Loss of Anticodon Wobble Uridine Modifications Affects tRNALys Function and Protein Levels in Saccharomyces cerevisiae

In eukaryotes, wobble uridines in the anticodons of tRNA^Lys _UUU, tRNA^Glu _UUC and tRNA^Gln _UUG are modified to 5-methoxy-carbonyl-methyl-2-thio-uridine (mcm⁵s²U). While mutations in subunits of the Elongator complex (Elp1-Elp6), which disable mcm⁵ side chain formation, or removal of components of the thiolation pathway (Ncs2/Ncs6, Urm1, Uba4) are individually tolerated, the combination of both modification defects has been reported to have lethal effects on Saccharomyces cerevisiae. Contrary to such absolute requirement of mcm⁵s²U for viability, we demonstrate here that in the S. cerevisiae S288C-derived background, both pathways can be simultaneously inactivated, resulting in combined loss of tRNA anticodon modifications (mcm⁵U and s²U) without a lethal effect. However, an elp3 disruption strain displays synthetic sick interaction and synergistic temperature sensitivity when combined with either uba4 or urm1 mutations, suggesting major translational defects in the absence of mcm⁵s²U modifications. Consistent with this notion, we find cellular protein levels drastically decreased in an elp3uba4 double mutant and show that this effect as well as growth phenotypes can be partially rescued by excess of tRNA^Lys _UUU. These results may indicate a global translational or protein homeostasis defect in cells simultaneously lacking mcm⁵ and s² wobble uridine modification that could account for growth impairment and mainly originates from tRNA^Lys _UUU hypomodification and malfunction.     

Low Abdominal NIRS Values and Elevated Plasma Intestinal Fatty Acid-Binding Protein in a Premature Piglet Model of Necrotizing Enterocolitis

To identify early markers of necrotizing enterocolitis (NEC), we hypothesized that continuous abdominal near-infrared spectroscopy (A-NIRS) measurement of splanchnic tissue oxygen saturation and intermittent plasma intestinal fatty-acid binding protein (pI-FABP) measured every 6 hours can detect NEC prior to onset of clinical symptoms. Premature piglets received parenteral nutrition for 48-hours after delivery, followed by enteral feeds every three hours until death or euthanasia at 96-hours. Continuous A-NIRS, systemic oxygen saturation (SpO₂), and heart rate were measured while monitoring for clinical signs of NEC. Blood samples obtained at 6-hour intervals were used to determine pI-FABP levels by ELISA. Piglets were classified as fulminant-NEC (f-NEC), non-fulminant-NEC (nf-NEC) and No-NEC according to severity of clinical and histologic features. Of 38 piglets, 37% (n=14) developed nf-NEC, 18% (n=7) developed f-NEC and 45% (n=17) had No-NEC. There were significant differences in baseline heart rate (p=0.008), SpO₂ (p<0.001) and A-NIRS (p<0.001) among the three groups. A-NIRS values of NEC piglets remained lower throughout the study with mean for f-NEC of 69±3.8%, 71.9±4.04% for nf-NEC, and 78.4±1.8% for No-NEC piglets (p<0.001). A-NIRS <75% predicted NEC with 97% sensitivity and 97% specificity. NEC piglets demonstrated greater variability from baseline in A-NIRS than healthy piglets (10.1% vs. 6.3%; p=0.04). Mean pI-FABP levels were higher in animals that developed NEC compared to No-NEC piglets (0.66 vs. 0.09 ng/mL;p<0.001). In f-NEC piglets, pI-FABP increased precipitously after feeds (0.04 to 1.87 ng/mL;p<0.001). pI-FABP levels increased in parallel with disease progression and a value >0.25ng/mL identified animals with NEC (68% sensitivity and 90% specificity). NIRS is a real-time, non-invasive tool that can serve as a diagnostic modality for NEC. In premature piglets, low A-NIRS in the early neonatal period and increased variability during initial feeds are highly predictive of NEC, which is then confirmed by rising plasma I-FABP levels. These modalities may help identify neonates with NEC prior to clinical manifestations of disease.     

Psychosocial Care Needs of Melanoma Survivors: Are They Being Met?

Patients who have survived malignant melanoma for more than five years may lack the opportunity to talk about their burden. As a consequence their psychosocial care needs remain undetected and available supportive interventions may not be utilised. Therefore, the psychosocial burden of this patient group needs to be assessed using specific screening instruments. The aim of this study was to investigate the psychosocial burden of long-term melanoma survivors, their psychosocial care needs and the determinants of these needs. We wanted to find out if the use of professional support corresponds to the care needs defined by experts. Using the cancer registry of Rhineland-Palatinate, melanoma patients diagnosed at least 5 years before the survey were contacted by physicians. N = 689 former patients completed the Hornheide Questionnaire (short form HQ-S) to identify psychosocial support need (scale cut off ≥ 16 or item-based cut-off score) and the potential psychosocial determinants of these needs. Additionally, they were asked about their utilisation of the professional support system. More than one third (36%) of them was in need for professional psychosocial support. The highest burden scores concerned worry about tumour progression. Younger age (< 50), higher general fatigue, higher symptom burden, lower general health, negative social interactions and unfulfilled information needs were significant predictors of the need for psychosocial intervention. Related to the percentage of survivors identified as ‘in need’, the professional support system was underused. Further studies should investigate whether using the HQ-S to routinely identify burdened melanoma patients could lead to better fulfilment of their intervention needs, ultimately enhancing health-related quality of life.     

Pre-Training Reversible Inactivation of the Basal Amygdala (BA) Disrupts Contextual,but Not Auditory,Fear Conditioning,in Rats

The basolateral amygdala complex (BLA), including the lateral (LA), basal (BA) and accessory basal (AB) nuclei, is involved in acquisition of contextual and auditory fear conditioning. The BA is one of the main targets for hippocampal information, a brain structure critical for contextual learning, which integrates several discrete stimuli into a single configural representation. Congruent with the hodology, selective neurotoxic damage to the BA results in impairments in contextual, but not auditory, fear conditioning, similarly to the behavioral impairments found after hippocampal damage. This study evaluated the effects of muscimol-induced reversible inactivation of the BA during a simultaneous contextual and auditory fear conditioning training on later fear responses to both the context and the tone, tested separately, without muscimol administration. As compared to control rats micro-infused with vehicle, subjects micro-infused with muscimol before training exhibited, during testing without muscimol, significant reduction of freezing responses to the conditioned context, but not to the conditioned tone. Therefore, reversible inactivation of the BA during training impaired contextual, but not auditory fear conditioning, thus confirming and extending similar behavioral observations following selective neurotoxic damage to the BA and, in addition, revealing that this effect is not related to the lack of a functional BA during testing.     

IL22/IL-22R Pathway Induces Cell Survival in Human Glioblastoma Cells

Interleukin-22 (IL-22) is a member of the IL-10 cytokine family that binds to a heterodimeric receptor consisting of IL-22 receptor 1 (IL-22R1) and IL-10R2. IL-22R expression was initially characterized on epithelial cells, and plays an essential role in a number of inflammatory diseases. Recently, a functional receptor was detected on cancer cells such as hepatocarcinoma and lung carcinoma, but its presence was not reported in glioblastoma (GBM). Two GBM cell lines and 10 primary cell lines established from patients undergoing surgery for malignant GBM were used to investigate the expression of IL-22 and IL-22R by using quantitative RT-PCR, western blotting and confocal microscopy studies. The role of IL-22 in proliferation and survival of GBM cell lines was investigated in vitro by BrdU and ELISA cell death assays. We report herein that the two subunits of the IL-22R complex are expressed on human GBM cells. Their activation, depending on exogenous IL-22, induced antiapoptotic effect and cell proliferation. IL-22 treatment of GBM cells resulted in increased levels of phosphorylated Akt, STAT3 signaling protein and its downstream antiapoptotic protein Bcl-xL and decreased level of phosphorylated ERK1/2. In addition, IL-22R subunits were expressed in all the 10 tested primary cell lines established from GBM tumors. Our results showed that IL-22R is expressed on GBM established and primary cell lines. Depending on STAT3, ERK1/2 and PI3K/Akt pathways, IL-22 induced GBM cell survival. These data are consistent with a potential role of IL-22R in tumorigenesis of GBM. Since endogenous IL-22 was not detected in all studied GBM cells, we hypothesize that IL-22R could be activated by immune microenvironmental IL-22 producing cells.     

Retinal Glia Promote Dorsal Root Ganglion Axon Regeneration

Axon regeneration in the adult central nervous system (CNS) is limited by several factors including a lack of neurotrophic support. Recent studies have shown that glia from the adult rat CNS, specifically retinal astrocytes and Müller glia, can promote regeneration of retinal ganglion cell axons. In the present study we investigated whether retinal glia also exert a growth promoting effect outside the visual system. We found that retinal glial conditioned medium significantly enhanced neurite growth and branching of adult rat dorsal root ganglion neurons (DRG) in culture. Furthermore, transplantation of retinal glia significantly enhanced regeneration of DRG axons past the dorsal root entry zone after root crush in adult rats. To identify the factors that mediate the growth promoting effects of retinal glia, mass spectrometric analysis of retinal glial conditioned medium was performed. Apolipoprotein E and secreted protein acidic and rich in cysteine (SPARC) were found to be present in high abundance, a finding further confirmed by western blotting. Inhibition of Apolipoprotein E and SPARC significantly reduced the neuritogenic effects of retinal glial conditioned medium on DRG in culture, suggesting that Apolipoprotein E and SPARC are the major mediators of this regenerative response.     

Muscle Histidine-Containing Dipeptides Are Elevated by Glucose Intolerance in Both Rodents and Men

Objective

Muscle carnosine and its methylated form anserine are histidine-containing dipeptides. Both dipeptides have the ability to quench reactive carbonyl species and previous studies have shown that endogenous tissue levels are decreased in chronic diseases, such as diabetes.

Design and Methods

Rodent study: Skeletal muscles of rats and mice were collected from 4 different diet-intervention studies, aiming to induce various degrees of glucose intolerance: 45% high-fat feeding (male rats), 60% high-fat feeding (male rats), cafeteria feeding (male rats), 70% high-fat feeding (female mice). Body weight, glucose-tolerance and muscle histidine-containing dipeptides were assessed. Human study: Muscle biopsies were taken from m. vastus lateralis in 35 males (9 lean, 8 obese, 9 prediabetic and 9 newly diagnosed type 2 diabetic patients) and muscle carnosine and gene expression of muscle fiber type markers were measured.

Results

Diet interventions in rodents (cafeteria and 70% high-fat feeding) induced increases in body weight, glucose intolerance and levels of histidine-containing dipeptides in muscle. In humans, obese, prediabetic and diabetic men had increased muscle carnosine content compared to the lean (+21% (p>0.1), +30% (p<0.05) and +39% (p<0.05), respectively). The gene expression of fast-oxidative type 2A myosin heavy chain was increased in the prediabetic (1.8-fold, p<0.05) and tended to increase in the diabetic men (1.6-fold, p = 0.07), compared to healthy lean subjects.

Conclusion

Muscle histidine-containing dipeptides increases with progressive glucose intolerance, in male individuals (cross-sectional). In addition, high-fat diet-induced glucose intolerance was associated with increased muscle histidine-containing dipeptides in female mice (interventional). Increased muscle carnosine content might reflect fiber type composition and/or act as a compensatory mechanism aimed at preventing cell damage in states of impaired glucose tolerance.