Maximum likelihood estimates of <i>d_N</i> and <i>d_S</i> using codon-based models.

1)<p>Assuming that M₀ is true null hypothesis (<i>Η</i>₀) with <i>p</i>₀ parameters and that M₃ is the alternative hypothesis (<i>Η</i>₁) with <i>p</i>₁ parameters. The log likelihood difference <b><i>2Δℓ = 2(ℓ₁−ℓ₀)</i></b> follows asymptotically a χ² distribution with (<i>p</i>₁−<i>p</i>₀) degrees of freedom.</p>*)<p>Best-fitted model according to the likelihood ratio test (LRT) between of M₀ vs. M₃ with 4 degrees of freedom.</p>$)<p>Best-fitted model according to the likelihood ratio test (LRT) between of M₂ vs. M₃ with 1 degree of freedom.</p>#)<p>Positively selected sites were detected according to the Naive Empirical Bayes method with p≥0.99. Sites 33 and 44 (underlined) correspond respectively to sites 11 and 25 of V3 loop; which are related with R5-to-X4 change.</p>∶)<p>This result was obtained using unique sequences and X4-strains because exclusion of them did not affect on the model preference or the parameter values.</p

Maximum a posteriori tree of the blood donor and the recipients.

<p>This tree was constructed using molecular clones of the donor (DO) and the blood recipients. Sequences from all time points were included. The sampling time of clones are indicated in the sequences names (last two digits). Highlighted areas designate clones isolated the late stages of infection. Numbers above the branches indicate the Bayesian posteriori statistical support for the tree clades. A) The sequences of the donor DO are colored in magenta (GPGR variant) and in green (GSGR variants). The sequences of the recipient RA are colored in orange. Branches colored in orange indicate viruses that spread over the infection time in the RA. B) All clones generated from 1987 to 1990 from the HIV infection of the recipient RB were also included. The sequences of the recipient RB are colored in blue. The blue branches indicate viruses that spread over time in the RB. The red branches in the tree indicate the X4 variants.</p

Adaptive evolution during the natural HIV-1 infection.

<p>A maximum likelihood tree was inferred with unique V3 region sequences of the blood recipient RB. Branch-site model was used to evaluate if specific regions (clusters corresponding to distinct chronological times) of the tree were under distinct selective pressures. The tree depicts selective pressures (dN/dS) in distinct moments (years) of HIV-1 infection. The ratio dN/dS was estimated in isolates sampled from 1986 to 1989 (blue branches), then in isolates from 1989 to 1990 (pink branches) and finally in the cluster where X4 viruses appeared (red branches, X4 variants are indicated by arrows within this cluster). Dotted lines delineate each group of sequences where (dN/dS) was estimated. The last two digits in each name designate the year that a specific sequence was sampled. The panel in the bottom of the figure shows the estimated parameters of model M3 (null hypothesis) and model D (alternative hypothesis) (see the manuscript for details).</p

Schematic representation of the blood-transmission cluster.

<p>Each column represents all samples of one patient. DO: donor; RA: recipient A; RB: recipient B; SC: sexual partner of the RB. Gray rectangles indicate each time-point with sampling date, number of clones generated and the mean of the pairwise diversity plus standard error. Filled circles indicate number of X4-variant sequences. Arrows indicate the date and the transmission route.</p

Donor axons regenerate and become myelinated equally well either side of cross-bridges placed between the donor tibial (TIB) nerve and the recipient denervated common peroneal (CP) whether or nor the distal nerve stump is isolated from denervated targets.

<p>A. Cross-sections of regenerated TIB axons within the recipient denervated CP nerve stump, proximal and distal to the insertion of 3 cross-bridges between the nerves 3 months previously (Experiment #1, No Repair: <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127397#pone.0127397.t001" target="_blank">Table 1</a>). TIB axons that grew within the CP nerve stump distal to the cross-bridges tended to be larger than B. those in the CP nerve stump that was ligated, consistent with findings that regenerated axons recover size only after making functional contacts. C. Mean (±SE) TIB axon numbers in the recipient denervated CP nerve stump were the same proximal and distal to 3 cross-bridges, whether or not the CP nerve stump was isolated from denervated targets. This negates possible neurotrophic target influences on donor TIB nerves growing into a recipient denervated CP nerve stump.</p

<i>In vivo</i> recording of muscle isometric contractile forces to determine numbers of reinnervated motor units.

<p>A. In the second set of experiments (Repair, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127397#pone.0127397.t001" target="_blank">Table 1</a>), bipolar electrodes placed on the regenerated common peroneal (CP) nerve were used to electrically stimulate the CP nerve supramaximally to evoke extensor digitorum longus (EDL) muscle twitch (B) and tetanic isometric contractions or to stimulate the CP nerve incrementally in order to evoke all-or-none increments in twitch force. Motor unit (MU) number was calculated as the ratio of the muscle and average MU twitch forces—motor unit number estimation (MUNE).</p

Retrograde labeling of motor and sensory tibial (TIB) neurons that grow axons from a donor TIB nerve into a recipient denervated common peroneal (CP) nerve and, of CP neurons that regenerate their axons after delayed CP nerve repair.

<p>In the first set of experiments (No Repair: <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127397#pone.0127397.t001" target="_blank">Table 1</a>) A. Fluorogold (FG) and fluororuby (FR) retrograde dyes were applied to the CP nerve stump 5 mm either side of 3.2 mm long cross-bridges in order to identify the TIB neurons that had grown axons through the cross-bridges into the recipient denervated CP nerve stump. B,C. In a second set of experiments (Repair, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127397#pone.0127397.t001" target="_blank">Table 1</a>), 5 months after delayed repair of CP nerve, FG or FR was applied to the regenerated CP axons to enumerate the CP neurons that had regenerated through the denervated CP nerve stump ‘protected’ by 3 (B) and 9 (C) bridges, as examples. By cutting through the cross-bridges at the same time as the dye application, retrograde labelling was confined to only the CP neurons that had regenerated their axons through the cross-bridges (B,C).</p

Regenerated tibial (TIB) axons lie outside as well as inside perineurium in the recipient common peroneal (CP) nerve stump proximal to the 10 mm length of the donor TIB nerve and the recipient CP nerve stump where the perineurium was stripped to place 5, 7, or 9 cross-bridges between the donor TIB nerve and recipient CP nerve stump.

<p>Cross-sections of the recipient denervated CP nerve either side of A. 3, B. 7 and C. 9 cross-bridges that were placed between the donor TIB nerve and recipient denervated CP nerve stump (Experiment #1, No Repair: <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127397#pone.0127397.t001" target="_blank">Table 1</a>). Regenerated and myelinated TIB axons distal to the cross-bridges were multi-fasciculated when the perineurium was removed to place 5–9 cross-bridges (B,C). D. The means (±SE) of the numbers of TIB axons that regenerated proximal and distal to the cross-bridges were not statistically different irrespective of the number of cross-bridges that were placed whilst E. the same numbers (means ± SE) of TIB axons regenerated within and outside of the perineurium of the CP nerve stump proximal but not distal to 5, 7 and 9 cross-bridges.</p

Donor tibial (TIB) axons 'protect' recipient denervated common peroneal (CP) nerve stumps to promote regeneration of CP axons and reinnervation of denervated muscles within 5 months after a 3 month delayed CP nerve repair.

<p>A. Examples of maximal twitch and tetanic contractions of extensor digitorum longus (EDL) muscle. These were recorded in response to 0.5 Hz and 100 Hz repetitive CP nerve stimulation at 2X threshold showing increased contractile forces when 3 as compared to 0 bridges were placed between donor TIB nerve and the recipient CP nerve stump 3 months prior to the delayed CP nerve repair (coaptation). (Note the smaller Y-axes for 0 as compared to 3 cross-bridges). The mean (±SE) values of muscle B. wet weight, C. twitch and D. tetanic forces and of the numbers of E. CP motoneurons that regenerated their axons and F. motor units (measured as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127397#pone.0127397.g004" target="_blank">Fig 4</a>) were significantly increased when 3 cross-bridges were placed as compared to no cross-bridges placed. G. The mean (±SE) values of motor unit twitch forces were not significantly different, the return of all the regenerating motor axons reinnervating all the denervated muscle fibers.</p

Donor tibial (TIB) neurons grew their axons across cross-bridges into a recipient denervated common peroneal (CP) distal nerve stump where they proceeded to grow both proximal and distal to the cross-bridges.

<p>In Experiment #1, No Repair: <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127397#pone.0127397.t001" target="_blank">Table 1</a>, motor and sensory neurons were backlabelled with two fluorescent dyes applied proximal and distal to the cross-bridges (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127397#pone.0127397.g002" target="_blank">Fig 2A</a>). The numbers (±SE) of TIB motoneurons and sensory neurons that regenerated their axons into the denervated CP distal nerve stump both proximal and distal to 3, 5, 7 and 9 cross-bridges (as illustrated) were not significantly different. Very few neurons regenerated axons both proximal and distal to the cross-bridges as shown by the few neurons that were double-labelled with two retrograde dyes.</p