Annealing of ssDNA and compaction of dsDNA by the HIV-1 nucleocapsid and Gag proteins visualized using nanofluidic channels

The nucleocapsid protein NC is a crucial component in the human immunodeficiency virus type 1 life cycle. It functions both in its processed mature form and as part of the polyprotein Gag that plays a key role in the formation of new viruses. NC can protect nucleic acids (NAs) from degradation by compacting them to a dense coil. Moreover, through its NA chaperone activity, NC can also promote the most stable conformation of NAs. Here, we explore the balance between these activities for NC and Gag by confining DNA-protein complexes in nanochannels. The chaperone activity is visualized as concatemerization and circularization of long DNA via annealing of short single-stranded DNA overhangs. The first ten amino acids of NC are important for the chaperone activity that is almost completely absent for Gag. Gag condenses DNA more efficiently than mature NC, suggesting that additional residues of Gag are involved. Importantly, this is the first single DNA molecule study of full-length Gag and we reveal important differences to the truncated Δ-p6 Gag that has been used before. In addition, the study also highlights how nanochannels can be used to study reactions on ends of long single DNA molecules, which is not trivial with competing single DNA molecule techniques

A transgenic mouse expressing CHMP2B intron5 mutant in neurons develops histological and behavioural features of amyotrophic lateral sclerosis and frontotemporal dementia

International audienceMutations in the charged multivesicular body protein 2B (CHMP2B) are associated with frontotemporal dementia (FTD), amyotrophic lateral sclerosis (ALS), and with a mixed ALS-FTD syndrome. To model this syndrome, we generated a transgenic mouse line expressing the human CHMP2Bintron5 mutant in a neuron-specific manner. These mice developed a dose-dependent disease phenotype. A longitudinal study revealed progressive gait abnormalities, reduced muscle strength and decreased motor coordination. CHMP2Bintron5 mice died due to generalized paralysis. When paralyzed, signs of denervation were present as attested by altered electromyographic profiles, by decreased number of fully innervated neuromuscular junctions, by reduction in size of motor endplates and by a decrease of sciatic nerve axons area. However, spinal motor neurons cell bodies were preserved until death. In addition to the motor dysfunctions, CHMP2Bintron5 mice progressively developed FTD-relevant behavioural modifications such as disinhibition, stereotypies, decrease in social interactions, compulsivity and change in dietary preferences. Furthermore, neurons in the affected spinal cord and brain regions showed accumulation of p62-positive cytoplasmic inclusions associated or not with ubiquitin and CHMP2Bintron5 As observed in FTD3 patients, these inclusions were negative for TDP-43 and FUS. Moreover, astrogliosis and microgliosis developed with age. Altogether, these data indicate that the neuronal expression of human CHMP2Bintron5 in areas involved in motor and cognitive functions induces progressive motor alterations associated with dementia symptoms and with histopathological hallmarks reminiscent of both ALS and FTD

The nucleic acid chaperone activity of the HIV-1 Gag polyprotein is boosted by its cellular partner RPL7: a kinetic study

10.1093/nar/gkaa659Nucleic Acids Researc

Systemic down-regulation of delta-9 desaturase promotes muscle oxidative metabolism and accelerates muscle function recovery following nerve injury.

The progressive deterioration of the neuromuscular axis is typically observed in degenerative conditions of the lower motor neurons, such as amyotrophic lateral sclerosis (ALS). Neurodegeneration in this disease is associated with systemic metabolic perturbations, including hypermetabolism and dyslipidemia. Our previous gene profiling studies on ALS muscle revealed down-regulation of delta-9 desaturase, or SCD1, which is the rate-limiting enzyme in the synthesis of monounsaturated fatty acids. Interestingly, knocking out SCD1 gene is known to induce hypermetabolism and stimulate fatty acid beta-oxidation. Here we investigated whether SCD1 deficiency can affect muscle function and its restoration in response to injury. The genetic ablation of SCD1 was not detrimental per se to muscle function. On the contrary, muscles in SCD1 knockout mice shifted toward a more oxidative metabolism, and enhanced the expression of synaptic genes. Repressing SCD1 expression or reducing SCD-dependent enzymatic activity accelerated the recovery of muscle function after inducing sciatic nerve crush. Overall, these findings provide evidence for a new role of SCD1 in modulating the restorative potential of skeletal muscles

Blood Cell Palmitoleate-Palmitate Ratio Is an Independent Prognostic Factor for Amyotrophic Lateral Sclerosis.

Growing evidence supports a link between fatty acid metabolism and amyotrophic lateral sclerosis (ALS). Here we determined the fatty acid composition of blood lipids to identify markers of disease progression and survival. We enrolled 117 patients from two clinical centers and 48 of these were age and gender matched with healthy volunteers. We extracted total lipids from serum and blood cells, and separated fatty acid methyl esters by gas chromatography. We measured circulating biochemical parameters indicative of the metabolic status. Association between fatty acid composition and clinical readouts was studied, including ALS functional rating scale-revised (ALSFRS-R), survival, disease duration, site of onset and body mass index. Palmitoleate (16:1) and oleate (18:1) levels, and stearoyl-CoA desaturase indices (16:1/16:0 and 18:1/18:0) significantly increased in blood cells from ALS patients compared to healthy controls. Palmitoleate levels and 16:1/16:0 ratio in blood cells, but not body mass index or leptin concentrations, negatively correlated with ALSFRS-R decline over a six-month period (p<0.05). Multivariate Cox analysis, with age, body mass index, site of onset and ALSFRS-R as covariables, showed that blood cell 16:1/16:0 ratio was an independent prognostic factor for survival (hazard ratio=0.1 per unit of ratio, 95% confidence interval=0.01-0.57, p=0.009). In patients with high 16:1/16:0 ratio, survival at blood collection was extended by 10 months, as compared to patients with low ratio. The 16:1/16:0 index is an easy-to-handle parameter that predicts survival of ALS patients independently of body mass index. It therefore deserves further validation in larger cohorts for being used to assess disease outcome and effects of disease-modifying drugs

SCD1 expression in ALS patient muscle and after nerve injury.

<p>(A) Expression of SCD1 and SCD5 in deltoid muscle biopsies from ALS patients and healthy subjects (CT, white columns), as identified by microarray analysis of the database deposited at <a href="http://www.ebi.ac.uk/arrayexpress/(accession" target="_blank">http://www.ebi.ac.uk/arrayexpress/(accession</a> number E-MEXP-3260) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064525#pone.0064525-Pradat1" target="_blank">[12]</a>. ALS samples were obtained from muscle not clinically or electromyography affected (Unaff, orange columns) and from muscle with advanced pathology, characterized by reduced strength and neurogenic electromyography pattern (Aff, brown columns). *<i>P</i><0.05 (1-way ANOVA followed by Tukey's multiple comparison test, n = 4–10). (B) Expression of SCD1 in gastrocnemius following sciatic nerve axotomy (Axo) or crush at indicated post-operation days. Contralateral muscle expression is represented by 100% baseline. **<i>P</i><0.01, ***<i>P</i><0.001 (One sample t-test, n = 4–10).</p

Gene expression specific to the motor end plate in SCD1 knockout mice.

<p>Expression of AChR-α, AChR-γ, AChR-ε and MuSK in gastrocnemius and tibialis anterior from SCD1 knockout mice (brown columns) and wild-type littermates (white columns). *<i>P</i><0.05, ***<i>P</i><0.001 (Unpaired t-test, n = 4–11).</p

Muscle function recovery in SCD-deficient mice submitted to nerve crush.

<p>Restoration of muscle grip strength in SCD1 knockout mice (A) or MF-438 treated mice (C) (black circles) and corresponding control littermates (white circles) at the indicated post-operation times. ***<i>P</i><0.001 (2-way ANOVA, n = 4–12). Percentage of electromyography episodes of spontaneous activity in SCD1 knockout mice (Inset A) or MF-438 treated mice (Inset C) (KO or MF, brown columns) and corresponding control littermates (WT or CT, white columns) two weeks after sciatic nerve crush. *<i>P</i><0.05 (Unpaired t-test, n = 4–7). (B) Relative density of muscle fiber types in ipsilateral and contralateral tibialis anterior from SCD1 knockout mice and wild-type littermates two weeks after sciatic nerve crush. According to SDH histochemistry, fibers were classified as dark brown colored fibers with high metabolic oxidative capacity (brown columns), pale brown colored fibers with medium oxidative capacity (orange columns) and non-satined fibers (white columns). *<i>P</i><0.05 and ***<i>P</i><0.001 (1-way ANOVA followed by Tukey's multiple comparison test, (n = 4–6). (D) Kaplan-Meier curves showing the percentage of MF-438 treated mice (black circles) and control littermates (white circles) that started to exhibit a grip strength distinct from zero after initial total paralysis. Logrank test (n = 10–11). Inset D, averaged time at start of recovery in MF-438 treated mice (MF, brown column) and control littermates (CT, white column). *<i>P</i><0.05 (Unpaired t-test, n = 10–11).</p

Effects of MF-438 on metabolism and muscle function.

<p>(A) C16:1/C16:0 and C18:1/C18:0 fatty acid ratio in plasma from MF-438 treated mice (brown columns) and control littermates (CT, white columns). ***<i>P</i><0.001 (Unpaired t-test, n = 4–6). (B) Time course of respiratory quotient (RQ) before and after treatment with MF-438 at a dose of 10 mg/kg body mass/day (indicated by the black bar) (n = 4). Time course of body mass (C), muscle grip strength expressed as a percentage of day 0 (D), and specific grip strength, as determined by normalizing peak force to body mass (E), in mice fed regular chow (white circles) and mice fed regular chow supplemented with MF-438 (black circles). **<i>P</i><0.01 (2-way ANOVA followed by Bonferroni test, n = 5–6). (F) Expression of PGC1-α, AChR-α, and MuSK in gastrocnemius from MF-438 treated mice (brown columns) and control littermates (white columns). *<i>P</i><0.05 (Unpaired t-test, n = 3–9).</p

Metabolic phenotype of muscle from SCD1 knockout mice.

<p>(A) Expression of PGC1-α, PPARα and PDK4 in gastrocnemius and tibialis anterior from SCD1 knockout mice (brown columns) and wild-type littermates (white columns). *<i>P</i><0.05, **<i>P</i><0.01 (Unpaired t-test, n = 3–11). (B) Number of muscle fibers in tibialis anterior from SCD1 knockout mice (KO, brown column) and wild-type littermates (WT, white column). *<i>P</i><0.05 (Unpaired t-test, n = 7–10). (C) Distribution of the calibers of muscle fibers in tibialis anterior from SCD1 knockout mice (327 fibers, black circles) and wild-type littermates (283 fibers, white circles). Representative microphotographs of wild-type and knockout tibialis anterior are shown. (D) Averaged cross-sectional area of SDH-positive and SDH-negative fibers in tibialis anterior from SCD1 knockout mice (brown columns) and wild-type littermates (white columns). *<i>P</i><0.05, **<i>P</i><0.01 (Unpaired t-test, n = 7–10). (E) Number of SDH-positive (orange bars) and SDH-negative fibers (white bars) in tibialis anterior from SCD1 knockout mice (KO) and wild-type littermates (WT). ***<i>P</i><0.001 (Chi-square test, n = 283–327).</p