Plum pudding random medium model of biological tissue toward remote microscopy from spectroscopic light scattering

Biological tissue has a complex structure and exhibits rich spectroscopic behavior. There is \emph{no} tissue model up to now able to account for the observed spectroscopy of tissue light scattering and its anisotropy. Here we present, \emph{for the first time}, a plum pudding random medium (PPRM) model for biological tissue which succinctly describes tissue as a superposition of distinctive scattering structures (plum) embedded inside a fractal continuous medium of background refractive index fluctuation (pudding). PPRM faithfully reproduces the wavelength dependence of tissue light scattering and attributes the "anomalous" trend in the anisotropy to the plum and the powerlaw dependence of the reduced scattering coefficient to the fractal scattering pudding. Most importantly, PPRM opens up a novel venue of quantifying the tissue architecture and microscopic structures on average from macroscopic probing of the bulk with scattered light alone without tissue excision. We demonstrate this potential by visualizing the fine microscopic structural alterations in breast tissue (adipose, glandular, fibrocystic, fibroadenoma, and ductal carcinoma) deduced from noncontact spectroscopic measurement

Wnt4 interacts with MuSK and increases MuSK level of phosphorylation.

<p>(<b>A</b>) Domain structure of MuSK, MuSKΔCRD and Wnt4-HA proteins. SS, signal sequence; TM, transmembrane. (<b>B</b>) Quantification of AChR cluster numbers in control or agrin treated myotubes transfected with MuSK or MuSKΔCRD. The deletion of MuSK CRD domain did not affect agrin-induced AChR clustering. (<b>C, D</b>) Coimmunoprecipitation of MuSK/Wnt4 in COS 7 cells. COS 7 cells were cotransfected with Wnt4-HA and MuSK or MuSKΔCRD. Western blot using HA antibodies was performed on cell lysates to assess the expression of Wnt4-HA (C, WCL, Whole Cell Lysate). Western blot of MuSK or HA immunoprecipitates probed with HA or MuSK antibodies showed that Wnt4 interacted with MuSK but not with MuSKΔCRD. (<b>E</b>) MuSK phosphorylation induced by Wnt4. HEK 293T cells were cotransfected with HA-MuSK or HA-MuSKΔCRD with or without Wnt4-HA. HA-MuSK, HA-MuSKΔCRD and Wnt4-HA were immunoprecipitated with HA antibodies. Western blots of HA immunoprecipitates were probed with HA or phosphotyrosine (pTyr) antibodies to assess HA-MuSK or HA-MuSKΔCRD tyrosine phosphorylation level. (<b>F</b>) Quantification of HA-MuSK or HA-MuSKΔCRD phosphorylation levels normalized to the total amount of MuSK or MuSKΔCRD proteins expressed as the +Wnt4/−Wnt4 ratio. Wnt4 induced MuSK but not MuSKΔCRD phosphorylation. Error bars show means ± SEM from three independent experiments. *: non phosphorylated MuSK, **: phosphorylated MuSK.</p

Wnt4 does not alter muscle structure but modifiy fiber type composition.

<p>(<b>A</b>) Histological analysis of hind limb muscle cross sections from stage E18.5 control littermate (wild type) or Wnt4−/− embryos stained with heamatoxylin/eosin. The muscle gross organization was not affected in the Wnt4−/− embryos (N = 3 for Wnt4 mutants and N = 4 for control littermate embryos). (<b>B</b>) Measurement of muscle fibers perimeter. No significant difference in limb muscle section perimeter from Wnt4−/− mutant compared to wild type was detected. (<b>C</b>) Hind limb muscle cross sections (soleus level) from stage E18.5 control littermates (wild type) or Wnt4−/− embryos stained with myosin heavy chain I (MyHCI) antibodies (N = 2 for Wnt4 mutants and control littermate embryos). (<b>D</b>) Measurement of the number of MyHCI postive cells. The number of MyHCI positive cells was increased in Wnt4−/− mutant compared to wild type. Error bars show means ± SEM. **<i>P</i><0.001; Mann-Whitney <i>U</i> test. NS, non significant. Scale bars: in A, 30 µm; in C, 10 µm.</p

Wnt4 expression during neuromuscular junction development.

<p>(<b>A</b>) Table showing results of Affymetrix microarrays data comparing relative Wnt4 mRNA expression during myotube differentiation, between stages T1/T2 and T2/T3 (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029976#s4" target="_blank">Materials and Methods</a>). Relative Wnt4 mRNA is upregulated more than three fold between stage T1/T2 and downregulated more than one fold between stages T2/T3. (<b>B and C</b>) Real time RT-PCR quantification of relative Wnt4 mRNA expression during myotube differentiation (B, stages T1, T2 and T3) and hind limb development (C, embryonic stages E13.5, E14, E16 and newborn mice P0, N = 6 embryos tested for each stage). Relative Wnt4 mRNA expression is significantly increased between stages T1/T2 and further downregulated between stages T2/T3 and decreases as the limb developed. (<b>D</b>) Real time RT-PCR quantification of relative MuSK and Wnt4 mRNA expression in synaptic and extrasynaptic regions of diaphragms from stage E18.5 embryos. Relative MuSK and Wnt4 expression are three and two fold increased in synaptic compared to extrasynaptic regions respectively. Results are represented as relative expression (2^{−Δ<i>C</i>t} versus reference gene ×100, N = 3). (<b>E and F</b>) In situ hybridization with probes for Wnt4 mRNAs in E11.5 and E13.5 spinal cord sections (thoracic level) of wild type mice embryos (N = 3 embryos tested for each condition). Wnt4 mRNA is expressed in the floor plate and dorsal spinal cord but not in motoneurons (MN). Error bars show means ± SEM from three independent experiments. *<i>P</i><0.05; **<i>P</i><0.001; Mann-Whitney <i>U</i> test. Scale bar: in E, 20 µm for E and F.</p

Aberrant neuromuscular junction innervation in muscles of Wnt4−/− embryos.

<p>(<b>A–F</b>) Confocal images of whole mount diaphragm (A and B), intercostal muscles (C and D) or cross sections of hind limb muscles (E and F) from stage E18.5 control littermates (wild type, A, C and E) or Wnt4−/− embryos (B, D and F) stained with neurofilament (NF, red) and synaptophysin (Syn, red) antibodies together with α-bungarotoxin (AChRs, green). Examples of nerve terminals passing through and projecting beyond the central band of AChR clusters in mutant diaphragm or intercostal muscles are indicated by white arrows in the merged image in B and D. Examples of non innervated synapses in mutant limb muscles are indicated by white stars in the merged image in F. (<b>G</b>) Measurement of AChR endplate band width, AChR clusters surface, α-bungarotoxin fluorescence signal intensity (numbers of AChR clusters tested: 95 in control and 76 in Wnt4−/−), AChR cluser number and number of non innervated synapses (%) in limb muscle cross sections (numbers of synapses counted: 35 in control and 28 in Wnt4−/−; N = 3 for Wnt4 mutants and N = 4 for control littermates embryos). Error bars show means ± SEM. *<i>P</i><0.05; **<i>P</i><0.001; Mann-Whitney <i>U</i> test. NS, non significant. Scale bars: in the merged image in A, 60 µm for A and B; in the merged image in D, 30 µm for C, D, E and F.</p

Synaptic markers are localized at the NMJ in Wnt4−/− embryos.

<p>(<b>A–F</b>) Hind limb muscle cross sections from stage E18.5 control littermates (A–C) or Wnt4−/− embryos (D–F) stained with AChE (red, A and D), MuSK (red, B and E) or rapsyn (red, C and F) antibodies together with α-bungarotoxin (AChR, green). AChE, MuSK and rapsyn colocalized with AChR at the NMJ of wild type and Wnt4−/− mutant embryos (15 cross sections from 2 Wnt4 mutants and control littermates were analyzed for each condition). (<b>G</b>) Confocal images of hind limb muscles cross sections from stage E18.5 Wnt4−/− embryos stained with neurofilament (NF, red), synaptophysin (Syn, red) and rapsyn (blue) antibodies together with α-bungarotoxin (AChRs, green). Examples of innervated and non innervated synapses are indicated by white stars and arrowhead respectively. Non innervated synapses still expressed the rapsyn protein (15 cross sections from 2 for Wnt4 mutants and control littermate embryos). Scale bar: in the merged image in A, 20 µm.</p

Muscle phenotype of MCK-UCP1 mice.

<p>A: body weight of male (empty diamonds) and female (empty triangles) wild-type mice, and male (filled diamonds) and female (filled triangles) MCK-UCP1 mice as a function of time. *, p<0.05 vs corresponding MCK-UCP1 (n = 7 mice per group). B: grip strength of mice as in A. *, p<0.05 vs corresponding MCK-UCP1 C: representative photomicrographs showing hematoxylin and eosin staining of gastrocnemius from wild-type mice (Wt) or MCK-UCP1 mice at 3 months (3 m) and 7 months (7 m) of age. The right panel shows the quantification of mean fiber area. *, p<0.05 (n = 4 mice per group).</p

Mild late onset motor neuron degeneration in MCK-UCP1 mice.

<p>A: representative photomicrographs showing ventral roots from wild-type (Wt) and MCK-UCP1 mice. Note the presence of two degenerating axons in the MCK-UCP1 picture (arrows). B: distribution of the calibers of axons in the ventral roots from wild-type (empty circles) or MCK-UCP1 (filled circles) mice. Small-caliber axons represent gamma-axons, which innervate muscle spindles, whereas large-caliber axons represent alpha-axons, which innervate skeletal muscles. Note the decrease in the population of the largest caliber axons in MCK-UCP1 ventral roots. C: size distribution of axons in the ventral roots from 7-month-old wild-type (empty columns) and MCK-UCP1 (filled columns) mice. *, p<0.05 vs wild-type (n = 3 mice per genotype). D: quantification of the number of motor neurons in the lumbar spinal cord form wild-type (Wt) and MCK-UCP1 (MCK-UCP1) mice at 3 months (3 m) and 7 months (7 m) of age, after toluidine blue-staining (left panel) and ChAT immunoreactivity (right panel). *, p<0.05 vs wild-type (n = 5 mice per group). E: representative photomicrographs showing GFAP immunoreactivity in the lumbar spinal cord from 7-month-old wild-type (Wt) or MCK-UCP1 mice. Ventral horns are indicated by dashed lines. Scale bar, 20 µm.</p

MCK-UCP1 do not display muscle dystrophy.

<p>A: serum creatine kinase (left panel) and lactate (right panel) concentrations in wild-type (Wt) or MCK-UCP1 mice at 2 months (2 m) and 7 months (7 m) of age. Non-significant differences (n = 8 mice per group). B: mRNA levels of atrogin-1 and M-cadherin in gastrocnemius from wild-type (empty circles) or MCK-UCP1 (black circles) mice at perinatal (PN), 1 month (1 m) and 7 months (7 m) of age. *, p<0.05 vs corresponding wild-type (n = 3–8 mice per group). C: representative photomicrographs showing hematoxylin and eosin staining of gastrocnemius from cardiotoxin-injured (48 hours after injury, CTX), wild-type (Wt) or MCK-UCP1 mice at 3 months (3 m) and 7 months (7 m) of age. Three mice per group were analyzed. A necrotic myofiber is indicated by an arrow. D: Evans blue dye staining as visualised by fluorescence in gastrocnemius from cardiotoxin-injured (48 hours after injury, CTX), wild-type (Wt) and MCK-UCP1 mice at 3 months (3 m) and 7 months (7 m) of age. Three mice per group were analyzed. Only a few necrotic myofibers could be detected in 7-month-old MCK-UCP1 mice (arrow).</p

UCP1 overexpression exacerbates motor neuron disease in mice.

<p>A–C: Kaplan-Meier curves showing the cumulative probability of disease onset (age at the peak of body mass prior to decline, A), progression through early disease stage (when maximal body mass decreases by 10%, B), and total survival (C). The p-values, as calculated using the logrank test, are shown. D: duration of early disease stage (left) and total duration of disease (right) in SOD1(G86R) mice (empty columns) and compound SOD1(G86R)/MCK-UCP1 mice (filled columns). *, p<0.05 vs corresponding SOD1(G86R).</p