[Advancements and current research in orofacial tissue regeneration].

The orofacial system, an intricate assembly of diverse tissues that underpin the unique biologic and morphological identity of an individual, presents a formidable challenge in the realm of tissue regeneration within oral and maxillofacial surgery. This review conducts a retrospective appraisal of advancements in the field of orofacial tissue regeneration, elucidating the current research landscape while critically addressing the persisting challenges. It underscores the pivotal roles of orofacial mesenchymal stem cells in orchestrating regenerative processes, offering an insightful outlook on future developments. The objective is to demarcate innovative therapeutic avenues and clinical implications by fostering a comprehensive understanding of this domain among dental practitioners. As such, it aspires to serve as a valuable reference for prospective investigations and to elevate the knowledge base pertaining to orofacial tissue regeneration.

Thermally-assisted milling and hydrogenolysis for synthesizing ultrafine MgH2with destabilized thermodynamics.

A novel process has been developed to synthesize MgH2nanoparticles by combining ball milling and thermal hydrogenolysis of di-n-butylmagnesium (C4H9)2Mg, denoted as MgBu2. With the aid of mechanical impact, the hydrogenolysis temperature of MgBu2in heptane and cyclohexane solution was considerably lowered down to 100 °C, and the MgH2nanoparticles with an average particle size ofca.8.9 nm were obtained without scaffolds. The nano-size effect of the MgH2nanoparticles causes a notable decrease in the onset dehydrogenation temperature of 225 °C and enthalpy of 69.78 kJ mol-1 · H2. This thermally-assisted milling and hydrogenolysis process may also be extended for synthesizing other nanomaterials.

Visible red and infrared light alters gene expression in human marrow stromal fibroblast cells.

OBJECTIVES: This study tested whether or not gene expression in human marrow stromal fibroblast (MSF) cells depends on light wavelength and energy density. MATERIALS AND METHODS: Primary cultures of isolated human bone marrow stem cells (hBMSC) were exposed to visible red (VR, 633 nm) and infrared (IR, 830 nm) radiation wavelengths from a light emitting diode (LED) over a range of energy densities (0.5, 1.0, 1.5, and 2.0 Joules/cm2) Cultured cells were assayed for cell proliferation, osteogenic potential, adipogenesis, mRNA and protein content. mRNA was analyzed by microarray and compared among different wavelengths and energy densities. Mesenchymal and epithelial cell responses were compared to determine whether responses were cell type specific. Protein array analysis was used to further analyze key pathways identified by microarrays. RESULT: Different wavelengths and energy densities produced unique sets of genes identified by microarray analysis. Pathway analysis pointed to TGF-beta 1 in the visible red and Akt 1 in the infrared wavelengths as key pathways to study. TGF-beta protein arrays suggested switching from canonical to non-canonical TGF-beta pathways with increases to longer IR wavelengths. Microarrays suggest RANKL and MMP 10 followed IR energy density dose-response curves. Epithelial and mesenchymal cells respond differently to stimulation by light suggesting cell type-specific response is possible. CONCLUSIONS: These studies demonstrate differential gene expression with different wavelengths, energy densities and cell types. These differences in gene expression have the potential to be exploited for therapeutic purposes and can help explain contradictory results in the literature when wavelengths, energy densities and cell types differ.

Viral and cellular proteins involved in coronavirus replication.

As the largest RNA virus, coronavirus replication employs complex mechanisms and involves various viral and cellular proteins. The first open reading frame of the coronavirus genome encodes a large polyprotein, which is processed into a number of viral proteins required for viral replication directly or indirectly. These proteins include the RNA-dependent RNA polymerase (RdRp), RNA helicase, proteases, metal-binding proteins, and a number of other proteins of unknown function. Genetic studies suggest that most of these proteins are involved in viral RNA replication. In addition to viral proteins, several cellular proteins, such as heterogeneous nuclear ribonucleoprotein (hnRNP) A1, polypyrimidine-tract-binding (PTB) protein, poly(A)-binding protein (PABP), and mitochondrial aconitase (m-aconitase), have been identified to interact with the critical cis-acting elements of coronavirus replication. Like many other RNA viruses, coronavirus may subvert these cellular proteins from cellular RNA processing or translation machineries to play a role in viral replication.

Hepatitis C viral RNA: challenges and promises.

Hepatitis C virus (HCV), a positive-sense, single-stranded RNA virus of the Flaviviridae family, is a major cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma worldwide. Its RNA is difficult to study because biological materials are scarce and RNA replication is of low efficiency. This review focuses on the structure and functions of HCV RNA along with their biological and clinical significance. Despite the challenging characteristics of HCV, significant progress has been made in understanding the properties of HCV RNA and developing viral replication systems toward the improvement of antiviral therapies.

Heterogeneous nuclear ribonucleoprotein A1 regulates RNA synthesis of a cytoplasmic virus.

Heterogeneous nuclear ribonucleoprotein (hnRNP A1) is involved in pre-mRNA splicing in the nucleus and translational regulation in the cytoplasm. In the present study, we demonstrate that hnRNP A1 also participates in the transcription and replication of a cytoplasmic RNA virus, mouse hepatitis virus (MHV). Overexpression of hnRNP A1 accelerated the kinetics of viral RNA synthesis, whereas the expression in the cytoplasm of a dominant-negative hnRNP A1 mutant that lacks the nuclear transport domain significantly delayed it. The hnRNP A1 mutant caused a global inhibition of viral mRNA transcription and genomic replication, and also a preferential inhibition of the replication of defective-interfering RNAs. Similar to the wild-type hnRNP A1, the hnRNP A1 mutant complexed with an MHV polymerase gene product, the nucleocapsid protein and the viral RNA. However, in contrast to the wild-type hnRNP A1, the mutant protein failed to bind a 250 kDa cellular protein, suggesting that the recruitment of cellular proteins by hnRNP A1 is important for MHV RNA synthesis. Our findings establish the importance of cellular factors in viral RNA-dependent RNA synthesis.

Hepatitis C virus and interferon resistance.

Interferon plays a critical role in the host's natural defense against viral infections and in their treatment. It is the only therapy for hepatitis C virus (HCV) infection; however, many virus isolates are resistant. Several HCV proteins have been shown to possess properties that enable the virus to evade the interferon-mediated cellular antiviral responses.

Hepatitis C virus RNA polymerase and NS5A complex with a SNARE-like protein.

Hepatitis C virus (HCV) NS5A is a phosphoprotein that possesses a cryptic trans-activation activity. To investigate its potential role in viral replication, we searched for the cellular proteins interacting with NS5A protein by yeast two-hybrid screening of a human hepatocyte cDNA library. We identified a newly discovered soluble N-ethylmaleimide-sensitive factor attachment protein receptor-like protein termed human vesicle-associated membrane protein-associated protein of 33 kDa (hVAP-33). In vitro binding assay and in vivo coimmunoprecipitation studies confirmed the interaction between hVAP-33 and NS5A. Interestingly, hVAP-33 was also shown to interact with NS5B, the viral RNA-dependent RNA polymerase. NS5A and NS5B bind to different domains of hVAP-33: NS5A binds to the C-terminus, whereas NS5B binds to the N-terminus of hVAP-33. Immunofluorescent staining showed a significant colocalization of hVAP-33 with both NS5A and NS5B proteins. hVAP-33 contains a coiled-coil domain followed by a membrane-spanning domain at its C-terminus. Cell fractionation analysis revealed that hVAP-33 is predominantly associated with the ER, the Golgi complex, and the prelysosomal membrane, consistent with its potential role in intracellular membrane trafficking. These interactions provide a mechanism for membrane association of the HCV RNA replication complex and further suggest that NS5A is a part of the viral RNA replication complex.

Inhibition of the interferon-inducible protein kinase PKR by HCV E2 protein.

Most isolates of hepatitis C virus (HCV) infections are resistant to interferon, the only available therapy, but the mechanism underlying this resistance has not been defined. Here it is shown that the HCV envelope protein E2 contains a sequence identical with phosphorylation sites of the interferon-inducible protein kinase PKR and the translation initiation factor eIF2alpha, a target of PKR. E2 inhibited the kinase activity of PKR and blocked its inhibitory effect on protein synthesis and cell growth. This interaction of E2 and PKR may be one mechanism by which HCV circumvents the antiviral effect of interferon.

Loss of heterozygosity of the Rb gene correlates with pRb protein expression and associates with p53 alteration in human esophageal cancer.

To understand the alterations of Rb tumor suppressor gene and the relationship between defects in the Rb and p53 pathways in human esophageal carcinogenesis, we examined the loss of heterozygosity (LOH) of the Rb gene and immunohistochemical staining of pRb protein in 56 esophageal squamous cell carcinoma specimens and related the results to the p53 gene alterations. Using four introgenic polymorphic markers as probes, we observed LOH of the Rb gene in 30 of the 55 informative tumor samples. Immunohistochemical analysis revealed different patterns of pRb expression among the tumor samples. In the 56 cases, 16 displayed extensive pRb staining comparable to that of the adjacent normal epithelia, whereas 33 showed either significantly decreased or no pRb staining and 7 had a focal staining pattern reflecting heterogeneous cancer nests in the tumor with respect to Rb status. In the tumor samples containing Rb LOH, 90% showed low or no pRb expression, whereas in samples without Rb LOH, only 20% had altered pRb expression. There was a strong association between LOH of the Rb gene and alteration of pRb expression in our samples (P < 0.0001), suggesting LOH is a main event leading to Rb inactivation. We found that Rb LOH was more frequent in tumors with p53 mutations (P < 0.05), which occurred in 31 of the 49 cases analyzed. When the status of Rb and p53 alterations was evaluated by the combined results of immunohistochemical and genetic analyses, we found that alteration of Rb and p53 had an even stronger association in our esophageal squamous cell carcinoma samples (P = 0.0015). Among the 51 cases in which both the Rb and p53 status were determined, 31 contained alterations in both genes, and only 5 and 6 cases were altered in only Rb and only p53, respectively. Our results suggest that defects in the Rb and p53 pathways and their potential synergistic effect in deregulating cell cycle and apoptosis are major mechanisms for esophageal carcinogenesis.

Colocalization and membrane association of murine hepatitis virus gene 1 products and De novo-synthesized viral RNA in infected cells.

Murine hepatitis virus (MHV) gene 1, the 22-kb polymerase (pol) gene, is first translated into a polyprotein and subsequently processed into multiple proteins by viral autoproteases. Genetic complementation analyses suggest that the majority of the gene 1 products are required for viral RNA synthesis. However, there is no physical evidence supporting the association of any of these products with viral RNA synthesis. We have now performed immunofluorescent-staining studies with four polyclonal antisera to localize various MHV-A59 gene 1 products in virus-infected cells. Immunoprecipitation experiments showed that these antisera detected proteins representing the two papain-like proteases and the 3C-like protease encoded by open reading frame (ORF) 1a, the putative polymerase (p100) and a p35 encoded by ORF 1b, and their precursors. De novo-synthesized viral RNA was labeled with bromouridine triphosphate in lysolecithin-permeabilized MHV-infected cells. Confocal microscopy revealed that all of the viral proteins detected by these antisera colocalized with newly synthesized viral RNA in the cytoplasm, particularly in the perinuclear region of infected cells. Several cysteine and serine protease inhibitors, i.e., E64d, leupeptin, and zinc chloride, inhibited viral RNA synthesis without affecting the localization of viral proteins, suggesting that the processing of the MHV gene 1 polyprotein is tightly associated with viral RNA synthesis. Dual labeling with antibodies specific for cytoplasmic membrane structures showed that MHV gene 1 products and RNA colocalized with the Golgi apparatus in HeLa cells. However, in murine 17CL-1 cells, the viral proteins and viral RNA did not colocalize with the Golgi apparatus but, instead, partially colocalized with the endoplasmic reticulum. Our results provide clear physical evidence that several MHV gene 1 products, including the proteases and the polymerase, are associated with the viral RNA replication-transcription machinery, which may localize to different membrane structures in different cell lines.

Role of p53 gene mutations in human esophageal carcinogenesis: results from immunohistochemical and mutation analyses of carcinomas and nearby non-cancerous lesions.

In order to characterize p53 alterations in esophageal cancer and to study their roles in carcinogenesis, we performed gene mutation and immunohistochemical analysis on 43 surgically resected human esophageal specimens, which contain squamous cell carcinoma (SCC) and adjacent non-cancerous lesions, from a high-incidence area of Linzhou in Henan, China. A newly developed immunohisto-selective sequencing (IHSS) method was used to enrich the p53 immunostain-positive cells for mutation analysis. p53 gene mutations were detected in 30 out of 43 (70%) SCC cases. Among 29 SCC cases that were stained positive for p53 protein, 25 (86%) were found to contain p53 mutations. In five cases of SCC with homogeneous p53 staining, the same mutation was observed in samples taken from four different positions of each tumor. In a well differentiated cancer nest, p53 mutation was detected in only the peripheral p53-positive cells. In tumor areas with heterogeneous p53 staining, either the area stained positive for p53 had an additional mutation to the negatively stained area or both areas lacked any detectable p53 mutation. In the p53-positive non-cancerous lesions adjacent to cancer, p53 mutations were detected in seven out of 16 (47%) samples with basal cell hyperplasia (BCH), eight out of 12 (67%) samples with dysplasia (DYS), and six out of seven (86%) samples with carcinoma in situ (CIS). All mutations found in lesions with DYS and CIS were the same as those in the nearby SCC. In seven cases of BCH containing mutations, only three had the same mutations as the nearby SCC. The results suggest that p53 mutation is an early event in esophageal carcinogenesis occurring in most of the DYS and CIS lesions, and cells with such mutations will progress to carcinoma, whereas the role of p53 mutations in BCH is less clear.

Immunohistoselective sequencing (IHSS) of p53 tumor suppressor gene in human oesophageal precancerous lesions.

Accumulation of p53 protein occurs in human oesophageal precancerous lesions and even in near-normal oesophageal epithelium. In some instances, p53 gene mutations have been detected. In many of the cases of p53 protein accumulation in early lesions, however, p53 mutations were not detected due to either the lack of mutation or the low abundance of cells with a mutation. In order to enrich p53 immunostain-positive cells for single strand conformation polymorphism (SSCP) analysis and DNA sequencing, an immunohisto-selective sequencing (IHSS) method was developed. Anti-p53 antibody-peroxidase stained oesophageal tissue sections were subjected to ultraviolet (UV) irradiation to damage the DNA in p53 immunostain-negative cells. The immunostain protected p53 immunostain-positive cells from the UV light and thus preserved the DNA in those cells for PCR amplification. Comparison of the SSCP results from sections with and without UV treatment showed that the IHSS method selectively enriched p53 immunostain-positive cells. With this method, we could analyse mutations in samples with as few as 30 p53 immunostain-positive cells per tissue section. Analysis was carried out on tissues with precancerous lesions from six surgically-resected oesophageal specimens and 13 oesophageal biopsies from symptom-free subjects. The results of mutation analysis for some of the samples were confirmed by microdissection to enrich the p53-positive cells. The mutations in tissues with precancerous lesions were compared with those in the corresponding squamous cell carcinomas. The IHSS method is shown to be a simple and effective way to analyse mutations in p53 immunostain-positive cells. IHSS may also be a general method for molecular analysis of biological specimens after immunohistochemical staining.

Changes in p53 and cyclin D1 protein levels and cell proliferation in different stages of human esophageal and gastric-cardia carcinogenesis.

The objective of this study was to quantify the changes in p53 and cyclin D1 protein levels in different stages of human esophageal and gastric cardia carcinogenesis in a high-risk population in Henan, China. Immunoreactivity of p53, cyclin D1 and proliferating-cell nuclear antigen (PCNA) was observed in the cell nuclei of esophageal and gastric cardia biopsies. The number of p53-immunostaining-positive cells was low in normal epithelia, slightly increased in basal-cell hyperplasia (BCH), markedly increased in dysplasia (DYS) (10-fold), and further increased in squamous-cell carcinoma (SCC) (40-fold). This pattern of change was similar to that of cell proliferation as indicated by PCNA immunostaining. On the other hand, the number of cyclin D1-immunostaining-positive cells did not increase from BCH to DYS, although a slight increase from DYS to SCC was noted. In the gastric cardia, again, the pattern of change of p53-positive cells in different stages of lesions paralleled the pattern of cell proliferation. The number of p53-positive cells was very low, much lower than that of PCNA-positive cells, in normal, chronic superficial gastritis (CSG) and chronic atrophic gastritis (CAG); therefore, the increase of p53-positive cells from CAG to DYS was more dramatic (100-fold). From DYS to adenocarcinoma (AC), the p53-positive and the PCNA-positive cells increased 4-fold. On the other hand, the number of cyclin D1-positive cells did not increase in pre-cancerous lesions, but increased slightly in AC. This study demonstrates that p53 protein accumulation increased with the progression of pre-cancerous lesions, especially in the genesis of dysplasia, both in the esophagus and in the gastric cardia. Our approach of quantitative immunohistochemistry sheds light on the mechanisms of genesis of esophageal and gastric-cardia cancers, which frequently occur together in many high-incidence areas.

Effects of green tea and black tea on 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone bioactivation, DNA methylation, and lung tumorigenesis in A/J mice.

Previous studies in our laboratory showed that decaffeinated green tea and black tea extracts inhibited 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced tumorigenicity in A/J female mice. In order to understand the mechanism of the inhibitory action, we examined the effects of decaffeinated green tea, black tea, and tea components on the metabolic activation of NNK in vitro and in vivo in this animal model. When added to incubation mixtures containing mouse lung microsomes, decaffeinated green tea and black tea extracts and their fractions, at concentrations up to 0.4 mg/ml, inhibited NNK oxidation and NNK-induced DNA methylation. Among the tea components examined, (-)-epigallocatechin-3-gallate was the most potent inhibitor with 50% inhibitory concentrations of about 0.12 mM for both NNK oxidation and DNA methylation. At these concentrations, (-)-epigallocatechin-3-gallate inhibited the catalytic activities of several P450 enzymes and was more potent against P450 1A and 2B1 than 2E1. When decaffeinated green or black tea extracts were given to female A/J mice as the sole source of drinking fluid before an i.p. injection of NNK (100 mg/kg body weight), a statistically significant inhibition of lung DNA methylation, however, was not observed, although a significant reduction in lung tumor multiplicity was observed. The results suggest that, although inhibition of the metabolic activation of NNK and the subsequent DNA alkylation by tea extracts can be demonstrated in vitro, this mechanism may not be important for the inhibitory action of tea against lung tumorigenesis.

Crystallin mRNA concentrations and distribution in lens of normal and galactosemic rats. Implications in development of sugar cataracts.

It is well established that high concentrations of sugar in the lens of the eye eventually lead to fiber cell destruction and cataracts. In these studies the decrease in crystallin mRNAs was quantified as a result of influx of high concentrations of galactose into the lens of rats. The alpha A-, alpha B1-, and gamma-crystallin mRNA concentrations were assessed in normal lens and in lens undergoing development of sugar cataracts by northern blot and in situ hybridization methods. In a normal, 28-day-old lens, alpha A-crystallin mRNA accumulated to high levels throughout the fiberplasm, and alpha B-crystallin mRNA was present at low levels in epithelial cells, with increased expression in elongating epithelial and fiber cells. The beta B1-crystallin mRNA was distributed to about the same grain density throughout the fiberplasm but at significantly lower levels than alpha A-crystallin mRNA. The gamma-crystallin mRNA first emerged in the terminally differentiated fiber cell, with insignificant amounts detected in the elongating epithelial and fiber cells at the bow. Measurements of hybridization levels on the same RNA population isolated from a single lens showed that in the controls, alpha A-crystallin mRNA comprised about ten times the level of alpha B-crystallin mRNA and twice the level of beta B1- and gamma-crystallin mRNAs. In the cataractous lens the rate of decrease in the concentrations of alpha A-, alpha B- and beta B1-crystallin mRNAs was the same; the decrease in gamma-crystallin mRNA was far more severe. By 20 days of feeding of galactose, at the age of 48 days, gamma-crystallin mRNA diminished to about 9% of the control levels, alpha A-crystallin mRNA to 49%, alpha B-crystallin mRNA to 55%, and beta B1-crystallin mRNA to 65%. In the normal lens, at 48 days of age, the levels of alpha A-, alpha B-, and beta B1-crystallin mRNAs showed no significant changes; the gamma-crystallin mRNA level decreased significantly, to about 70% of the day-28 level, the time at which galactose feeding began. Overall, these data suggest that the loss in crystallin mRNAs in response to the development of galactose cataracts follows this order of decline: gamma greater than alpha B greater than alpha A greater than beta B1.

Management of aldose reductase mRNA abundance in rat lens undergoing reversal of galactose induced cataracts. A model for gene response to changes in the environment.

Aldose reductase (AR) mRNA concentration in rat lens was quantitated by hybridization of a RNA transcript from a previously described AR cDNA clone to mRNA found in epithelial and cortical cytosols. This was done on normal rat lens and on lens initially made cataractous by feeding of a diet of Purina Chow containing 50% galactose, followed by reversal of the cataracts due to the removal of the galactose from the diet. Recent data from this laboratory has shown that AR mRNA was increased in lens epithelial cells upon administration of galactose; while in the cortex it was reduced to insignificant levels when fiber cell damage became extensive by day 20 on galactose. Present data reveals that, upon removal of galactose from the diet, the lens epithelial AR mRNA was gradually reduced from the high levels found at day 20 of galactose feeding to low levels by day 30 of reversal. On the other hand, the cortex exhibited an initial sudden increase in AR mRNA at days 1 to 6 of reversal and by day 30 it was reduced to levels below those found in the untreated lens. DNA content in the epithelium also began to decrease to normal levels by day 16 following reversal of the cataracts. The data demonstrate that the concentration of AR mRNA in lens of reversed cataracts appears to faithfully reflect the loss of epithelial cellular need for AR mRNA in favor of enhanced differentiation of epithelial cells to secondary fiber cells.

[Caries inhibiting effect of ammonium fluoride varnish in vitro].

The reaction of incorporation of fluoride into tooth enamel from NH4F varnish, and Duaphat were measured using SEMq2 in vitro. Level of enamel uptake of fluoride was highest in teeth treated with NH4F varnish. Average depth of fluoride penetrated into enamel was more than 80 microns from the two varnishes. Prolonged coating duration from 24 hours to 1 week did not increase uptake and penetration of fluoride from both varnishes. The NH4F varnish was found to be superior to Duraphat in terms of inhibiting artificial caries lesion formation (P less than 0.001).

The dental health status of 6 and 12 year-old Beijing schoolchildren in 1987.

There have been few epidemiological studies in China which have randomly selected subjects for examination. This study randomly selected 240 subjects aged 6 and 12 years from six schools in the Haidian District of Beijing, China. Examiners were calibrated and used standardised light sources and methods. Five per cent of the 6 year old children and 54 per cent of the 12 year old children were caries free. The dental caries treatment needs for 6 year old children were substantially greater in quantity and complexity than the needs of those aged 12 years. School based preventive and treatment programmes directed against the differing requirements of the age groups and supported by community-wide strategies for prevention appear as dental health matters of urgency in China.