8 y 10-10 M), and the dimethyl sulfoxide 10-4 M incremented in a significant manner the weight of the foliage and root matter (fresh and dry), the root volume, and the foliar area. The effect of the salicylic acid was notorious in the induction of the blooming treatments: 10-8 y 10-10 M were obtained the blooming at 113 PTD and it also obtained the greatest flower diameter (13.6 and 12.6 cm) respectively. The N, P and K concentrations were different and the treatments with AS and DMSO surpassed the control. The N and K concentrations in the

The leaf and fruit essential oils of Eugenia monteverdensis Barrie (Myrtaceae), from Monteverde, Costa Rica, were isolated by hydrodistillation and analyzed by GC-MS. A total of 18 compounds were identified in the leaf oil and 19 compounds were identified in the fruit oil, accounting for 100 % of the total compositions. The leaf oil of E. monteverdensis was dominated by α-pinene (92.0 %) with a small amount of linalool (2.1 %) and (E)-caryophyllene (1.1 %). The major components in the fruit essential oil were α-pinene (55.1 %) and linalool (22.7 %) with lesser amounts of limonene (7.7 %), (E)-caryophyllene (4.7 %), β-pinene (2.3 %), and α-terpineol (2.0 %). The essential oils were screened for cytotoxic and antibacterial activity, but were found to be inactive.

Pancreatic ductal adenocarcinoma (PDA) develops predominantly through pancreatic intraepithelial neoplasia (PanIN) and intraductal papillary mucinous neoplasm (IPMN) precursor lesions. Pancreatic acinar cells are reprogrammed to a “ductal-like” state during PanIN-PDA formation. Here, we demonstrate a parallel mechanism operative in mature duct cells during which functional cells undergo “ductal retrogression” to form IPMN-PDA. We further identify critical antagonistic roles for Brahma-related gene 1 (Brg1), a catalytic subunit of the SWI/SNF complexes, during IPMN-PDA development. In mature duct cells, Brg1 inhibits the dedifferentiation that precedes neoplastic transformation, thus attenuating tumor initiation. In contrast, Brg1 promotes tumorigenesis in full-blown PDA by supporting a mesenchymal-like transcriptional landscape. We further show that JQ1, a drug that is currently being tested in clinical trials for hematological malignancies, impairs PDA tumorigenesis by both mimicking some and inhibiting other Brg1-mediated functions. In summary, our study demonstrates the context-dependent roles of Brg1 and points to potential therapeutic treatment options based on epigenetic regulation in PDA.

Se probaron varias concentraciones (1,000, 2,000, 3,000, 4,000 y 5,000 mg·litro-1) de daminozide sobre plantas de crisantemo cv. Polaris White, en maceta y se midieron el incremento del diámetro del tallo, altura de la planta, número de nudos, longitud de entrenudos, diámetro de la inflorescencia, biomasa y área foliar para comparar su efecto contra un testigo. La concentración de 1,000 mg·litro-1 provocó que los tallos incrementaran su diámetro 10.08 % en comparación con el testigo en tanto que con 4,000 mg·litro-1 el diámetro del tallo se redujo 8 %. Los entrenudos disminuyeron su longitud hasta 76 % con 4,000 mg·litro-1 en comparación con el testigo. Concentraciones iguales o mayores a 2,000 mg·litro-1 redujeron hasta en 34.3 % la altura de la planta. El daminozide favoreció el crecimiento de las inflorescencias incrementando su diámetro 31.22 % en promedio

Aqueous solutions of 1.0 μM to 0.1 nM concentrations of salicylic acid (SA) were sprayed on African violet grown under greenhouse conditions to estimate its effect on the flower expression of the plant. These solutions were sprayed on the shoots of the plant on three occasions, 21, 28 and 35 days after being potted. Salicyliclate at 0.1 nM increased the number of leaves from 16 to 19, the number of flower primordia from 8 to 14, the rosette diameter from 130 to 177 mm in comparison with the control. The same concentration induce flowering at 74 days of plant age whilest the control plants last 89 days to flower.

Beet necrotic yellow vein virus (BNYVV) causes rhizomania of sugar beet (Beta vulgaris), which is characterized by stunting, leaf necrosis, constriction of the taproot, and extensive lateral- and feeder-root proliferation. Beet soilborne mosaic virus (BSBMV) causes similar but typically less severe symptoms than those of BNYVV. Both viruses are widely distributed in sugar beet-growing regions of the United States. Both viruses are vectored by the soilborne plasmodiophorid Polymyxa betae Keskin and are very similar in morphology and biology, sharing many characteristics in common. In 1999, soil samples were collected from sugar beet fields in Colorado, Minnesota, North Dakota, and Texas to determine the spatial association and covariation of the viruses in sugar beet fields. In 2000, additional samples were collected from fields in Minnesota and North Dakota. Over the 2-year period, soil samples were collected from 11 fields in various quadrat sizes. The viruses were assayed by growing sugar beet (cv. Beta 1395) in the soil samples and their incidence was determined using the double-antibody sandwich enzyme-linked immunosorbent assay. Both viruses were detected in samples from all fields but were in greater frequencies singly than in association. Association of the two viruses (where both viruses were detected in the same sample or bait plant) varied among fields, ranging from 1 to 42%. Geostatistical analysis revealed that both viruses, in large part, exhibited similar spatial patterns. In all but two fields, there was no spatial dependence among the sampling locations at sampled grid sizes. Their semivariances were constant at all separation distances in all directions indicating random spatial patterns. Overall, the spatial pattern of BNYVV appeared to be a little more structured than that of BSBMV. Even though both viruses are transmitted by the same vector and also exhibited similar distribution patterns, the incidence of one virus may not be estimated from that of the other due to lack of strong association and spatial dependence. However, similarity in spatial patterns of the two suggests that a similar sampling method can be employed for both viruses.

Mechanical strain is an important determinant of bone mass and architecture, and the aim of this investigation was to further understand the role of the cell–cell signaling molecules, IL-1β, TNF-α, and IL-6 in the mechanobiology of bone. Mouse calvarial osteoblasts in monolayer culture were subjected to a cyclic out-of-plane deformation of 0.69% for 6 s, every 90 s for 2–48 h, and the levels of each cytokine plus their downstream targets RANKL and OPG measured in culture supernatants by ELISAs. Mouse osteoblasts constitutively synthesized IL-1β, TNF-α, and IL-6, the production of which was significantly up-regulated in all three by cyclic mechanical strain. RANKL and OPG were also constitutively synthesized; mechanical deformation however, resulted in a down-regulation of RANKL and an up-regulation OPG synthesis. We next tested whether the immunoreactive RANKL and OPG were biologically active in an isolated osteoclast resorption pit assay – this showed that culture supernatants from mechanically deformed cells significantly inhibited osteoclast-mediated resorptive activity across the 48 h time-course. These findings are counterintuitive, because IL-1β, TNF-α, and IL-6 have well-established reputations as bone resorptive agents. Nevertheless, they are pleiotropic molecules with multiple biological activities, underlining the complexity of the biological response of osteoblasts to mechanical deformation, and the need to understand cell–cell signaling in terms of cytokine networks. It is also important to recognize that osteoblasts cultured in vitro are deprived of the mechanical stimuli to which they are exposed in vivo – in other words, the cells are in a physiological default state that in the intact skeleton leads to decreased bone strains below the critical threshold required to maintain normal bone structure.

AIM: To describe the variation that divalent metal transporter 1 (DMT1) shows in patients after Roux-en-Y gastric bypass (RYGB) surgery. METHODS: Prospective and analytical study of DMT1 level at the brush border of proximal jejunum in patients having undergone RYGB surgery. The mucosa of proximal jejunum forming the gastrojejunal anastomosis was biopsied during surgery and after 6 mo later with an endoscopic biopsy. All the patients received precise instructions regarding feeding and nutritional supplementation. Both samples were processed at the same time by immunohistochemistry and western blot. Samples were analysed by a pathologist. For statistical analysis, the χ2 and Wilcoxon tests were used. RESULTS: Sixteen patients were recruited, 13 of whom completed the study. Twelve were women. Average age and body mass index (BMI) were 44.1 and 40.4, respectively. Both body weight and BMI decreased significantly during the study period, with an average percent excess weight loss (%EWL) of 60% ± 13.3% and an average percent excess BMI loss (%EBMIL) of 79.6% ± 21.6%. Only two patients presented with mild anaemia 6 mo after surgery, but their ferritin levels stayed within normal ranges. Staining for DMT1 showed a significant increase in the cytoplasm of enterocytes located at the tips of the villi (χ2 = 6.03; P = 0.049). Nevertheless, the total quantity of DMT1 decreased significantly (Z = 2.04; P = 0.04). Associated with these results, we observed a significant increase in goblet cells in the villi 6 mo postoperatively (Z = -2.47; P = 0.013). CONCLUSION: Six months after RYGB surgery, patients exhibit an increase in DMT1 expression in the enterocytes of the tips of the villi at the proximal jejunum.

Background Arthropods are the most diverse animal phylum, but their genomic resources are relatively few. While the genome of the branchiopod Daphnia pulex is now available, no other large-scale crustacean genomic resources are available for comparison. In particular, genomic resources are lacking for the most tractable laboratory model of crustacean development, the amphipod Parhyale hawaiensis. Insight into shared and divergent characters of crustacean genomes will facilitate interpretation of future developmental, biomedical, and ecological research using crustacean models. Results To generate a transcriptome enriched for maternally provided and zygotically transcribed developmental genes, we created cDNA from ovaries and embryos of P. hawaiensis. Using 454 pyrosequencing, we sequenced over 1.1 billion bases of this cDNA, and assembled them de novo to create, to our knowledge, the second largest crustacean genomic resource to date. We found an unusually high proportion of C2H2 zinc finger-containing transcripts, as has also been reported for the genome of the pea aphid Acyrthosiphon pisum. Consistent with previous reports, we detected trans-spliced transcripts, but found that they did not noticeably impact transcriptome assembly. Our assembly products yielded 19,067 unique BLAST hits against nr (E-value cutoff e-10). These included over 400 predicted transcripts with significant similarity to D. pulex sequences but not to sequences of any other animal. Annotation of several hundred genes revealed P. hawaiensis homologues of genes involved in development, gametogenesis, and a majority of the members of six major conserved metazoan signaling pathways. Conclusions The amphipod P. hawaiensis has higher transcript complexity than known insect transcriptomes, and trans-splicing does not appear to be a major contributor to this complexity. We discuss the importance of a reliable comparative genomic framework within which to consider findings from new crustacean models such as D. pulex and P. hawaiensis, as well as the need for development of further substantial crustacean genomic resources.

OBJECTIVETumor necrosis factor ligand family members B-cell–activating factor (BAFF) and a proliferation-inducing ligand (APRIL) can exert powerful effects on B-cell activation and development, type 1 T-helper cell (Th1) immune responses, and autoimmunity. We examined the effect of blocking BAFF and APRIL on the development of autoimmune diabetes.RESEARCH DESIGN AND METHODSFemale NOD mice were administered B-cell maturation antigen (BCMA)-Fc from 9 to 15 weeks of age. Diabetes incidence, islet pathology, and T- and B-cell populations were examined.RESULTSBCMA-Fc treatment reduced the severity of insulitis and prevented diabetes development in NOD mice. BCMA-Fc–treated mice showed reduced follicular, marginal-zone, and T2MZ B-cells. B-cell reduction was accompanied by decreased frequencies of pathogenic CD4+CD40+ T-cells and reduced Th1 cytokines IL-7, IL-15, and IL-17. Thus, T-cell activation was blunted with reduced B-cells. However, BCMA-Fc–treated mice still harbored detectable diabetogenic T-cells, suggesting that regulatory mechanisms contributed to diabetes prevention. Indeed, BCMA-Fc–treated mice accumulated increased CD4+CD25+ regulatory T-cells (Tregs) with age. CD4+CD25+ cells were essential for maintaining euglycemia because their depletion abrogated BCMA-Fc–mediated protection. BCMA-Fc did not directly affect Treg homeostasis given that CD4+CD25+Foxp3+ T-cells did not express TACI or BR3 receptors and that CD4+CD25+Foxp3+ T-cell frequencies were equivalent in wild-type, BAFF−/−, TACI−/−, BCMA−/−, and BR3−/− mice. Rather, B-cell depletion resulted in CD4+CD25+ T-cell–mediated protection from diabetes because anti-CD25 monoclonal antibody treatment precipitated diabetes in both diabetes-resistant NOD.μMT−/− and BCMA-Fc–treated mice.CONCLUSIONSBAFF/APRIL blockade prevents diabetes. BCMA-Fc reduces B-cells, subsequently blunting autoimmune activity and allowing endogenous regulatory mechanisms to preserve a prehyperglycemic state.

Serum from patients with paracoccidioidomycosis (PARA) did not block digestive abilities of neutrophils (PMNs) from healthy individuals against Paracoccidioides brasiliensis. Conversely, serum from healthy donors did not enhance digestive capacities of PMNs from patients with PARA vis á vis the causative organism. We conclude that the specific digestive defect present in PMNs from patients with PARA is not mediated by serum factors.

Purpose: Vascular endothelial growth factor (VEGF) isoforms play different roles in the temporal sprouting of endothelial-lined vessels in a nude mouse peritoneal model as cells respond to nontreated control and gas-plasma-treated bioresorbable poly-d,l-lactide acid 3D scaffolds with human aortic endothelial cells (HAEC). Methods and materials: Nude mice peritoneums were incubated with HAEC (CW=control; TW=gas-plasma treated) or polymer scaffolds (C p =control; T p =treated) for 12, 24 and 72 days. Cytoplasmic and nuclear protein fractions were isolated using NER, electrophoresized using NuPAGE–MES and analyzed by WesternBreeze Chemiluminescent. Results: Prominent VEGF bands included 28, 45 and 62 kDa; 52-kDa VEGF observed in cytoplasmic TW fractions contributed about 18.6% at 12 days, 20.0% at 24 days and 13.1% at 72 days of the total VEGF signal. Yet, it was only noted in CW at 72 days where it accounted for 6.9%. A unique 32-kDa band appeared in both C p (24.6%) and T p (18.3%). Significant differences between band densities occurred for cytoplasmic nuclear CW24–TW24 ( P =.022), CW72–TW72 ( P =.011) and, also, cytoplasmic C p 24–T p 24 ( P =.038). Conclusions: The temporal and spatial organization of the TW isoforms results in more angiogenesis.

The gene coding for the immunity protein ( mceB ) and the structural gene of microcin E492 ( mceA ), a low-molecular-weight channel-forming bacteriocin produced by a strain of Klebsiella pneumoniae , have been characterized. The microcin gene codes for a precursor protein of either 99 or 103 amino acids. Protein sequencing of the N-terminal region of microcin E492 unequivocally identified this gene as the microcin structural gene and indicated that this microcin is synthesized as a precursor protein that is cleaved at either amino acid 15 or 19, at a site resembling the double-glycine motif. The gene encoding the 95-amino-acid immunity protein ( mceB ) was identified by cloning the DNA segment that encodes only this polypeptide into an expression vector and demonstrating the acquisition of immunity to microcin E492. As expected, the immunity protein was found to be associated with the inner membrane. Analysis of the DNA sequence indicates that these genes belong to the same family as microcin 24, and they do not share structural motifs with any other known channel-forming bacteriocin. The organization of the microcin- and immunity protein-encoding genes suggests that they are coordinately expressed.

Microcin E492 is a polypeptide antibiotic that is produced and excreted by Klebsiella pneumoniae RYC492. The genetic determinants for microcin synthesis and immunity were cloned in Escherichia coli VCS257 into the cosmid vector pHC79, starting from total DNA of K. pneumoniae RYC492. The microcin E492 expressed in E. coli had the same properties as that of K. pneumoniae, i.e., the same molecular weight, the ability to form ionic channels in planar phospholipid bilayers, and essentially identical biological properties. Microcin E492 expression in E. coli, like that in K. pneumoniae, was mainly in the exponential phase of growth, declining in the stationary phase. The immunity determinant was subcloned into the same vector, and its expression was found to disappear in the stationary phase. This phenomenon is not dependent on rpoS, the stationary-phase sigma factor.

Amplification by the Polymerase Chain Reaction (PCR) and subsequent electrophoresis of the amplified products have become a useful approach for typing variable number of tandem repeats (VNTR) loci. Currently there is an increasingly number of data on VNTR and especially on the STR loci (i.e., Edwards et al, 1992), regarding their allele frequencies and genotype distribution in various population.

The essential oil from the aerial parts of Blumea perrottetiana was obtained by hydrodistillation and analyzed by GC-MS. The volatile oil is dominated by 2,5-dimethoxy- p-cymene (30.0%) and 1,8-cineole (11.0%) with lesser amounts of sabinene (8.1%), δ-cadinene (5.3%) and ( E)-caryophyllene (3.9%). The essential oil demonstrated notable insecticidal activity against the red flour beetle, Tribolium castaneum, consistent with traditional uses of the plant as an insecticide and anthelmintic.

The seasonal variation in the chemical composition of the leaf essential oil of Liriodendron tulipifera has been analyzed by GC-MS. Two individual trees were sampled five times during the course of the growing season. Twenty components were identified in the leaf oils, which were dominated by sesquiterpene hydrocarbons, principally germacrene D and β-elemene, in the early part of the season (42-44% and 18-23%, respectively,) but monoterpene hydrocarbons, largely ( Z)-β-ocimene, dominated the later season leaf oils (40-60%). The leaf oils exhibited in-vitro antibacterial activity against Bacillus cereus and Staphylococcus aureus as well as cytotoxic activity on MDA-MB-231 and Hs 578T human breast tumor cells.

Sporotrichosis is a mycosis which in our country has been described as only producing cutaneous and subcutaneous infections; in other countries it has been reported as causing pulmonary, bone, joint and meningeal infections. Possibly the systemic form also occurs in Venezuela since, along with the causative agent, all other factors considered as predisposing to systemic infection exist here. Through immunodiffusion (ID) and immunoelectrophoresis (IEP) and using a filamentous form metabolic antigen from Sporothrix schenckii, we were able to demonstrate precipitating antibodies against this fungus both in patients with cutaneous sporotrichosis, diagnosed by culture of the infecting agent, and in patients with pulmonary lesions. The IEP test showed the presence of an anodic arc which we have called the 'S' arc, which could be one of the specific antigens of S. schenckii.

Inhibition of paraoxon hydrolase (paraoxonase) activity by 'in vitro' exposure to EDTA, Mg2+, Co2+, Ba2+, La3+, Zn2+, Cu2+, Hg2+, p-hydroxymercuribenzoate (p-OH-MB) and phenyl mercuric acetate (PMA) was investigated in human liver microsomes. Enzyme activity was totally inhibited by 1 mM EDTA in a time-dependent manner, in contrast to previous data obtained in rat liver where an EDTA-resistant fraction was detected. The possible influence of postmortem changes in these results was checked in a parallel experiment using rat livers with different postmortem intervals. From our results the existence in human liver of an EDTA-resistant fraction cannot be discarded. Ba, La and PMA showed immediate inhibition. By contrast the other compounds tested were time-dependent inhibitors. Ba and Zn showed the highest IC50 values. Cu and mercurials (Hg, p-OH-MB, PMA) were the most potent inhibitors of human liver paraoxonase. Kinetic analysis (Lineweaver-Burk and Dixon plots) indicated that different inhibitors exhibit different inhibition patterns: competitive (EDTA, Ba, La, Cu, p-OH-MB and PMA), non competitive (Zn) and mixed (Hg). The pretreatment of sample with dithiothreitol (DTT) protects against the inhibitory effect of mercurials. Furthermore after inhibition by mercurials the activity was restored by DTT. These results confirmed the essential role of the -SH groups to maintain the catalytic activity of paraoxonase and suggest the existence of two types of -SH groups that could differ in their localization.

The effects of three different enzyme-inducing agents (phenobarbital, 3-methylcholanthrene and rifampicin) on plasma and liver microsomal fraction paraoxonase and arylesterase were studied in rats. Although phenobarbital and 3-methylcholanthrene each increased the esterase activities in microsomal fraction, only 3-methylcholanthrene was capable to increase them in plasma. By contrast, the administration of rifampicin decreased both enzyme activities in liver and plasma. The results indicate that at least there exists two esterase activities in rat liver microsomes which hydrolyse both paraoxon and phenylacetate, but only one of them is released into the blood.