Generation and analysis of miR-206 and miR-133b double knockout mice.

<p>(<b>A</b>) <i>Trans</i>-allelic targeted meiotic recombination was used to generate mice lacking both miR-206 and miR-133b. MiR-206 and miR-133b heterozygous mice each containing a loxP site in place of the miRNA stemloop were bred together and with mice expressing Cre recombinase in the germline. Zygotes produced from sperm that underwent <i>trans</i>-allelic recombination contained one chromosome lacking miR-206 and miR-133b and one chromosome with a miR-206 and miR-133b duplication. These animals were then bred to obtain miR-206 and miR-133b double knockout mice, i.e. 7H4 knockout mice. P1, forward primer upstream of miR-206. P2, reverse primer downstream of miR-133b. (<b>B</b>) PCR using P1 and P2 primers (in A) gives a detectable product (550 bp) only in 7H4 heterozygous and knockout mice, demonstrating that the 7H4 genomic region containing the miR-206 and miR-133b stem loops is completely missing from the 7H4 null allele. (<b>C</b>) PCR using primers specific for the miR-133b allele yields a 600 bp band only when the WT allele is present, but no band for the 7H4 null allele. (<b>D</b>) Quantitative RT-PCR for the stemloop regions of miR-206 and miR-133b. As expected, miR-206 and miR-133b are absent in 7H4 knockout mice.</p

Lack of both miR-206 and miR-133b delays NMJ regeneration.

<p>(<b>A–D</b>) To determine whether both miRNAs, miR-206 and miR-133b (7H4), act in concert to affect muscle reinnervation, the peroneal nerve was crushed in control (A) and 7H4 knockout mice (B) and reinnervation of the extensor digitorium longus was examined 9 days post injury. In 7H4 muscles, the incidence of partially and completely denervated NMJs is higher than that in muscles from control animals (C, D). At least 6 mice were examined per genotype and 50 NMJs per mouse visualized. FI, fully innervated; PI, partially innervated; FD, fully denervated NMJs. Error bar  =  SEM. P-value (*) <0.02. Scale bar  = 50 μm. (<b>E, F</b>) Quantitative mRNA expression of pre-miR-1-1, pre-miR-1-2, pre-miR-133a-1, and pre-miR-133a-2 in EDL (E) and soleus (F) muscle of adult WT (black circles represent individual values and black line the mean) and 7H4 knockout (red circles represent individual values and red line the mean) mice. Gene expression is normalized to Gapdh and results are scaled to the average value of the WT samples.</p

Normal NMJ development in miR-133b knockout mice.

<p>(<b>A</b>) Immunofluorescence staining of axonal neurofilaments and vesicular synaptophysin (green) and BTX staining of postsynaptic nAChRs (red) to visualize axons innervating synaptic sites. Filled white arrowheads, NMJs with multiple axon innervation; empty arrowheads, retraction bulbs. Scale bar  = 20 μm. (<b>B</b>) The proportion of sternomastoid NMJs with multiple innervation decreases at a similar rate in control and knockout mice. (<b>C</b>) Proportion of developing sternomastoid NMJs with single, double or triple innervation is similar in control and knockout mice.</p

MiR-133b does not regulate muscle reinnervation or ALS disease progression.

<p>(<b>A</b>) Semi-quantitative RT-PCR of cDNA from control or denervated hindlimb muscle 2 and 4 days after unilateral sciatic nerve cut. Levels of pre-miR-133b and AChRγ increase dramatically in denervated muscle, while levels of pre-miR-133a-1, pre-miR-133a-2 and GAPDH are unchanged, suggesting differential regulation of miR-133a and miR-133b. (<b>B–E</b>) Analysis of muscle reinnervation in tibialis anterior muscle from control (B) and miR-133b null mice (C) 3 weeks following nerve cut. (<b>D</b>) Percentage of tibialis anterior NMJs that were reinnervated. (<b>E</b>) Percentage of NMJs that were denerverated, partially reinnervated, or fully reinnervated. (<b>F,G</b>) Analysis of sternomastoid muscle reinnervation 9 days following accessory nerve crush. At least 6 mice were analyzed and 200 NMJs were examined per animal. Error bars indicate SEM. Scale bar  = 20 μm. (<b>H–J</b>) In the SOD1-G93A mouse model for ALS, loss of miR-133b does not exacerbate symptoms; disease onset (H), survival rate (I), and disease progression (J) are unchanged in the absence of miR-133b. Data were obtained from: 8 female, 8 male SOD1G93A; 10 female, 8 male miR-133b+/−;SOD1G93A; and 6 female, 9 male R-133b−/−;SOD1G93A mice. Error bars indicate SEM.</p

Development of NMJs in 7H4 mice.

<p>(<b>A–D</b>) Both miR-206 and miR-133b are dispensable for development of the NMJ. There is no obvious difference in the transformation of the postsynapse (stained using f-BTX, red) from a small plaque into a large pretzel between 7H4 knockout (B and D) and control mice of the same age (A and C). The formation of the presynaptic apparatus is also indistinguishable between 7H4 knockout mice and control mice of the same age, visualized using antibodies against synaptotagmin-2, green, and neurofilament, blue, in young animals (A and B) and YFP expressed in motor axons (C and D). Scale bar  = 10 μm for P9 and 20 μm for adult NMJs.</p

The specific degree-of-polymerization of A-type proanthocyanidin oligomers impacts <i>Streptococcus mutans</i> glucan-mediated adhesion and transcriptome responses within biofilms

<div><p>Cranberry A-type proanthocyanidins (PACs) have been recognized for their inhibitory activity against bacterial adhesion and biofilm-derived infections. However, the precise identification of the specific classes of degree-of-polymerization (DP) conferring PACs bioactivity remains a major challenge owing to the complex chemistry of these flavonoids. In this study, chemically characterized cranberries were used in a multistep separation and structure-determination technique to isolate A-type PAC oligomers of defined DP. The influences of PACs on the 3D architecture of biofilms and <i>Streptococcus mutans</i>-transcriptome responses within biofilms were investigated. Treatment regimens that simulated topical exposures experienced clinically (twice-daily, 60 s each) were used over a saliva-coated hydroxyapatite biofilm model. Biofilm accumulation was impaired, while specific genes involved in the adhesion of bacteria, acid stress tolerance, and glycolysis were affected by the topical treatments (<i>vs</i> the vehicle-control). Genes (<i>rmpC</i>, <i>mepA</i>, <i>sdcBB</i>, and <i>gbpC</i>) associated with sucrose-dependent binding of bacteria were repressed by PACs. PACs of DP 4 and particularly DP 8 to 13 were the most effective in disrupting bacterial adhesion to glucan-coated apatitic surface (>85% inhibition <i>vs</i> vehicle control), and gene expression (eg <i>rmpC</i>). This study identified putative molecular targets of A-type cranberry PACs in <i>S. mutans</i> while demonstrating that PAC oligomers with a specific DP may be effective in disrupting the assembly of cariogenic biofilms.</p> </div

Dendritic Arbors of Pyramidal Neurons Are Stable

<p>(A) MZPs near the cell body of the pyramidal cell “dow” acquired over 9 wk.</p> <p>(B) Two-dimensional projections of three-dimensional skeletal reconstructions of “dow.”</p> <p>(C) High-magnification view of branch tip (green arrow) in region outlined by green box in (B).</p> <p>Scale bars: (A and B), 50 μm; (C), 10 μm.</p

The Change in BTL is Plotted for Each Individual Monitored Branch Tip of Every Imaged Cell

<p>Three-letter code, top right.</p> <p>(A–F) pyramidal cells; (G–N) non-pyramidal cells. Triangles and dashed lines denote the minimum length of the branch tip as it exceeds the border of the imaging volume.</p

Dendritic Growth in Multiple Branches of a Non-Pyramidal Neuron

<p>(A) MZPs near the cell body of the (∼118-μm deep) non-pyramidal cell “nmr” acquired over 4 wk. Two examples of dendritic branch growth are indicated by red arrowheads.</p> <p>(B) Two-dimensional projections of three-dimensional skeletal reconstructions of the non-pyramidal neuron “nmr.”</p> <p>(C) High-magnification view of one growing branch tip (#20) (red box in [A and B]). Red arrowhead marks the approximate distal end of the branch tip at 11 wk.</p> <p>(D) Three-dimensional isosurface reconstructions of branch tips in (C).</p> <p>(E) High-magnification view of branch tip #15 (green box in [A and B]).</p> <p>(F) Three-dimensional isosurface reconstructions of branch tips in (E).</p> <p>(G) Plot of change in BTL of dynamic branch tips as a function of age. Number to the right denotes branch tip number.</p> <p>Scale bars: (A and B), 25 μm; (B–F), 5 μm.</p

Branch Extensions, Retractions, and De Novo Branch Tip Addition in a Non-Pyramidal Neuron

<p>(A) Three-dimensional skeletal reconstructions of the sparsely spinous non-pyramidal neuron “zen” from images acquired over 5 wk.</p> <p>(B) High-magnification MZP view of region outlined by purple box in (A). Red arrowheads indicate examples of structural remodeling. Three-dimensional isosurface reconstructions show</p> <p>(C), the elongation and retraction of a spine toward an axon (yellow overlay).</p> <p>(D) Structural change in a cluster of branch tips (#50, far right) (red box in [B]).</p> <p>(E) Higher-magnification MZP view of region outlined by green box in (A). Examples of process retraction and branch-tip (#3) addition are labeled with yellow and green arrowheads, respectively.</p> <p>(F) Three-dimensional isosurface reconstructions of (E) with axon in yellow overlay.</p> <p>(G) MZP view of region outlined by cyan box in (A) shows branch-tip (#16) addition on a different dendrite.</p> <p>(H) Three-dimensional isosurface reconstructions of (G).</p> <p>(I) Plot of change in BTL of dynamic branch tips as a function of age. Number to the right denotes branch tip number.</p> <p>Scale bars: (A), 50 μm; (B–H), 5 μm.</p