Single time frame overview of the genetic changes in conjunctival melanoma from, intraepithelial disease to invasive melanoma. A study of 4 exenteration specimens illustrating the potential role of Cyclin D1.

Introduction: Despite advances in the understanding of the molecular pathogenesis of cutaneous melanoma, relatively little is known about the genetic changes that occur in the progression of conjunctival melanocytic from intraepithelial lesions to invasive conjunctival melanoma. Methods: We exposed four exenteration specimens that each contained varying grades of intraepithelial conjunctival melanocytic neoplasia and invasive neoplasia to a combination of various techniques, including array comparative genomic hybridisation (aCGH), RNA seq, fluorescence in-situ hybridisation (FISH) and immunohistochemistry. Results: Three out of four of the invasive melanomas showed gains in 11q13 (CCND1 locus) by aCGH. FISH demonstrated CCND1 gain in invasive melanoma and in conjunctival melanocytic intraepithelial lesions of all grades (Low grade CMIL and in-situ melanoma) and this was paralleled by increased expression of Cyclin D1 protein within the atypical melanocytes by immunohistochemistry, using a double staining method with a red end point for Melan A cytoplasmic staining and a brown end-point for nuclear Cyclin D1 expression. Higher grades of melanocytic intraepithelial lesions showed more cells expressing Cyclin D1 compared to lower grade melanocytic intraepithelial lesions. The Cyclin D1 protein expression was in the same location as the amplified CCND1 signal by FISH. One out of three of these cases also showed amplification of the 12q13-15 locus corresponding to MDM2 and FISH confirmed gains in the conjunctival melanocytic intraepithelial neoplasia and invasive melanoma. The remaining fourth case showed a homozygous deletion of 9p21 (CDKN2A) by aCGH only, with immunohistochemistry showing clonal loss of p16 protein expression in the invasive and conjunctival melanocytic intraepithelial lesion. Two out of four of the invasive melanomas harboured classical driver mutations in NRAS and NF-1respectively. None of the cases showed mutations in BRAF, KIT and TERT mutations. RNAseq data showed secondary mutations in ARAF, PLCB4, MET, EZH2, MAP2K2, CTNNB1, CIITA, NF2, TP53 and MEN1, some of which are implicated in the MAPK pathway. Conclusion: Conjunctival melanocytic intraepithelial lesions harbour amplifications of CCND1 (3 cases), MDM2 (1 case) and loss of CDKN2A (1 case), which are also present when the lesion progresses to invasive melanoma, implicating these amplifications in the early pathogenesis of conjunctival melanocytic intraepithelial lesions. This study represents the first attempt to capture the mutational landscape of at all stages of conjunctival melanoma in a single tissue excision.

p53, p21, and cyclin d1 protein expression patterns in patients with breast cancer

Background and Aim: The mutation in the wild-type tumor suppressor gene p53 is the most common genetic change in human tumors. In addition, the normal function of p21, which is both antiproliferative and an inhibitor of the cell cycle, is disrupted in some types of cancer. Meanwhile, cyclin D1 is a member of the cyclin protein family that is involved in regulating cell cycle progression. This study aimed to assess the expressions of the cell cycle inhibitory proteins p21, cyclin D1, and tumor suppressor gene p53, as well as their influence on the expressed histopathological changes in breast cancer tissues. Materials and Methods: Overall, 40 breast tissue specimens were investigated in this study, 30 of which were cancerous, while 10 were healthy tissues. p53, p21, and cyclin D1 expression patterns were detected using an immunohistochemistry (IHC) system. Results: The IHC reactions for p53 were positively observed in 27/30 (90%) cancerous tissues, compared with 2/10 (20%) normal breast tissues. For p21, reactions were observed in 28/30 (93.33%) cancerous tissues and 3/10 (30%) control tissues. For cyclin D1, reactions were observed in 25/30 (83.33%) cancerous tissues and 1/10 (10%) control tissues. The differences between the breast cancer tissues and the control tissues were statistically highly significant (p<0.01). Conclusion: The high expression rates of p21, cyclin D1, and p53 in malignant breast cancer cells with little or no regulatory role might imply mutational events in these proteins operating in concert with a variety of other genetic mutations in these tissues, which may play a molecular role in the development and/or progression of breast carcinogenesis.

Quantitative and Qualitative Structure Activity Relationship Study for Development of the Pet- Inhibitory Activity of 3-Nitro-2,4,6- Trihydroxy Benzamides

The aim in this paper of these work present two components analyses quantitative and qualitative which consisted in the development and evaluation of the first component quantitative and structure activity relationships (QSAR) for the prediction In fact, various compounds inhibiting photosynthesis constitute the largest class of commercial herbicides. All of those inhibitors, including ureas, triazines, bis carbamates and phenols, interrupt photosynthetic electron transport (PET) by binding to quinine-binding protein (D1-protein). Various physicochemical descriptors were used in multiple linear regressions method (MLR) to develop the theoretical models, than using a cross-validation with leave-one-out method to optimize the model as well as possible to fit with the biological data, the most common approach used to study. The second quality of the compound compared with criteria for their power, these rules Lu Propose the four basic characteristics that Lipinski has identified.

The influence of nutrient availability on the thermotolerance of symbiotic dinoflagellates of family Symbiodiniaceae

<p><b>Coral reefs are the most biodiverse ocean ecosystems on the planet, providing essential habitat for over 25% of the world’s marine organisms. Their structural complexity and stability contribute to essential coastline defence against erosion, as well as provide billions of dollars per year in economic value in the form of tourism and artisanal fishing. Fundamental to this unique and indispensable habitat is the symbiotic relationship between cnidarian corals and algal dinoflagellates of family Symbiodiniaceae. Reef-building corals gain a crucial majority of their daily energy needs from their endosymbiotic dinoflagellates, which facilitates coral growth, reproduction and formation of the reef structure on which countless other organisms thrive. However, this symbiosis has come under threat from warming oceans as a consequence of anthropogenic climate change. Under thermal stress the cnidarian-dinoflagellate symbiosis breaks down, resulting in expulsion of the dinoflagellate (‘coral bleaching’), followed by the eventual death of the coral animal. Tolerance of elevated temperatures is known to vary among coral and Symbiodiniaceae species, and may be influenced by the interaction of nutrient availability and photosynthetic function of the endosymbiont. </b></p> <p>The aim of this thesis was to investigate photophysiological mechanisms in thermotolerant and thermally sensitive Symbiodiniaceae to determine how they are affected by thermal and nutritive stress, both in and out of symbiosis. </p> <p>Cultured Symbiodiniaceae phylotypes A4 (thermotolerant) and B2 (thermally sensitive) were subjected to high and low nutrient and temperature treatments. The photosynthetic health (quantum yield, FV/FM), rate of chloroplastic protein synthesis (D1 protein) and photophysiological response to light (NPQ) of each phylotype was monitored to determine if thermotolerance was related to nutrient utilization under heat stress. Phylotype A4 showed considerably increased D1 synthesis regardless of nutrient treatment when compared to phylotype B2, but only minor differences in FV/FM. Also, correlation between D1 concentration and FV/FM was observed in A4, but not B2 during recovery from heat stress. Responses to short term light exposure contrasted significantly between the two phylotypes under all conditions, indicating marked differences in the photophysiological apparatus. </p> <p>To examine nutrient use and thermotolerance in symbiosis, the model symbiotic anemone Aiptasia pallida (commonly Aiptasia) was inoculated with either Symbiodiniaceae species Breviolum minutum, phylotype A4 or phylotype B2. The holobionts (host and symbiont) were fed or starved for a period of six weeks and then subjected to heat stress. D1protein concentration and FV/FM was similar in all fed holobionts, regardless of symbiont type and heat treatment. Following heat stress, all starved holobionts showed extremely low concentrations of D1 protein, but comparable FV/FM, while in low temperature starved treatments, only Aiptasia hosting B. minutum showed any recovery of D1 protein. The study shows that efficiency of nutrient utilization in photosynthetic pathways is not necessarily an indicator of thermotolerance, nor does it dictate the ability of the symbiont to confer physiological benefits to the host under conditions of heat or nutrient stress. Rather, host-symbiont pairings most likely reflect responses to external pressures dictated by the local environment. The implications of the physiological disparities between the Symbiodiniaceae types tested are discussed in the context of environmental adaptations and host-symbiont nutrient dynamics. The complexity of symbiotic interactions highlighted by this study reinforces the imperative necessity of further investigations into cnidarian-dinoflagellate symbioses, particularly in regard to thermotolerance and photophysiology. Only through understanding the physiological effects of rising ocean temperatures on this essential partnership can we begin the work of protecting the coral reef habitat for future generations.</p>

The influence of nutrient availability on the thermotolerance of symbiotic dinoflagellates of family Symbiodiniaceae

<p><b>Coral reefs are the most biodiverse ocean ecosystems on the planet, providing essential habitat for over 25% of the world’s marine organisms. Their structural complexity and stability contribute to essential coastline defence against erosion, as well as provide billions of dollars per year in economic value in the form of tourism and artisanal fishing. Fundamental to this unique and indispensable habitat is the symbiotic relationship between cnidarian corals and algal dinoflagellates of family Symbiodiniaceae. Reef-building corals gain a crucial majority of their daily energy needs from their endosymbiotic dinoflagellates, which facilitates coral growth, reproduction and formation of the reef structure on which countless other organisms thrive. However, this symbiosis has come under threat from warming oceans as a consequence of anthropogenic climate change. Under thermal stress the cnidarian-dinoflagellate symbiosis breaks down, resulting in expulsion of the dinoflagellate (‘coral bleaching’), followed by the eventual death of the coral animal. Tolerance of elevated temperatures is known to vary among coral and Symbiodiniaceae species, and may be influenced by the interaction of nutrient availability and photosynthetic function of the endosymbiont. </b></p> <p>The aim of this thesis was to investigate photophysiological mechanisms in thermotolerant and thermally sensitive Symbiodiniaceae to determine how they are affected by thermal and nutritive stress, both in and out of symbiosis. </p> <p>Cultured Symbiodiniaceae phylotypes A4 (thermotolerant) and B2 (thermally sensitive) were subjected to high and low nutrient and temperature treatments. The photosynthetic health (quantum yield, FV/FM), rate of chloroplastic protein synthesis (D1 protein) and photophysiological response to light (NPQ) of each phylotype was monitored to determine if thermotolerance was related to nutrient utilization under heat stress. Phylotype A4 showed considerably increased D1 synthesis regardless of nutrient treatment when compared to phylotype B2, but only minor differences in FV/FM. Also, correlation between D1 concentration and FV/FM was observed in A4, but not B2 during recovery from heat stress. Responses to short term light exposure contrasted significantly between the two phylotypes under all conditions, indicating marked differences in the photophysiological apparatus. </p> <p>To examine nutrient use and thermotolerance in symbiosis, the model symbiotic anemone Aiptasia pallida (commonly Aiptasia) was inoculated with either Symbiodiniaceae species Breviolum minutum, phylotype A4 or phylotype B2. The holobionts (host and symbiont) were fed or starved for a period of six weeks and then subjected to heat stress. D1protein concentration and FV/FM was similar in all fed holobionts, regardless of symbiont type and heat treatment. Following heat stress, all starved holobionts showed extremely low concentrations of D1 protein, but comparable FV/FM, while in low temperature starved treatments, only Aiptasia hosting B. minutum showed any recovery of D1 protein. The study shows that efficiency of nutrient utilization in photosynthetic pathways is not necessarily an indicator of thermotolerance, nor does it dictate the ability of the symbiont to confer physiological benefits to the host under conditions of heat or nutrient stress. Rather, host-symbiont pairings most likely reflect responses to external pressures dictated by the local environment. The implications of the physiological disparities between the Symbiodiniaceae types tested are discussed in the context of environmental adaptations and host-symbiont nutrient dynamics. The complexity of symbiotic interactions highlighted by this study reinforces the imperative necessity of further investigations into cnidarian-dinoflagellate symbioses, particularly in regard to thermotolerance and photophysiology. Only through understanding the physiological effects of rising ocean temperatures on this essential partnership can we begin the work of protecting the coral reef habitat for future generations.</p>

Methyl Jasmonate Protects the PS II System by Maintaining the Stability of Chloroplast D1 Protein and Accelerating Enzymatic Antioxidants in Heat-Stressed Wheat Plants

The application of 10 µM methyl jasmonate (MeJA) for the protection of wheat (Triticum aestivum L.) photosystem II (PS II) against heat stress (HS) was studied. Heat stress was induced at 42 °C to established plants, which were then recovered at 25 °C and monitored during their growth for the study duration. Application of MeJA resulted in increased enzymatic antioxidant activity that reduced the content of hydrogen peroxide (H2O2) and thiobarbituric acid reactive substances (TBARS) and enhanced the photosynthetic efficiency. Exogenous MeJA had a beneficial effect on chlorophyll fluorescence under HS and enhanced the pigment system (PS) II system, as observed in a JIP-test, a new tool for chlorophyll fluorescence induction curve. Exogenous MeJA improved the quantum yield of electron transport (ETo/CS) as well as electron transport flux for each reaction center (ET0/RC). However, the specific energy fluxes per reaction center (RC), i.e., TR0/RC (trapping) and DI0/RC (dissipation), were reduced by MeJA. These results indicate that MeJA affects the efficiency of PS II by stabilizing the D1 protein, increasing its abundance, and enhancing the expression of the psbA and psbB genes under HS, which encode proteins of the PS II core RC complex. Thus, MeJA is a potential tool to protect PS II and D1 protein in wheat plants under HS and to accelerate the recovery of the photosynthetic capacity.

Nitric Oxide Functions as a Downstream Signal for Melatonin-Induced Cold Tolerance in Cucumber Seedlings

Melatonin (MT) and nitric oxide (NO) are two multifunctional signaling molecules that are involved in the response of plants to abiotic stresses. However, how MT and NO synergize in response to cold stress affecting plants is still not clear. In this study, we found that endogenous MT accumulation under cold stress was positively correlated with cold tolerance in different varieties of cucumber seedlings. The data presented here also provide evidence that endogenous NO is involved in the response to cold stress. About 100 μM MT significantly increased the nitrate reductase (NR) activity, NR-relative messenger RNA (mRNA) expression, and endogenous NO accumulation in cucumber seedlings. However, 75 μM sodium nitroprusside (SNP, a NO donor) showed no significant effect on the relative mRNA expression of tryptophan decarboxylase (TDC), tryptamine-5-hydroxylase (T5H), serotonin-N-acetyltransferase (SNAT), or acetylserotonin O-methyltransferase (ASMT), the key genes for MT synthesis and endogenous MT levels. Compared with H2O treatment, both MT and SNP decreased electrolyte leakage (EL), malondialdehyde (MDA), and reactive oxygen species (ROS) accumulation by activating the antioxidant system and consequently mitigated cold damage in cucumber seedlings. MT and SNP also enhanced photosynthetic carbon assimilation, which was mainly attributed to an increase in the activity and mRNA expression of the key enzymes in the Calvin–Benson cycle. Simultaneously, MT- and SNP-induced photoprotection for both photosystem II (PSII) and photosystem I (PSI) in cucumber seedlings, by stimulating the PsbA (D1) protein repair pathway and ferredoxin-mediated NADP+ photoreduction, respectively. Moreover, exogenous MT and SNP markedly upregulated the expression of chilling response genes, such as inducer of CBF expression (ICE1), C-repeat-binding factor (CBF1), and cold-responsive (COR47). MT-induced cold tolerance was suppressed by 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO, a specific scavenger of NO). However, p-chlorophenylalanine (p-CPA, a MT synthesis inhibitor) did not affect NO-induced cold tolerance. Thus, novel results suggest that NO acts as a downstream signal in the MT-induced plant tolerance to cold stress.

6-Ethoxy-4-N-(2-Morpholin-4-Ylethyl)-2-N-Propan-2-yl-1,3,5-Triazine-2,4-Diamine Endows Herbicidal Activity Against Phalaris Minor a Weed of Wheat Crop Field: An in-Silico and Experimental Approaches of Herbicide Discovery

P. minor is a major weed of wheat crop which has evolved resistance against herbicides. Isoproturon is the most accepted herbicide developed in 1992. Later, introduced herbicides also developed resistance and cross-resistance to their respective binding sites. Isoproturon binds at the QB binding site of the D1 protein of photosystem-II (PS-II) which blocks the electron transfer in photosynthesis. In this work, a series of computational studies have been implemented to discover herbicides against the D1 protein of P. minor. Through computational study, twenty-four lead molecules are reported which have shown a higher binding affinity and inhibition constant compared to the reference ligand. The conformational stability of docked complexes was evaluated by molecular dynamics simulation and MM/PBSA method. Ala225, Ser226, Phe227, and Asn229 present in the binding site of protein play an important role in the stability of the protein-lead complex via hydrogen bond interaction and pi-pi interaction. Moreover, interactions and binding free energy between protein-lead complexes have been calculated using MM/PBSA methods. It has revealed that binding was energetically favourable and driven by electrostatic interactions. Among 24 leads, computational results have uncovered eight promising compounds as potential herbicides which have shown comparable physiochemical profile, better docking scores, system stability, H-bond occupancy, and binding free energy than terbutryn a reference molecule. Prioritized molecules were custom synthesized and evaluated for their herbicidal activity and specificity through whole plant assay under laboratory-controlled conditions. The prioritized lead molecule ELC5 (6-ethoxy-4-N-(2-morpholin-4-ylethyl)-2-N-propan-2-yl-1,3,5-triazine-2,4-diamine) has shown comparable activity to the reference isoproturon herbicide against P. minor.

Binding Properties of Photosynthetic Herbicides with the QB Site of the D1 Protein in Plant Photosystem II: A Combined Functional and Molecular Docking Study

Photosystem II (PSII) is a multi-subunit enzymatic complex embedded in the thylakoid membranes responsible for the primary photosynthetic reactions vital for plants. Many herbicides used for weed control inhibit PSII by interfering with the photosynthetic electron transport at the level of the D1 protein, through competition with the native plastoquinone for the QB site. Molecular details of the interaction of these herbicides in the D1 QB site remain to be elucidated in plants. Here, we investigated the inhibitory effect on plant PSII of the PSII-inhibiting herbicides diuron, metobromuron, bentazon, terbuthylazine and metribuzin. We combined analysis of OJIP chlorophyll fluorescence kinetics and PSII activity assays performed on thylakoid membranes isolated from pea plants with molecular docking using the high-resolution PSII structure recently solved from the same plant. Both approaches showed for terbuthylazine, metribuzin and diuron the highest affinity for the D1 QB site, with the latter two molecules forming hydrogen bonds with His215. Conversely, they revealed for bentazon the lowest PSII inhibitory effect accompanied by a general lack of specificity for the QB site and for metobromuron an intermediate behavior. These results represent valuable information for future design of more selective herbicides with enhanced QB binding affinities to be effective in reduced amounts.

CCND1 copy number increase and cyclin D1 expression in acral melanoma: a comparative study of fluorescence in situ hybridization and immunohistochemistry in a Chinese cohort

Background CCND1 copy number increase is characteristic of acral melanoma and is useful in distinguishing benign and malignant acral melanocytic lesions. Increase of the gene copy number may result in protein overexpression. This raises the possibility that detection of high expression of cyclin D1 by immunohistochemistry (IHC) may be used as a surrogate for direct evaluation of increase in the CCND1 gene copy number. Methods We examined increases in CCND1 copy number with fluorescence in situ hybridization (FISH), and examined cyclin D1 protein expression with IHC in 61 acral melanomas. Results Using FISH, 29 acral melanomas (29/61, 47.5%) showed increase in the CCND1 copy number, including 8 (8/61, 13.1%) which showed low-level increase in the CCND1 copy number and 21 (21/61, 34.4%) with high-level increase in the CCND1 copy number. By analysis of IHC, the median IHC score was 15% (range: 1–80%) in acral melanomas with no CCND1 copy number alteration. In acral melanomas with low-level CCND1 copy number increase, the median IHC score was 25% (range: 3–90%). In acral melanomas with high-level CCND1 copy number increase, the median IHC score was 60% (range: 1–95%). Comparing FISH and IHC, cyclin D1 protein expression level has no corelation with the CCND1 copy number in acral melanomas which have no CCND1 copy number alteration and low-level CCND1 copy number increase (P = 0.108). Cyclin D1 protein expression level correlated positively with CCND1 copy number in acral melanomas with high-level CCND1 copy number increase (P = 0.038). The sensitivity, specificity and positive predictive value of using cyclin D1 IHC to predict CCND1 FISH result was 72.4, 62.5 and 63.6%. Increase in CCND1 copy number was associated with Breslow thickness in invasive acral melanoma. Conclusion High-level increase in the CCND1 copy number can induce high cyclin D1 protein expression in acral melanomas. However low-level increase and normal CCND1 copy number have no obvious correlation with protein expression. Cyclin D1 IHC cannot serve as a surrogate for CCND1 FISH in acral melanomas.