3-D neurohistology of transparent tongue in health and injury with optical clearing

Tongue receives extensive innervation to perform taste, sensory, and motor functions. Details of the tongue neuroanatomy and its plasticity in response to injury offer insights to investigate tongue neurophysiology and pathophysiology. However, due to the dispersed nature of the neural network, standard histology cannot provide a global view of the innervation. We prepared transparent mouse tongue by optical clearing to reveal the spatial features of the tongue innervation and its remodeling in injury. Immunostaining of neuronal markers, including PGP9.5 (pan-neuronal marker), calcitonin gene-related peptide (sensory nerves), tyrosine hydroxylase (sympathetic nerves), and vesicular acetylcholine transporter (cholinergic parasympathetic nerves and neuromuscular junctions), was combined with vessel painting and nuclear staining to label the tissue network and architecture. The tongue specimens were immersed in the optical-clearing solution to facilitate photon penetration for 3-dimensiontal (3-D) confocal microscopy. Taking advantage of the transparent tissue, we simultaneously revealed the tongue microstructure and innervation with subcellular-level resolution. 3-D projection of the papillary neurovascular complex and taste bud innervation was used to demonstrate the spatial features of tongue mucosa and the panoramic imaging approach. In the tongue injury induced by 4-nitroquinoline 1-oxide administration in the drinking water, we observed neural tissue remodeling in response to the changes of mucosal and muscular structures. Neural networks and the neuromuscular junctions were both found rearranged at the peri-lesional region, suggesting the nerve-lesion interactions in response to injury. Overall, this new tongue histological approach provides a useful tool for 3-D imaging of neural tissues to better characterize their roles with the mucosal and muscular components in health and disease

Rho Kinases Regulate the Renewal and Neural Differentiation of Embryonic Stem Cells in a Cell Plating Density–Dependent Manner

[[abstract]]BACKGROUND: Rho kinases (ROCKs) mediate cell contraction, local adhesion, and cell motility, which are considered to be important in cell differentiation. We postulated that ROCKs are involved in controlling embryonic stem (ES) cell renewal and differentiation. METHODOLOGY/PRINCIPAL FINDINGS: CCE, a murine ES cell, was treated with Y-27632 for 48 to 96 hours and colony formation was evaluated. Y-27632 blocked CCE colony formation and induced CCE to grow as individual cells, regardless of the initial seeding cell density either at 10(4)/cm(2) ("high" seeding density) or 2x10(3)/cm(2) ("low" density). However, at high seeding density, Y-27632-treated cells exhibited reduction of alkaline phosphatase (AP) staining and Oct3/4 expression. They expressed SOX-1, nestin, and MAP2c, but not betaIII-tubulin or NG-2. They did not express endoderm or mesoderm lineage markers. After removal of Y-27632, the cells failed to form colonies or regain undifferentiated state. Silencing of ROCK-1 or ROCK-2 with selective small interference RNA induced CCE morphological changes similar to Y-27632. Silencing of ROCK-1 or ROCK-2 individually was sufficient to cause reduction of AP and Oct3/4, and expression of SOX-1, nestin, and MAP2c; and combined silencing of both ROCKs did not augment the effects exerted by individual ROCK siRNA. Y-27632-treated CCE cells seeded at 2x10(3) or 6.6x10(3) cells/cm(2) did not lose renewal factors or express differentiation markers. Furthermore, they were able to form AP-positive colonies after removal of Y-27632 and reseeding. Similar to ROCK inhibition by Y-27632, silencing of ROCK-1 or ROCK-2 in cells seeded at 2x10(3)/cm(2) did not change renewal factors. CONCLUSIONS/SIGNIFICANCE: We conclude that ROCKs promote ES cell colony formation, maintain them at undifferentiated state, and prevent them from neural differentiation at high seeding density. ROCK inhibition represents a new strategy for preparing large numbers of neural progenitor cells

Quantitative analysis of nanoparticle internalization in mammalian cells by high resolution X-ray microscopy

<p>Abstract</p> <p>Background</p> <p>Quantitative analysis of nanoparticle uptake at the cellular level is critical to nanomedicine procedures. In particular, it is required for a realistic evaluation of their effects. Unfortunately, quantitative measurements of nanoparticle uptake still pose a formidable technical challenge. We present here a method to tackle this problem and analyze the number of metal nanoparticles present in different types of cells. The method relies on high-lateral-resolution (better than 30 nm) transmission x-ray microimages with both absorption contrast and phase contrast -- including two-dimensional (2D) projection images and three-dimensional (3D) tomographic reconstructions that directly show the nanoparticles.</p> <p>Results</p> <p>Practical tests were successfully conducted on bare and polyethylene glycol (PEG) coated gold nanoparticles obtained by x-ray irradiation. Using two different cell lines, EMT and HeLa, we obtained the number of nanoparticle clusters uptaken by each cell and the cluster size. Furthermore, the analysis revealed interesting differences between 2D and 3D cultured cells as well as between 2D and 3D data for the same 3D specimen.</p> <p>Conclusions</p> <p>We demonstrated the feasibility and effectiveness of our method, proving that it is accurate enough to measure the nanoparticle uptake differences between cells as well as the sizes of the formed nanoparticle clusters. The differences between 2D and 3D cultures and 2D and 3D images stress the importance of the 3D analysis which is made possible by our approach.</p

Large expert-curated database for benchmarking document similarity detection in biomedical literature search

Document recommendation systems for locating relevant literature have mostly relied on methods developed a decade ago. This is largely due to the lack of a large offline gold-standard benchmark of relevant documents that cover a variety of research fields such that newly developed literature search techniques can be compared, improved and translated into practice. To overcome this bottleneck, we have established the RElevant LIterature SearcH consortium consisting of more than 1500 scientists from 84 countries, who have collectively annotated the relevance of over 180 000 PubMed-listed articles with regard to their respective seed (input) article/s. The majority of annotations were contributed by highly experienced, original authors of the seed articles. The collected data cover 76% of all unique PubMed Medical Subject Headings descriptors. No systematic biases were observed across different experience levels, research fields or time spent on annotations. More importantly, annotations of the same document pairs contributed by different scientists were highly concordant. We further show that the three representative baseline methods used to generate recommended articles for evaluation (Okapi Best Matching 25, Term Frequency-Inverse Document Frequency and PubMed Related Articles) had similar overall performances. Additionally, we found that these methods each tend to produce distinct collections of recommended articles, suggesting that a hybrid method may be required to completely capture all relevant articles. The established database server located at https://relishdb.ict.griffith.edu.au is freely available for the downloading of annotation data and the blind testing of new methods. We expect that this benchmark will be useful for stimulating the development of new powerful techniques for title and title/abstract-based search engines for relevant articles in biomedical research.Peer reviewe

Measurement equivalence between the original and estimated Mini-Mental State Examination in people with dementia

[[abstract]]Background: The Cognitive Abilities Screening Instrument (CASI) is increasingly used to assess general cognitive function in people with dementia. The Mini-Mental State Examination (MMSE) score can be converted from the CASI (i.e., the estimated MMSE). Recognizing that measurement equivalence is critical to meaningfully representing one with the other, we aimed to determine whether the estimated MMSE score obtained from the CASI was equivalent to the original MMSE in people with dementia. Methods: We obtained 110 data points for the MMSE and CASI scores in people with dementia. The intraclass correlation coefficient (ICC), Pearson's r, percent of standard error of measurement (SEM%), paired t-test, and effect size (Cohen's d) were used to investigate the equivalence. Results: To examine the equivalence between the original and estimated MMSE score, the ICC and Pearson's r of the total score and six domains were 0.62-0.95 and 0.62-0.96, respectively. The SEM% of the total score and six domains were 0.6-8.9%. The paired t-test results showed a significant difference (p < 0.05) between the total score and the three domains. The Cohen's d of the total score and six domains were 0.06-0.27. Conclusions: The estimated MMSE score was found to have moderate to excellent equivalence to the original MMSE score. The three domains (i.e., registration, attention and calculation, and visual-constructional ability) with moderate equivalence should be used cautiously to interchange with the original MMSE in people with dementia

Uric Acid Spherulites in the Reflector Layer of Firefly Light Organ

<div><p>Background</p><p>In firefly light organs, reflector layer is a specialized tissue which is believed to play a key role for increasing the bioluminescence intensity through reflection. However, the nature of this unique tissue remains elusive. In this report, we investigated the role, fine structure and nature of the reflector layer in the light organ of adult <i>Luciola cerata</i>.</p> <p>Principal Findings</p><p>Our results indicated that the reflector layer is capable of reflecting bioluminescence, and contains abundant uric acid. Electron microscopy (EM) demonstrated that the cytosol of the reflector layer's cells is filled with densely packed spherical granules, which should be the uric acid granules. These granules are highly regular in size (∼700 nm in diameter), and exhibit a radial internal structure. X-ray diffraction (XRD) analyses revealed that an intense single peak pattern with a d-spacing value of 0.320 nm is specifically detected in the light organ, and is highly similar to the diffraction peak pattern and d-spacing value of needle-formed crystals of monosodium urate monohydrate. However, the molar ratio evaluation of uric acid to various cations (K⁺, Na⁺, Ca²⁺ and Mg²⁺) in the light organ deduced that only a few uric acid molecules were in the form of urate salts. Thus, non-salt uric acid should be the source of the diffraction signal detected in the light organ.</p> <p>Conclusions</p><p>In the light organ, the intense single peak diffraction signal might come from a unique needle-like uric acid form, which is different from other known structures of non-salt uric acid form. The finding of a radial structure in the granules of reflector layer implies that the spherical uric acid granules might be formed by the radial arrangement of needle-formed packing matter.</p> </div

Light Intensity measurement of dorsal and ventral light organs.

<p>A) Ventral view (left panel) and dorsal view (right panel) of an isolated luminescent light organ. The light organ (including photogenic layer and reflector layer) was dissected from the 6^th and 7^th body segments of a dying <i>L. cerata</i>. B) Evaluation of light intensity from the ventral and dorsal luminescent light organs. The averaged light intensity (optical density, O.D) of ventral or dorsal light organs was evaluated from at least three images using ImageJ, and normalized by the averaged light intensity of ventral light organ.</p

The XRD patterns of the light organ and other artificial uric acid or urate crystals.

<p>Needle-formed crystals of monosodium urate monohydrate (MSUM) and plate-formed crystals of uric acid dihydrate (UAD) were prepared as described in Method and Material. The d-spacing values for the intense peak of MSUM and UAD, similar to that of the light organ of <i>L. cerata</i> (LO), are indicated, respectively.</p

SEM micrographs of the reflector layer and the photogenic layer in the light organ.

<p>A) Light organ (LO), located at the abdominal tissue section (the 6^th body segment), is a slab-like tissue with a thickness about 240 µm. B) The photogenic layer (P) located at the ventral light organ is a 40 µm thick tissue, and shows a morphology distinct from that of the reflector layer (R). C) In the reflector layer (R), densely packed spherical granules are found, and some of them had become hollowed (indicated as arrows). DO: Dorsal organ.</p

The X-ray diffraction (XRD) patterns of the light organ and other tissues of <i>L. cerata</i>.

<p>Homogenized tissues of the light organ (LO), the dorsal organ (DO), the thorax (T) and the head (H) were prepared for XRD analyses. The d-spacing value of 0. 320 nm, corresponding to the diffraction peak of light organ, is indicated.</p