Relationship Between Dual-Energy X-Ray Absorptiometry, Ultrasonography, and Anthropometry Methods to Estimate Muscle Mass and Muscle Quality in Older Adults.

Decreased muscle quality (MQ) may explain functional capacity impairments during aging. Thus, it is essential to verify the interaction between MQ and functional capacity in older adults. We investigated the relationship between MQ and functional capacity in older adults (n = 34; 66.3 ± 4.6 year). MQ was estimated by maximum strength of knee extensors normalized to thigh muscle mass. Maximum strength was assessed on an isokinetic dynamometer (peak torque), while dual-energy X-ray absorptiometry (DXA), ultrasonography, and anthropometry were used to determine thigh muscle mass. Functional capacity was verified by 30-s sit to stand and timed up and go tests. Significant correlations were found between MQ assessed by DXA with 30-s sit to stand (r = .35; p < .05) and timed up and go (r = -.47; p < .05), and MQ assessed by anthropometry with timed up and go (r = -.41; p < .05), but not between MQ assessed by ultrasonography with functional capacity (p > .05). No significant relationship between muscle mass with functional capacity was observed. Thus, MQ assessed by DXA and MQ assessed by anthropometry may partially explain functional capacity in older adults. Interestingly, muscle mass alone did not explain performance in functional tests in this population.

Can supplemental protein to low-protein containing meals superimpose on resistance-training muscle adaptations in older adults? A randomized clinical trial.

PURPOSE: To evaluate the effects of supplementing protein to the low-protein containing meals on selected parameters of muscle mass, strength, and functional capacity in older individuals undergoing resistance training. METHODS: Thirty-one male and female older individuals (60 to 80 years) were randomized into either a whey protein/WP (n = 15, 20 g at breakfast + 20 g at dinner) or placebo-maltodextrin/PL (n = 16, 20 g at breakfast + 20 g at dinner) group. Both groups underwent a supervised, progressive resistance training (2×/week) program for 12 weeks. Maximal isometric voluntary contraction (MIVC) of knee extensors, muscle thickness (MT) of knee extensors and elbow flexors, rectus femoris muscle quality (MQ), body composition (as measured by DXA) and functional capacity [as measured by 30-s sit-to-stand (30ss) and timed-up-and-go tests (TUG)] were evaluated at baseline and after the 12-week intervention. RESULTS: Knee extensor MIVC (WP ∆ = 11.9 ± 11.4% and PL ∆ = 12.9 ± 9.9%) was significantly increased over time, with no between-group differences (all p < 0.05 for main effect of time). Upper- and lower-limb MT were significantly increased over time, with no effect of supplementation (WP: ∆ = 7.0 ± 7.3%, PL: ∆ = 9.5 ± 10.3%; and WP: ∆ = 4.5 ± 5.8%, PL: ∆ = 14.7 ± 28.9%, respectively; all p = 0.001 for main effect of time, respectively). Total and upper-limb lean mass were significantly increased, irrespective of the dietary intervention (WP: ∆ = 0.2 ± 6.3%, PL: ∆ = 1.8 ± 2.9%; and WP: ∆ = 0.10 ± 0.03%, PL: ∆ = 0.15 ± 0.02%, respectively; all p < 0.05 for main effect of time). Main effects of time (all p < 0.05) were also found for 30SS and TUG (fast and usual speeds) (WP: ∆ = 18.2 ± 34.4%, PL: ∆ = 10.4 ± 16.9%; WP: ∆ = 5.4 ± 6.7%, PL: ∆ = 0.7 ± 6.0% and WP: ∆ = 3.3 ± 6.1%, PL: ∆ = 2.3 ± 5.2%, respectively). CONCLUSION: Supplementing additional whey protein to the lowest-protein containing meals (i.e., ~20 g at breakfast and ~20 g at dinner, daily) did not further augment resistance training-induced neuromuscular adaptations (i.e. muscle strength and mass) in healthy older individuals.

Identification and characterization of short leader and trailer RNAs synthesized by the Ebola virus RNA polymerase.

Transcription of non-segmented negative sense (NNS) RNA viruses follows a stop-start mechanism and is thought to be initiated at the genome's very 3'-end. The synthesis of short abortive leader transcripts (leaderRNAs) has been linked to transcription initiation for some NNS viruses. Here, we identified the synthesis of abortive leaderRNAs (as well as trailer RNAs) that are specifically initiated opposite to (anti)genome nt 2; leaderRNAs are predominantly terminated in the region of nt ~ 60-80. LeaderRNA synthesis requires hexamer phasing in the 3'-leader promoter. We determined a steady-state NP mRNA:leaderRNA ratio of ~10 to 30-fold at 48 h after Ebola virus (EBOV) infection, and this ratio was higher (70 to 190-fold) for minigenome-transfected cells. LeaderRNA initiation at nt 2 and the range of termination sites were not affected by structure and length variation between promoter elements 1 and 2, nor the presence or absence of VP30. Synthesis of leaderRNA is suppressed in the presence of VP30 and termination of leaderRNA is not mediated by cryptic gene end (GE) signals in the 3'-leader promoter. We further found different genomic 3'-end nucleotide requirements for transcription versus replication, suggesting that promoter recognition is different in the replication and transcription mode of the EBOV polymerase. We further provide evidence arguing against a potential role of EBOV leaderRNAs as effector molecules in innate immunity. Taken together, our findings are consistent with a model according to which leaderRNAs are abortive replicative RNAs whose synthesis is not linked to transcription initiation. Rather, replication and transcription complexes are proposed to independently initiate RNA synthesis at separate sites in the 3'-leader promoter, i.e., at the second nucleotide of the genome 3'-end and at the more internally positioned transcription start site preceding the first gene, respectively, as reported for Vesicular stomatitis virus.

RNA secondary structure at the transcription start site influences EBOV transcription initiation and replication in a length- and stability-dependent manner.

Ebola virus (EBOV) RNA has the potential to form hairpin structures at the transcription start sequence (TSS) and reinitiation sites of internal genes, both on the genomic and antigenomic/mRNA level. Hairpin formation involving the TSS and the spacer sequence between promotor elements (PE) 1 and 2 was suggested to regulate viral transcription. Here, we provide evidence that such RNA structures form during RNA synthesis by the viral polymerase and affect its activity. This was analysed using monocistronic minigenomes carrying hairpin structure variants in the TSS-spacer region that differ in length and stability. Transcription and replication were measured via reporter activity and by qRT-PCR quantification of the distinct viral RNA species. We demonstrate that viral RNA synthesis is remarkably tolerant to spacer extensions of up to ~54 nt, but declines beyond this length limit (~25% residual activity for a 66-nt extension). Minor incremental stabilizations of hairpin structures in the TSS-spacer region and on the mRNA/antigenomic level were found to rapidly abolish viral polymerase activity, which may be exploited for antisense strategies to inhibit viral RNA synthesis. Finally, balanced viral transcription and replication can still occur when any RNA structure formation potential at the TSS is eliminated, provided that hexamer phasing in the promoter region is maintained. Altogether, the findings deepen and refine our insight into structure and length constraints within the EBOV transcription and replication promoter and suggest a remarkable flexibility of the viral polymerase in recognition of PE1 and PE2.

Regulation of VP30-Dependent Transcription by RNA Sequence and Structure in the Genomic Ebola Virus Promoter.

Viral transcription and replication of Ebola virus (EBOV) is balanced by transcription factor VP30, an RNA binding protein. An RNA hairpin at the transcription start site (TSS) of the first gene (NP hairpin) in the 3'-leader promoter is thought to mediate the VP30 dependency of transcription. Here, we investigated the constraints of VP30 dependency using a series of monocistronic minigenomes with sequence, structure and length deviations from the native NP hairpin. Hairpin stabilizations decreased while destabilizations increased transcription in the absence of VP30, but in all cases, transcription activity was higher in the presence versus absence of VP30. This also pertains to a mutant that is unable to form any RNA secondary structure at the TSS, demonstrating that the activity of VP30 is not simply determined by the capacity to form a hairpin structure at the TSS. Introduction of continuous 3'-UN5 hexamer phasing between promoter elements PE1 and PE2 by a single point mutation in the NP hairpin boosted VP30-independent transcription. Moreover, this point mutation, but also hairpin stabilizations, impaired the relative increase of replication in the absence of VP30. Our results suggest that the native NP hairpin is optimized for tight regulation by VP30 while avoiding an extent of hairpin stability that impairs viral transcription, as well as for enabling the switch from transcription to replication when VP30 is not part of the polymerase complex.IMPORTANCE A detailed understanding is lacking how the Ebola virus (EBOV) protein VP30 regulates activity of the viral polymerase complex. Here, we studied how RNA sequence, length and structure at the transcription start site (TSS) in the 3'-leader promoter influence the impact of VP30 on viral polymerase activity. We found that hairpin stabilizations tighten the VP30 dependency of transcription but reduce transcription efficiency and attenuate the switch to replication in the absence of VP30. Upon hairpin destabilization, VP30-independent transcription - already weakly detectable at the native promoter - increases, but never reaches the same extent as in the presence of VP30. We conclude that the native hairpin structure involving the TSS (i) establishes an optimal balance between efficient transcription and tight regulation by VP30, (ii) is linked to hexamer phasing in the promoter, and (iii) favors the switch to replication when VP30 is absent.

Hexamer phasing governs transcription initiation in the 3'-leader of Ebola virus.

The genomic, bipartite replication promoter of Ebola virus (EBOV) consists of elements 1 (PE1) and 2 (PE2). PE1 (55 nt at the 3'-terminus) is separated from PE2 (harboring eight 3'-UN5 hexamers) by the transcription start sequence (TSS) of the first nucleoprotein (NP) gene plus a spacer sequence. Insertions or deletions in the spacer were reported to support genome replication if comprising 6 or 12, but not 1/2/3/5/9 nt. This gave rise to the formulation of the "rule of 6" for the EBOV replication promoter. Here, we studied the impact of such hexamer phasing on viral transcription using a series of replication-competent and -deficient monocistronic minigenomes, in which the spacer of the NP gene was mutated or replaced with that of internal EBOV genes and mutated variants thereof. Beyond reporter gene assays, we conducted qRT-PCR to determine the levels of mRNA, genomic and antigenomic RNA. We demonstrate that hexamer phasing is also essential for viral transcription, that UN5 hexamer periodicity extends into PE1 and that the spacer region can be expanded by 48 nt without losses of transcriptional activity. Making the UN5 hexamer phasing continuous between PE1 and PE2 enhanced the efficiency of transcription and replication. We show that the 2 nt preceding the TSS are essential for transcription. We further propose a role for UN5 hexamer phasing in positioning NP during initiation of RNA synthesis, or in dissociation/reassociation of NP from the template RNA strand while threading the RNA through the active site of the elongating polymerase during replication and transcription.

Impact of RNA isolation protocols on RNA detection by Northern blotting.

We prepared total RNA from the Gram-positive soil bacterium Bacillus subtilis by different RNA extraction procedures to compare their suitability for Northern blot detection of tiny RNAs (~14-mers) or RNAs of intermediate size (100-200 nt) in terms of signal quality, intensity, and reproducibility. Our analysis included two hot phenol methods and two TRIzol extraction procedures. We found that signal intensity/detection sensitivity makes the key difference. Total RNAs prepared by the hot phenol method comprise the length spectrum from tRNAs to large ribosomal RNAs. Larger RNAs are less abundant in TRIzol preparations which instead enrich for RNAs of tRNA size and smaller. Thus, hot phenol methods are the choice for the detection of intermediate-sized and longer RNAs, whereas TRIzol extraction procedures are more suited for the detection of tiny RNAs.

Improved Northern blot detection of small RNAs using EDC crosslinking and DNA/LNA probes.

Successful detection of very small RNAs (tiny RNA, ~14 nt in length) by Northern blotting is dependent on improved Northern blot protocols that combine chemical crosslinking of RNA with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) to positively charged membranes, the use of native polyacrylamide gels, and the development of highly sensitive and specific probes modified with locked nucleic acids (LNA). In this protocol, we show that Northern blot detection of tiny RNAs with 5'-digoxigenin-labeled DNA/LNA mixmer probes is a highly sensitive and specific method and, in our hands, more sensitive than using a corresponding DNA/LNA mixmer probe with a 5'-(32)P-end label.

Dietary omega-3 deficiency reduces BDNF content and activation NMDA receptor and Fyn in dorsal hippocampus: implications on persistence of long-term memory in rats.

Omega-3 (n-3) fatty acids are important for adequate brain function and cognition. The aim of the present study was to evaluate how n-3 fatty acids influence the persistence of long-term memory (LTM) in an aversive memory task and to explore the putative mechanism involved. Female rats received isocaloric diets that included n-3 (n-3 group) or not (D group). The adult litters were subjected to an inhibitory avoidance task (0.7 mA, 1.0 seconds foot shock) to elicit persistent LTM. Twelve hours after the training session, the fatty acid profile and the brain derived neurotrophic factor (BDNF) content of the dorsal hippocampus were assessed. In addition, we measured the activation of the NR2B subunit of the N-methyl-d-aspartate (NMDA) receptor and the SRC family protein Fyn. Despite pronounced learning in both groups, the persistence of LTM was abolished in the D group 7 days after the training session. We also observed that the D group presented reductions in hippocampal DHA (22:6 n-3) and BDNF content. Twelve hours after the training session, the D group showed decreased NR2B and Fyn phosphorylation in the dorsal hippocampus, with no change in the total content of these proteins. Further, there was a decrease in the interaction of Fyn with NR2B in the D group, as observed by co-immunoprecipitation. Taken together, these data suggest that n-3 fatty acids influence the persistence of LTM by maintaining adequate levels of DHA and BDNF as well as by influencing the activation of NR2B and Fyn during the period of memory formation.

Regulatory T cells suppress the late phase of the immune response in lymph nodes through P-selectin glycoprotein ligand-1.

Regulatory T cells (Tregs) maintain tolerance toward self-antigens and suppress autoimmune diseases, although the underlying molecular mechanisms are unclear. In this study, we show that mice deficient for P-selectin glycoprotein ligand-1 (PSGL-1) develop a more severe form of experimental autoimmune encephalomyelitis than wild type animals do, suggesting that PSGL-1 has a role in the negative regulation of autoimmunity. We found that Tregs lacking PSGL-1 were unable to suppress experimental autoimmune encephalomyelitis and failed to inhibit T cell proliferation in vivo in the lymph nodes. Using two-photon laser-scanning microscopy in the lymph node, we found that PSGL-1 expression on Tregs had no role in the suppression of early T cell priming after immunization with Ag. Instead, PSGL-1-deficient Tregs lost the ability to modulate T cell movement and failed to inhibit the T cell-dendritic cell contacts and T cell clustering essential for sustained T cell activation during the late phase of the immune response. Notably, PSGL-1 expression on myelin-specific effector T cells had no role in T cell locomotion in the lymph node. Our data show that PSGL-1 represents a previously unknown, phase-specific mechanism for Treg-mediated suppression of the persistence of immune responses and autoimmunity induction.

Adipose-derived mesenchymal stem cells ameliorate chronic experimental autoimmune encephalomyelitis.

Mesenchymal stem cells (MSCs) represent a promising therapeutic approach for neurological autoimmune diseases; previous studies have shown that treatment with bone marrow-derived MSCs induces immune modulation and reduces disease severity in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. Here we show that intravenous administration of adipose-derived MSCs (ASCs) before disease onset significantly reduces the severity of EAE by immune modulation and decreases spinal cord inflammation and demyelination. ASCs preferentially home into lymphoid organs but also migrates inside the central nervous system (CNS). Most importantly, administration of ASCs in chronic established EAE significantly ameliorates the disease course and reduces both demyelination and axonal loss, and induces a Th2-type cytokine shift in T cells. Interestingly, a relevant subset of ASCs expresses activated alpha 4 integrins and adheres to inflamed brain venules in intravital microscopy experiments. Bioluminescence imaging shows that alpha 4 integrins control ASC accumulation in inflamed CNS. Importantly, we found that ASC cultures produce basic fibroblast growth factor, brain-derived growth factor, and platelet-derived growth factor-AB. Moreover, ASC infiltration within demyelinated areas is accompanied by increased number of endogenous oligodendrocyte progenitors. In conclusion, we show that ASCs have clear therapeutic potential by a bimodal mechanism, by suppressing the autoimmune response in early phases of disease as well as by inducing local neuroregeneration by endogenous progenitors in animals with established disease. Overall, our data suggest that ASCs represent a valuable tool for stem cell-based therapy in chronic inflammatory diseases of the CNS.

Avaliação do desenvolvimento neuropsicomotor em crianças de um bairro da periferia de Porto Alegre / Assessment of the neuropsicomotor development of children living in the outskirts of Porto Alegre

Objetivos: Avaliar o desenvolvimento neuropsocomotor em crianças com desnutrição, crianças com alto risco para desnutrição e crianças com peso e altura ideal para a idade ( eutróficas), residentes da periferia de Porto Alegre (RS, Brasil). Métodos: A avaliação do desenvolvimento foi realizada através do Teste de Triagem de Denver II, onde foram analisados os domínios pessoal-social, linguagem, área motora fino-adaptativa e área motora ampla. Vinte crianças (idade: 1 a 6 anos) desnutridas ou com alto risco de desnutrição formaram o grupo desnutrição. Dezesseis crianças eutróficas, pareadas em relação a idade, formaram o grupo controle. Resultados: Os resultados obtidos com o teste de Triagem de Denver II demonstraram que os dois grupos apresentaram suspeita de atraso ou anormalidade no desenvolvimento. O percentual de crianças com atraso no desenvolvimento foi maior no grupo de desnutrição em três dos quatro domínios avaliados. Entretanto, não foi encontrada diferença significativa entre os grupos (p<0,05). Conclusões: a despeito da falta de poder estatístico do presente estudo, os resultados apontam para o fato de que fatores biológicos, assim como condições ambientais socioeconômicas podem determinar atraso no desenvolvimento neuropsicomotor. Esses dados apontam para a necessidade de triagem sistemática do desenvolvimento infantil e programas de intervenção precoce em comunidades carentes, ambas as ações vinculadas a um eficaz programa de saúde pública voltado para essa faixa etária.