Development of a Scoring Rubric Assessing Medical Students' Explanations of Pathology Reports.

CONTEXT.­: With increasing availability of immediate patient access to pathology reports, it is imperative that all physicians be equipped to discuss pathology reports with their patients. No validated measures exist to assess how pathology report findings are communicated during patient encounters. OBJECTIVE.­: To pilot a scoring rubric evaluating medical students' communication of pathology reports to standardized patients. DESIGN.­: The rubric was iteratively developed using the Pathology Competencies for Medical Education and Accreditation Council for Graduate Medical Education pathology residency milestones. After a brief training, third- and fourth-year medical students completed 2 standardized patient encounters, presenting simulated benign and malignant pathology reports. Encounters were video recorded and scored by 2 pathologists to calculate overall and item-specific interrater reliability. RESULTS.­: All students recognized the need for pathology report teaching, which was lacking in their medical curriculum. Interrater agreement was high for malignant report scores (intraclass correlation coefficient, 0.65) but negligible for benign reports (intraclass correlation coefficient, 0). On malignant reports, most items demonstrated good interrater agreement, except for discussing the block (cassette) summary, explaining the purpose of the pathology report, and acknowledging uncertainty. Participating students (N = 9) felt the training was valuable given their limited prior exposure to pathology reports. CONCLUSIONS.­: This pilot study demonstrates the feasibility of using a structured rubric to assess the communication of pathology reports to patients. Our findings also provide a scalable example of training on pathology report communication, which can be incorporated in the undergraduate medical curriculum to equip more physicians to facilitate patients' understanding of their pathology reports.

Overexpression of a novel small auxin-up RNA gene, OsSAUR11, enhances rice deep rootedness.

BACKGROUND: Deep rooting is an important factor affecting rice drought resistance. However, few genes have been identified to control this trait in rice. Previously, we identified several candidate genes by QTL mapping of the ratio of deep rooting and gene expression analysis in rice. RESULTS: In the present work, we cloned one of these candidate genes, OsSAUR11, which encodes a small auxin-up RNA (SAUR) protein. Overexpression of OsSAUR11 significantly enhanced the ratio of deep rooting of transgenic rice, but knockout of this gene did not significantly affect deep rooting. The expression of OsSAUR11 in rice root was induced by auxin and drought, and OsSAUR11-GFP was localized both in the plasma membrane and cell nucleus. Through an electrophoretic mobility shift assay and gene expression analysis in transgenic rice, we found that the transcription factor OsbZIP62 can bind to the promoter of OsSAUR11 and promote its expression. A luciferase complementary test showed that OsSAUR11 interacts with the protein phosphatase OsPP36. Additionally, expression of several auxin synthesis and transport genes (e.g., OsYUC5 and OsPIN2) were down-regulated in OsSAUR11-overexpressing rice plants. CONCLUSIONS: This study revealed a novel gene OsSAUR11 positively regulates deep rooting in rice, which provides an empirical basis for future improvement of rice root architecture and drought resistance.

Natural variation of Alfin-like family affects seed size and drought tolerance in rice.

The Alfin-like (AL) family is a group of small plant-specific transcriptional factors involved in abiotic stresses in dicotyledon. In an early study, we found an AL gene in rice that was associated with grain yield under drought stress. However, little information is known about the AL family in rice. In this study, AL genes in the rice genome were identified, and the OsAL proteins were found to locate in the nucleus and have no transcriptional self-activation activity. The expression of the OsALs was regulated by different environmental stimulations and plant hormones. Association and domestication analysis revealed that natural variation of most OsALs was significantly associated with yield traits, drought resistance and divergence in grain size in indica and japonica rice varieties. Hap1 of OsAL7.1 and Hap7 of OsAL11 were favorable haplotypes of seed weight and germination under osmotic stress. Furthermore, osal7.1 and osal11 mutants have larger seeds and are more sensitive to abscisic acid and mannitol during germination stage. Overexpressing of OsAL7.1 and OsAL11 in rice weakened the tolerance to drought in the adult stage. Thus, our work provides informative knowledge for exploring and harnessing haplotype diversity of OsALs to improve yield stability under drought stress.

Akkermansia muciniphila and its outer membrane protein Amuc_1100 prophylactically attenuate 5-fluorouracil-induced intestinal mucositis.

5-Fluorouracil (5-FU) is a chemotherapy drug used to treat tumors. Previous studies have shown that Akkermansia muciniphila (A. muciniphila) and its outer membrane protein, Amuc_1100, alleviate dextran sodium sulfate (DSS)-induced colitis in mice. We investigated the effects of both A. muciniphila and Amuc_1100 on 5-FU-induced intestinal mucosal damage in mice. C57BL/6 mice were gavaged with A. muciniphila or Amuc_1100 daily before, during, and after 5-FU injection for a total of 14 days. By evaluating diarrheal toxicity scores, body weight changes, colonic anatomy images, and histopathology of intestinal injury in these mice, we found that A. muciniphila and Amuc_1100 alleviated 5-FU-induced intestinal mucositis. Quantitative polymerase chain reaction assays of intestinal cytokine mRNA levels demonstrated that both A. muciniphila and Amuc_1100 attenuated the upregulation of intestinal Tumor Necrosis Factor-α (TNF-α) and interleukin-6 (IL-6) induced by 5-FU treatment. In addition, they both reduced 5-FU-induced the NLR family pyrin domain containing 3 (NLRP3) inflammatory vesicle activation. Furthermore, by monitoring the mRNA expression of tight junction proteins in the intestine, we found that A. muciniphila and Amuc_1100 were capable of restoring the damaged intestinal barrier. Notably, A. muciniphila and Amuc_1100 also played a role in altering the structure of the intestinal microbial community. The present study revealed the protective role of both A. muciniphila and Amuc_1100 in the intestinal mucositis caused by 5-FU, providing new insights into treatment options.

Overexpressing PpBURP2 in Rice Increases Plant Defense to Abiotic Stress and Bacterial Leaf Blight.

Mosses are one of the earliest diverging land plants that adapted to living on land. The BURP domain-containing proteins (BURP proteins) are plant-specific proteins that appeared when plants shifted from aquatic environments to land. Phylogenetic analysis revealed that the BURP domain of higher plants is originated from lower land plants and divergent because of motif conversion. To discover the function of BURP protein in moss, rice transgenics with ectopic expression of PpBURP2 were subjected to different abiotic stresses treatments. The results revealed that the ectopic expression of PpBURP2 enhanced the tolerance to osmotic and saline stresses at the seedling stage and drought stress at the adult stage. Further ectopic expression of PpBURP2 improved the cadmium (2+) (Cd2+) tolerance and reduced Cd2+ accumulation in rice leaves. Transcriptomic analysis of the transgenic PpBURP2 plants showed that the differentially expressed genes were involved in the metabolism of secondary metabolites, energy, oxidation-reduction process, and defense-related genes. Further experiments showed that the photosynthetic efficiency and resistance against bacterial leaf blight were obviously improved in transgenic plants. Yeast two-hybrid and bimolecular fluorescence complementation (BiFC) assays revealed the physical interaction of BURP domain protein from rice and moss with mitogen-activated protein kinase kinase (MKK) from rice. Therefore, our findings demonstrate that overexpressing PpBURP2 in rice confers resistance to abiotic stresses and bacterial leaf blight. They also suggested that the regulatory role of BURP-like proteins across lower and higher plants was evolutionary conservation of responses of different classes of plants to different environmental challenges.

Ubiquitin ligase OsRINGzf1 regulates drought resistance by controlling the turnover of OsPIP2;1.

Water is crucial for plant growth and survival. The transcellular water movement is facilitated by aquaporins (AQPs) that rapidly and reversibly modify water permeability. The abundance of AQPs is regulated by its synthesis, redistribution and degradation. However, the molecular mechanism of proteasomal degradation of AQPs remains unclear. Here, we demonstrate that a novel E3 ligase, OsRINGzf1, mediated the degradation of AQPs in rice. OsRINGzf1 is the candidate gene from a drought-related quantitative trait locus (QTL) on the long arm of chromosome 4 in rice (Oryza sativa) and encodes a Really Interesting New Gene (RING) zinc finger protein 1. OsRINGzf1 possesses the E3 ligase activity, ubiquitinates and mediates OsPIP2;1 degradation, thus reducing its protein abundance. The content of OsPIP2;1 protein was decreased in OsRINGzf1 overexpression (OE) plants. The degradation of OsPIP2;1 was inhibited by MG132. The OsRINGzf1 OE plants, with higher leaf-related water content (LRWC) and lower leaf water loss rate (LWLR), exhibited enhanced drought resistance, whereas the RNAi and knockout plants of OsRINGzf1 were more sensitive to drought. Together, our data demonstrate that OsRINGzf1 positively regulates drought resistance through promoting the degradation of OsPIP2;1 to enhance water retention capacity in rice.

A natural allele of OsMS1 responds to temperature changes and confers thermosensitive genic male sterility.

Changes in ambient temperature influence crop fertility and production. Understanding of how crops sense and respond to temperature is thus crucial for sustainable agriculture. The thermosensitive genic male-sterile (TGMS) lines are widely used for hybrid rice breeding and also provide a good system to investigate the mechanisms underlying temperature sensing and responses in crops. Here, we show that OsMS1 is a histone binding protein, and its natural allele OsMS1wenmin1 confers thermosensitive male sterility in rice. OsMS1 is primarily localized in nuclei, while OsMS1wenmin1 is localized in nuclei and cytoplasm. Temperature regulates the abundances of OsMS1 and OsMS1wenmin1 proteins. The high temperature causes more reduction of OsMS1wenmin1 than OsMS1 in nuclei. OsMS1 associates with the transcription factor TDR to regulate expression of downstream genes in a temperature-dependent manner. Thus, our findings uncover a thermosensitive mechanism that could be useful for hybrid crop breeding.

A ß-N-acetylhexosaminidase Amuc_2109 from Akkermansia muciniphila protects against dextran sulfate sodium-induced colitis in mice by enhancing intestinal barrier and modulating gut microbiota.

Inflammatory bowel disease (IBD) is associated with the microbial composition of the gut and its metabolites. Akkermansia muciniphila is a probiotic that exerts a significant alleviative or therapeutic effect on host enteritis. This study was designed to determine the protective effect and potential mechanism underlying the secretion of ß-acetylaminohexosidase (Amuc_2109) by A. muciniphila against dextran sulfate sodium (DSS)-induced colitis in mice. C57BL/6 mice were gavaged with Amuc_2109 for 21 days, and during the last seven days of treatment, they drank DSS dissolved in their drinking water to induce colitis. Our results showed that supplementation with Amuc_2109 improved DSS-induced colitis as evidenced by lowered disease activity index (DAI) scores, reduced weight loss, increased colon length, and inhibited oxidative stress. In addition, Amuc_2109 inhibited the overexpression of inflammatory cytokines (TNF-α, IL-1ß, IL-6) and the NLR family pyrin domain containing 3 (NLRP3) inflammasome in DSS-induced colitis. Furthermore, supplementation with Amuc_2109 also restored the mRNA expression of tight junction proteins (ZO-1, occludin, claudin-1). Further analysis of fecal microbial 16S rRNA sequences showed that Amuc_2109 reshaped the intestinal microbiota. While the anti-inflammatory effects of Amuc_2109 were only manifested with the wild-type protein, the anti-inflammatory effects were completely lost after the mutation of its key catalytic amino acids rendered Amuc_2109 inactive. In summary, these findings demonstrate the potential of Amuc_2109, as a therapeutic agent for ulcerative colitis. We posit that it will provide additional assistance in the prevention and treatment of mucus layer-related diseases such as ulcerative colitis.

Overexpression of OsHMGB707, a High Mobility Group Protein, Enhances Rice Drought Tolerance by Promoting Stress-Related Gene Expression.

Drought stress adversely affects crop growth and productivity worldwide. In response, plants have evolved several strategies in which numerous genes are induced to counter stress. High mobility group (HMG) proteins are the second most abundant family of chromosomal proteins. They play a crucial role in gene transcriptional regulation by modulating the chromatin/DNA structure. In this study, we isolated a novel HMG gene, OsHMGB707, one of the candidate genes localized in the quantitative trait loci (QTL) interval of rice drought tolerance, and examined its function on rice stress tolerance. The expression of OsHMGB707 was up-regulated by dehydration and high salt treatment. Its overexpression significantly enhanced drought tolerance in transgenic rice plants, whereas its knockdown through RNA interference (RNAi) did not affect the drought tolerance of the transgenic rice plants. Notably, OsHMGB707-GFP is localized in the cell nucleus, and OsHMGB707 is protein-bound to the synthetic four-way junction DNA. Several genes were up-regulated in OsHMGB707-overexpression (OE) rice lines compared to the wild-type rice varieties. Some of the genes encode stress-related proteins (e.g., DREB transcription factors, heat shock protein 20, and heat shock protein DnaJ). In summary, OsHMGB707 encodes a stress-responsive high mobility group protein and regulates rice drought tolerance by promoting the expression of stress-related genes.

An APETALA2/ethylene responsive factor, OsEBP89 knockout enhances adaptation to direct-seeding on wet land and tolerance to drought stress in rice.

Water stress is the most important adverse factor limiting rice production. Too much water leads to flood and too little leads to drought. Floods and droughts can severely damage crop at different times of the rice life cycle. So the research on submergence tolerance and drought resistance of rice is particularly urgent. In this study, we reported that OsEBP89 (Oryza sativa Ethylene-responsive element binding protein, clone 89), a member of the AP2/ERF subfamily, is involved in a novel signal transduction associated with the tolerance to drought and submergence stress. OsEBP89 was found to be strongly inhibited by drought stress and promoted by submergence. The OsEBP89 protein was located at the nucleus in the rice protoplast. Loss of OsEBP89 was found to improve the seed germination under submerged conditions and also enhanced the tolerance to drought stress throughout growth stage. Additionally, OsEBP89 knockout rice plants increased the accumulation of proline, improved the ability to scavenge ROS compared to overexpression lines and wild type after PEG treatment. Transcriptome data indicates that knockout of OsEBP89 improved the expression of specific genes in response to adverse factors, such as OsAPX1, OsHsfA3, and OsP5CS. Further results indicate that OsEBP89 can interact with and be phosphorylated by SnRK1α (sucrose non-fermenting-1-related protein kinase-1 gene). These findings provide insight into the mechanism of abiotic stress tolerance, and suggest OsEBP89 as a new genetic engineering resource to improve abiotic stress tolerance in rice.

A stress-responsive bZIP transcription factor OsbZIP62 improves drought and oxidative tolerance in rice.

BACKGROUND: Drought is a major abiotic stress factor that influences the yield of crops. Basic leucine zipper motif (bZIP) transcription factors play an important regulatory role in plant drought stress responses. However, the functions of a number of bZIP transcription factors in rice are still unknown. RESULTS: In this study, a novel drought stress-related bZIP transcription factor, OsbZIP62, was identified in rice. This gene was selected from a transcriptome analysis of several typical rice varieties with different drought tolerances. OsbZIP62 expression was induced by drought, hydrogen peroxide, and abscisic acid (ABA) treatment. Overexpression of OsbZIP62-VP64 (OsbZIP62V) enhanced the drought tolerance and oxidative stress tolerance of transgenic rice, while osbzip62 mutants exhibited the opposite phenotype. OsbZIP62-GFP was localized to the nucleus, and the N-terminal sequence (amino acids 1-68) was necessary for the transcriptional activation activity of OsbZIP62. RNA-seq analysis showed that the expression of many stress-related genes (e.g., OsGL1, OsNAC10, and DSM2) was upregulated in OsbZIP62V plants. Moreover, OsbZIP62 could bind to the promoters of several putative target genes and could interact with stress/ABA-activated protein kinases (SAPKs). CONCLUSIONS: OsbZIP62 is involved in ABA signalling pathways and positively regulates rice drought tolerance by regulating the expression of genes associated with stress, and this gene could be used for the genetic modification of crops with improved drought tolerance.

Metastatic TFE3-overexpressing renal clear cell carcinoma with dense granules: a histological, immunohistochemical, and ultrastructural study.

This is a case report of a 46-year-old white male who presented with dyspnea. Thoracic and abdominal examinations showed a heterogeneously enhancing mass in the right kidney, multiple pulmonary nodules, and left pleural thickening with large pleural effusion. Pleura biopsy revealed a malignant neoplasm composed of cells with predominantly clear cytoplasm. Considering the large mass in the right kidney, clear cell renal cell carcinoma (RCC) was the main differential diagnosis. The diagnosis in this case was not definitive by histology alone since clear cell RCC markers such as RCC and AE1/AE3 were negative, and CD10 was only focally positive. Transcription factor E3 (TFE3) immunohistochemistry was positive, while the XP11.2 translocation testing was negative. Electron microscopy demonstrated that the tumor cells had abundant cytoplasmic glycogen and lipid, focal long microvilli lining rare lumina, and adjacent interdigitating cell membranes joining the neoplastic cells, indicating a diagnosis of renal clear cell carcinoma. In addition, numerous crystalline-like dense granules were identified in the cytoplasm of the neoplastic cells, which are reminiscent of those typically seen in alveolar soft part sarcoma and rarely described in XP11.2 translocation RCC. Overall, this renal tumor likely represents a variant of XP11.2 translocation RCC, overexpressing TFE3 with dense granules.

Pyrrolizidine Alkaloid Secondary Pyrrolic Metabolites Construct Multiple Activation Pathways Leading to DNA Adduct Formation and Potential Liver Tumor Initiation.

Pyrrolizidine alkaloids (PAs) and their N-oxide derivatives are hepatotoxic, genotoxic, and carcinogenic phytochemicals. PAs induce liver tumors through a general genotoxic mechanism mediated by a set of four (±)-6,7-dihydro-7-hydroxy-1-hydroxymethyl-5 H-pyrrolizine (DHP)-derived DNA adducts. To date, the primary pyrrolic metabolites dehydro-PAs, their hydrolyzed metabolite DHP, and two secondary pyrrolic metabolites 7-glutathione-DHP (7-GS-DHP) and 7-cysteine-DHP are the known metabolites that can generate these DHP-DNA adducts in vivo and/or in PA-treated cells. Secondary pyrrolic metabolites are formed from the reaction of dehydro-PAs with glutathione, amino acids, and proteins. In this investigation, we determined whether or not more secondary pyrrolic metabolites can bind to calf thymus DNA and to cellular DNA in HepG2 cells resulting in the formation of DHP-DNA adducts using a series of secondary pyrrolic metabolites (including 7-methoxy-DHP, 9-ethoxy-DHP, 9-valine-DHP, 7-GS-DHP, 7-cysteine-DHP, and 7,9-diglutathione-DHP) and synthetic pyrroles for study. We found that (i) many secondary pyrrolic metabolites are DNA reactive and can form DHP-DNA adducts and (ii) multiple activation pathways are involved in producing DHP-DNA adducts associated with PA-induced liver tumor initiation. These results suggest that secondary pyrrolic metabolites play a vital role in the initiation of PA-induced liver tumors.

Metastatic Colonic Adenocarcinoma to the Extrahepatic Common Bile Duct: The Critical Role of the Pathologist and Immunohistochemistry in Guiding Patient Care Decisions.

BACKGROUND Adenocarcinoma of the colon frequently invades adjacent organs, spreads intraperitoneal, and metastasizes to intestinal lymph nodes, lungs, and the liver. Metastasis solely to the extrahepatic bile duct is extremely rare and has only been previously reported on 15 occasions. The accurate determination that an extrahepatic common bile duct lesion is of colonic origin has critical therapeutic implications. CASE REPORT The patient was a 50-year-old male with a history of colon cancer S/P surgical resection in September 2014. At that time, the tumor extended to the serosal margin, and was staged as pT4a N0 MX stage II. In April of 2016, the patientwas admitted to our facility and an ERCP was performed. A biopsy was performed during the ERCP followed by sphincterotomy and metal stent placement. The surgical pathology revealed an adenocarcinoma with surrounding benign glandular structures. The surrounding benign glands served as an appropriate control when compared to the malignant glands. The benign glands were positive for CK-7 and the malignant glands were negative for CK-7. The malignant glands were also positive for CK-20 and CDX-2, and the benign glands were negative for CK-20 and CDX-2. This profile was consistent with an adenocarcinoma metastasis from colon primary tumor. CONCLUSIONS This case superbly illustrates the critical role of the pathologist, and their knowledge and understanding of immunohistochemistry, in arriving at the correct diagnosis and in assisting surgeons and oncologists in guiding the care, management, and appropriate therapeutic decisions regarding patients. In patients with a history of colorectal carcinoma (CRC), immunohistochemistry is required to arrive at the correct diagnosis as treatment options can be very different based on diagnosis.

Genome-Wide Association Study for Plant Height and Grain Yield in Rice under Contrasting Moisture Regimes.

Drought is one of the vitally critical environmental stresses affecting both growth and yield potential in rice. Drought resistance is a complicated quantitative trait that is regulated by numerous small effect loci and hundreds of genes controlling various morphological and physiological responses to drought. For this study, 270 rice landraces and cultivars were analyzed for their drought resistance. This was done via determination of changes in plant height and grain yield under contrasting water regimes, followed by detailed identification of the underlying genetic architecture via genome-wide association study (GWAS). We controlled population structure by setting top two eigenvectors and combining kinship matrix for GWAS in this study. Eighteen, five, and six associated loci were identified for plant height, grain yield per plant, and drought resistant coefficient, respectively. Nine known functional genes were identified, including five for plant height (OsGA2ox3, OsGH3-2, sd-1, OsGNA1, and OsSAP11/OsDOG), two for grain yield per plant (OsCYP51G3 and OsRRMh) and two for drought resistant coefficient (OsPYL2 and OsGA2ox9), implying very reliable results. A previous study reported OsGNA1 to regulate root development, but this study reports additional controlling of both plant height and root length. Moreover, OsRLK5 is a new drought resistant candidate gene discovered in this study. OsRLK5 mutants showed faster water loss rates in detached leaves. This gene plays an important role in the positive regulation of yield-related traits under drought conditions. We furthermore discovered several new loci contributing to the three investigated traits (plant height, grain yield, and drought resistance). These associated loci and candidate genes significantly improve our knowledge of the genetic control of these traits in rice. In addition, many drought resistant cultivars screened in this study can be used as parental genotypes to improve drought resistance of rice by molecular breeding.

Research progress of photoperiod regulated genes on flowering time in rice.

Rice flowering regulation is an extremely complex process, which is controlled by genetic factors and external environment. Photoperiodic regulatory pathway is pivotal to control flowering in rice, in which florigen genes Hd3a and RTF1 are at the core and they are regulated by upstream Hd1-dependent, Ehd1-dependent, as well as both Hd1- and Ehd1-independent pathways. The three pathways bring a variety of light signal information together to Hd3a and RTF1 for further integration, and then transmit the signals in the form of florigen to the downstream flowering related genes. In this review, we summarize the research progress of photoperiod regulated genes on flowering time in rice, including the photoreceptors and circadian rhythm genes, the florigens, its upstream, downstream and interacting genes. We hope to provide a reference for in-depth study of rice flowering regulation.

OsGRAS23, a rice GRAS transcription factor gene, is involved in drought stress response through regulating expression of stress-responsive genes.

BACKGROUND: Drought is a major abiotic stress factors that reduces agricultural productivity. GRAS transcription factors are plant-specific proteins that play diverse roles in plant development. However, the functions of a number of GRAS genes identified in rice are unknown, especially the GRAS genes related to rice drought resistance have not been characterized. RESULTS: In this study, a novel GRAS transcription factor gene named OsGRAS23, which is located in a drought-resistant QTL interval on chromosome 4 of rice, was isolated. The expression of OsGRAS23 was induced by drought, NaCl, and jasmonic acid treatments. The OsGRAS23-GFP fused protein was localized in the nucleus of tobacco epidermal cells. A trans-activation assay in yeast cells demonstrated that the OsGRAS23 protein possessed a strong transcriptional activation activity. OsGRAS23-overexpressing rice plants showed improved drought resistance and oxidative stress tolerance as well as less H2O2 accumulation compared with the wild-type plants. Furthermore, microarray analysis showed that several anti-oxidation related genes were up-regulated in the OsGRAS23-overexpressing rice plants. The yeast one hybrid test indicated that OsGRAS23 could bind to the promoters of its potential target genes. CONCLUSIONS: Our results demonstrate that OsGRAS23 encodes a stress-responsive GRAS transcription factor and positively modulates rice drought tolerance via the induction of a number of stress-responsive genes.

Regulation of corneal stroma extracellular matrix assembly.

The transparent cornea is the major refractive element of the eye. A finely controlled assembly of the stromal extracellular matrix is critical to corneal function, as well as in establishing the appropriate mechanical stability required to maintain corneal shape and curvature. In the stroma, homogeneous, small diameter collagen fibrils, regularly packed with a highly ordered hierarchical organization, are essential for function. This review focuses on corneal stroma assembly and the regulation of collagen fibrillogenesis. Corneal collagen fibrillogenesis involves multiple molecules interacting in sequential steps, as well as interactions between keratocytes and stroma matrix components. The stroma has the highest collagen V:I ratio in the body. Collagen V regulates the nucleation of protofibril assembly, thus controlling the number of fibrils and assembly of smaller diameter fibrils in the stroma. The corneal stroma is also enriched in small leucine-rich proteoglycans (SLRPs) that cooperate in a temporal and spatial manner to regulate linear and lateral collagen fibril growth. In addition, the fibril-associated collagens (FACITs) such as collagen XII and collagen XIV have roles in the regulation of fibril packing and inter-lamellar interactions. A communicating keratocyte network contributes to the overall and long-range regulation of stromal extracellular matrix assembly, by creating micro-domains where the sequential steps in stromal matrix assembly are controlled. Keratocytes control the synthesis of extracellular matrix components, which interact with the keratocytes dynamically to coordinate the regulatory steps into a cohesive process. Mutations or deficiencies in stromal regulatory molecules result in altered interactions and deficiencies in both transparency and refraction, leading to corneal stroma pathobiology such as stromal dystrophies, cornea plana and keratoconus.

UVA photoirradiation of benzo[a]pyrene metabolites: induction of cytotoxicity, reactive oxygen species, and lipid peroxidation.

Benzo[a]pyrene (BaP) is a prototype for studying carcinogenesis of polycyclic aromatic hydrocarbons (PAHs). We have long been interested in studying the phototoxicity of PAHs. In this study, we determined that metabolism of BaP by human skin HaCaT keratinocytes resulted in six identified phase I metabolites, for example, BaP trans-7,8-dihydrodiol (BaP t-7,8-diol), BaP t-4,5-diol, BaP t-9,10-diol, 3-hydroxybenzo[a]pyrene (3-OH-BaP), BaP (7,10/8,9)tetrol, and BaP (7/8,9,10)tetrol. The photocytotoxicity of BaP, 3-OH-BaP, BaP t-7,8-diol, BaP trans-7,8-diol-anti-9,10-epoxide (BPDE), and BaP (7,10/8,9)tetrol in the HaCaT keratinocytes was examined. When irradiated with 1.0 J/cm(2) UVA light, these compounds when tested at doses of 0.1, 0.2, and 0.5 µM, all induced photocytotoxicity in a dose-dependent manner. When photoirradiation was conducted in the presence of a lipid (methyl linoleate), BaP metabolites, BPDE, and three related PAHs, pyrene, 7,8,9,10-tetrahydro-BaP trans-7,8-diol, and 7,8,9,10-tetrahydro-BaP trans-9,10-diol, all induced lipid peroxidation. The formation of lipid peroxides by BaP t-7,8-diol was inhibited by NaN3 and enhanced by deuterated methanol, which suggests that singlet oxygen may be involved in the generation of lipid peroxides. The formation of lipid hydroperoxides was partially inhibited by superoxide dismutase (SOD). Electron spin resonance spin trapping experiments indicated that both singlet oxygen and superoxide radical anion were generated from UVA photoirradiation of BPDE in a light dose responding manner.