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Background Industry payments made to health care providers can create competing interests. The purpose of this study was to define the overall financial relationships between industry and academic endodontics faculty members, detail any variation in such payment data as related to individual faculty member characteristics and leadership position by institution type, and comment on the potential impacts from conflicts of interest (COIs) created by such relationships. Methods The author identified and characterized academic endodontists from information on their institutional websites. The author obtained reported industry payments from 2013 through 2019 from the Centers for Medicare & Medicaid Services Open Payments database. The author also noted the distributions of academic endodontists and industry payments by institution, academic rank, sex, and residency program director position. The author subjected the data to descriptive and nonparametric analyses. Results Of the 302 academic endodontists included, 240 (80%) accepted reported industry payments totaling $4,260,316.97. Overall, the median of total industry payments for all 302 faculty members was $217.89 (interquartile range [IQR], $34.06-$3,070.00). Among those accepting payments, the median amount was $382.80 (IQR, $110.40-$6,234.00). The top decile of paid academic endodontists received $3,669,291.47 in industry payments (86% of the total), with a median payment of $24,013 (IQR, $17,043-$91,190). Significant sex-associated industry payment differences were seen among the overall faculty and among those with the residency program director position. Conclusions Most academic endodontists accept industry payments. Significant sex differences exist in overall faculty member academic rank distribution, leadership role, and accepted median industry payment amounts. COI issues have the potential to arise among academic endodontists when such industry payments are accepted. Practical Implications Existing sex disparities in academic endodontics within the United States ideally should be acknowledged. COI issues can arise when academic faculty members accept industry payments. Public knowledge of these conflicts could negatively affect individual faculty members, their institutions, and related areas such as academic publishing. Appropriate faculty member COI disclosure, attestation, annual updates, and transparency are important mitigation measures.

OBJECTIVES The author conducted a study to determine the frequency and types of adverse events associated with endodontic ultrasonic devices as reported to the U.S. Food and Drug Administration (FDA) Manufacturer and User Facility Device Experience (MAUDE) database. METHODS Endodontic ultrasonic device-related adverse events reported to the MAUDE database from January 1, 2016, to October 31, 2020, were accessed and reviewed. RESULTS A total of 1258 adverse event reports were submitted to the FDA MAUDE database, as classified under FDA product code ELC (ultrasonic scaler) during the study period. Among these reports, 403 were specific to the dedicated use of the 2 main types of ultrasonic devices used in endodontic treatment: ultrasonic tip devices and irrigation-related devices. Device malfunction-associated events, consisting primarily of device breakage, comprised 393 of the 1258 adverse event reports, whereas the remaining 10 reports were identified as being patient injury-related reports. CONCLUSIONS The frequency, root causes, and economic costs of ultrasonic tip device breakage remain largely unstudied. Ultrasonic endodontic device-related adverse events and patient injuries occurring within clinical practice may be underreported at the present time. Consequently, the risks and ultimate impacts to patients from ultrasonic endodontic device breakage, malfunction and unknown cause-related adverse events, and patient injuries during their clinical usage remain largely unknown at the present time. Eight of the 10 patient injury-related reports made to the FDA MAUDE database during the period under study, containing descriptions of varying degrees of injury severity, were associated with an irrigation-related device.

Introduction Several studies in radiology and medicine have evaluated the satisfaction of search (SOS) error effect in chest radiography, abdominal radiography, osteoradiology, and patients with multiple trauma. No research to date has been published evaluating the possible existence of the SOS error phenomenon made during dental periapical radiograph interpretations. The purpose of the present pilot study was to determine if an SOS error effect exists when dental clinicians interpret periapical radiographs. The null hypothesis was that the detection accuracy will be the same or will improve for the detection of native lesions in the presence of an added abnormality. The alternative hypothesis is that there will be a decrease in detection accuracy for native lesions in the presence of an added abnormality. Methods Six images were selected to be part of the present experiment. One of the 6 images served as the positive control, and another image served as the negative control. Four images, each including a single subtle carious lesion, were selected to represent the experimental images. The single subtle carious lesion present within the 4 experimental radiographs served as the native pathology, and an abnormality such as a periapical radiolucency, resorption, inadequate nonideal root canal obturation material, or recurrent carious lesion was artificially inserted into the image as the added pathology. Thus, the second set of images consisted of the same 4 images containing the native pathology including an added pathology that was inserted into the image using Adobe Photoshop CS6 (Adobe, Inc, San Jose, CA). Purposive sampling was obtained from 16 examiners including residents from endodontics and periodontics as well as alumni and faculty from the Saint Louis University Center for Advanced Dental Education, St Louis, MO. Each observer participated as a subject during 2 time-separated sessions. Each session was separated by a minimum period of 3 months' duration in order to prevent memory bias. Before starting each interpretation session, the participants were given verbal instructions. Subjects were instructed to provide a location (by tooth number), identify, and rate the presence of all suspected pathology using a Likert scale of 1–5 (1: definitely normal, 2: probably normal, 3: possibly abnormal, 4: probably abnormal, and 5: definitely abnormal). In the second session, the radiographs that were initially presented containing only the native lesion were presented again with the added abnormality, and vice versa. The observers' reports and confidence ratings were recorded and analyzed. Ratings of 3–5 were considered as being positive for the presence of pathology. Results A true SOS error occurs when the presence of the native lesion is reported correctly without an added abnormality but is not reported (missed) in the presence of an added abnormality. In our study, a true SOS error occurred in 13 of the 64 interpretation sets (20.31%). There was a total of 64 expected native lesions present within the 4 native images viewed by 16 observers. In the 4 added images, there was a total of 64 expected added findings. In the images containing only native lesions, the observers reported 30 of the 64 expected native lesions. In the images containing an artificially added abnormality, the observers reported 58 of the 64 expected added abnormalities and 25 of the 64 expected native lesions. Observers reported fewer native lesions in the presence of an added abnormality. Conclusions The current investigation demonstrated the existence of the SOS effect during periapical radiographic interpretations. In 20.31% of interpretations, a true SOS error occurred. This study is clinically relevant because it can help clinicians in reducing false-negative errors made during radiographic interpretation, thus preventing misdiagnosis.

Introduction Cone-beam computed tomography (CBCT) machines produce relatively low levels of harmful ionizing radiation, as compared with the computed tomography devices used in medical practices. The Carestream CS9600 CBCT imaging device has been recently introduced into the marketplace, and the manufacturer reports the use of an increased x-ray tube voltage (120 kVp) for the device, along with a reduced patient dose that is achieved using added filtration. Independent dosimetry studies are performed to ensure appropriate radiation exposure dose levels are within recommended safety guidelines.The purpose of this study is to independently evaluate and measure the radiation exposure dose performance parameters of the CS9600 CBCT, including its multiple field of view, exposure settings, and filtration options. Methods A thimble ionization chamber and PMMA phantom were used to characterize dose index using the established SEDENTEXTCT evaluation method. Results The phantom-obtained radiation dose index measures ranged from 0.128782–13.848 milligrays (mGy) for the various scanning options evaluated. The field of view, type of filter used, and phantom size all had a direct impact on the relationship between the experimentally obtained dose index measures and the dose area product values reported by the manufacturer. Conclusions A strong linear correlation was observed between the experimentally obtained dose index measures and the manufacturer-reported dose area product values. The 0.7 mm Cu filter that has been added to the CS9600 reduced the exposure dose index measures even with the x-ray tube kilovoltage peak (kVp) being increased to 120 kVp, as compared with the 0.15 mm Cu filter at 90 kVp.

Dental enamel, the hardest tissue in the human body, is derived from dental epithelial cell ameloblast-secreted enamel matrices. Enamel mineralization occurs in a strictly synchronized manner along with ameloblast maturation in association with ion transport and pH balance, and any disruption of these processes results in enamel hypomineralization. G protein–coupled receptors (GPCRs) function as transducers of external signals by activating associated G proteins and regulate cellular physiology. Tissue-specific GPCRs play important roles in organ development, although their activities in tooth development remain poorly understood. The present results show that the adhesion GPCR Gpr115 (Adgrf4) is highly and preferentially expressed in mature ameloblasts and plays a crucial role during enamel mineralization. To investigate the in vivo function of Gpr115, knockout (Gpr115-KO) mice were created and found to develop hypomineralized enamel, with a larger acidic area because of the dysregulation of ion composition. Transcriptomic analysis also revealed that deletion of Gpr115 disrupted pH homeostasis and ion transport processes in enamel formation. In addition, in vitro analyses using the dental epithelial cell line cervical loop–derived dental epithelial (CLDE) cell demonstrated that Gpr115 is indispensable for the expression of carbonic anhydrase 6 (Car6), which has a critical role in enamel mineralization. Furthermore, an acidic condition induced Car6 expression under the regulation of Gpr115 in CLDE cells. Thus, we concluded that Gpr115 plays an important role in enamel mineralization via regulation of Car6 expression in ameloblasts. The present findings indicate a novel function of Gpr115 in ectodermal organ development and clarify the molecular mechanism of enamel formation.

Tooth development involves the coordinated transcriptional regulation of extracellular matrix proteins produced by ameloblasts and odontoblasts. In this study, whole-genome ChIP-seq analysis was applied to identify the transcriptional regulatory gene targets of Sp6 in mesenchymal cells of the developing tooth. Bioinformatic analysis of a pool of Sp6 target peaks identified the consensus nine nucleotide binding DNA motif CTg/aTAATTA. Consistent with these findings, a number of enamel and dentin matrix genes including amelogenin (Amelx), ameloblastin (Ambn), enamelin (Enam) and dental sialophosphoprotein (Dspp), were identified to contain Sp6 target sequences. Sp6 peaks were also found in other important tooth genes including transcription factors (Dlx2, Dlx3, Dlx4, Dlx5, Sp6, Sp7, Pitx2, and Msx2) and extracellular matrix-related proteins (Col1a2, Col11a2, Halpn1). Unsupervised UMAP clustering of tooth single cell RNA-seq data confirmed the presence of Sp6 transcripts co-expressed with many of the identified target genes within ameloblasts and odontoblasts. Lastly, transcriptional reporter assays using promoter fragments from the Hapln1 and Sp6 gene itself revealed that Sp6 co-expression enhanced gene transcriptional activity. Taken together these results highlight that Sp6 is a major regulator of multiple extracellular matrix genes in the developing tooth.

Link protein is encoded by the Hapln1 gene and is a prototypical protein found in the cartilage matrix. It acts as an important component of the endochondral skeleton during early development. To study its transcriptional regulation, promoter fragments derived from the link protein gene were coupled to the β-galactosidase reporter and used to study in vivo transgene expression in mice. In day 15.5 mouse embryos, a link promoter fragment spanning −1020 to +40 nucleotides demonstrated highly specific β-galactosidase staining of skeletal structures, including the appendicular and axial cartilaginous tissues. Two shorter promoter fragments, spanning −690 to +40 and −315 to +40 nucleotides, demonstrated limb- and genitalia-specific expression resembling that of homeodomain-regulated tissues. Bioinformatic analysis revealed a highly conserved, Hox-like binding site (HLBS) at approximately −220 bp of the promoter, shared by both constructs, which contained the Hox-core consensus sequence TAATTA. Electromobility shift assays demonstrated binding of Hox-B4 recombinant protein to the HLBS, which was eliminated with nucleotide substitutions within the core-binding element. Co-transfection analysis of the HLBS demonstrated a 22-fold transcriptional activation by HoxA9 expression, which was ablated with a substitution within the core HLBS element. Together these findings establish promoter regions within the link protein gene that are important for in vivo expression and identify the potential role of homeodomain-containing proteins in controlling cartilage and limb gene expression.

The development of ectodermal organs, such as teeth, requires epithelial–mesenchymal interactions. Basic helix–loop–helix (bHLH) transcription factors regulate various aspects of tissue development, and we have previously identified a bHLH transcription factor, AmeloD, from a tooth germ cDNA library. Here, we provide both in vitro and in vivo evidence that AmeloD is important in tooth development. We created AmeloD-knockout (KO) mice to identify the in vivo functions of AmeloD that are critical for tooth morphogenesis. We found that AmeloD-KO mice developed enamel hypoplasia and small teeth because of increased expression of E-cadherin in inner enamel epithelial (IEE) cells, and it may cause inhibition of the cell migration. We used the CLDE dental epithelial cell line to conduct further mechanistic analyses to determine whether AmeloD overexpression in CLDE cells suppresses E-cadherin expression and promotes cell migration. Knockout of epiprofin (Epfn), another transcription factor required for tooth morphogenesis and development, and analysis of AmeloD expression and deletion revealed that AmeloD also contributed to multiple tooth formation in Epfn-KO mice by promoting the invasion of dental epithelial cells into the mesenchymal region. Thus, AmeloD appears to play an important role in tooth morphogenesis by modulating E-cadherin and dental epithelial–mesenchymal interactions. These findings provide detailed insights into the mechanism of ectodermal organ development.

Dental epithelial stem cells give rise to four types of dental epithelial cells: inner enamel epithelium (IEE), outer enamel epithelium (OEE), stratum intermedium (SI), and stellate reticulum (SR). IEE cells further differentiate into enamel-forming ameloblasts, which play distinct roles, and are essential for enamel formation. These are conventionally classified by their shape, although their transcriptome and biological roles are yet to be fully understood. Here, we aimed to use single-cell RNA sequencing to clarify the heterogeneity of dental epithelial cell types. Unbiased clustering of 6,260 single cells from incisors of postnatal day 7 mice classified them into two clusters of ameloblast, IEE/OEE, SI/SR, and two mesenchymal populations. Secretory-stage ameloblasts expressed Amel and Enam were divided into Dspp + and Ambn + ameloblasts. Pseudo-time analysis indicated Dspp + ameloblasts differentiate into Ambn + ameloblasts. Further, Dspp and Ambn could be stage-specific markers of ameloblasts. Gene ontology analysis of each cluster indicated potent roles of cell types: OEE in the regulation of tooth size and SR in the transport of nutrients. Subsequently, we identified novel dental epithelial cell marker genes, namely Pttg1, Atf3, Cldn10, and Krt15. The results not only provided a resource of transcriptome data in dental cells but also contributed to the molecular analyses of enamel formation.

Basic-helix-loop-helix (bHLH) transcription factors play an important role in various organs’ development; however, a tooth-specific bHLH factor has not been reported. In this study, we identified a novel tooth-specific bHLH transcription factor, which we named AmeloD, by screening a tooth germ complementary DNA (cDNA) library using a yeast 2-hybrid system. AmeloD was mapped onto the mouse chromosome 1q32. Phylogenetic analysis showed that AmeloD belongs to the achaete-scute complex-like ( ASCL) gene family and is a homologue of ASCL5. AmeloD was uniquely expressed in the inner enamel epithelium (IEE), but its expression was suppressed after IEE cell differentiation into ameloblasts. Furthermore, AmeloD expression showed an inverse expression pattern with the epithelial cell-specific cell–cell adhesion molecule E-cadherin in the dental epithelium. Overexpression of AmeloD in dental epithelial cell line CLDE cells resulted in E-cadherin suppression. We found that AmeloD bound to E-box cis-regulatory elements in the proximal promoter region of the E-cadherin gene. These results reveal that AmeloD functions as a suppressor of E-cadherin transcription in IEE cells. Our study demonstrated that AmeloD is a novel tooth-specific bHLH transcription factor that may regulate tooth development through the suppression of E-cadherin in IEE cells.

Introduction The rapid antibiotic sensitivity test (RAST) is a novel in-office culture and sensitivity system for endodontic infections. The purpose of this research was to validate the RAST system as a viable, in-office alternative to antibiotic sensitivity testing using turbidity to determine antibiotic sensitivities of endodontic infections. Methods Aspirates were taken from the root canals of 9 necrotic human teeth at the initiation of root canal therapy. These samples were cultured in the RAST medium, and antibiotic sensitivity to 6 antibiotics was tested. Further analysis was performed using 16S ribosomal RNA (rRNA) gene sequencing. Results Thirty-one bacterial phyla were identified as well as 2 phyla of the kingdom Archaea. Augmentin (Dr. Reddy's Laboratories Ltd, Hyderabad, India) and ampicillin performed identically at 24 hours, inhibiting turbidity in 100% of the samples. At 48 hours in anaerobic conditions, Augmentin outperformed ampicillin by 13%. Ciprofloxacin was the least efficacious antibiotic. At 48 hours, only 22% of anaerobic ciprofloxacin cultures affectively inhibited bacterial growth. Conclusions The RAST medium is a viable in-office alternative to antibiotic susceptibility testing in an off-site laboratory. It is able to support the growth of a wide variety of microorganisms in both aerobic and anaerobic environments, and, in combination with 16S rRNA gene sequencing, it led to the identification of a new archaebacterial phylum, Crenarchaeota, as part of the endodontic infection microbiome.

The common way of speaking of patristic thought is as theology. Disuse of the appellation ‘patristic philosophy’ is the result of separationist taxonomies in both philosophy and theology. Returning to the meanings of the terms theologia and philosophia in ancient and late ancient thought, this paper argues, with an eye toward Orthodox thought, for the reasonableness of speaking of patristic thought as philosophy.

Introduction: The blood-brain barrier (BBB) breakdown occurs when the integrity of BBB components is lost as a consequence of ischemic stroke. Perlecan, a major heparan sulfate proteoglycan of basement membranes, is expressed by endothelial cells (ECs) and is adjacent to pericytes (PCs), suggesting supportive functions in the BBB. Recent studies highlight the importance of PCs in the process of repairing BBB functions, which are triggered by the upregulation of platelet-derived growth factor receptor β (PDGFRβ). We hypothesized that perlecan may play a protective role in BBB maintenance through the interaction with PCs during the repair process of the BBB disruption. Methods: We induced a 60-minute middle cerebral artery occlusion (MCAO) in adult conditional perlecan -deficient ( Perlecan -/- -Tg) mice in a C57BL/6 background, which express the perlecan transgene only in cartilage to rescue the perinatal lethality of Perlecan -/- mice. Recombinant perlecan C-terminal domain V (DV) was used for in vitro assays. Results: Perlecan -/- -Tg mice exhibited larger infarct volumes and more BBB leakage than the control mice on post-surgery day (PSD) 2 after MCAO. While the control mice showed increased numbers of PDGFRβ-positive PCs around the ischemic lesion on PSD 3, such upregulation was not observed in Perlecan -/- -Tg mice, suggesting that perlecan may participate in PC activation against the BBB breakdown. In wild-type mice, the expression of integrin α5, a potential receptor for perlecan, was upregulated both in PCs and in ECs in the ischemic lesion. In culture, PCs showed attachment activity to DV through integrin α5β1. DV induced well-assembled actin stress fibers and focal adhesions in PCs and promoted the PC migration induced by PDGF-BB. At molecular level, DV enhanced the PDGF-BB-induced phosphorylation of PDGFRβ, in addition to that of SHP-2 and FAK, suggesting that DV enhanced both PDGFRβ and integrin signaling. Conclusions: These results revealed that perlecan regulates the activation of PCs through the cooperative function of PDGFRβ and integrin α5β1, contributing to the repair process of the BBB. Perlecan DV may provide a potential therapeutic effect, based on a new approach from extracellular matrix to the disrupted BBB in ischemic stroke.

Introduction: The disruption of the blood-brain barrier (BBB) is a contributing factor for the deterioration of brain damages in ischemic stroke. Recent studies revealed that microvascular pericytes are involved in the maintenance of the BBB and in the repair process through platelet-derived growth factor receptor β (PDGFRβ), the expression of which is increased around the ischemic lesion. Here we focus on perlecan, the major heparan sulfate proteoglycan in the basement membrane (BM). It is expressed by endothelial cells in the brain and is implicated in many biological functions. In this report, we have studied the role of Perlecan in the BBB breakdown and in the subsequent repair process after ischemic stroke in a mouse model. We hypothesized that perlecan may play a protective role in the disruption of BBB through the interaction with pericytes after ischemic stroke. Methods: To elucidate the role of perlecan in the brain vasculature, we induced a 60-minute transient middle cerebral artery occlusion (MCAO) in adult conditional perlecan -deficient ( Perlecan -/- -Tg) mice, which express the perlecan transgene only in the cartilage to rescue the perinatal lethality of perlecan -deficient mice. Results: Although the BBB formation and function in the brain vasculature appeared to be unaffected in Perlecan -/- -Tg mice under healthy condition, Perlecan -/- -Tg mice demonstrated larger infarct volumes and more BBB leakage than control mice on post-surgery day (PSD) 2 after MCAO. Perlecan -/- -Tg mice exhibited less PDGFRβ-positive pericytes around the ischemic lesion on PSD 3 to 7 than control mice, suggesting that the perlecan deficiency suppressed pericyte activation. At a mechanistic level, integrin α5, a potential receptor for perlecan, was detectable in both endothelial cells and pericytes in the ischemic lesion, suggesting that endothelial cell-derived perlecan may regulate pericyte activation in response to ischemia through integrin α5. Conclusions: Our results suggest that perlecan is required for the activation of pericytes and thereby, contributing to the endothelial cell-pericyte integrity in the BBB maintenance after ischemic stroke.

Pseudo-Dionysius’ first principle is hyperousios. By definition, that concept is not theistic. In his oeuvre, however, Pseudo-Dionysius promotes Trinitarianism. A majority of Pseudo-Dionysius’ interpreters have maintained that these concepts are compatible. This article makes a case for the incoherence of that position.

Mineral trioxide aggregate (MTA) has been found to be very biocompatible in a large number of studies. However, the handling properties can be challenging and research on modified materials to enhance placement are few. The purpose of this study is to compare a new faster setting, and more easily placed preparation with classic MTA in an animal model. Canine premolar teeth from two dogs were randomized and received quadrant surgery timed to allow 50 day and 98 day comparisons. Histologic and radiographic comparisons were made. Results were essentially equal in healing, even close to the materials.

This essay is an interdisciplinary study of beauty that attempts to bridge the gap between religion/theology and science in some measure by drawing from Dionysius the Areopagite (c. 500) a notion of being that I argue is consonant with the notion of the sense of the beautiful, which I develop using Steven Weinberg's and Werner Heisenberg's discussions of empirical beauty. I use the term ikon to refer concisely to Dionysius' theophanic notion of being, namely, that the beyond-being is nonsubstantially present in being.

Background There is an urgent need for a simple and accurate test for the diagnosis of human Mycobacterium tuberculosis, the infectious agent causing tuberculosis (TB). Here we describe a serological test based on light emitting recombinant proteins for the diagnosis of pulmonary Mycobacterium tuberculosis infection. Methods Luciferase Immunoprecipitation Systems (LIPS), a fluid-phase immunoassay, was used to examine antibody responses against a panel of 24 different M. tuberculosis proteins. Three different strategies were used for generating the constructs expressing the recombinant fusion M. tuberculosis proteins with luciferase: synthetic gene synthesis, Gateway recombination cloning, and custom PCR synthesis. A pilot cohort of African pulmonary TB patients was used for initial antibody screening and confirmatory studies with selected antigens were performed with a cohort from Thailand and healthy US blood donors. In addition to testing M. tuberculosis antigens separately, a mixture that tested seven antigens simultaneously was evaluated for diagnostic performance. Results LIPS testing of a pilot set of serum samples from African pulmonary TB patients identified a potential subset of diagnostically useful M. tuberculosis antigens. Evaluation of a second independent cohort from Thailand validated highly significant antibody responses against seven antigens (PstS1, Rv0831c, FbpA, EspB, bfrB, HspX and ssb), which often showed robust antibody levels up to 50- to 1000-fold higher than local community controls. Marked heterogeneity of antibody responses was observed in the patients and the combined results demonstrated 73.5 % sensitivity and 100 % specificity for detection of pulmonary TB. A LIPS test simultaneously employing the seven M. tuberculosis antigen as a mixture matched the combined diagnostic performance of the separate tests, but showed an even higher diagnostic sensitivity (90 %) when a cut-off based on healthy US blood donors was used. Conclusion A LIPS immunoassay employing multiple M. tuberculosis antigens shows promise for the rapid and quantitative serological detection of pulmonary TB. Electronic supplementary material The online version of this article (doi:10.1186/s12866-015-0545-y) contains supplementary material, which is available to authorized users.

Quantitative humoral profiling of recent samples from a human immunodeficiency virus (HIV)-infected adult who was cured following a delta32/delta32 CCR5 stem cell transplant in 2007 revealed no antibodies against p24, matrix, nucleocapsid, integrase, protease, and gp120, but low levels of antibodies against reverse transcriptase, tat, and gp41. Antibody levels to these HIV proteins persisted at high and stable levels in most noncontrollers, elite controllers, and antiretroviral-treated subjects, but a rare subset of controllers had low levels of antibodies against matrix, reverse transcriptase, integrase, and/or protease. Comprehensive HIV antibody profiles may prove useful for monitoring curative interventions.

Although HIV-positive patients are at higher risk for developing a variety of infection-related cancers, the prevalence of infections with the seven known cancer-associated viruses has not been studied. Luciferase immunoprecipitation systems were used to evaluate antiviral antibodies in four 23-person groups: healthy blood donors and HIV-infected patients with oral hairy leukoplakia (OLP), Kaposi's sarcoma (KS), or non-Hodgkin lymphoma (NHL). Antibody profiling revealed that all HIV-positive individuals were strongly seropositive for anti-gp41 and antireverse transcriptase antibodies. However, anti-p24 HIV antibody levels were highly variable and some OLP and KS patients demonstrated weak or negative responses. Profiling two EBV antigens revealed no statistical difference in antibody levels among the three HIV-infected groups. A high frequency of KSHV infection was detected in HIV patients including 100% of KS, 78% of OLP, and 57% of NHL patients. Most HIV-infected subjects (84%) showed anti-HBV core antibodies, but only a few showed antibodies against HCV. MCV seropositivity was also common (94%) in the HIV-infected individuals and KS patients showed statistically higher antibody levels compared to the OLP and NHL patients. Overall, 68% of the HIV-infected patients showed seropositivity with at least four cancer-associated viruses. Antibody profiles against these and other infectious agents could be useful for enhancing the clinical management of HIV patients. © 2012 Peter D. Burbelo et al.

Patients with pseudoachondroplasia have a skeletal dysplasia with marked short stature. The most common cause of this condition is an autosomal dominant mutation, although autosomal recessive inheritance has been reported. Linkage to 2 cartilage-specific candidate genes, type II collagen (COL2A1) and proteoglycan link protein genes (CRTL1), was tested in 9 autosomal dominant families with pseudoachondroplasia. Tight linkage to these candidate genes was excluded with LOD scores for COL2A1 of −2.45 at θ= 0.05 and for CRTL1 of −7.28 at θ= 0.001. Discordant inheritance of the disease phenotype with each of these genes was also observed. Thus, these 2 candidate genes can be excluded as the cause of disease in these families. © Wiley-Liss, Inc.

The type II collagen gene (Col2a1) is expressed primarily in chondrocytes. Transcription of Col2a1 is mediated by cell-specific regulatory elements located within the promoter and first intron. Here, we map a minimal enhancer and identify elements that determine cartilage-specific Col2a1 expression by analyzing the activity of a series of chimeric genes consisting of rat Col2a1 first intron deletion mutants ligated to the chloramphenicol acetyltransferase reporter gene. We show that a 100-base pair (bp) segment within the first intron is the minimum size necessary for high level, cell type-specific expression of Col2a1. Sequence analysis of this 100-bp Col2a1 enhancer revealed several sequence motifs similar to motifs present within the regulatory region of the link protein gene, another cartilage gene. These motifs include an AT-rich element, a C1 motif and a C3 motif. Deletion of any of these elements reduced Col2a1 enhancer activity in chick embryo chondrocytes. We also tested enhancer-mediated activity in CFK2 cells which differentiate to a chondrogenic phenotype and begin to express type II collagen mRNA after extended culture. In stably transfected CFK2 cells, constructs containing the 100-bp enhancer were activated during the transition from prechondrogenic to chondrogenic cell populations and deletions within the enhancer strongly down-regulated activity. Chondrocyte-specific DNA-protein complexes were identified using nuclear extracts prepared from chick embryo chondrocytes and 32P-labeled oligonucleotides from these regions of the first intron. These results suggest that interaction of chondrocyte specific nuclear factors with multiple core elements from a small region within the first intron are important for cell-type specific Col2a1 enhancer activity.

The cartilage matrix is composed of characteristic components including type II collagen, aggrecan and link protein. In this paper, we report two DNA elements that regulate the link protein gene. Using transient transfection assays with link protein gene constructs in chondrocytes, chloramphenicol acetyl transferase (CAT) assays were used to measure the transcriptional activity of the link protein gene. Previously, we identified an enhancer-like activity within the first intron of the gene. In this paper, we report an active 34 bp (+1390 to +1424) fragment within this region that contains a glucocorticoid-like response element (GRE). Both deletion of, and site-specific mutations within this sequence motif reduced the dexamethasone-inducible activity. The GRE-like sequence from the rat link protein gene, or the homologous sequence from the human link protein gene were included in vectors containing the thymidine kinase promoter linked to the CAT gene (tkCAT). Both human and rat elements transferred the ability to respond to dexamethasone and hydrocortisone with a > 10-fold induction. Deletions through the promoter from -923 to -900 identified a second site required for both glucocorticoid and serum responsiveness. A four base substitution at this site resulted in a loss of serum responsiveness. This region contains an AT-rich element, similar to the AT-rich elements involved in homeotic protein regulation of the growth hormone gene and the muscle creatine kinase gene. Southwestern analysis using oligonucleotides containing the AT-rich element from the link protein gene or the muscle creatine kinase gene, identified a 32 kDa protein band from nuclear extracts of chick chondrocytes. Using these AT-rich oligonucleotides in band-shift analyses, nuclear extracts of chick sternal muscle, rat chondrosarcoma and chick sternal chondrocytes each showed formation of different complexes suggesting cell specificity. AT-rich elements have been identified as binding sites for homeodomain-containing proteins and can contribute to gene regulation by serum response factors. The identification of an AT-rich element in the link protein gene suggests similar functions for this element.

Publisher Summary This chapter discusses the molecular biology of cartilage matrix. Cartilage is a highly specialized connective tissue with distinct morphological and biochemical characteristics. It is characterized by isolated round-shaped chondrocytes surrounded by extensive areas of extracellular matrix. Chondrocytes are differentiated from other mesenchymal cells by the production and secretion of large amounts of cartilage-specific matrix components. Transcription of the collagen type II gene can be modulated by exogenous factors that are present in serum. In a study discussed in the chapter, three steroid hormones were found to affect transcriptional activity of the collagen type II gene. Dexamethasone and retinoic acid inhibited the collagen type II promoter enhancer mediated CAT activity, whereas vitamin D increased the activity. Because these steroid hormones modulate the transcriptional activity through receptor-mediated DNA binding, these receptors may be directly binding to the sequences of the collagen type II gene. Hormones and vitamins have a role in cartilage expression and maturation. Interaction of these factors with specific receptors can mediate transcriptional activity through direct binding of the complex to regulatory sequences of cartilage genes.

We have isolated the 5'-end of the gene for the rat and human link protein by screening genomic libraries with oligonucleotides corresponding to the 5'-cDNA sequence. Several overlapping clones were isolated for the human link protein gene, while only one clone was obtained for the rat. All the clones contained a single exon of which the sequence was identical to the most 5'-end of the rat and human cDNAs. Transcription initiation sites for the rat link gene were identified by primer extension and S1 protection analysis using total RNA from the rat Swarm chondrosarcoma. Transcriptional initiation sites for the human link gene were determined by specific primer extension of RNA from human fetal cartilage. Comparison of 1500 bp of 5'-flanking sequence between the rat and human link protein genes showed strong sequence conservation near the start site of transcription with 80% overall identity. Analysis of the 5'-flanking regions also revealed a large inverted repeat consisting of repeating purine-pyrimidine, which has the potential to form left-handed Z-DNA. Transcriptional regulation of the link protein gene was studied by coupling either 7.0 kb or 0.85 kb of 5'-flanking rat DNA to the chloramphenicol acetyltransferase (CAT) gene followed by transfection into chick embryonic chondrocytes (CEC) and HeLa cells. Both constructs had considerable CAT activity in CEC cells and less activity in HeLa cells. Furthermore, inclusion of a DNA fragment from the first intron increased relative CAT activity in both of these cell types. The increased activity from the first intron was shown to be orientation independent in CEC. These results indicate the presence of positive cisacting regulatory elements in both the promoter and first intron of the rat gene for link protein.

Previously we isolated a cDNA clone which encoded the carboxyl-terminal portion of the rat link protein (Doege, K., Hassell, J.R., Caterson, B., and Yamada, Y. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 3761-3765). Here we report the isolation of cDNA clones encoding the remainder of the protein. Sequence analysis revealed two classes of cDNA clones identical except for the occurrence of an additional internal nucleotide segment of 159 base pairs coding for 53 amino acids in one of the clones. Genomic clones were then isolated using the cDNA as a probe. Sequence analysis of genomic clones revealed that the internal 159-base pair cDNA segment was contained in a single exon. The protein sequence deduced from these two cDNAs were Mr = 44,779 (link 45) and Mr = 38,570 (link 39). Northern blotting of chondrosarcoma RNA revealed that both the alternate exon specific to link 45 and an exon common to both link 45 and 39 hybridized to the same size classes of link protein transcripts (1.5-5.5 kilobases), although link 45 transcripts are much less prevalent than link 39 transcripts. The link 39 sequence corresponds exactly with the predominant form of chondrosarcoma link protein. The similarity in size between link 45 and the larger form of link protein (LP1) observed in other species appears fortuitous.