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Ratings 7,2 / 10. Wing-Cheong Law. 2019. &ref(https://m.media-amazon.com/images/M/MV5BN2YzZGZkNTQtOTkyMS00MTgwLWEzODgtYWEzNWVjY2JiOWE3XkEyXkFqcGdeQXVyNjgwNTk4Mg@@._V1_SY1000_CR0,0,629,1000_AL_.jpg). Writers Susan Chan. description Xiao Q is a movie starring Simon Yam, Gigi Leung, and Him Law. Adapted from the Japanese novel Goodbye, Khoru, following a guide dog and his conflicted master. 盡情熬夜吧 反正你想的那個人很可能已經睡了 被子蓋得很好，夢也很甜 這個夜?你除了收穫到猝死的可能性以外 沒有任何東西留給你了. Little Q Free full review. 喜歡朴樹的嗓音和吐字，更欣賞他的「不忘初心」和才華 ? ?? ?. 哇，大??人呀，炒?喜?大??人?. 好听！新年快?. Little Q free full text. Little q free full episodes.
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Little q free full games. Little q free full fight. Little q free full length. Writer Xiao Q Resume 凡人看世界。 Release date 2019; In Hong Kong, China, grumpy pastry chef Lee is losing his sight because of eye disease. People around him have always wanted to help him, but he has repeatedly refused people's help. Until a guide dog named littleQ came to him, Lee gradually adapted and fell in love with the guide dog, the two of them rely on each other until littleQ died of cancer; Director Wing-Cheong Law; Genres Drama; duration 107Minutes; You don't want to see dogs because they die. yes animals do die, so do humans. do you just stay inside all the time because he you see anyone they will die and you will cry haha. weirdo. Bao quang temple santa ana. Xiao quan photographer. Xiao qing. Bao que huong. Im from china??. Soo cute couple ? fang leng & xiaq. Me recuerda a Baekhyun tratando de conquistar a Sehun ahahaha. Bao qing tian. Had to punch a wall to fell like a man again Xiao qu. Đang d?ch mà dân ít đeo kh?u trang nh?. Xiao q li md. Xiao qiang water. Here before this gets picked up by Youtubes algoritm... My heart is melting?. Xiao qiao. Xiao qin pommel horse. Xiao q. t. r. I like her very much??i saw her show everyday i wish my girlfriend should look like her. Xiao qiang kung fu. Bao quang tri moi nhat. John q. xiao. Xiao q and fang ling. This one is awesome. ai zhongguo. I love you from thai land. Xiao yu cun seafood restaurant. 我的心空空如也，只想跟你?我愛你. Xiaoqing. Kerennn. Tq for upload this song. Me encanta ??. Gosh she's so pretty and he's so freaking handsome ??. Xiao quan & sun yusai. Xiao q 2019. Xiao-qing yang. Look at that smile! she is living her best life, thankyou for taking such great care of her <3 I miss the couple but i love you fang lie love u so muchhhhh want to meet u once in life love from India I wish I could be a part of this series. ?????????. Xiao qiang water jump. Xiao hui. Xiao q1 nail & spa. ?了?了T_T好想全世界旅行多想去看看感??首歌. Xiao qi lai hao kan. Why you have not upload full episode with eng sub Bao quang tri. Xiao qin wang. OH MY GOD. Xiao qi ye. Porfavor suban en sub en esp quiero ver ya van varios dias que me quede esperando el 25 y los demas ????. Xiao qiao dynasty warriors. Xiao qing long. Xiao q u. Why do I keep on watching these. It makes me sad Fang leng?????. I want to take you to romantic Turkey?? ??????. Bao quang ngai moi nhat. I think i am the only one who came here everyday to listen his voice ?. Xiao qiao drama. A “republic” lol that denotes democracy, when China is infact some kind of weird communist-esq capitalist dictatorship. Bao quan doi nhan dan. Bao quang ninh. Xiao qing long wan. Que es xiao. ???????????? ???????????????. Xiao q full movie. 唱的真好听！???. That actually is a really nice haircut lol. Xiao qiang. Xiao qiao dynasty warriors 9. Xiao qin wang san diego map. 蜜��和席琳也很??. 很棒啊~加油?在多做一點歌?. Xiao qing model. Xiao qing ge karaoke. Bao quan. Xiao q1 nail. ぉ123????é?き Xiao Q Download Full 8. 0 stars - Demarcus Humen.Sad life his my dream husband. almost perfect... Little q free full version. Research Article [version 1; peer review: 1 approved, 1 approved with reservations] Siska Septiana 1, Boy Muchlis Bachtiar 2, Nancy Dewi Yuliana 1, 1 Siska Septiana 1, Boy Muchlis Bachtiar 2, Nancy Dewi Yuliana 1, Christofora Hanny Wijaya 1 OPEN PEER REVIEW REVIEWER STATUS Background: Cajuputs candy (CC), an Indonesian functional food, utilizes the bioactivity of Melaleuca cajuputi essential oil (MCEO) to maintain oral cavity health. Synergistic interaction between Candida albicans and Streptococcus mutans is a crucial step in the pathogenesis of early childhood caries. Our recent study revealed several alternative MCEOs as the main flavors in CC. The capacity of CC to interfere with the fungus-bacterium relationship remains unknown. This study aimed to evaluate CC efficacy to impair biofilm formation by these dual cariogenic microbes. Methods: The inhibition capacity of CC against mixed-biofilm comprising C. albicans and S. mutans was assessed by quantitative (crystal violet assay, tetrazolium salt [MTT] assay, colony forming unit/mL counting, biofilm-related gene expression) and qualitative analysis (light microscopy and scanning electron microscopy). Result: Both biofilm-biomass and viable cells were significantly reduced in the presence of CC. Scanning electron microscopy imaging confirmed this inhibition capacity, demonstrating morphology alteration of C. albicans, along with reduced microcolonies of S. mutans in the biofilm mass. This finding was related to the transcription level of selected biofilm-associated genes, expressed either by C. albicans or S. mutans. Based on qPCR results, CC could interfere with the transition of C. albicans yeast form to the hyphal form, while it suppressed insoluble glucan production by S. G2 derived from Mojokerto MCEO showed the greatest inhibition activity on the relationship between these cross-kingdom oral microorganisms (p < 0. 05). Conclusion: In general, all CC formulas showed biofilm inhibition capacity. Candy derived from Mojokerto MCEO showed the greatest capacity to maintain the commensal form of C. albicans and to inhibit extracellular polysaccharide production by S. Therefore, the development of dual-species biofilms can be impaired effectively by the CC tested. Keywords Cajuputs candy, essential oil, caries, mixed biofilm, Candida albicans, Streptococcus mutans Corresponding author: Christofora Hanny Wijaya Competing interests: IPB University is the patent holder for Cajuputs candy and CHW and BMB are named on the patent. However, the authors declare that they do not have any financial interest to disclose. Grant information: This study was funded by Directorate General of Resources for Science, Technology, and Higher Education, Ministry of Research, Technology and Higher Education, The Republic of Indonesia, through Program Pendidikan Magister menuju Doktor untuk Sarjana Unggul (PMDSU) 2018. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Copyright: © 2019 Septiana S et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. How to cite: Septiana S, Bachtiar BM, Yuliana ND and Wijaya CH. Cajuputs candy impairs Candida albicans and Streptococcus mutans mixed biofilm formation in vitro [version 1; peer review: 1 approved, 1 approved with reservations]. F1000Research 2019, 8:1923 () First published: 15 Nov 2019, 8:1923 () Latest published: 15 Nov 2019, 8:1923 () Abbreviations CC, Cajuputs candy; MCEO, Melaleuca cajuputi essential oil; G0, untreated biofilm, control group, without the addition of CC formula; G1, biofilm group treated with Cajuputs candy made with Melaleuca cajuputi essential oil from Pulau Buru; G2, biofilm group treated with Cajuputs candy made with Melaleuca cajuputi essential oil from Mojokerto; G3, biofilm group treated with Cajuputs candy made with Melaleuca cajuputi essential oil from Ponorogo; G4, biofilm group treated with Cajuputs candy made with Melaleuca cajuputi essential oil from Pasuruan; G5, biofilm group treated with Cajuputs candy made with Melaleuca cajuputi essential oil from Kuningan. Introduction Candida albicans is the most prevalent fungus in oral microbiota 1. This opportunistic fungus grows as yeast, pseudohyphae, and hyphae based on environmental conditions 2. The hyphal form is relevant for its virulence as it allows penetration and invasion of epithelial cells 3. Streptococcus mutans is a strong acidogenic and aciduric bacteria, defined as the major cause of dental caries. The critical virulence factor of S. mutans is its capacity to convert dietary sugars to produce an extracellular polysaccharide (EPS) matrix, mainly through glucosyltransferase enzymes (Gtfs) 4. EPS is the main building block of the biofilm. It can provide a binding site for colonization by other microbes and creates an acidic environment 5, 6. Several studies have reported that C. albicans is frequently found with S. mutans in early childhood caries (ECC) 7 ? 9. The presence of both microbes indicates cross-kingdom feeding 10. Furthermore, GtfB from S. mutans plays a significant role in mediating this dual-species interaction 11. Their co-species interaction enhanced cell accumulation, biofilm formation, and Gtf gene expression 8, 12. Therefore, targeting the synergism of C. mutans in mixed biofilms has become a promising strategy for oral antimicrobial exploration 13 ? 15. In accordance with the efficacy of essential oils as natural antimicrobial substances, Cajuputs candy (CC) has been developed using Melaleuca cajuputi essential oil (MCEO) as the main flavor ingredient. Previous work in our lab revealed the efficacy of CC in inhibiting biofilm formation by single oral microbes such as S. mutans (unpublished report) and C. albicans 16. As C. mutans have been noted for their synergistic relationship 5, 7, 8, 10, we evaluated the capacity of CC to impair their symbiotic interaction in this study. This finding will provide novel evidence for CC in interfering with the traits of cariogenic oral microorganisms. Methods Microbial strains and MCEO samples A C. mutans Xc were used for this study. C. albicans was obtained from the Oral Biology Laboratory stock culture previously isolated from the patients with their consent in the dental hospital of Universitas Indonesia 17. S. mutans Xc was kindly provided by Prof. Yoshihisa Yamashita, Department of Preventive Dentistry, Kyushu University, Japan 17. They were maintained as glycerol stocks at -80°C in our laboratory. albicans was grown in Sabouraud dextrose broth (SDB) (Oxoid, UK) for 24 hours at 37°C. mutans was cultured in brain heart infusion (BHI) (Himedia Laboratories, India) for 24 hours under anaerobic conditions (10% CO 2, 10% H 2, 10% N 2). The cell densities of each culture were quantified using total plate count on an agar medium. Five essential oils were obtained. MCEO from Mojokerto, Ponorogo, Pasuruan, and Kuningan were provided by Perhutani Indonesia, whereas MCEO from Pulau Buru was obtained from local villages where they produce the MCEO on a small scale by home distilling. For this, approximately 300kg of sun-dried leaves of Melaleuca cajuputi are placed in the boiler of the distilling apparatus and hydrodistillation is performed for six hours. After passing through the condenser, the extracted oil is collected and separated from the residual water. The essential oil is stored in a dark bottle. CC preparation The candies were prepared by mixing 98 g isomalt (Beneo-Palatinit GmbH, Germany), 0. 1 g Acesulfame K (Anhui Jinhe Industries, China) and 0. 1 g water 18. The mixed ingredients were heated to 150°C with continuous stirring. As the temperature decreased to 135°C, 820 ?L MCEO and 180 ?L peppermint oil (Brataco Chemika, Indonesia) was added and the dough was molded. Peppermint oil was used as a secondary flavor in addition to MCEO. To identify the most active MCEO, the MCEOs were varied among the candies. Pulau Buru was used as the targeted reference as it has been utilized from the beginning of our research series 16, 18 and needed to be replaced with other potent MCEOs due to its currently limited amounts. Four MCEOs were selected from our previous work as they had similar sensory characteristics to MCEO Pulau Buru (Siska Septiana, unpublished report). Five kinds of CCs were prepared using MCEO from different origins with Pulau Buru as the reference, and Mojokerto, Ponorogo, Pasuruan, and Kuningan as the alternative MCEOs. Mixed biofilm formation A mixed biofilm was prepared on a 96-well plate by inoculating approximately 2 × 10 4 colony forming units per milliliter (CFU/mL) of C. albicans suspended in SDB and 2 × 10 6 CFU/mL of S. mutans in BHI in an equal suspension volume (50 ?L). The well was previously coated with fetal bovine serum (Biosera, South America) with one-hour incubation at 37°C. Supernatants were removed after a 90-minute incubation under anaerobic conditions. Then, 140?L of tryptic soy broth (Oxoid, UK) supplemented with 1% sucrose was added to each well followed by 60 ?L of CC formula (each CC was dissolved in sterile distilled water (1:2 w/v) prior to the analysis). The biofilm group treated with CC made from Pulau Buru MCEO (as the reference) was represented as G1. Other treated groups G2, G3, G4, and G5 represented biofilms with the addition of CC made from Mojokerto, Ponorogo, Pasuruan, and Kuningan MCEOs, respectively. The untreated control (G0) was mixed biofilm without addition of the test CC formula. A light microscope equipped with a mobile camera (Primo Vert, Zeiss, Germany) was used to observe biofilm formation. Mixed biofilm analysis The plates mentioned previously were incubated for zero, three, and 24 hours a
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