Gallbladder polyps are common in the general population, but gallbladder metastasis of renal cell carcinoma (RCC) is very rare. In a patient with RCC diagnosed with a small gallbladder polyp that does not meet the traditional size criteria, the surgeon faces a dilemma of whether cholecystectomy should be performed given the possibility of metastasis.A 55-year-old man who had received a left nephrectomy for RCC presented with a gallbladder polyp that was noted at the time of the nephrectomy. Imaging showed the maximum diameter of the polyp had increased from 5 mm to 24 mm in the 40 months after the initial diagnosis.Pathological and immunohistology findings confirmed the gallbladder polyp as a metastasis of clear-cell RCC.: We performed a laparoscopic cholecystectomy.Even though the synchronous solitary gallbladder metastasis was left untreated and a cholecystectomy was not performed over the 40 months, no metastasis occurred in other sites. The patient is free from disease 10 months after the cholecystectomy.Solitary gallbladder metastasis of RCC may have more favorable outcomes than typical metastases. Although gallbladder metastasis of RCC occur rarely, it can occur, and any changes in gallbladder polyps in RCC patients should be managed under a strong suspicion of metastasis. Gallbladder metastases from RCC may also occur but are very rare.Herein, we report a case involving a gallbladder polyp that was identified as a synchronous metastasis of RCC found 40 months after the nephrectomy.2In February 2019, a 55-year-old man was referred to our department for further evaluation and treatment of a gallbladder mass. He had a history of nephrectomy for clear-cell RCC of the left kidney. Contrast-enhanced computed tomography (CT) performed just before the nephrectomy revealed a tumor measuring 9 cm at the upper pole of the left kidney that exhibited exophytic growth and had a heterogeneous solid and cystic consistency. Simultaneously, a small enhancing polyp measuring 5 mm in diameter was identified at the liver side of the gallbladder body (Fig. The patient was followed up with CT scanning in an outpatient clinic, which indicated no suspicious lesions suggesting the recurrence of RCC except for the gallbladder polyp, whose diameter grew progressively by 5 mm in the following 34 months. The last CT scan performed before the patient's cholecystectomy in February 2019 showed that the polypoid gallbladder mass had grown and had a maximum diameter of 22 mm (Fig. We performed a laparoscopic cholecystectomy in April 2019. Gross examination of the specimen showed a pink–red polypoid mass measuring 23 mm in the largest dimension that was detached from the inner mucosal layer. To decide on the extent of surgery needed, we checked an intraoperative frozen biopsy, which showed an RCC metastasis without involvement of the muscular layer. The final pathology diagnosis was gallbladder metastasis of clear-cell RCC. Microscopically, the lesion comprised malignant clear cells that were confined to the polyp and a negative margin Fig. . ImmunohTo detect other possible metastases, we requested positron emission tomography after the cholecystectomy, which showed no lesions of the metastasis. The patient is free from disease and is in a satisfactory condition 10 months after the cholecystectomy. The patient provided written informed consent for publication of this report.3In this case, a 5 mm gallbladder polyp showed enhancement on contrast CT and was identified at the body of the gallbladder before the nephrectomy. The polyp was confirmed as a synchronous solitary gallbladder metastasis of RCC 40 months after the nephrectomy. Bhatt et al also reported that patients aged >50 years, sessile polyps, and single polyps are independent risk factors for malignancy; for polyps measuring 4 to 10 mm, they recommended cholecystectomy according to the risk probability of malignancy.Cholecystectomy for polypoid lesions of the gallbladder is traditionally performed according to the size threshold of 10 mm. However, in clinical practice, there is controversy whether cholecystectomy should be performed in patients with a gallbladder polyp smaller than 1 cm. Several factors should be considered when deciding whether to perform a cholecystectomy given the potential risk of malignancy; these factors include the patient's age and ethnicity; the lesion's morphology, size, and growth; and the presence and number of gallstones. A European group has recommended performing a cholecystectomy in patients with polyps measuring 6 to 9 mm if the patient has risk factors for gallbladder malignancy such as age >50 years, primary sclerosing cholangitis, Indian ethnicity, or a sessile polyp, and follow-up ultrasound for polyps <6 mm or measuring 6 to 9 mm in patients with no risk factors. In a CT study, Choi et al analyzed the imaging features of gallbladder metastasis and reported that the characteristic pattern of gallbladder metastasis from RCC is the appearance of polypoid lesions with strong enhancement in the early and late arterial phase and wash-out in the portal phase. However, another study found no significant differences in enhancement pattern between nonneoplastic and neoplastic polyps. Moreover, for small polyps measuring <1 cm, imaging studies are limited in their ability to identify the characteristics associated with malignancy.Despite the variety of imaging modalities available, it can be difficult to distinguish neoplastic polyps from nonneoplastic polyps because of their significant overlap in appearance. Transabdominal ultrasonography is good for detecting gallbladder polyps, but it has a poor discriminating ability between true and pseudo polyps. In other words, there was no definitive reason to perform the cholecystectomy according to the conventional indications.Given the difficulties mentioned above, deciding to perform a cholecystectomy at the time of initial diagnosis presented as a dilemma for the surgeons because the polyp measured <6 mm and it was not perceptible on an unenhanced CT image, which is a suggestive feature of cholesterol polyps.However, there seems to have been a mistake in the management of the gallbladder polyp in this patient. The follow-up CT showed that the polyp had grown progressively to 10 mm 34 months after the initial presentation. Therefore, the cholecystectomy should have at least been performed at that time. reported a 5-year survival rate of 80% in RCC patient with solitary gallbladder metastasis and concluded that gallbladder metastasis from RCC is not always at an advanced stage with a poor outcome. We also presume that a solitary gallbladder metastasis of RCC may exhibit different disease behaviors compared with typical metastases because no concomitant metastases occurred during the 40-month follow-up in this patient.Distant metastasis from RCC usually have a poor outcome, and the 5-year survival rate is <10%. However, in contrast to typical metastases, solitary gallbladder metastasis of RCC have a more favorable patient survival. Shyr et al4Solitary gallbladder metastasis of RCC may have a more favorable outcome than typical metastases. Although gallbladder metastasis of RCC is very rare, it can occur, and any changes in a gallbladder polyp in RCC patients should be managed under a strong suspicion of metastasis.Conceptualization: Sung Hoon Cho, Hyung Jun Kwon, Jae Min Chun.Resources: Ja Ryung Han, Seock Hwan Choi, Hyun Tae Kim, Man-Hoon Han, Jae Min Chun.Supervision: Young Seok Han.Writing – original draft: Sung Hoon Cho.Writing –- review & editing: Sung Hoon Cho, Young Seok Han, Jae Min Chun.


Global change causes widespread decline of coral reefs. In order to counter the anticipated disappearance of coral reefs by the end of this century, many initiatives are emerging, including creation of marine protected areas (MPAs), reef restoration projects, and assisted evolution initiatives. Such efforts, although critically important, are locally constrained. We propose to build a “Noah's Ark” biological repository for corals that taps into the network of the world’s public aquaria and coral reef scientists. Public aquaria will serve not only as a reservoir for the purpose of conservation, restoration, and research of reef-building corals but also as a laboratory for the implementation of operations for the selection of stress-resilient and resistant genotypes. The proposed project will provide a global dimension to coral reef education and protection as a result of the involvement of a network of public and private aquaria. Global change is causing a widespread decline in coral reefs. This Community Page article proposes to build the World Coral Conservatory, a “Noah's Ark” biological repository that taps into the network of the world’s public aquaria and coral reef scientists, in order to preserve the fast-disappearing biodiversity of coral reefs. Reef-building corals play a pivotal role in marine ecosystems and constitute the foundation of coral reef ecosystems that represent the world’s most important biogenic structures. Coral reefs provide habitat and trophic support for one-third of all marine organisms, and their biodiversity rivals that of tropical rainforests . In addiBoth in situ and ex situ (on-land) actions can be taken to protect and/or restore coral reefs, and both approaches have their advantages and drawbacks as outlined in the following. The vast majority of conservation actions are in situ protection, either through the establishment of marine protected areas (MPAs) or through restoration of degraded coral reefs. The importance and benefits of MPAs are well documented, serving primarily to decrease local stress to increase the resilience of reefs –17. UnfoTo address the problem that not all reefs can be saved and to focus efforts on reefs that are most likely to survive in the future, Hoegh-Guldberg and colleagues conducted a global scale analysis to identify resilient regions for which long-term coral reef conservation may be an attainable goal, even under ongoing ocean warming . The idAn alternative for in situ conservation management would be to use ocean warming resilient/resistant specimens for reef restoration purposes. However, natural populations of resilient/resistant corals, such as those from the Persian Gulf, are locally adapted and likely do not prevail in other environments , 66. GivOn-land (ex situ) solutions aim to preserve corals outside their natural environment, with the benefit that such maintained corals are not subject to marine heatwaves or other natural events  that might destroy them. Rather, ex situ solutions provide a more controlled environment where corals are not subject to arbitrary environmental change. At the same time, we acknowledge the diversity of opinion around this topic, and the concern that aquaria conditions ultimately “select” for domestic rather than wild genetic profiles. In other words, corals that are well adapted to “benign” aquaria conditions will do better, which may make it challenging to maintain coral genotypes under aquaria settings that respond well to environmental extremes. The ultimate utility of aquaria as a coral conservatory (and seedbank) will have to be demonstrated, and the best way to do this is by incorporating “the science learned along the way”. Similar to what has been done with vertebrates  or cropsEx situ culturing builds on the conservation work already carried out by zoos and aquaria for endangered populations of vertebrates and invertebrates , 55. ThiPublic aquaria around the world now maintain and present live coral and ensure continuous replication/persistence through so-called cuttings. Emerging from discussions at the “International Coral Reef Symposium 2016” (ICRS2016) in Honolulu , we propose here the creation of the WCC. This endeavor would take advantage of combining 3 of the 4 safeguarding actions proposed by Allemand and Osborn , supportThe aims of the WCC are as follows:To create a repository of living coral colonies that represent the majority of extant species;To preserve coral genetic and species diversity from a variety of habitats and by this a mechanism to contribute to restore coral reef diversity through reintroduction and reef restoration approaches ;To contribute to protecting coral reef biodiversity by developing solution-based approaches that combine reef science, conservation, and management;To provide researchers from all over the world with referenced and trackable biological material, which also reduces the burden of collection of biological material from coral reef ecosystems and supports standardization via research on shared resources ;To use the available biological resources for "assisted evolution approaches" to increase stress tolerance and resilience/resistance; andSpecial Report on the Ocean and Cryosphere in a Changing Climate, approved in Monaco in September 2019, highlights education as an essential component as a solution for safeguarding ecosystems.To provide comprehensive information on corals and coral reefs to the general public and stakeholders in order to educate and enable people to participate in the collective effort of coral reef conservation. The IPCC The urgent need to preserve the unique biodiversity and natural heritage of corals and to minimize harvesting corals from the field for scientific or commercial purposes makes approaches such as the WCC a priority. Notably, we will prioritize coral species based on geographical distribution and population size. For the biological repository, which we term the “coral bank”, state-of-the-art genetic techniques will allow the precise identification of species and genotypes in order to trace colonies from their respective origin and to monitor distribution and spread of daughter colonies  for research and propagation. Preservation of genetic diversity within species and populations in the WCC via genotype identification, archiving, and tracing is a prerequisite for successful conservation measures and currently not widely implemented in coral restoration practices.The first meeting of the steering committee of the WCC was held in 2019 during the Monaco Ocean Week, and we defined a plan to sample, cultivate, investigate, develop, communicate, restore, and transfer .Tara Pacific Expedition [https://greatbarrierreeflegacy.org/), the Association of Zoos and Aquariums, the National Oceanic and Atmospheric Administration, and the Mote Marine Laboratory, which are participating in the Florida Reef Tract project (https://www.aza.org/the-florida-reef-tract); further, the Smithsonian Conservation Biology Institute in Hawai’i.The number of coral species currently kept in aquariums is on the order of 250, which represents only a small fraction, around 15%, of the estimated 1,600 coral species globally , highligpedition  and in oOur second goal is to perform a targeted collection of corals with specific traits. Endemic species should be part of the priority list, given their unique cultural value and potentially interesting genetics and physiology , 63, 64.Because it is paramount that samples are uniquely identified and tracked in their respective native environment, we propose to use transponders for this purpose, as is done, for example, for penguins . The barIn addition to the aquarium of Monaco, which was the first in the world to present a life-size living coral reef, public aquariums are now successfully maintaining and cultivating live corals, for example, Océanopolis (France), Nausicaa (France), and Burgers' Zoo (The Netherlands). In total, we anticipate about 50 aquariums to comprise the network of the WCC.For all coral species that are collected under the WCC, we will provide an assessment of genetic diversity based on collected coral colonies. Further, a reference transcriptome (and genomes where possible) will be produced for each species to address questions regarding the following:Biodiversity: with more than 13,000 species, the sequencing of nearly 10% of Cnidarians will provide us with a good overview of the species that comprise the phylum Cnidaria;Evolution: availability of 1,000+ transcriptomes allows us to address fundamental questions of coral evolutionary biology, such as the formation of new species, adaptive radiation, calcification, symbiosis, and the genetics of adaptability in light of climate change;Biomedical and cosmetic applications: corals offer very interesting aspects concerning aging and regeneration, for which the sequencing of 1,000 transcriptomes will improve our knowledge regarding candidate genes contributing to longevity and the extraordinary resistance to UV exposure. Further, corals can become ecological sentinels, and tests using coral cultures can become an industrial standard of ecotoxicity for cosmetics and other xenobiotics ;Biomonitoring: the recent use of environmental Deoxyribonucleic Acid (eDNA) is a major breakthrough  because The sequencing efforts will result in a massive amount of valuable data for the scientific community and will be made publicly available along with the tracking and metadata.The WCC is also interested in applying assisted evolution approaches  using thWe think that it is also important to have a network of non-governmental organizations (NGOs) working on reef restoration, such as the “50 Reefs initiative” , which aims to identify, preserve, and restore 50 coral reefs around the world as priority reefs for conservation . The WCCThe WCC, via its public aquarium network, can play a prominent role in educating various audiences about the state of scientific research, the specific biology of corals, the health state of reefs around the world, their contribution to the world's biodiversity, and the services they provide to humans.Finally, capacity building is an important aspect to this project; transfer of knowledge and techniques on coral culture  is neceThe WCC contains many areas of interest to industry and, in particular, holds promise for the development of biomedical and cosmetic applications. The involvement of industrial actors should provide financial resources to support research and development, which will provide a link for industry to follow their interests but, at the same time, share part of the responsibility to maintain coral reefs as an ecological and economical resource for future generations.https://u.osu.edu/grottoli.1/coral-bleaching-rcn/). These concerted efforts could then provide decision-makers , reef-area managers, and users with relevant tools in terms of information, methodologies, and decision support. The global network of public aquaria to house valuable coral reef resources provides an essential link between conservation, research, and education efforts.We here lay out the overall vision and mission of the WCC , an internationally open community of research laboratories, science centers, public aquaria, stakeholders, and national administrations that builds on the idea to use aquaria as a “Noah’s Ark” to preserve corals and to establish a global network and platform for sharing of biological material, and for knowledge generation and exchange to enhance and support conservation and restoration of coral reef ecosystems. This network is based on the principles of sharing of data, organization of conferences, symposia, seminars, congresses, temporary exhibitions, and internet forums and welcomes participation and contribution of industry, schools, associations, divers, and citizens. We set out a call to the community to facilitate the pooling of resources and federate stakeholders around common projects for the good of coral reefs, as proposed by the Coral Bleaching Research Coordination Network (


For instance, microarray has long been used to study the gene expression profiles of AML patients. The different profile of gene expression has enabled clinicians to tailor better treatment for patients and predict whether patients have the tendency to relapse  affects 20% of diagnosed hematological malignancies in pediatric patients . Standar relapse . In a re relapse . The stu relapse . Another relapse . Howeverquencing . A recenquencing . Previouquencing . NeverthAML is the second commonest hematological malignancy affecting children worldwide and more research at transcriptome level is needed to help to improve the survival rates.de novo and relapsed pediatric AML in the Malaysian population.This data is of value to understand further the molecular landscape underpinning Most of the data available only report the molecular profiles at diagnosis and relapse stages. In this study we performed the sequence at remission as well. This finding is important for researchers to understand the changes in the progression of the disease at all stages.Deep transcriptome sequencing will allow users to not only obtain the gene expression profile, but also for fusion gene identification and mutation analysis.In this experimental design, we successfully sequenced the RNA of three Malaysian pediatric AML patients at three different stages; diagnosis, remission, and relapse. One of the analyses that can be used with this data is the identification of differentially expressed genes. For instance, here, we compare the gene expression profile for PAML1, using three different group comparisons (1) Diagnosis vs. Relapse, (2) Diagnosis vs. Remission, and (3) Relapse vs, Remission  derived from the bone marrow of 3 patients were obtained. This study was reviewed and approved by the Medical Research Ethics Committee of Universiti Kebangsaan Malaysia. Written informed consent was obtained from all parents. The percentage of blasts at diagnosis and relapse were >50%, while at remission the blasts percentage were <5%. PAML1 had a cytogenetically normal karyotype, while PAML2 and PAML3 had t translocations. Mononuclear cells (MNC) were isolated using the Ficoll-Paque  method.Total RNA from isolated MNC was extracted and purified according to the standard protocol of AllPrep DNA/RNA/miRNA Universal Kit . The quality and quantity of the total RNA were checked using NanoDrop ND-1000  and Qubit 2.0 Fluorometer . Total RNA integrity was then assessed using the Agilent 2100 Bio-Analyzer . Only those bone marrow aspirate (BMA) with RNA integrity number (RIN) > 7.0 were included in transcriptome sequencing (RNA-Seq).One microgram of total RNA was used to remove ribosomal RNA (rRNA) using the Ribo-Zero rRNA Removal Kit . Purified rRNA-depleted RNA was subjected to RNA library preparation using the ScriptSeqTM v2 RNA-Seq Library Preparation Kit according to the manufacturer instructions. After library construction, the library was diluted to 1.5 ng/µl after preliminary quantitation by Qubit 2.0 and insert size by Agilent 2100. Qpcr was used to accurately quantify the library effective concentration (> 2nM), in order to ensure the library quality. The libraries were then sequenced on Illumina HiSeq 2500 .Paired-end sequences were individually obtained as FASTQ files from the images by CASAVA base recognition software. We later filtered the raw reads to remove adaptor sequences, reads containing undetermined bases >10%, low quality reads having QScore of over 50% bases of the read is < = 5. After obtaining the clean reads, we further mapped the reads against the human reference genome (GRCh38) using TopHat2 . For thehttps://www.ncbi.nlm.nih.gov/sra/?term=PRJNA509497).All of the sequencing reads from this project have been uploaded to NCBI with the BioProject ID PRJNA509497  provided by the Ministry of Higher Education Malaysia, with the grant ID FRGS/1/2015/SKK08/UKM/03/2.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Cnot6l), a deadenylase within the CCR4-NOT complex, results in mRNA decay defects during metaphase I (MI) entry. Knockout of B-cell translocation gene-4 (Btg4), an adaptor protein of the CCR4-NOT complex, results in mRNA decay defects following fertilization. Therefore, mechanisms controlling mRNA turnover have significant impacts on oocyte competence and early embryonic development. Post-transcriptional inosine RNA modifications can impact mRNA stability, possibly through a translation mechanism. Here, we assessed inosine RNA modifications in oocytes, eggs, and embryos from -/-Cnot6l and -/-Btg4 mice, which display stabilization of mRNA and over-translation of the stabilized transcripts. If inosine modifications have a role in modulating RNA stability, we hypothesize that in these mutant backgrounds, we would observe changes or a disruption in inosine mRNA modifications. To test this, we used a computational approach to identify inosine RNA modifications in total and polysomal RNA-seq data during meiotic maturation . We observed pronounced depletion of inosine mRNA modifications in samples from -/-Cnot6l, but not in -/-Btg4 mice. Additionally, analysis of ribosome-associated RNA revealed clearance of inosine modified mRNA. These observations suggest a novel mechanism of mRNA clearance during oocyte maturation, in which inosine-containing transcripts decay in an independent, but parallel mechanism to CCR4-NOT deadenylation. Mammalian oocytes must degrade maternal transcripts through a process called translational mRNA decay, in which maternal mRNA undergoes translational activation, followed by deadenylation and mRNA decay. Once a transcript is translationally activated, it becomes deadenylated by the CCR4-NOT complex. Knockout of CCR4-NOT Transcription Complex Subunit 6 Like ( Growing mammalian oocytes transcribe and store transcripts that support meiotic maturation, fertilization, and early embryo development ,2,3,4. TCnot6l) is uniquely expressed in mouse oocytes, and regulates deadenylation of transcripts undergoing translational mRNA decay during oocyte maturation [-/-Cnot6l mice are severely subfertile due to the over-translation of undegraded maternal transcripts, resulting in spindle defects during the completion of meiosis [-/-Zfp36l2 mice are infertile due to decreased oocyte numbers as well as decreased rates of oocyte maturation [-/-Btg4 mice are also infertile due to embryonic arrest at the 2-cell and 4-cell stages [An understanding of the individual components of the CCR4-NOT complex has illuminated specific features of mRNA transcripts leading to their specific and selective decay. The ribonuclease CCR4-NOT Transcription Complex Subunit 6 Like ; Mean ± SEM) were significantly lower than both WT samples and -/-Btg4 samples at all oocyte/egg/embryo stages . The results  at the GV, MI, and MII stages in WT and -/-Cnot6l oocytes/eggs. The polysome RNA-seq datasets we analyzed were prepared by isolating mRNA bound by multiple ribosomes  from groups of 500 whole oocytes . Polysome RNA-seq was not performed on Btg4-/- oocytes or eggs [-/-Cnot6l oocytes/eggs, significantly fewer transcripts were inosine-modified at the GV stage, and the number of inosine-modified transcripts increased, rather than decreased during oocyte maturation , compared to WT samples  and -/-Btg4 samples , compared to WT oocytes/eggs . Overall, the distribution of inosine RNA modifications is similar between all stages of oocyte maturation in WT and -/-Btg4 mice, with the majority of modifications occurring in the CDS and 3′ UTR. Conversely, samples from -/-Cnot6l mice display a higher proportion of intron inosine RNA modifications   to ident-/-Btg4 samples [-/-Cnot6l mice, the pattern of inosine RNA modifications was reversed, with more than 60% exhibiting nonsynonymous substitutions , however, other codons were not modified at all , as seen in -/-Cnot6l MI and MII oocytes (p < 0.05). Conversely, at the MII stage of -/-Cnot6l eggs, there is a negative association of RNA modification efficiency and RNA abundance . Thus, in the -/-Cnot6l oocytes/eggs where mRNA is stabilized, the inosine modified mRNA are degraded, while the unmodified mRNA are stabilized.Transcriptome-wide inosine RNA modification efficiency at various codons remained around 50% throughout oocyte maturation in WT samples  is linked to the catalytic activity of ADAR [-/-Cnot6l samples had similar levels of inosine RNA modifications at the GV stage , while in -/-Cnot6l oocytes/eggs, we observed a negative correlation . If CNOT7 or CNOT8 were compensating for an absence of CNOT6L, we would predict a similar correlation of mRNA abundance to inosine modification efficiency in both WT and -/-Cnot6l samples because the inosine modified transcripts would still be degraded in the absence of CNOT6L. However, we observed the opposite; mRNA stabilized in the absence of CNOT6L is targeted for degradation if it contains inosine modified codons. It is possible that CNOT7 or CNOT8 could be compensating for the absence of CNOT6L to mediate the decay of inosine containing transcripts. We observed that among common ribosome-associated inosine modified transcripts, in WT oocytes/eggs, the abundance of mRNA has a positive correlation with inosine modification efficiency  PRJNA486094 [WT GV, MI, MII, and Zygote RNA-seq data, and GV, MI, MII, and Zygote RNA-seq data from NA486094 . The autNA486094 . Mus musculus index built from GRCm38 containing common SNP annotation from dbSNP. We identified putative inosine RNA modifications utilizing a combination of the following software: HISAT2 aligner, the Genome Analysis Tool Kit (GATK), and Ensembl Variant Effect Predictor (VEP) ,33,44,45Default settings for HISAT2 were used for each type of library aligned  . Prior t


Metastasis is one of the main causes of death and treatment failure in ovarian cancer. Some small molecule inhibitors can effectively inhibit the metastasis of primary tumors. However, they do not kill the primary tumor cells, which may lead to continuous proliferation. Herein, we have prepared a multifunctional nanoparticles named TPD@TB/KBU2046, which consisted of three functional moieties: (1) KBU2046  that can inhibit the metastasis of the primary tumors, (2) TB (photodynamic-AIEgens) that may suppress the growth of the primary tumors, and (3) TPD, which contains TMTP1  that can enhance the TB/KBU2046 dosage in the tumor site.Methods: The TPD@TB/KBU2046 was prepared by nano-precipitation method. We linked the targeting peptide (TMTP1) to the nanoparticles via amidation reaction. TPD@TB/KBU2046 nanoparticles were characterized for encapsulation efficiency, particle size, absorption spectra, emission spectra and ROS production. The combinational efficacy in image-guided anti-metastasis and photodynamic therapy of TPD@TB/KBU2046 was explored both in vitro and in vivo.Results: The TPD@TB/KBU2046 showed an average hydrodynamic size of approximately 50 nm with good stability. In vitro, TPD@TB/KBU2046 not only inhibited the metastasis of the tumors, but also suppressed the growth of the tumors under AIEgens-mediated photodynamic therapy. In vivo, we confirmed that TPD@TB/KBU2046 has the therapeutic effects of anti-tumor growth and anti-metastasis through subcutaneous and orthotopic ovarian tumor models.Conclusion: Our findings provided an effective strategy to compensate for the congenital defects of some small molecule inhibitors and thus enhanced the therapeutic efficacy of ovarian cancer. The inherent properties of these small molecule inhibitors, including lack of cytotoxicity, make their biocompatibility excellent, but this is a defect in the treatment of tumors. Therefore, based on the defect of these small molecule inhibitors, it is necessary to introduce a therapeutic element to suppress the growth of the tumors.Recently, many small molecule inhibitors, such as tea polyphenols, curcumin, KBU2046, BENC-511, ZD6474, Photodynamic therapy (PDT) is a burgeoning, noninvasive and safe therapeutic assay that utilized a photosensitizer, light of particular wavelength and oxygen to achieve its therapeutic effect Scheme 1). In addition, TPD@TB/KBU2046 nanoparticles contain tumor targeting peptide (TMTP1), which specifically binds to highly metastatic tumor cells In vivo, our results showed that TPD@TB/ KBU2046 nanoparticles not only possess excellent tumor targeting, but also had cooperative effects of anti-metastasis and anti-growth, which provided a promising strategy for the treatment of ovarian cancer.Herein, we have successfully prepared a self-assembled nanoparticle (TPD@TB/KBU2046) containing small molecule inhibitor (KBU2046) and photodynamic-AIEgen (TB) (Dimethylsulfoxide (DMSO), tetrahydrofuran (THF), dimethyl formamide (DMF) was purchased from China National Medicines Co. Ltd . 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[succinimidyl(polyethylene-glycol)-2000] (NHS-PEG-DSPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N [methoxy(polyethylene glycol)-2000] (PEG-DSPE) were purchased from Xi'an Ruixi Biological Technology Co. Ltd . The peptide TMTP1 was purchased from GL Biochem Ltd . PVDF membrane was purchased from Millipore Corporation . Fetal bovine serum was purchased from Gibco Company . 2′,7′-dichlorofluorescin diacetate (DCFH-DA) and Rose Bengal (RB) were provided by Yeasen Co. Ltd. . 9,10-Anthracenediyl-bis(methylene)dim alonic acid (ABDA) and 5-chloromethylfluorescein diacetate (CMFDA) were provided by Sigma-Aldrich . Cell Counting Kit-8 (CCK8) was purchased from Beyotime Biotechnology .1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[succinimidyl(polyethylene glycol)-2000]  was linked with TMTP1 (T) by amidation reaction to yield the TMTP1-PEG-DSPE (TPD). In brief, NHS-PEG-DSPE  was added to a DMF solution (1 ml) of TMTP1 . The above solution was poured into ultrapure water and stirred for 24 h. Then, the reaction product was added to water solution and purified by dialysis (MWCO 3500) in flowing ultra-pure water for 48 h. After lyophilization, the product TPD was obtained with a yield of 73.4%.via a 0.2 μm filter membrane to get targeted nanoparticles. Nano-ZS ZEN3690  was used to detect the particle size at 25 °C. Particle size was measured in PBS buffer.NHS-PEG-DSPE , TMTP1-PEG-DSPE , 2,6-Bis(4-(diphenylamino)phenyl)-4,8-bis((2-ethylhexyl)oxy)benzodithiophene 1,1,5,5-tetraoxide , and 4'-fluoroisoflavanone  were simultaneously dissolved in tetrahydrofuran (THF) solution (1 mL). Sonicator with a microtip probe was used to stir the aforesaid solution for 5 min at 10 W. The mixed solution was added into the water, whereafter, the organic solvent THF in the mixed solution was blown dry with nitrogen. The solution was then filtered 6 SKOV-3 or EGFP- SKOV-3 cells were subcutaneously inoculated into the right anterior side of female BALB/c nude mice. Tumor growth was measured using a vernier caliper. The tumor volume was calculated as: volume = 0.5× ((tumor length) × (tumor width) × (tumor width)).The experimental animal scheme strictly complied with the requirements of the experimental animal ethics committee of Tongji Medical College and conformed to the principles of animal protection, animal welfare, and ethics. BALB/c nude mice were purchased from HFK Bioscience Co. . They were fed normally and provided unlimited access to water. Briefly, 1 ×103, SKOV-3 tumor-bearing mice were randomly split into five groups. Mice were injected with TPD@TB/KBU2046 ), PD@TB/KBU2046 , TPD@TB , TPD@KBU2046  and TPD  via tail vein, respectively. After 12 h, the nanoparticles were distributed into the tumor and then the tumor area was exposed to white light (200 mW cm-2) for 20 min. The weight and tumor volume of the mice were daily recorded. The tumors and major organs were collected for histological observation by standard hematoxylin & eosin and immunohistochemical staining.When the volume of SKOV-3 tumor reached around 20 mm3. Surgical treatment was performed after four consecutive injections of nanoparticles. After resection of the tumors, the abdominal cavity  of mice were treated with white light irradiation (Intensity: 200 mW cm-2) for 20 min. After treatment, the recurrence and metastasis of tumors in mice were observed by IVIS.15 female BALB/C nude mice (5-week-old) were operated under anesthesia to expose the abdominal cavity, and the EGFP-SKOV-3 cells were transplanted into right ovarian capsule. TPD@TB/KBU2046 , PD@TB/KBU2046 , TPD@TB , TPD@KBU 2046  and TPD  were injected into the tail vein when the primary tumor was about 200 mmvia a modified nano-precipitation approach using TPD to form nanoparticles According to our previous report, we synthesized the AIE-photosensitizer TPA-BDTO (TB)  48. The Five nanoparticles  were respectively incubated with SKOV-3 cells for 4 h. Strong red fluorescence (TB) can be observed in the SKOV-3 cells by CLSM after cultured with TPD@TB/KBU2046, PD@TB/KBU2046 and TPD@TB  was used to establish a tumor-bearing mice model, and then the tumors were localized according to the spontaneous fluorescence  TPD@TB/KBU2046; (2) PD@TB/KBU2046; (3) TPD@TB; (4) TPD@KBU2046; and (5) TPD. The mice in each group were exposed to white light. As shown in Figure In order to investigate the anti-tumor efficacy of the nanoparticles 046; and  TPD. The046; and  TPD. The046; and  TPD. TheThe anti-metastasis effect of nanoparticles was evaluated according to the tumor boundary and lymph node metastasis. The black arrow in Figure  in vivo.Ki-67 was a marker that reflected the proliferative capability of tumor cells  in vivo.Low toxicity or even no toxicity of biological materials is a prerequisite for the further application In order to test the universality of this strategy and further improve the therapeutic effect of PDT, we used an orthotopic ovarian tumor model, combined with surgical treatment to indirectly increase the penetration depth of light, thereby improving the curative effect of PDT. Before treatment, we found that TPD@TB/KBU2046 also had a good targeting effect on orthotopic ovarian tumor Figure B. 15 micin vitro and in vivo. All in all, these findings implied that using photodynamic-AIEgens to compensate for the congenital defects of small molecule inhibitors may be a simple and efficient strategy.In conclusion, we proposed an effective self- assembled TMTP1-targeted nanoparticle entrapping AIEgen and small molecule inhibitor for image- guided PDT and anti-metastasis therapy. The targeting effect of TPD@TB/KBU2046 nanoparticles based on TMTP1 can increase the concentration of AIEgen (TB) and small molecule inhibitor (KBU2046) in the tumor site and reduce toxic effects toward normal tissues. The nanoparticles were demonstrated to exhibit superb ROS generation efficiency as well as excellent anti-metastasis capability. TPD@TB/KBU2046 can achieve cooperative treatment of anti-metastasis therapy and image-guided anti-growth for ovarian cancer both Supplementary figures and tables.Click here for additional data file.


In addition, additional conditions, such as storage medium and storage temperature of human peripheral blood mononuclear cells were evaluated, i.e., 4 °C for RNAlater or -80 °C for QIAzol and for the respective cognate lysis buffers; easyMAG, RNeasy or RiboPure. RNA was extracted from aliquots that had been stored for one day (Run 1) or 83 days (Run 2). After DNase treatment, quantity and quality of RNA were assessed by means of a NanoDrop spectrophotometer, 2100 Bioanalyzer and RT-qPCR for the ACTB reference gene. We observed that high-quality RNA can be obtained using RNeasy and RiboPure, regardless of the storage medium, whereas samples stored in RNAlater resulted in the least amount of RNA extracted. In addition, RiboPure combined with storage of samples in its cognate lysis buffer yielded twice as much RNA as all other procedures. These results were supported by RT-qPCR and by the reproducibility observed for two independent extraction runs.RNA quality and quantity are important factors for ensuring the accuracy of gene expression analysis and other RNA-based downstream applications. Thus far, only a limited number of methodological studies have compared sample storage and RNA extraction procedures for human cells. We compared three commercially available RNA extraction kits,  RNA expression level is a good indicator of the physiological status of cells and reveals the cell response under different stress conditions such as those encountered during host-pathogen interactions. High quality and quantity of extracted RNA is crucial for downstream applications such as reliable gene expression quantification through quantitative PCR  and RNA-Prior to RNA extraction, the appropriate storage of samples is of utmost importance as well. First, RNA is prone to degradation and since different RNAs can have different stability, this may influence the gene expression pattern . On the e.g. the NucliSENS easyMAG platform .Obtaining high-quality RNA also depends on the RNA extraction kit. Some kits are based on phenol-chloroform extraction, such as the RiboPure RNA Purification Kit—blood (Thermo Fisher Scientific) and TRI Reagent , which contain guanidine thiocyanate to denature cellular components and to inhibit RNase activity. Other kits are based on silica spin columns, such as the NucleoSpin RNA Blood Kit  and the RNeasy Mini Kit , which also use guanidine thiocyanate and β-mercaptoethanol, respectively, to inactivate RNases. Finally, other extractions are based on magnetic silica particles that capture nucleic acids, Regardless of the storage condition and RNA extraction procedure used, it is necessary to determine not only the quality but also the quantity of RNA prior to downstream analysis. Although it has been shown that 500 picograms is enough for cDNA synthesis , it is rStudies comparing different storage conditions and RNA extraction procedures that comprehensively analyze both RNA quality and quantity are therefore needed. In addition, results need to be somehow quantified and directly measured in order to facilitate comparison with other studies. For example, one study previously analyzed RNA yield after different storage conditions, and quantified RNA yield indirectly with Cq values obtained during reverse transcription and amplification (RT-qPCR) , such thACTB gene. Finally, RNA quality and yield were compared to determine which combination procedure of storage and extraction kit provided the best results.In this study, we compared the effect of storage of human peripheral blood mononuclear cells (PBMCs) in different storage media during two different time periods (one day vs. 83 days). We also compared three different RNA extraction kits. We analyzed RNA quality (as RNA purity and RNA integrity) and RNA yield using a NanoDrop spectrophotometer ND-1000 , a 2100 Bioanalyzer, and through RT-qPCR of the All methods were carried out in accordance to relevant guidelines and regulations and all experimental protocols were approved by the ethical committee of the University of Ghent (EC/2016/0192).Raw data containing all measurements, averages and standards deviations of the main figures and tables used in this study are shown in 6 PBMCs for two different time periods (1 day and 83 days) in five different storage media, i.e., (1) at 4 °C in RNAlater (RNL) or at -80 °C in (2) QIAzol Lysis Reagent , or in the cognate lysis buffers, i.e., (3) easyMAG lysis buffer (EML) or (4) RNeasy Mini Kit lysis buffer (RLT) or (5) RiboPure RNA Purification Kit—blood, lysis buffer (RPL). In addition, three commercial RNA extraction kits were compared, i.e., NucliSENS easyMAG (EM), RNeasy Mini Kit (RE) and RiboPure RNA Purification Kit—blood (RP).The setup of this study is illustrated in Since RNAlater (RNL) is not a lysis buffer, it must be removed to avoid interference with the cognate lysis buffer and downstream procedures, such as RNA extraction. Therefore, after storage in RNL, the aliquots were centrifuged and RNA was extracted from the pellet (RNLp). RNA was also extracted from the supernatant (RNLs) to evaluate crossover of RNA to the supernatant during centrifugation of the aliquots, which would lead to underestimation of the RNA quantity.This resulted in a total of 12 procedures (4 sample storage conditions in combination with 3 RNA extraction kits), as indicated in All 12 procedures were carried out for triplicate aliquots, and after storage for one day (Run 1) and for 83 days (Run 2).ACTB (reference) gene after storage of the RNA for 398 days (Run 1) and 442 days (Run 2), respectively.RNA quantity and quality were assessed, after DNase treatment of the RNA extracts, using a NanoDrop ND-1000 and a 2100 Bioanalyzer for each RNA extract, after storage of the RNA for 83 days (Run 1) and for 127 days (Run 2), and by means of RT-qPCR for the PBMCs were isolated from a buffy coat derived from one donor from Rode Kruis Vlaanderen , as previously described in .g for 10 min, the supernatant was removed, and cells were resuspended in HBSS to reach a final concentration of 107 PBMCs/ml.Cells were centrifuged again at 350 7 PBMCs/ml was divided in five parts , at 106 PBMC/ml. The five batches were aliquoted in numbers and volumes that were appropriate for each protocol. All aliquots were stored at -80 °C, or at 4 °C for RNL, until the three RNA extraction procedures were carried out.The PBMCs were stored in five different storage media, whereby each of the five batches was divided into different aliquots prior to storage, in order to minimize the number of freeze-thaw cycles. Prior to storage, the suspension of 10ve parts . These fi.e., after one day (Run 1) and after 83 days (Run 2) of storage. Each RNA extraction kit was carried out in triplicate, i.e., starting from three separately stored aliquots per storage condition.The three RNA extraction kits  were each carried out on two separate dates, g to remove RNL and then the pellet of cells (RNLp) was resuspended in the cognate lysis buffer of each kit. Removal of RNL is needed to avoid dilution of and interference with the cognate lysis buffers, in order to maximize cell lysis. In addition, we also extracted RNA from the RNLs to assess to what extent RNA was lost into the supernatant (RNLs)–which is usually discarded.For each run, on the day of the RNA extractions, the RNL-stored aliquots were centrifuged for 10 min at 20,800 i.e., four series  of three replicates each, were added to the cartridges, to reach the required 2-ml starting volume. After incubation for 10 min at room temperature, 100 μl of silica v/v EM Extraction Buffer 3 was added to each cartridge and processed on the easyMAG. The RNA was eluted in 35 μl of EM elution buffer.For the EM RNA extraction kit, the manufacturer advises to start the extraction on the apparatus in 2-ml volumes. To achieve this volume for the RNLp, 1 ml of EML was added to the pellet. This was not needed for RNLs or for cells that had been stored in QZL or in the cognate lysis buffer (EML), which were already in a 1 ml volume. Subsequently, EM RNA extraction was started by adding 1 ml of EML to each of 12 empty cartridges. Thereafter, the 12 one-ml aliquots, For the RE RNA extraction kit, the cells in the RNLp were disrupted by mixing with 350 μl of RLT, that had been supplemented with 143 mM β-mercaptoethanol. The disrupted RNLp, as well as the RNLs, QZL and RLT aliquots, were further homogenized by pipetting directly onto a QIAshredder spin column, that was placed in a 2 ml collection tube, and by subsequent centrifugation for 2 min at full speed. All aliquots were mixed with 350 μl of 70% ethanol for RNLp and RLT, and with 1 ml of 70% ethanol for RNLs and QZL to the homogenized lysate, in order to keep a 1:1 ethanol/lysate ratio.For the RP RNA extraction kit, 300 μl of acid-Phenol:Chloroform was added to all 12 samples, with PBMCs in 1 ml of QZL, in 1 ml of RNLs or in 850 μl of RPL, respectively. Pellets from RNLp were disrupted by adding 850 μl of RPL and vortexed vigorously to resuspend and lyse the cells.Contaminating DNA was removed from each RNA extract by means of DNase treatment, according to the manufacturer’s recommendations.DNase digestion of the EM RNA extracts (35 μl)  was carrThe RE RNA extraction procedure comprises an on-column DNase treatment using RNase-free DNase (Qiagen), as described by the manufacturer.g, and the supernatant was transferred to a new RNase-free tube. The final volume after DNase treatment and inactivation was 126.5 μl.DNase digestion of the RP RNA extracts  was carr260 nm/A280 nm) using a NanoDrop.RNA purity was assessed by determination of the ratio for absorbance at 260 nm vs. absorbance at 280 nm  and by means of RT-qPCR for the 2 and LightCycler 480 High Resolution Melting Master Mix . Amplification was carried out on a LightCycler 480 (Roche) using the following program: pre-incubation for 30 s at 95 °C and amplification for 45 cycles of 30 s at 95 °C, 10 s at 56 °C and 30 s at 72 °C, after which a high resolution melting curve was generated, using the following protocol: 5 s at 95 °C, 1 min at 58 °C, followed by a gradual increase in temperature from 60 °C to 97 °C, using a ramp rate of 0.02 °C per s. Results were analyzed with the standard LightCycler 480 Software, version 1.5 (Roche).For RT-qPCR, cDNA was prepared by adding 5 μl of RNA extract to a final volume of 20 μl of the RevertAid RT Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. Two μl of the cDNA was subsequently added to a total reaction volume of 10 μl PCR mix, consisting of 0.5 μM of each β-actin primer , 2 mM ofi.e., the storage medium or the RNA extraction kit, was most important for the procedure that resulted in the highest RNA yield, i.e., the RPL-RP procedure. Therefore, we performed the EM RNA extraction kit after storage of the PBMCs in 2 ml of RPL instead of 2 ml of EML, and the RP RNA extraction kit after storage of the PBMCs in 850 μl of EML instead of 850 μl of RPL.We aimed to assess which part of the RNA extraction procedure t test for paired samples was applied to compare results from both runs, using the IBM SPSS Statistics software v 25.0 .For statistical comparisons of sample storage conditions, RNA extraction kits, and of instruments to analyze RNA , three independent extractions were considered and data were analyzed using the linear mixed model for repeated measures, followed by Bonferroni’s multiple testing correction. The 260 nm/A280 nm ratios close to 2.0  yielded RNA with Ae to 2.0 , indicatUsing the 2100 Bioanalyzer, RIN values higher than 7 were observed for the RE and RP extraction kits, except for QZL-RP in Run 1 , which ip < 0.05) than those obtained with the 2100 Bioanalyzer nucleotides, further comparisons were made between procedures and runs only on the basis of 2100 Bioanalyzer results.i.e. after one day and after 83 days, no significant differences were observed with regard to yield, except for the RPL-RP procedure, which however yielded most RNA in both runs .We next analyzed which storage medium provided the highest RNA yield for each of the RNA extraction kits considered . For EM,p < 0.05), while no statistical differences were observed between RE and RP, nor between EM and RP. For both, storage in RNL and storage in cognate buffer, RP yielded most RNA (p < 0.05), when compared to EM and RE, which were similar to each other.Because, with regard to RNA yield, the best storage medium ) also depended upon the RNA extraction kit, we enquired which RNA extraction procedure resulted in the highest RNA yield. For this purpose, the combinations of each of the storage media with each of the RNA extraction kits was compared . For stoTogether, these results indicate that RP was the most efficient RNA extraction kit. Moreover, for RP combined with its cognate lysis buffer (RPL), the RNA yield was twice as high as for all other RNA extraction procedures Tables  and 3.ACTB, a clear correlation between the Cq values and RNA concentration was observed, with the exception of QZL-EM Run 2 and QZL-RP Run 1, which resulted in very high Cq values (indicative of low amounts of target molecules), compared to moderate RNA concentrations , indicating substantial loss of RNA.When the extracted RNA was used as a template to amplify trations . These rAfter it had been determined that the highest RNA yield was obtained by the procedure that combined storage/lysis in RPL with the RP RNA extraction kit, we questioned which part of the procedure was most important to obtain high RNA yield. Because all three EM-based RNA extraction procedures yielded least RNA, we addressed this question by comparing the RNA yield obtained with the following four combinations: EML-EM, EML-RP, RPL-EM and RPL-RP.i.e., ~2000 ng RNA with EML and ~2250 ng with its cognate lysis buffer RPL, compared to the EM RNA extraction kit, i.e., less than 300 ng with RPL and almost 900 ng with its cognate lysis buffer EML.The highest RNA yield was always obtained using the RP RNA extraction kit, irrespective of the storage medium , i.e., ~Reliable quantification of gene expression is greatly affected by the quality and quantity of the extracted RNA. Therefore, the way samples are stored preceding RNA extraction as well as the kit of choice to extract RNA are crucial. Thus far, only a limited number of studies compared storage and extraction of RNA from human samples in a methodological manner ,23,24. IRNA integrity and purity must be considered in order to obtain reliable quantification of gene expression . RNA intRNA purity is commonly evaluated by measuring the ratio between the absorbance at 260 nm and 280 nm (A260/A280) absorbance, whereby a value of ~2.0 is generally accepted as indicating that the RNA is free of proteins.Our study shows that high-quality RNA can be obtained with RE and RP RNA extraction kits regardless of the sample storage medium. All EM extractions also resulted in pure RNA, but with low RIN values (RIN 1). Since RNA analysis was performed with the RNA 6000 Nano kit (Agilent), it may be possible that these results were due to the low concentration of RNA in EM extracts and not due to degradation. For this reason, the analysis was repeated using the RNA 6000 Pico kit (Agilent). Some improvement of the quality of the samples was observed, but RNA was still degraded, with RIN values of 6.6 after QZL storage and less than 2.5 with RNL and EML .In agreement with our results, RE has been reported to provide high-quality RNA from T lymphocytes  , from ba260/A280 ratios [Several studies do not consider RNA integrity, and instead assess RNA quality indirectly, through microarray experiments , RT-qPCR0 ratios .260/A280 ratios [The limited number of studies that addressed EM and RP based RNA extraction determined quality of RNA from viruses through RT-qPCR ,41, and 0 ratios .i.e., 0.34 mM), for an A260/A280 value of 1.8.Both NanoDrop and the 2100 Bioanalyzer correlated well with regard to the relative values obtained for the different procedures that were compared. However, in most cases, NanoDrop indicated RNA concentrations that were 1.1- to 2.5-fold higher than those obtained with the 2100 Bioanalyzer. This is in agreement with the results of , reportiIn addition, the very similar RNA yields, obtained after separate runs of RNA extraction after one day and after 83 days of storage of PBMCs, lends strong support for the reproducibility of our results.After having shown that purity and integrity of RNA extracts was excellent, with the exception of RNA integrity after EM RNA extraction, and that both runs yielded comparable amounts of RNA, we determined which of three storage conditions yielded most RNA for each of three RNA extraction kits. Together, the results suggest that the optimal storage medium also depends on the subsequent RNA extraction kit. In particular, samples stored in RNAlater resulted in the least amount of RNA extracted. Indeed, according to Qiagen guidelines, RNAlater is not recommended for storage of animal cells–although this is not mentioned in the guidelines of Thermo Fisher Scientific, for the brand of RNAlater that we used. In addition, we stored samples in RNAlater at 4 °C for 83 days, whereas -80 °C is recommended for storage during prolonged time. Despite of prolonged storage at 4 °C, a considerable amount of RNA was still obtained.Since, according to the manufacturer´s instructions, the RNL supernatant is to be discarded, we assessed possible loss of RNA in the RNLs, which can result from a) prolonged storage of the cells in RNL (at 4 °C), b) from lysis of cells during the centrifugation step needed to remove the RNLs, or c) from incomplete precipitation of cells during centrifugation. For this purpose, RNA was extracted from the RNLs without prior cell lysis.ACTB was negative (Cq > 40). The observed difference between both approaches might be due to the presence of extracellular and/or noncoding RNA, not resulting from cell lysis.Quantification of total RNA in the RNLs by means of the 2100 Bioanalyzer indicated 216.7 ng for RE and 274.1 for RP, whereas RT-qPCR for mRNA of the ACTB signal (Cq ~ 28.5). This could be due to lysis of cells still present in the RNLs, because in the case of EM, 1 ml of cognate lysis buffer is added, in accordance with the manufacturers’ instructions.However, for EM, we observed higher quantities of RNA in the RNLs (410.7 ng) and moreover, a positive This would indicate that some cells are not precipitated during centrifugation of RNL and that cellular RNA is lost as well, not due to lysis of cells during storage, but due to incomplete recuperation of cells into the RNLp during centrifugation. Indeed, microscopy indicated that many cells were still present in the RNLs .i.e., storage in RNL, QZL or cognate lysis buffer  combined with the EM, RE or RP RNA extraction kits, the RPL-RP extraction procedure was found to yield twice as much RNA as all other procedures.When comparing the RNA yield for all nine RNA extraction procedures, Candida cells in RNL, the RiboPure RNA Purification Kit also outperformed RE and EM RNA extraction kits, with RP, RE and EM yielding 5.8, 2.6 and 2.2 μg of RNA respectively. Other RNA extraction kits have been shown to provide higher RNA yield. Recently, [6 PBMCs, using the miRNeasy Mini Kit (Qiagen), the Total RNA Purification Kit (Norgen Biotek Corp) and the NucleoSpin miRNAs Kit (Macherey-Nagel). This result is in agreement with our study in which we obtained between 200 and 1800 ng RNA using different RNA extraction kits and storage conditions. We obtained up to 800 ng with the RNeasy Mini Kit which is a bit lower than the miRNeasy Mini Kit they used, which might be explained by the fact that the RNeasy Mini Kit selectively excludes all small RNAs (< 200 nt).In our previous study , using secently,  comparedecently,  obtainedecently,  extractei.e. the storage/lysis medium or the RNA extraction kit, we compared the RNA yields obtained with the four possible procedures composed of storage in EML versus RPL buffer combined with EM versus RP RNA extraction kit. The results indicated that the RNA extraction kit has the greatest influence on RNA yield, because whenever RP was used, the highest yield was obtained. Storage in the lysis buffer (EML vs. RPL) also contributed, because storage in the cognate RPL buffer further increased the yield (borderline significantly) from 2004 ng (EML) to 2258 ng (RPL).To assess which part of the procedure was most important to improve the RNA yield, In conclusion, when samples can be frozen immediately, this is preferable over storage in RNL at 4 °C. However, a shortcoming of this study is that we failed to assess the performance of RNL storage at -80 °C, as recommended.In addition, frozen samples  are preferably stored in the cognate lysis buffer of the RNA extraction kit. On the other hand, we recently reported that using RNL for the storage of yeast cells gave excellent results , which mIn conclusion, in this study, RPL-RP offered high-quality RNA and the highest yield of RNA and was the most effective procedure to extract RNA from stored PBMCs. Our results are supported not only by the reproducibility of the experiments in which two different runs of extractions were carried out, but also by RT-qPCR results.S1 Tablei.e. 6 aliquots were stored per each of 12 combination procedures. PBMC Storage medium: EML: easyMAG lysis buffer; QZL: QIAzol; RLT: RNeasy lysis buffer; RNL: RNAlater pellet; RNLs: RNAlater supernatant; RPL: RiboPure RNA Purification Kit–blood lysis buffer. RNA extraction kits: EM: NucliSENS easyMAG; RE: RNeasy Mini Kit; RP: RiboPure RNA Purification Kit–blood. A: Respectively EML, RLT and RPL for RNA extraction kits EM, RE and RP. B: To add up to a starting volume of 2 ml, as prescribed by the manufacturer. C: No addition of cognate lysis buffer: to check whether RNA could be detected in RNLs supernatant, as a control for possible loss of RNA due to spontaneous lysis of cells during centrifugation of RNL. D: No addition of cognate lysis buffer, because already added prior to storage. E: See All kits were carried out in triplicate for each of two extraction runs, (XLSX)Click here for additional data file.S2 Table6 cells. b: Results are means and standard deviations of three independent experiments. c: Pellets were resuspended in 1 ml aliquots of saline before counting.a: Starting material consists of 1-ml aliquots PBMCs containing 10(DOCX)Click here for additional data file.S1 Fig6 PBMCs was measured with a 2100 Bioanalyzer and results are shown as A) gel-like image and B) electropherogram profiles. The RIN value is reported on a scale of 1 to 10, whereby values above 7 are considered to represent high quality and non-degraded RNA. vQZL: QIAzol, RNL: RNAlater, RPL: Lysis buffer from RiboPure RNA Purification Kit–blood, EM: NucliSENS easyMAG extraction, RE: RNeasy Mini Kit, RP: RiboPure RNA purification Kit–blood, RLT: Lysis buffer from RNeasy Mini Kit, EML: Lysis buffer from NucliSENS easyMAG extraction.RNA integrity from 10(TIF)Click here for additional data file.S2 Fig6 PBMCs was measured with a 2100 Bioanalyzer and results are shown as A) gel-like image and B) electropherogram profiles. The RIN value is reported on a scale of 1 to 10, whereby values above 7 are considered to represent high quality and non-degraded RNA. QZL: QIAzol, RNL: RNAlater, RPL: Lysis buffer from RiboPure RNA Purification Kit–blood, EM: NucliSENS easyMAG extraction, RE: RNeasy Mini Kit, RP: RiboPure RNA purification Kit–blood, RLT: Lysis buffer from RNeasy Mini Kit, EML: Lysis buffer from NucliSENS easyMAG extraction.RNA integrity from 10(TIF)Click here for additional data file.S1 File(XLSX)Click here for additional data file.S2 File(XLSX)Click here for additional data file.


Sexual and reproductive health (SRH) communication is most likely to promote healthy sexual practices and to reduce risky sexual behavior among adolescents. Communication is the principal means for parents to transmit sexual values and knowledge to their children. Although there are few studies conducted on parent-adolescent communication, there is no study conducted in the town of Ambo. This study was aimed at assessing the level of parent-adolescent communication on SRH issues and its associated factors among school students in Ambo town, Oromia, Ethiopia. th to March 9th, 2019. A systematic sampling technique was used to select the study subject. Data were collected through self-administered questionnaires, and FGD was conducted with parents of students. Data was entered using EpiData version 3.1 and exported to SPSS version 23.0 for statistical analysis. Binary and multivariable logistic regression analyses were used to ascertain the association using a 95% confidence interval (CI) and p value (<0.05).  An institution-based concurrent mixed-method cross-sectional study was conducted among 591 secondary and preparatory school students in Ambo town from February 24 The proportion of students who had communication on sexual and reproductive health issues with their parents was 222 (37.6%). Being female , private school , a father with secondary education  and diploma and above , considering sex education necessary , got information about SRH issues from school  and media , and mother's openness to communicate about SRH issues  were found to be significantly associated with parent-adolescent communication on SRH issues.  The study showed that parent-adolescent communication on SRH issues is low. Being female, those from a private school, father's education, perceived importance of sex education, source of information about SRH issues (school and media), and mother's openness to communicate about SRH issues were identified to be factors associated with the communication. Therefore, the concerned body should consider the identified factors to improve the current level of parent-adolescent communication and adolescent reproductive health. Globally, more than 1.1 million adolescents aged 10-19 years died in 2016, over 3000 every day, mostly from preventable or treatable causes .Sexual health is a state of physical, mental, and social wellbeing concerning sexuality across the life span that involves physical, emotional, mental, social, and spiritual dimensions . Today, Adolescence is a time when many young people experience critical and life-defining challenges such as their first sexual experience, marriage, pregnancy, and parenthood . Of all The health status of adolescents is strongly connected to several risk behaviors, which are often established during the adolescent years that end in chronic and nonchronic diseases , 13. TheIn Ethiopia, the highest prevalence of HIV infection was reported in the age group 15 to 24 (12.1%). About 60% of adolescent pregnancies in Ethiopia are unwanted or unintended pregnancies resulting from unprotected sexual intercourse and mostly end up in an unsafe abortion [A study conducted in Ghana, Accra, reported that 73.6% had talked about HIV/AIDS with parents or other family members . In EthiStudies conducted in Ethiopia show that cultural factors including cultural taboos, shame, lack of communication skill, embarrassment, fear of parents, nonresponsiveness of parents, and unwelcoming nature of parents to accept young people constrained by lack of adequate knowledge, sociocultural norms, and parental belief that discussion of such issues promote premarital sexual practice were reasons that hinder communication between parent and adolescents , 22. ParLack of accurate information about reproductive health and sexuality, lack of access to health services including contraception, and vulnerability to sexual abuse put adolescents at the highest risk . With reSex is a subject many are uncomfortable discussing in a meaningful way, especially with children . ParentaParents have an important role in protecting their children from risk by supervising and providing information about sexual-related risk, including the formation of attitudes and values about sexuality and the reduction of risky behaviors . The finth to March 9th, 2019. Ambo town is located in the central part of Oromia regional state, and it is the capital of the West Shoa zone. It is located 114 km to the west of Addis Ababa on the main road that leads to Wollega. The town had three urban and three rural kebeles . According to Ambo town administration office 2018 data, the total population of the town was 108,000 of which 53,400 were males and 54,600 were females [An institution-based concurrent mixed-method cross-sectional study was conducted among secondary and preparatory school students in Ambo town from February 24 females .There are 4 secondary schools, 2 (one public and one private) preparatory schools, and 21 (10 public and 11 private) elementary schools found in Ambo town. The total numbers of students attending grades 9-12 during the 2018/19 calendar year were 9,457; of these, 5,033 and 4,224 were males and females, respectively. There were 3 youth service centers in the town .The source populations were students from grade 9 to grade 12 attending secondary and preparatory schools in Ambo town in the academic year of 2018/2019. Students from grades 9 to 12 attending secondary and preparatory schools in Ambo town and who were selected by systematic random sampling from secondary and preparatory schools found in Ambo town in the academic year of 2018/2019 were our study population. For the qualitative part, the study subjects (discussants) were purposively selected parents who have adolescent students in preparatory and high schools and were involved in the focus group discussions to explore communication on SRH issues. All students aged 10-19 years and who were married or have no parents were excluded from the study.The sample size for the quantitative part was determined using the single population proportion formula with the assumption of 95% two-sided confidence level (CI), 36.9% of the proportion of parent-adolescent communicating on sexual and reproductive health in Debre Markos Town (2014)  and 5% mn = (Zα/2)2P(1 − P)/d2 = (1.96)2∗(0.369)∗(1 − 0.369)/(0.05)2 = 358.By considering a 10% nonresponse rate and 1.5 design effect, the final sample size was 591.th student was selected after randomly identifying the first respondent from the list of the first 9 students on the roster, then every 9th respondent was selected. For the qualitative part, FDG was conducted among purposively selected parents who had adolescent students in preparatory and high schools.A multistage sampling technique was employed to recruit study participants. In the beginning, schools in the town were stratified into public and private ones. Then, three out of four governmental schools and one out of two private schools were randomly selected. Then, schools were further stratified by the grade of students. Then, 75% of sections from all grades were selected by simple random sampling technique using the lottery method. Furthermore, the number of students selected from each section was proportionally allocated according to the number of students in the class. Finally, a systematic random sampling technique was used to select the study unit from each selected section by using the list of students from the roster as a sampling frame. Accordingly, every 9Parent-adolescent communication on sexual and reproductive health: when students discussed SRH issues of at least two topics with their parents  in the last 12 months [2 months .Knowledgeable on SRH: those students who score greater than the mean of sexual and reproductive health-related questions while those students who score the mean or below the mean were considered as not knowledgeable on SRH issues [H issues .Parent-adolescent communication on SRH issues was our dependent variable. Variables, such as Sociodemographic characteristics, knowledge on SRH, sexual attitude and behaviors, and sociocultural factors were our independent variables. Four experienced data collectors who have completed grade 12 and fluent in the Afan Oromo language were recruited. Two nurse professionals were recruited as supervisors, and they were responsible for leading the whole situation of data collection processes. Data were collected through a self-administered questionnaire by using a structured Afan Oromo version questionnaire. The questionnaires were originally adapted by reviewing different related literature , 30–35 iFor the qualitative part, four focus group discussions stratified into male groups and female groups were carried out among purposively selected parents who had adolescent students in preparatory and high schools. A semistructured interview guideline was used to lead the discussion. Two trained health workers moderated the FGD sessions. The FGD consisting of eight to ten discussant mothers and fathers were conducted separately to let them freely express their ideas which also helps to increase the quality of information that can be generated. During the data collection process, the researcher took notes in written form and recorded the voice of the respondent depending on their willingness to be recorded, and the collected data was transcribed for further analysis. The data collection process continued till all the research questions were answered and the point of saturation was reached.The training was given to both data collectors and supervisors for two days by investigators. The questionnaire was pretested on 5% of students at secondary and preparatory schools in Guder town, which is located 15 kilometers away from the study area, after which, necessary modifications were made. Daily supervision was conducted throughout the data collection period.p value of ≤0.25 in the bivariate analysis were entered into a multivariable logistic regression analysis to determine the independently associated factor of adolescent-parent communication on sexual and reproductive health issues. Variables with a p value less than 0.05 in the multivariate analysis were considered as significantly associated. The qualitative data were analyzed according to its thematic area and were triangulated with the quantitative results.The collected data were checked for completeness, coded and entered into EpiData version 3.1, and exported to SPSS windows version 23.0 for statistical analysis. Descriptive statistical analysis was used to compute frequency, percentage, and mean for independent and dependent variables. Binary logistic regression analysis was used to ascertain the association between explanatory variables and outcome. Variables with a For qualitative data, data analysis was started alongside the data collection process through transcribing, noticing important and developing new issues that were needed to be included in the process, reviewing the collected data and memos, and cleaning data throughout the process. The data that were collected through focus group discussions were transcribed, coded, and thematically analyzed based on the procedure. After the data collection in Afan Oromo, the researchers transcribed it into English and the coding process followed by being guided by the research questions. After coding, categorization followed, and the coded data were categorized into similar and related categories. The coded data were categorized based on their similarity or relatedness. The researchers categorized the coded data depending on the similarity and relationship of codes and summarized it manually.Ethical clearance was obtained from the research and ethical review committee of the College of Medicine and Health Sciences, Ambo University, before the data collection. A support letter was also obtained from the Ambo town educational office to Ambo preparatory and secondary school administrations. Participants were told that they have the right to discontinue or refuse to participate in the study. Written assent was obtained from students who were under 18 years and consent was also obtained from their parents. Written informed consent was obtained from adolescents who were 18 to 19 years of age.Five hundred ninety-one students participated in the study, of which five hundred ninety (590) participants completed the questionnaire fully with a response rate of 99.8%. More than half 315 (53.4%) of the respondents were female. Three-fourths, 438 (74.24%), of students were between 17 and 19 years old. The mean age of the study participants was 17.27 with a standard deviation of ±1.22 years. Almost half, 317 (53.73%), of the respondents were from grades 9 and 10. Nearly all, 566 (95.93%), of them were from the Oromo ethnic group. Furthermore, about 469 (80.17%) of adolescents' mothers were private employees. The majority, 470 (79.66%), of respondents were living with both of their parents. Four hundred forty-one (74.75%) of study participants reported that they did not receive pocket money monthly from their parents. In about 174 (30.16%) of respondents, their mothers had no formal education .Overall, 296 (53.62%) of the respondents discussed at least two topics about SRH issues at least with the father, mother, sister, peer, teacher, and other individuals in the past 12 months, whereas 256 (46.38%) did not discuss the issue with anyone. The proportion of adolescents who had communicated with their parents regarding sexual and reproductive health issues on at least two topics in the last 12 months was 222 (37.6%). Two hundred seventy-nine (47.29%) of the respondents reported that they had discussed contraceptive methods. Out of 590 respondents, 231 (39.15%) of the students had discussed sexual intercourse. However, 160 (69.26%) of these respondents had discussed with their friends and 50 (21.65%) with their mother. Near to half of the participants, 263 (44.58%), had discussed unwanted pregnancy. One hundred seventy (64.64%) of the respondents had discussed with their friends/peers and 89 (33.84%) with their mother .The data generated from FGD discussants indicated that they have communicated on various components of sexual and reproductive health issues, though the communication varies among families. Some parents communicate transparently with their children. A 36-year-old male participant said, “I transparently convey my communication and advice to my sons who are 16 and 18 years old. I openly and transparently convey my message regarding SRH issues at an appropriate age, conditions, and environment. I always transparently and adequately inform and give them feedback and information. Usually, when we are around the table during the evening, I communicate with them the risks and problems associated with teenage pregnancy, early sexual engagement, peer pressure, and risky behaviors based on existing problems in our country and facts in the society. We do this to help our children to be successful in their education and future life.”On the contrary, the data generated from mothers' FGD discussants revealed they communicate about SRH issues with adolescents; however, they do not openly and transparently communicate as needed and they only focus on few topics. A 35-year-old mother described her experience as “I have not shared with my children my experience even I have not discussed with them in detail about sexual and reproductive health issues, my discussion mostly focused on the impact of negative sexual and reproductive health outcome and changes during puberty”.The data generated indicated that the way parents used to grow up their children influences sexual and reproductive health communication. During focus group discussions, most of the discussants agreed and noted that children should be encouraged to talk, express their ideas, and listened to starting from when they were kids. One father participant explained that “Previously when my children were kids, I was not caring, supportive, and close to them. I did not know the importance of these during that time. My children are not open and free, even to communicate with my other issues. They are not close to issues and even academic issues too. I encourage them to communicate with me openly but they don't. These resulted from the way we used to rear up them.”Out of 263 (44.58%) respondents who had not discussed contraceptive methods, 109 (43.5%) and 89 (28.62%) reported their reason as do not know and shameful to discuss such issues with parents, respectively. Nearly half, 275 (46.69), of the respondents did not discuss sexual intercourse. The reasons they mentioned for not discussing sexual intercourse with their parents were shamefulness, 140 (38.89%), and do not know about the topic, 63 (17.50%) .The data generated from FGD participant parents identified (explored) the following hindering factors for parent-adolescent communication on SRH issues.Parents stated that in the past, communication on sexual and reproductive health issues was considered culturally taboo, and adolescents were not allowed to openly talk about SRH-related issues. One female participant said that “Cultural values and norms are the most hindering conditions, I ashamed to rise in such a way that not interested to discuss the issues of sexuality topics. And these have created an influence on SRH communication I have with my children”.The data generated from FGD with parents specified that still many people are ashamed to talk about SRH issues especially about sexual intercourse and opposite-sex relationship. Moreover, it was indicated that our cultural practice and attitudes have not allowed such communication and have an influence on SRH communication. One male participant illustrated that “Some of our cultural attitudes are negatively associated with such communication. Surprisingly, it is not common in our culture to openly talk about SRH issues, even to call or orally describe our reproductive organs by their names. Such practices of the society negatively impact discussion on SRH issues in a family context.”Business or lack of time to communicate and spend time with adolescents was the other hindering condition identified on FGDs. Parents indicated that sometimes, they were very busy with different income-generating and household activities/workload. And these prevented them from communicating and giving adequate time to their children.Around two-thirds, 385(65.25%), of adolescents have gotten information about SRH from different sources. Only 28 (4.75%) reported do not know where to get the information. Regarding the source of information on reproductive health issues, 247 (59.95%) of respondents get the information from school, 282(68.45%) from media , 72 (17.48%) from family, and 165 (40.05%) from their peers. The majority of adolescents, 486 (82.37%), prefer school as a source of information on SRH issues .The majority of respondents, 489 (82.88%), knew about STI/HIV/AIDS. HIV/AIDS was the most commonly known STI, 423 (86.50%), followed by syphilis, 262 (53.58%). Nearly two-thirds, 388(65.76%), of students knew about at least one contraceptive method . AccordiOne hundred ninety-six (33.22%) of the students believed that it is normal and acceptable to have sexual feelings during the adolescent period. Regarding their practice, 76 (12.88%) of the students had a history of sexual intercourse, of which 30 (39.48%) had more than one sexual partner. Of those who had a history of sexual intercourse, 49 (64.47%) of them made sex without a condom.Three hundred eighty-eight (65.76%) of students strongly agreed sex education is necessary . The datIn bivariate analysis, variables such as age group, sex, father's educational status, type of school, father's occupation, knowledge of age at which menses starts, knowledge of STDs, accepting the necessity of sex education, source of information about SRH , parent communication skills on SRH issues, and mother's openness to communicate about SRH issues were found to be significantly associated with parent-adolescent communication on SRH issues .The result of the multivariable logistic regression model revealed that sex, school type, father's educational status, accepting the necessity of sex education, source of information about SRH issues (school and media), and mother's openness to communicate about SRH issues were found to be significantly associated with discussing SRH issues.The sex of the respondents is found to be affecting their discussion on SRH issues. The odds of discussing SRH issues with parents were twice more likely in female students than males . Compared to students learning in government schools, private school students were 2.78 times more likely to have parent-adolescent SRH communication with their parents . Students with fathers having secondary education and college diploma and above were two and three times more likely to communicate with their parents on SRH issues than students with their father who could not read and write (AOR 2.93 (95% CI: 1.05-8.12) and AOR 3.27 (95% CI: 1.23-87), respectively). Adolescents, who had agreed on the importance (necessity) of sex education, were 2.83 times more likely to discuss SRH issues than those who did not agree .Those students who got SRH information from schools were 2 times more likely to communicate on SRH issues with their parents than those who never got SRH information . Similarly, students who got SRH information from mass media were almost 3 times more likely to communicate on SRH issues with their parents than those who did not get SRH information . This study also revealed that those adolescents who perceived their mothers were open to communicate on SRH issues were 3.3 times more likely to communicate on SRH issues with their parents than those whose mothers were not open to communicating on SRH issues  .This study assessed parent-adolescent communications on sexual and reproductive health issues and associated factors among preparatory and high school students in Ambo town, Oromia region, Ethiopia. The current study found that 37.6% of respondents had discussed at least two SRH issues with their parents in the last 12 months. This finding was in line with studies conducted in Debre Markos 36.9%) , Addis A.9% , AddThese differences may be due to demographic and sociocultural differences and a difference in accessing SRH information. There is also a time-period gap among studies.The findings from the qualitative data of the present study substantiate the quantitative results in which the majority of the FGD participants mentioned that they did not openly discuss SRH-related issues. Parents simply pick words or subjects related to sexuality and human reproductive health causally from mass media (television and radio) and then attempted to chat about these matters and also provide vague warnings rather than direct open discussions. FGD discussants also raised that parents are not discussing SRH issues with their adolescent children; instead, they attempted to probe into their life/experiences regarding sexuality and reproductive health which are referred to by adolescents as queries.Concerning factors associated with parent-adolescent communication, this study has shown that female students were more likely to discuss SRH issues compared to male students. This finding is similar to the study conducted in Addis Ababa, Ayer Tena high school, and Dire Dawa town in eastern Ethiopia , 34. ThiThis study is also consistent with the qualitative findings. FGD discussant parents agreed that female students should discuss SRH matters more than males, since females are more susceptible to reproductive health problems than males. Parents believe/agreed that girls should maintain their virginity, and it is not acceptable according to their culture and religion to have premarital sex and the daughter should keep her virginity until marriage.Government (public) school students were less likely to communicate on SRH issues compared to students learning in private schools. This may be due to socioeconomic differences, the educational background of parents, and the accessing of SRH information of the parents which may influence communication between parent and adolescents.Adolescents, whose fathers attended secondary school –12, wereAdolescents, who perceived sex education as important (necessary), were more likely to discuss SRH issues than those who did not. This could be due to knowledge of SRH issues and the perceived importance that sex education and reproductive health may matter. Those students who had gotten SRH information from schools were more likely to communicate on SRH issues with their parents than those who did not get SRH information. Similarly, students who had gotten SRH information from media were more likely to communicate on SRH issues with their parents than those who never got SRH information from media. This study was in line with a study conducted in Debra Markos town, Bullen district, and Woldia town, southern Ethiopia , 19, 32.The findings from the qualitative data also support this finding. According to data obtained from children's parent's FGD, the information that they get from religious organizations and mass media in different forms helped/paved the way for them to communicate with their adolescents on SRH issues. This shows that parents who had gotten SRH information communicated more with their adolescents.This study also revealed that those adolescents whose mothers had been open to communicating on SRH issues were more likely to communicate on SRH issues with their parents than those whose mothers were not open to communicating on SRH issues. This may be due to the difference in knowledge; most of the mothers were primary caregivers and did not feel shy when discussing sexual matters.The implications of this study for different concerned stakeholders were though age-appropriate comprehensive sexuality education is recommended to begin in early childhood and continue through a person's lifespan, the adolescent-parent communication on SRH issues is low. The study also highlighted that there is a variation with gender, school type, father's education status, perceived importance of sex education, source of information, and mother's openness to communicate about SRH issues. Therefore, the finding calls for initiating comprehensive family life education for the adolescents and parents using information education communication and behavioral change communication materials and through mass media both at schools and in the community, promoting parent-adolescent communication on sexuality and promoting school-based clubs to enhance communication on SRH matters.Since respondents were asked about communication on SRH issues in the last twelve months, it is not free from recall bias, as they might not remember what they had discussed in the last twelve months. Since the study touches on sensitive and intimate issues, the possibility of underestimation cannot be ruled out. Finally, this study is based on cross-sectional data, which implies that the direction of causal relationships cannot always be determined.This study finding showed that parent-adolescent communication on sexual and reproductive health issues in the study area is low. Adolescents discussed sexual matters more with peers than with a parent. Being female, adolescents from a private school, educational status of fathers, perceived importance of sex education, source of information about SRH issues (school and media), and mother's openness to communicate about SRH issues were found to be significantly associated with communication of adolescents with their parents about sexual and reproductive health matters.Therefore, the stakeholders should have to work on initiating comprehensive family life education for the adolescents and parents using information education communication and behavioral change communication materials and through mass media both at schools and in the community, promoting parent-adolescent communication on sexuality and promoting school-based clubs to enhance communication on SRH matters. Encouraging open discussion among family members in general and between parents and adolescents focusing on the identified predictor variables was also recommended.