Does a black light lens aid in composite removal?

OBJECTIVE: To evaluate the effectiveness of a black light lens as visual aid in composite restoration removal. Lost tooth structure, residual composite, and removal time were compared for operators with different levels of experience. METHODS: Occlusal preparations in 24 matched-pair extracted molars were etched, bonded, restored with composite, and thermocycled. The restored teeth were radiographed and two faculty and two student doctors removed the restorations with or without a black light lens while time was recorded. Digital scans of the cavity before and after restoration removal were used to calculate lost tooth structure and residual composite. RESULTS: Removal of restorations resulted in tooth structure loss and left residual composite. The use of the black light lens had no significant effect (two-way ANOVA; p value >0.05). However, operator experience significantly affected operating times and average depth of tooth structure loss (two-way ANOVA; p value <0.05). Student doctors assisted by the black light lost less tooth structure than experienced operators and improved their operating times (multiple comparisons; p value <0.05). CONCLUSIONS: The black light lens did not conserve tooth structure or avoid composite remnants compared to routine operation, nor affected the operating time. However, less-experienced operators did benefit from the black light in conserving tooth structure and time. CLINICAL SIGNIFICANCE: Replacement of defective composite restorations is a regular practice in restorative dentistry. When existing composite restorations are removed, loss of tooth structure is unavoidable. A black light lens might improve the ability of operators with less experience to conserve tooth structure even though it did not provide benefits for the experienced operators.

Molecular Characterization of Mycoplasma pneumoniae Isolates in the United States from 2012 to 2018.

Mycoplasma pneumoniae is a major cause of community-acquired pneumonia. There are limited data in the United States on the molecular epidemiological characteristics of M. pneumoniae We collected 446 M. pneumoniae-positive specimens from 9 states between August 2012 and October 2018. Culture, antimicrobial susceptibility testing, P1 subtyping, and multilocus VNTR (variable-number tandem repeats) analysis (MLVA) were performed to characterize the isolates. Macrolide-resistant M. pneumoniae (MRMp) was detected in 37 (8.3%) specimens. P1 subtype 2 (P1-2) was the predominant P1 subtype (59.8%). P1 subtype distribution did not change significantly chronologically or geographically. The macrolide resistance rate in P1 subtype 1 (P1-1) samples was significantly higher than that in P1-2 (12.9% versus 5.5%). Six P1-2 variants were identified, including two novel types, and variant 2c was predominant (64.6%). P1-2 variants were distributed significantly differently among geographic regions. Classical P1-2 was more frequent in lower respiratory tract specimens and had longer p1 trinucleotide repeats. Classical P1-2 was most common in MRMp (35.7%), while variant 2c was most common in macrolide-susceptible M. pneumoniae (67.5%). Fifteen MLVA types were identified; 3-5-6-2 (41.7%), 4-5-7-2 (35.3%), and 3-6-6-2 (16.6%) were the major types, and four MLVA clusters were delineated. The distribution of MLVA types varied significantly over time and geographic location. The predominant MLVA type switched from 4-5-7-2 to 3-5-6-2 in 2015. MLVA type was associated with P1 subtypes and P1-2 variant types but not with macrolide resistance. To investigate the M. pneumoniae genotype shift and its impact on clinical presentations, additional surveillance programs targeting more diverse populations and prolonged sampling times are required.

Multicenter Clinical Evaluation of the Automated Aries Group A Strep PCR Assay from Throat Swabs.

Group A Streptococcus (GAS) is one of the leading causes of bacterial pharyngitis. Early GAS diagnosis is critical for appropriate antibiotic administration that reduces the risk of GAS sequelae and limits spread of the infection. The Aries Group A Strep (GAS) assay (Luminex, Austin, TX) is a fully automated PCR assay for direct detection of GAS in throat swab specimens in less than 2 h with minimum hands-on time. This multicenter prospective study evaluated the clinical performance of the Aries GAS assay compared to that of Streptococcus pyogenes culture. Subjects with symptoms consistent with pharyngitis were enrolled across four sites in the United States, and a throat swab in liquid Amies medium was obtained. Aries and reference testing was performed within 72 and 48 h after sample collection, respectively. Of 623 throat swab specimens from patients with pharyngitis (93.6% <18 years old, 54.3% female), the reference method yielded valid results for 618 specimens. Reference and Aries assay testing showed GAS-positive results for 160 (25.9%) and 166 (26.9%) specimens, respectively. Compared to the reference method, Aries assay sensitivity was 97.5% (95% confidence interval [CI], 93.7% to 99.0%), specificity was 97.8% (95% CI, 96.0 to 98.8%), positive predictive value was 94.0% (95% CI, 89.3% to 96.7%), and negative predictive value was 99.1% (95% CI, 97.7% to 99.7%). There were 10 false-positive and four false-negative detections with the Aries assay. Discrepant analysis with bidirectional sequencing yielded concordant results with the Aries assay for nine of 14 discordant samples. The Aries assay had high sensitivity and specificity for qualitative detection of group A Streptococcus from patients with pharyngitis.

Macrolide-Resistant Mycoplasma pneumoniae in the United States as Determined from a National Surveillance Program.

There are sparse data to indicate the extent that macrolide-resistant Mycoplasma pneumoniae (MRMp) occurs in the United States or its clinical significance. Between 2015 and 2018, hospitals in 8 states collected and stored respiratory specimens that tested positive for M. pneumoniae and sent them to the University of Alabama at Birmingham, where real-time PCR was performed for detection of 23S rRNA mutations known to confer macrolide resistance. MRMp was detected in 27 of 360 specimens (7.5%). MRMp prevalence was significantly higher in the South and East (18.3%) than in the West (2.1%). A2063G was the predominant 23S rRNA mutation detected. MICs for macrolide-susceptible M. pneumoniae (MSMp) were ≤0.008 µg/ml, whereas MICs for MRMp were 16 to 32 µg/ml. Patients with MRMp infection were more likely to have a history of immunodeficiency or malignancy. Otherwise, there were no other significant differences in the clinical features between patients infected with MRMp and those infected with MSMp, nor were there any differences in radiographic findings, hospitalization rates, viral coinfections, the mean duration of antimicrobial treatment, or clinical outcomes. There was no significant change in MRMp incidence over time or according to age, sex, race/ethnicity, or status as an inpatient or an outpatient. Patients with MRMp were more likely to have received a macrolide prior to presentation, and their treatment was more likely to have been changed to a fluoroquinolone after presentation. This is the first national surveillance program for M. pneumoniae in the United States. Additional surveillance is needed to assess the clinical significance of MRMp and to monitor changes in MRMp prevalence.

Evaluation of Oxacillin and Cefoxitin Disk and MIC Breakpoints for Prediction of Methicillin Resistance in Human and Veterinary Isolates of Staphylococcus intermedius Group.

Staphylococcus pseudintermedius is a coagulase-positive species that colonizes the nares and anal mucosa of healthy dogs and cats. Human infections with S. pseudintermedius range in severity from bite wounds and rhinosinusitis to endocarditis; historically, these infections were thought to be uncommon, but new laboratory methods suggest that their true incidence is underreported. Oxacillin and cefoxitin disk and MIC tests were evaluated for the detection of mecA- or mecC-mediated methicillin resistance in 115 human and animal isolates of the Staphylococcus intermedius group (SIG), including 111 Staphylococcus pseudintermediusand 4 Staphylococcus delphini isolates, 37 of which were mecA positive. The disk and MIC breakpoints evaluated included the Clinical and Laboratory Standards Institute (CLSI) M100-S25 Staphylococcus aureus/Staphylococcus lugdunensis oxacillin MIC breakpoints and cefoxitin disk and MIC breakpoints, the CLSI M100-S25 coagulase-negative Staphylococcus (CoNS) oxacillin MIC breakpoint and cefoxitin disk breakpoint, the CLSI VET01-S2 S. pseudintermedius oxacillin MIC and disk breakpoints, and the European Committee on Antimicrobial Susceptibility Testing (EUCAST) S. pseudintermedius cefoxitin disk breakpoint. The oxacillin results interpreted by the VET01-S2 (disk and MIC) and M100-S25 CoNS (MIC) breakpoints agreed with the results of mecA/mecC PCR for all isolates, with the exception of one false-resistant result (1.3% of mecA/mecC PCR-negative isolates). In contrast, cefoxitin tests performed poorly, ranging from 3 to 89% false susceptibility (very major errors) and 0 to 48% false resistance (major errors). BD Phoenix, bioMérieux Vitek 2, and Beckman Coulter MicroScan commercial automated susceptibility test panel oxacillin MIC results were also evaluated and demonstrated >95% categorical agreement with mecA/mecC PCR results if interpreted by using the M100-S25 CoNS breakpoint. The Alere penicillin-binding protein 2a test accurately detected all mecA-positive isolates, although for four isolates, cefoxitin induction was required prior to testing. These data demonstrate that the cefoxitin surrogate test does not reliably detect the presence of mecA in S. pseudintermedius isolates and that laboratories should perform oxacillin disk or MIC tests of these isolates when they are encountered.

Long-term monitoring of exposure to toxic and essential metals and metalloids in the tawny owl (Strix aluco): Temporal trends and influence of spatial patterns.

As a result of regulatory decisions, atmospheric deposition of most toxic metals and metalloids (MEs) has decreased in Europe over the past few decades. However, little is known about how this reduction translates into exposure at higher trophic levels in the terrestrial environment where temporal trends may be spatially heterogeneous due to local current or legacy sources of emissions (e.g., industry) or long-range transport of elements (e.g., marine transport). The aim of this study was to characterize temporal and spatial trends of exposure to MEs in terrestrial food webs using a predatory bird, the tawny owl Strix aluco, as a biomonitor. Toxic (Al, As, Cd, Hg, Pb) and essential/beneficial (B, Co, Cu, Mn, Se) elemental concentrations were measured in feathers of nest-captured females from 1986 to 2016, extending a previous study published over the time-series 1986-2005 (n = 1051), in a breeding population in Norway. A drastic decline over time was shown for the toxic MEs (-97 % for Pb, -89 % for Cd, -48 % for Al, and -43 % for As) except Hg. The beneficial elements B, Mn, and Se showed oscillations but an overall decline (-86 %, -34 %, and -12 %, respectively) whereas the essentials Co and Cu did not exhibit significant trends. The distance to potential sources of contamination influenced both the spatial patterns of concentrations in owl feathers and their temporal trends. The accumulation of As, Cd, Co, Mn and Pb was overall higher in the vicinity of sites recorded as polluted, and a greater temporal decrease of As, B, and Cd concentrations was found in the areas of further distance to polluted sites. The decrease of Pb concentrations was sharper further from the coast during the 1980s than in coastal areas, while the opposite was observed for Mn. The levels of Hg and Se were higher in coastal areas, and Hg temporal trends differed according to the distance to the coast. This study highlights the valuable insights provided by long-term survey of wildlife exposure to pollutants and landscape indicators to reveal regional or local patterns and detect unexpected events, data that are crucial for regulation and conservation of ecosystem health.

High-Resolution Transcriptomic Landscape of the Human Submandibular Gland.

Saliva-secreting and transporting cells are part of the complex cellular milieu of the human salivary gland, where they play important roles in normal glandular physiology and diseased states. However, comprehensive molecular characterization, particularly at single-cell resolution, is still incomplete, in part due to difficulty in procuring normal human tissues. Here, we perform an in-depth analysis of male and female adult human submandibular gland (SMG) samples by bulk RNA sequencing (RNA-seq) and examine the molecular underpinnings of the heterogeneous cell populations by single-cell (sc) RNA-seq. Our results from scRNA-seq highlight the remarkable diversity of clusters of epithelial and nonepithelial cells that reside in the SMG that is also faithfully recapitulated by deconvolution of the bulk-RNA data sets. Our analyses reveal complex transcriptomic heterogeneity within both the ductal and acinar subpopulations and identify atypical SMG cell types, such as mucoacinar cells that are unique to humans and ionocytes that have been recently described in the mouse. We use CellChat to explore ligand-receptor interactome predictions that likely mediate crucial cell-cell communications between the various cell clusters. Finally, we apply a trajectory inference method to investigate specific cellular branching points and topology that offers insights into the dynamic and complex differentiation process of the adult SMG. The data sets and the analyses herein comprise an extensive wealth of high-resolution information and a valuable resource for a deeper mechanistic understanding of human SMG biology and pathophysiology.

Evaluation of digestive tolerance of a soluble corn fibre.

BACKGROUND: To assess consumers' acceptance of a new fibre, it is essential to evaluate its digestive tolerance after ingestion. We aimed to determine the tolerance of increasing dosages of Promitor™ Soluble Gluco Fibre (SGF; Tate&Lyle, Hoffman Estates, IL, USA) up to 70 g fibre per day using a validated gastrointestinal composite score. METHODS: A composite score of gastrointestinal tolerance integrating gastrointestinal symptoms, stool frequency and consistency was applied. To statistically validate this composite score, the gastrointestinal tolerance of inulin (10 g versus 20 g containing, respectively, 9 g versus 18 g of fibre) was assessed in 18 healthy volunteers in a randomised double-blind placebo-controlled cross-over study. Second, in a double-blind placebo-controlled cross-over study with 20 healthy volunteers, the gastrointestinal tolerance of SGF in both acute and 'spread over the day' conditions of consumption was assessed. RESULTS: By contrast to 10 g, 20 g of inulin demonstrated a significant difference in composite score compared to placebo [P < 0.001, difference = 7.6; 95% confidence interval (CI) = 3.8-11.3]. These values were considered as reference during the second study. In acute conditions, 40 g of SGF fibre was the highest (threshold) dose tested that indicates the digestive tolerance criteria (difference from placebo on the composite score <7.6 and upper limit of the 95% CI <11.3); this is twice the amount tolerated for inulin. In 'spread over the day' conditions, 65 g of SGF fibre was the threshold dose (P < 0.001, difference = 6.5; 95% CI = 3.4-9.5). CONCLUSIONS: The results of the present study demonstrate that 40 g of SGF fibre, when consumed as a single dose, and 65 g of SGF fibre, when consumed in multiple-doses, across the day are well-tolerated by healthy volunteers.

A Global Vista of the Epigenomic State of the Mouse Submandibular Gland.

The parotid, submandibular, and sublingual glands represent a trio of oral secretory glands whose primary function is to produce saliva, facilitate digestion of food, provide protection against microbes, and maintain oral health. While recent studies have begun to shed light on the global gene expression patterns and profiles of salivary glands, particularly those of mice, relatively little is known about the location and identity of transcriptional control elements. Here we have established the epigenomic landscape of the mouse submandibular salivary gland (SMG) by performing chromatin immunoprecipitation sequencing experiments for 4 key histone marks. Our analysis of the comprehensive SMG data sets and comparisons with those from other adult organs have identified critical enhancers and super-enhancers of the mouse SMG. By further integrating these findings with complementary RNA-sequencing based gene expression data, we have unearthed a number of molecular regulators such as members of the Fox family of transcription factors that are enriched and likely to be functionally relevant for SMG biology. Overall, our studies provide a powerful atlas of cis-regulatory elements that can be leveraged for better understanding the transcriptional control mechanisms of the mouse SMG, discovery of novel genetic switches, and modulating tissue-specific gene expression in a targeted fashion.

Hyaluronidase-sensitive halos around adherent cells. Their role in blocking lymphocyte-mediated cytolysis.

A variety of adherent sarcoma, carcinoma and normal cells are surrounded in vitro by thick, transparent zones (approximately equal to 9 micron thick) that spleen cells and a variety of other cells and particles cannot penetrate. Seven lymphoblastoid cell lines did not possess such halos. The presence of these halos around adherent fibrosarcoma cells appeared to protect them from lymphocyte-mediated cytolysis. Hyaluronidase treatment, which destroyed the halo and allowed lymphocytes to approach the tumor cell membrane, enhanced the cytotoxic action of immune but not of normal spleen cells. These observations, in addition to highlighting a little-known feature of the cell surface, may also be of general relevance to the in vitro and in vivo killing of tumor cells by immune effector cells.

Developmental and communicative interrelations of Ly123 and Ly1 cell sets.

To distinguish and define the differentiative and communicative relations of Ly123 and Ly1 cells in generating specific helper-effector (HE) (Ly1:HE) and specific suppression-inducing (SI) (Ly1:SI) cells, these two functional sets were generated from various combinations of congenic genetically marked sets of cortisone-resistant nylon-purified thymocytes (CRNPT) by culture on antigen-primed macrophages (M phi) (the (T-M phi culture system). It was thus shown that Ly1:HE and Ly1:SI cells are produced by differentiation from antecedent Ly123 cells. Ly1:HE and Ly1:SI are separate Ly1 populations; generationof Ly1:HE cells requires the presence of Ly1 cells, whereas the generation of Ly1:SI cells does not. Although the Ly23 CRNPT set, which is included when Ly123 cells are positively selected with Lty-2 antiserum is ruled out as a precursor source of Ly1:SI cells, the possibility of a communicative role for Ly23 cells in generating Ly1:SI cells remains to be investigated. The role of the Ly1 set required for the generation of Ly1:HE cells from CRNPT is communicative, not differential; and it is not a precursor source of Ly1:HE or Ly1:SI cells in the CRNPT population. It remains to be seen whether the use of additional phenotypic markers will distinguish subsets of Ly123 and Ly1 cells engaged in these several functions.

Two Ly-2 T helper cell subsets distinguished by Qa-1 phenotype. The priming environment determines whether one or both subsets will be generated.

The Qa-1 cell surface phenotype reportedly distinguishes two Ly-1 T cell subsets conjointly required for T helper effector activity. Ly-1 cells, obtained from several different priming regimens, were negatively selected with anti-Qa-1 plus complement and compared with unselected Ly-1 cells for helper cell activity. Priming isolated T cells on antigen-pulsed macrophages in the absence of B cells favors the generation of the Ly-1:Qa1- subset, which is capable of efficient helper activity in the absence of the Ly-1:Qa-1+ subset. Priming T cells in an environment containing B cells generates both Ly-1:Qa-1- helper effector cells and Ly-1:Qa-1+ cells which contribute to the helper effect. Whether Ly-1:Qa-1+ cells are capable of independent helper activity cannot be determined, and, as such, Ly-1:Qa-1+ cells are more appropriately termed "help associated" rather than "helper effector." Our results assign a membrane phenotype, Qa-1, which distinguishes an Ly-1 help-associated B cell requiring subset in our system and may prove to be a general marker in a number of systems of Ly-1 inducer cell subsets which functionally require or recognize B cells or their products.

T cell development. Precursors and inducer requirements of helper effector and feedback suppression inducer cell sets.

The surface phenotypes and differentiative history of specific helper-effector (HE) and specific feedback suppression-inducer (FBSI) cell sets were further defined in reference to the Qa-1 and I-J marker systems by culture of selected sets of cortisone-resistant nylon-purified thymocytes with antigen on primed macrophages. The generation of Ly-1:HE and Ly-1:FBSI cell sets required, in each case, two initiating sets: a precursor set and a differentiation-inducing set. Precursor sets were distinguished from inducer sets by genetic markers. Accordingly, HE cells, phenotype Ly-1:Qa-1-:I-J-, differentiated from Ly-123:Qa-1- cells in the presence of Ly-1:Qa-1+:I-J+ inducer cells; and FBSI cells, phenotype Ly-1:Qa-1+:I-J+, differentiated from Ly-123:Qa-1- in the presence of Ly-1:Qa-1+:I-J+ inducer cells. The Ly-123:Qa-1-precursors of HE and FBSI cells have been distinguished from one another previously but there is as yet no evidence whether differentiation of these precursor sets requires the same or different Ly-1:Qa-1+:I-J+ inducer sets.

Recognition among mice. Evidence from the use of a Y-maze differentially scented by congenic mice of different major histocompatibility types.

Previous studies of mating preference signified that mice can sense one another's major histocompatibility complex (MHC) types, probably by olfaction. This conclusion has now been substantiated by the use of a Y-maze whose two arms were differentially scented with currents of air conducted through boxes occupied by B6 (H-2b) males and by B6-H-2k congenic males. Four B6 mice, two males and two females, were successfully trained, by water deprivation and reward, to enter the arm scented by B6 or B6-H-2k males. One of the males and one of the females were trained to select the B6-scented arm; the other male and female were trained to select the B6-H-2k-scented arm. Untrained mice showed no MHC discrimination in the maze. The performance of the trained mice in distinguishing between MHC congenic homozygous F2 segregants derived from a cross of B6-H-2k with B6 was as good as their performance in distinguishing the respective inbred strains, thus essentially eliminating alternative and significant additional explanations of MHC-associated sensory discrimination. The data further indicate that chemosensory discrimination of MHC types can be entirely dissociated from sex differences and from the circumstances of mating.

Roseomonas mucosa isolated from bloodstream of pediatric patient.

We report a case of catheter-related bacteremia associated with Roseomonas mucosa isolated from an immunocompromised pediatric patient with a history of multiple episodes of urinary tract infection and bacteremia.

Signal transducer and activator of transcription 3 is a transcriptional factor regulating the gene expression of SALL4.

Both signal transducer and activator of transcription 3 (STAT3) and SALL4 are important in maintaining the pluripotent and self-renewal state of embryonic stem cells. We hypothesized that STAT3, a latent transcriptional factor, may regulate the gene expression of SALL4. In support of this hypothesis, DNA sequence analysis of the SALL4 gene promoter revealed four putative STAT3-binding sites. Using a SALL4-luciferase reporter gene assay, we found that modulation of the STAT3 activity significantly up-regulated the luciferase activity. By chromatin immunoprecipitation, the segment of the SALL4 promoter showing the highest affinity to STAT3 was localized to -366 to -163, in which there was only one putative STAT3 binding site starting at -199. Site-directed mutagenesis of all four putative STAT3-binding sites in the SALL4 promoter significantly reduced its responsiveness to STAT3, although the most dramatic effect was seen at the binding site starting at -199. We further tested the functional relationship between STAT3 and SALL4 using MDA-MB-231, a breast cell line carrying constitutive SALL4 expression and STAT3 activity. Down-regulation of the STAT3 activity using a dominant-negative construct resulted in a significant decrease in the expression of SALL4. To conclude, our data suggest that STAT3 and SALL4 probably cooperate in both physiological and pathological states.

A fully validated simple new method for environmental monitoring by surface sampling for cytotoxics.

A wipe sampling procedure followed by a simple ultra-performance liquid chromatography - tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for simultaneous quantification of six cytotoxic drugs: 5-fluorouracil (5FU), doxorubicin (DOXO), epirubicin (EPI), ifosfamide (IF), cyclophosphamide (CP) and gemcitabine (GEM), as surrogate markers for occupational exposure. After a solid-phase extraction of wiping filter on 10 × 10 cm surface, the separation was performed within 6.5 min, using a gradient mobile phase and the analytes were detected by mass spectrometry in the multiple reaction ion monitoring mode. The method was validated according to the recommendations of the US Food and Drug Administration. The method was linear (r2 > 0.9912) between 2.5 and 200 ng per wiping sample (25 to 2000 pg/cm2) for 5FU, doxorubicin and epirubicin and between 0.2 and 40 ng per wiping sample (2 to 400 pg/cm2) for cyclophosphamide, ifosfamide and gemcitabine. The lower limits of quantification were 2.5 ng (25 pg/ cm2) for 5FU, doxorubicin and epirubicin, and 0.2 ng (2 pg/cm2) for CP, IF and GEM. Within-day and between-day imprecisions were <14.0, 10.6, 11.1, 8.7, 11.2 and 10.9% for 5-fluorouracil, doxorubicin, epirubicin, ifosfamide cyclophosphamide and gemcitabine, respectively. The inaccuracies did not exceed 2.7, 10.9, 1.1, 4.5, 1.6 and 2.9% for the studied molecules, respectively. This new sensitive validated method for surface contamination studies of cytotoxics was successfully applied on different localizations in hospital. This approach is particularly suitable to assess occupational exposure risk to cytotoxic drugs.

Crystal structure of an ephrin ectodomain.

Eph receptor tyrosine kinases and their membrane-associated ligands, the ephrins, are essential regulators of axon guidance, cell migration, segmentation, and angiogenesis. There are two classes of vertebrate ephrin ligands which have distinct binding specificities for their cognate receptors. Multimerization of the ligands is required for receptor activation, and ephrin ligands themselves signal intracellularly upon binding Eph receptors. We have determined the structure of the extracellular domain of mouse ephrin-B2. The ephrin ectodomain is an eight-stranded beta barrel with topological similarity to plant nodulins and phytocyanins. Based on the structure, we have identified potential surface determinants of Eph/ephrin binding specificity and a ligand dimerization region. The high sequence similarity among ephrin ectodomains indicates that all ephrins may be modeled upon the ephrin-B2 structure presented here.

Fibroblast-collagen interactions in the formation of the secondary stroma of the chick cornea.

Fibroblasts invade the primary corneal stroma of the 6-day-old chick embryo eye. The way in which these cells build the secondary stroma has been studied by microscope examination of the stroma during the subsequent 8 Days. Eyes were embedded in low viscosity nitrocellulose, and 30-micrometer tangential sections of cornea were cut and stained with azan (giving blue collagen and red cells). These sections were sufficiently thick to include enough cells and collagen for stromal organization to be visible under Nomarski optics. Three days after invasion, the fibroblasts extend along collagen bundles in the posterior region of the stroma; surprisingly, fibroblasts near the epithelium are more rounded. The collagen itself is organized in orthogonal bundles rather than in sheets. Measurements show that posterior bundles increase in size with time while anterior stroma si similar in diameter to primary stroma. These observations confirm that the epithelium continues to deposit primary stroma up to at least the 14th day. They show, moreover, that fibroblasts deposit collagen fibrils on extant stroma and that the farther a bundle is from the epithelium, and hence the longer the period since it was first laid down, the wider it is likely to be. Analysis of the results and existing data on hyaluronic acid levels in the stroma suggests that Bowman's membrane, the region of anterior stroma that remains uncolonized by cells, is, during this period at least, primary stroma laid down but as yet unswollen.

Collagen substrata for studies on cell behavior.

A simple technique is described for the preparation of collagen substrata containing 0 1% of collagen by weight, in the form of native bundles with a 640 A period, the substrata are similar in these respects to soft-tissue matrices These substrate are hydrated collagen lattices (HCLs) in which the watery milieu is held within a fibrous collagen net mainly by capillary forces. HCLs have been characterized in terms of the course of collagen precipitation and aggregation, ultrastructure, and their stability under various conditions. The ways in which HCLs can be employed as both two- and three-dimensional substrata in cell behavioral studies are illustrated with some preliminary observations on the form, motility, adhesion, and growth of human diploid cells and two lines of malignant cells.