Long non-coding RNAs (lncRNAs) are implicated in many pathophysiological processes, including cancers. In particular, lncRNA DANCR is regarded as a cancer-associated lncRNA exerting various regulatory mechanisms. However, the expressions, functions, and mechanisms of action of DANCR in cervical cancer are still unclear.The expressions of DANCR in cervical cancer tissues and cell lines were evaluated using qRT-PCR. Correlations between DANCR expression and clinicopathological features and prognosis were analyzed. The roles of DANCR in cervical cancer growth were evaluated by in vitro CCK-8 and EdU assay, and in vivo xenograft assay. The regulatory effects of DANCR on Wnt/β-catenin signaling pathway were evaluated using nuclear proteins extraction, western blot, and qRT-PCR.DANCR is increased in cervical cancer tissues and cell lines. Increased expression of DANCR is associated with large tumor size, advanced FIGO stage, and poor overall survival of cervical cancer patients. Functional experiments showed that enhanced expression of DANCR promotes cervical cancer cell proliferation in vitro and xenograft growth in vivo. Conversely, DANCR knockdown inhibits cervical cancer cell proliferation in vitro and xenograft growth in vivo. Mechanistic investigation demonstrated that DANCR upregulates the expressions of FRAT1 and FRAT2 and activates the Wnt/β-catenin signaling pathway. Blocking the Wnt/β-catenin signaling pathway abolishes the pro-proliferative roles of DANCR overexpression and anti-proliferative roles of DANCR knockdown.Our findings suggest DANCR as an oncogenic lncRNA in cervical cancer through activating the Wnt/β-catenin signaling pathway, and imply that DANCR may be a promising prognostic biomarker and therapeutic target for cervical cancer. Cervical cancer is the second most common cancer in female, next to breast cancer, accounting for approximately 527,000 new cancer cases and 265,000 cancer deaths each year worldwide . With grAccumulating evidences revealed that many non-coding RNAs, including microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) are frequently deregulated and have critical roles in multiple cancers –10. ThesLncRNA differentiation antagonizing non-protein coding RNA (DANCR), previously termed as ANCR, was first revealed to suppress progenitor differentiation . SubsequIn the present study, we determined DANCR expression in cervical cancer tissues and cell lines, analyzed the correlation between DANCR expression and cervical cancer patients’ clinicopathological features, including prognosis. Furthermore, gain-of-function and loss-of-function assays were performed to investigate the biological roles of DANCR in cervical cancer growth. Finally, using public available dataset, combined with experimental verification, we investigated the mechanisms underlying the biological effects of DANCR in cervical cancer. We demonstrated that DANCR, which is an oncogenic lncRNA in cervical cancer through activating the Wnt/β-catenin signaling pathway, might be a promising prognostic biomarker and therapeutic target for cervical cancer.A total of 82 pairs of cervical cancer and adjacent noncancerous cervix tissues were obtained from cervical cancer patients with informed written consent who underwent potentially curative surgery in the First Affiliated Hospital of Zhengzhou University  from 2014 to 2016. All tissue samples were diagnosed by histopathological examination. Freshly resected tissue specimens were immediately frozen in liquid nitrogen and stored at − 80 ℃ until use. The Medical Ethics Committee of the First Affiliated Hospital of Zhengzhou University reviewed and approved this program in accordance with Helsinki Declaration.2 at 37 ℃.The human normal cervical epithelial cell line HCerEpiC was purchased from ScienCell Research Laboratories . The cervical cancer cell lines HeLa, SiHa, C-33A, and ME-180 were purchased from American Type Culture Collection . HCerEpiC cells were grown in Cervical Epithelial Cell Growth Supplement . HeLa, SiHa, and C-33A cells were grown in Eagle’s Minimum Essential Medium . ME-180 cells were grown in McCoy’s 5A Medium . All media were added with 10% fetal bovine serum . Where indicated, the cervical cancer cells were treated with 5 μM ICG-001 . All cell lines were grown in a humidified incubator with 5% COTotal RNA was extracted from indicated tissues and cells with TRIzol Regent (Invitrogen) following the protocol. After being treated with DNase I  to remove genomic DNA, the isolated RNA was subjected to reverse transcription using a PrimeScript RT Reagent Kit (Takara) following the protocol. Next, the cDNA was subjected to quantitative Real-Time PCR (qRT-PCR) using SYBR Premix Ex Taq (Takara) and gene specific primers. Primer sequences are as follows: for DANCR, 5′-GCGCCACTATGTAGCGGGTT-3′ (sense) and 5′-TCAATGGCTTGTGCCTGTAGTT-3′ (antisense); for FRAT1, 5′-TGGAAGCGAGAGTAAAAAGC-3′ (sense) and 5′-GGTCACGCCAAATAAGGAG-3′ (antisense); for FRAT2, 5′-TACCTCACTTAGCCCTTGG-3′ (sense) and 5′-ATGCGTGTCGTTAGTTTTCA-3′ (antisense); for C-myc, 5′-GCTGCTTAGACGCTGGATTT-3′ (sense) and 5′-CTCCTCCTCGTCGCAGTAGA-3′ (antisense); for Cyclin D1, 5′-TTCCTGTCCTACTACCGC-3′ (sense) and 5′-CTCCTCCTCTTCCTCCTC-3′ (antisense); and for β-actin, 5′-GGGAAATCGTGCGTGACATTAAG-3′ (sense) and 5′-TGTGTTGGCGTACAGGTCTTTG-3′ (antisense). The quantification of RNA expression was calculated following the comparative Ct method. β-actin was used as endogenous control.DANCR expressing plasmid pcDNA3.1-DANCR was constructed as previously described . BrieflyFor construction of DANCR stably overexpressed and control HeLa cells, DANCR expressing plasmid pcDNA3.1-DANCR or control empty plasmid pcDNA3.1 was transfected into HeLa cells. After 48 h, the transfected HeLa cells were treated with neomycin for 4 weeks. To construct DANCR stably depleted and control C-33A cells, DANCR specific shRNA or control scrambled shRNA was transfected into C-33A cells. After 48 h, the transfected C-33A cells were treated with neomycin for 4 weeks. The efficiencies of overexpression and knockdown were determined by qRT-PCR.®643 In Vitro Imaging Kit  according to the instructions. Results were analyzed by Zeiss fluorescence photomicroscope  via randomly counting ten fields.Cell proliferation was assessed by Cell Counting Kit-8 (CCK-8) and Ethynyl deoxyuridine (EdU) assays. For CCK-8 assay, DANCR stably overexpressed or depleted cervical cancer cells were resuspended and plated into 96-well plates at a density of 3000 cells per-well. After culture for indicated time, CCK-8 solution  was added into the wells. After 2 h of continued culture, absorbance values of optical density (OD) at 450 nm for each well were detected using automatic enzyme-linked immune detector. EdU assay was performed with the Cell-Light™ EdU Apollo6 DANCR stably overexpressed or depleted cervical cancer cells were resuspended in 100 μl phosphate buffered saline containing 50% matrigel (Invitrogen) and then subcutaneously injected into the flanks of female BALB/c-nu mice of 6 weeks old. The mice were maintained in a sterile environment on a daily 12-h light/12-h dark cycle. Subcutaneous tumor volumes were measured every 3 days using caliper and calculated following the formula: tumor volumes = 0.5 × length × width2. At the twenty-first day after injection, the mice were sacrificed and subcutaneous tumors were resected and weighed. The Animal Ethics Committee of the First Affiliated Hospital of Zhengzhou University reviewed and approved this program. Ki67 immunohistochemical staining was carried out as we previously described with Ki67 primary antibody  [1 × 10MA, USA) .Nuclear proteins were extracted from DANCR stably overexpressed or depleted cervical cancer cells using CelLytic™ NuCLEAR™ Extraction Kit (Sigma-Aldrich). Total proteins were extracted from indicted cervical cancer cells using RIPA buffer (Beyotime). Next, nuclear proteins or total proteins were subjected to 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). After being transferred onto polyvinylidene fluoride (PVDF) microporous membrane , the blots were incubated with primary antibodies against β-catenin , histone H3 (Abcam), FRAT1 (Abcam), FRAT2 (Sigma-Aldrich), C-myc , Cyclin D1 , or GAPDH . Histone H3 was employed as a loading control for nuclear protein, and GAPDH was employed as a loading control for total protein. The blots were visualized with chemiluminescence.t-test, Mann–Whitney test, and Pearson correlation analysis were carried put as indicated. Results were considered as statistically significant when p < 0.05.All statistical analyses were carried out using the GraphPad Prism 5.0 version . For comparisons, Wilcoxon signed-rank test, Pearson Chi square test, log-rank test, Student’s p = 0.015) and advanced FIGO stage (p = 0.025) (Table p = 0.022)  or control empty plasmid (pcDNA3.1). The overexpression efficiency was detected via qRT-PCR Fig. a. CCK-8 To further validate the functional relevance of DANCR in cervical cancer, we constructed DANCR stably depleted and control C-33A cells via transfecting DANCR specific shRNA (KD-DANCR) or control scrambled shRNA (KD-control). The knockdown efficiency was detected via qRT-PCR . Among the genes positively associated with DANCR  data using TANRIC (Next, we investigated the effects of DANCR on the Wnt/β-catenin signaling pathway in cervical cancer. Nuclear proteins from DANCR stably overexpressed and control HeLa cells, and DANCR stably depleted and control C-33A cells were extracted. Nuclear β-catenin levels were determined using western blot. The results displayed that enhanced expression of DANCR upregulated nuclear β-catenin levels, and while knockdown of DANCR downregulated nuclear β-catenin levels in cervical cancer cells Fig. a, b. FurTo investigate whether the activation of the Wnt/β-catenin signaling pathway mediates the pro-proliferative roles of DANCR in cervical cancer, we treated DANCR stably overexpressed and control HeLa cells, and DANCR stably depleted and control C-33A cells with the canonical Wnt/β-catenin pathway inhibitor ICG-001. CCK-8 and EdU assays displayed that treatment with ICG-001 abolished the pro-proliferative roles of DANCR overexpression Fig. a, b. ConPIK3CA, EP300, FBXW7, PTEN, HLA-A, ARID1A and so on in different cervical cancer tissues [Genomic and molecular features of cervical cancer are complex , 47. Acc tissues , 46. TheIn the present study, we focused on a lncRNA DANCR, which is located on chromosome 4q12. Although DANCR has been investigated in several cancers, and been regarded as a cancer-associated lncRNA , the funIn this study, we identified a novel mechanism mediating the oncogenic roles of DANCR in cervical cancer, which is the activation of the Wnt/β-catenin signalling pathway via upregulation of FRAT1 and FRAT2. Both public available TCGA data and cervical cancer tissues we collected display that the expression of FRAT1 and FRAT2 are positively associated with the expression of DANCR in cervical cancer tissues, supporting the positive regulation of FRAT1 and FRAT2 by DANCR. FRAT1 and FRAT2 belong to the GSK-3-binding protein family, inhibit GSK-3-mediated β-catenin phosphorylation and degradation, promote nuclear translocation of β-catenin, and activate Wnt/β-catenin signaling pathway . IndocyaIn conclusion, our findings demonstrate that lncRNA DANCR is upregulated in cervical cancer, positively associated with tumor size and FIGO stage, and a prognostic indicator of poor survival of cervical cancer patients. DANCR promotes cervical cancer growth via activating FRAT1/FRAT2-Wnt/β-catenin signaling pathway. Our results imply that DANCR may be a promising prognostic biomarker to evaluate cervical cancer progression and useful therapeutic target for cervical cancer treatment.Additional file 1: Table S1. The genes associated with DANCR in cervical cancer from TCGA data.


A limited number of studies assessed prevalence of clinically important AMR, including carbapenem resistance (n = 3), third generation cephalosporin resistance (n = 7), colistin resistance (n = 2), extended spectrum beta-lactamase (ESBL) production (n = 4), or multidrug resistance (n = 4). This review highlights significant gaps in our current understanding of CA-ARE in Central America, most notably a general dearth of research, which requires increased investment and research on CA-ARE as well as AMR more broadly.Community-acquired antimicrobial resistant Enterobacteriaceae (CA-ARE) are an increasingly important issue around the world. Characterizing the distribution of regionally specific patterns of resistance is important to contextualize and develop locally relevant interventions. This systematic review adopts a One Health framework considering the health of humans, animals, and the environment to describe CA-ARE in Central America. Twenty studies were identified that focused on antimicrobial resistance (AMR) in Enterobacteriaceae. Studies on CA-ARE in Central America characterized resistance from diverse sources, including humans ( Antimicrobial resistant Gram-negative bacteria belonging to the Enterobacteriaceae family pose a significant risk to human health, and are categorized through identification of priority groups as “critical”, “high” and “medium” by the World Health Organization (WHO) . The sprCA-ARE are attributed to many drivers, including antimicrobial misuse in humans and animals that create selective pressure for drug resistance, as well as poor water and sanitation systems that allow for the organisms to spread. In human health, the improper use of antimicrobials leads to a selection of CA-ARE . OutsideGiven the interconnecting factors contributing to increasing CA-ARE, understanding and responding to this issue necessitates the use of a One Health framework, which focuses on issues that occur at human, animal, and environmental interfaces ,11. UphoThis review of CA-ARE focuses on Central America, a region composed of seven countries: Belize, Costa Rica, El Salvador, Guatemala, Honduras, Nicaragua, and Panama, which are in varying stages of development in terms of demographics, health and economics . There iRegional surveillance networks, global reporting systems, and scientific research provide current knowledge of AMR in Central America. All seven Central American countries are members of the Latin American Network for Antimicrobial Resistance Surveillance (ReLAVRA), which was established by the Pan American Health Organization (PAHO) and WHO in 1996, “to inform AMR prevention and control policies and interventions in the region, through the ongoing collection of reliable, comparable, and reproducible AMR data” . Member Though coordinated international reporting systems are still developing, some Central American countries have national surveillance working groups or action plans. Costa Rica, for example, developed a national 2018–2025 action plan on AMR that includes five strategic objectives aimed at increasing AMR education, enhancing surveillance, improving sanitation and antimicrobial use procedures, and investing in vaccine and drug development . GuatemaThis systematic review identifies relevant research from the Central American region that characterizes CA-ARE from non-clinical human and animal (domestic and wild) samples, where there is high human–animal or human–environment overlap with the potential for human exposures.www.covidence.org [Salmonella, E. coli, Klebsiella) because of their important role in human infections and their ability to spread AMR genes that can be acquired in the community [We conducted a systematic literature review following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines . PubMed ence.org ), for inommunity ,25,26.Of 868 non-duplicate results, 795 studies were found to be irrelevant based on title and abstract . The mosAfter full-text inclusion and exclusion, 20 full texts were included for data extraction. All were reviewed for a standard set of factors, including study timeframe, study location, sample size, and type of resistance studied . Though Of the 20 papers included, 12 studied community-acquired AMR in humans, four presented isolates from wildlife or animal food products, two focused on isolates from environmental samples, and two studied isolates from several categories . As shown in Of the 12 studies of human isolates, 10 were categorized by sample type and symptoms experienced by study participants . Six studies used stool samples to investigate diarrhea ,30,31,32Salmonella infection was the first to demonstrate CTX-M and AmpC enzymes, which convey resistance to antimicrobials, in the Americas [Salmonella serotype Infantis, and the first report of blaCTX-M-15 in the genus Salmonella. Isolates from urine and stool samples were resistant to all 23 antimicrobial agents studied -producing Enterobacteriaceae [K. pneumoniae strain was resistant to all of the beta-lactams tested, trimethoprim/sulfamethoxazole and minocycline and had reduced susceptibility to ciprofloxacin, gentamicin and chloramphenicol; it was susceptible to amikacin, nalidixic acid, levofloxacin, tigecycline, colistin and fosfomycin.In Guatemala, eriaceae . The K. Hemidactylus frenatus, n = 115) were collected from geckos found in homes [Salmonella isolates were found to be resistant to trimethoprim–sulfamethoxazole, ciprofloxacin, or cefotaxime, which are frequently used to treat human salmonellosis in the region. Intermediate resistance to streptomycin and to sulfonamides was detected in 50% of the isolates. In raccoons (Procyon lotor) from urban Costa Rica, Salmonella was isolated from 42 (49%) of 86 viable fecal samples and were tested for antibiotic susceptibility [Of the four studies that took place at the human–animal interface, two focused on wildlife. In two regions of Costa Rica, intestinal samples from Asian house geckos , and humans [K. pneumoniae and E. coli: humans 27%, poultry 13%, and wild birds 8%. Isolates from wild birds and poultry were also found to have AMR genes (blaCTX-M) of human origin. In El Salvador, researchers conducted a metagenomics analysis, which is the untargeted sequencing of genetic material from uncultured microorganisms [A study conducted in Nicaragua investigated the fecal carriage of ESBL-producing d humans . From earganisms . They chThe articles included in this review studied isolates of several Enterobacteriaceae from different sources with resistance to different antimicrobials. For a more quantitative comparison, we reviewed each study for the highest percent resistance reported across classes of antimicrobials in the most prevalent bacterial species . In studBased on the WHO Priority Pathogens  and the n = 7), Nicaragua (n = 6), and Guatemala (n = 4), while Belize, El Salvador, and Honduras only had one study and Panama had none. The majority of research was in humans only (n = 12), while fewer studies focused on animals at the human–animal interface (n = 4) or the environment at the human–environment interface (n = 2). Two additional studies were interdisciplinary in design and included multiple One Health components . Researchers have noted that there are a number of challenges in conducting One Health research from the design stage, to execution, and through to monitoring and evaluation due to the poor availability of resources and personnel, which may be why we found such a low number of One Health studies in Central America [This review identified 20 studies of CA-ARE in Central America. There were differences in the number of studies conducted in each country; the majority of CA-ARE research was conducted in Costa Rica ( America .n = 3), third generation cephalosporins (n = 7), colistin (n = 2), and multi-drug resistance (n = 4). Only four studies identified whether CA-ARE were ESBL-producing. Quinolones were the most frequently studied antimicrobials of clinical importance (n = 10). Along with differences in the number of studies conducted in each country, this review identified variations in classes of antimicrobials studied. Over time and across countries, there are many differences in which classes of antimicrobials were studied. Some identified resistance across several classes while other studies focused on only one class. When considering clinically important AMR, there were a limited number of studies on prevalence of resistance to carbapenems , but not whether patients were outpatient or hospitalized for an extended period. Without the ability ascertain the specific source of AMR, it is difficult to determine whether the resistance measured in a study is a reflection of AMR in healthcare settings or in the community, which convey different implications for AMR in a larger One Health context. Salmonella, while only two studies focused on humans studied Salmonella. Research on CA-ARE and AMR in general needs greater uniformity in reporting and priority setting before significant comparisons can be made between AMR in clinical settings and AMR in the community, animals, and the environment.While the studies included in this review elucidate aspects of CA-ARE in Central America, this review was limited due to a lack of available research on the subject. One Health research was sparse. When One Health research was conducted, the bacterial species studied often did not align with bacterial species of concern in clinical settings . All of the studies on animals in this review studied Investment in local capacity will be essential for developing a One Health AMR framework in Central America. This review has shown that there are enormous knowledge gaps in the region that hinder global and regional efforts to reduce AMR spread, and international efforts, like GLASS and ReLAVRA, do not fully characterize the current AMR situation in Central America. The only country in the region with a national AMR action plan, Costa Rica, was also the country best described by existing literature on CA-ARE. It is clear that international and regional investment in understanding AMR is critical, while also improving systems that help mitigate the impact of AMR, such as healthcare access and services, patient and clinician education, expanded laboratory capacity, and regulation of antimicrobial use in all settings . Major improvements to One Health surveillance of AMR are greatly needed in Central America, and there is a significant need to apply this approach with multiple sectors involved at the early stages of development. Dedicated resources for the One Health approach will likely strengthen regional capacity to prevent and control the spread of AMR by building new knowledge and understanding of the drivers for AMR locally . HoweverStakeholders working in the animal, environmental and human health sectors have often found it difficult to understand each other’s objectives, and this has led to a lack of mutual understanding of the shared benefits of collaboration. Development and implementation of One Health frameworks that incorporate multidisciplinary goals and outcomes could augment this effort. Focusing on AMR that is clinically relevant , will likely be an important start for One Health AMR surveillance programs at the early stage of development. In this review, we highlight the limited scope of information about community-acquired AMR in Central America, particularly for last-line antimicrobials like carbapenems and colistin. This review draws attention to the gaps related to the lack of studies and surveillance with the One Health approach, especially in Enterobacteriaceae ,51,52.The 2016 WHO global containment plan includes, among its five pillars, the improvement of knowledge of AMR through communication and education, as well as the strengthening of the scientific base through surveillance and research. Outlining the state of knowledge about AMR, especially community-acquired AMR, in Central American countries is a starting point for regional plans. This review contributes to the knowledge of this state and to the consideration of the One Health approach as a way of addressing the problem. Effective design and implementation of strategies to decrease AMR will require the consideration of risk factors encompassing interactions between humans, animals and the environment. Further, greater transparency and uniformity in data collection strategies and dissemination of results will improve cohesion and comparability of findings across the region. These efforts will require greater coordination of efforts across sectors, and among countries. This study highlighted findings on CA-ARE in Central America based on 20 studies that were conducted over the past fifty years (since 1970). The limited amount of research highlights that there is a need to strengthen multi-sectorial AMR research and surveillance. Future steps in Central America should be to strengthen the relationships among the human health, animal health and environmental sectors and create an inclusive approach to AMR, supported by professional network activities that can facilitate partnerships and create cross-disciplinary awareness and participation.


The ortho-pentaphenylene derivatives with anchoring groups adopt multiple folded conformers and undergo conformational interconversion in solutions. Solvent-sensitive multiple conductance originating from different conformers is recorded by scanning tunneling microscopy break junction technique. These pseudo-elastic folded molecules can be stretched and compressed by mechanical force along with a variable conductance by up to two orders of magnitude, providing an impressively higher switching factor (114) than the reported values (ca. 1~25). The multichannel conductance governed by through-space and through-bond conducting pathways is rationalized as the charge transport mechanism for the folded ortho-pentaphenylene derivatives. These findings shed light on exploring robust single-molecule potentiometers based on helical structures, and are conducive to fundamental understanding of charge transport in higher-order helical molecules.Molecular potentiometers that can indicate displacement-conductance relationship, and predict and control molecular conductance are of significant importance but rarely developed. Herein, single-molecule potentiometers are designed based on  Molecular potentiometers that can indicate displacement-conductance relationship, and predict and control molecular conductance are of significant importance but rarely developed. Here, the authors design a robust single-molecule potentiometers based on helical structures and give insight in fundamental understanding of charge transport in higher-order helical molecules. Plenty of molecules are extensively studied and employed as molecular wires, molecular switches, and molecular transistors3. Certain special electronic components like potentiometers that can reveal the displacement-conductance relationship, and ultimately predict and control molecular conductance by external mechanical force, are of high importance but rarely developed. The very limited molecular potentiometers4 in the literature are mainly created based on linear molecules. The alkanes5, silanes6, and other linear systems with cis–trans isomers are common structures used as mechanical triggered switches because their conductance can be modulated by mechanical force via the deformation of the flexible alkane chains or cis–trans isomerization. The switching factor, defined as the ratio of the conductance of the highest conductance state to that of the lowest conductance state, is an important parameter to quantify modulation ratio7. The most-reported molecules usually have low mechanical switching factors ~107. Although it is reported that precise control of tilted angles of molecular wires can lead to a conductance increase of an order of magnitude up to 25-folds10, these switching factors are still far from satisfactory. Giant variations in molecular conformation, as well as charge transport mechanism, are desired to greatly alter conductance and achieve a controllable conductance–displacement relationship, which is conducive to realizing efficient molecular potentiometers with large switching factors.Advances of miniaturization in electronic devices unveil the possibility of adopting molecular electronics as primary elements in electronic circuityortho-phenylenes and their derivatives have been extensively studied in various research fronts13. They usually adopt a well-defined secondary structure, in which three repeating monomeric phenylene units form a helical turn15. Moreover, ortho-phenylenes often show dynamic conformational interconversion18 in solutions and exhibit pseudo-elastic properties that allow elongations without permanent deformation19. Such kind of backbone flexibility differentiates ortho-phenylenes from conventional linear molecules, making it possible to respond sensitively to mechanical stimuli and further control the charge transport behavior by stimulation. The superposition of offset π–π stacking interaction parallel to the helix axis and through-bond conjugation can endow ortho-phenylenes with multichannel charge transport in multidimensional degree21. With these merits, ortho-phenylenes have been theoretically predicted as potential candidates for molecular electronics long ago22, working as single-molecule solenoids, molecular springs, pressure sensors, and single-molecule potentiometers. However, there is scarcely any research that has literally applied ortho-phenylenes as molecular electronics in practice.Owing to the considerable potential as functional organic nanostructures, ortho-Pentaphenylene (o-PP) consisting of five ortho-position-linked phenyl rings is an intriguing and representative molecule for ortho-phenylenes. It holds the helical feature of ortho-phenylenes but with relatively less conformers, which makes it the most suitable model to study the optical and electronic characteristics of helical molecules. In this work, we wish to report the feasibility of ortho-phenylenes as molecular electronics for the first time based on o-PP. These o-PP derivatives can undergo conformational interconversion in solutions, rendering different kinds of conformers and thus multiple conductances as revealed by scanning tunneling microscopy break-junction (STM-BJ) technique25. In addition, their conformers are sensitive to solvents, leading to feasible conductance modulation that has not been found in other systems. More importantly, these pseudo-elastic molecules can be stretched and compressed by the mechanical force of the Au tip along with a dramatically variable conductance by up to two orders of magnitude, demonstrating the great potential as angstrom-scale single-molecule potentiometers , cyano (CN), and pyridines (Py), respectively, that can interact with Au atom, are designed and constructed with details in Supplementary Information  in these structures  into the shielding zone of π–π stacked phenyl rings and gives rise to distinctive upfield-shifted doublet peaks at ~6.15 ppm29. The minor signals are assigned to the co-parallelly folded AB conformer. The 2D exchange spectroscopy (EXSY) exhibits clear cross-peaks and illustrates that the protons giving rise to these minor signals are in exchange with those for major signals 30. But when these intramolecular rotations are suppressed by spatial constraint in the aggregated state, the nonradiative relaxation of the excited state is suppressed and the radiative decay is strengthened31, which will give rise to enhanced PL for these molecules. To validate this hypothesis, the PL behaviors of these molecules in tetrahydrofuran (THF) solutions and aggregates are investigated. In dilute THF solution, they show weak PL peaks at 388‒392 nm 18. Low absolute PL quantum yields (ΦPLs) of 6.6%, 21.6%, and 9.0% are measured for o-PP-2SMe, o-PP-2CN, and o-TP-2Py, respectively. Interestingly, when these molecules form aggregates by adding a large amount of poor solvent water into their THF solutions, the PL intensities are enhanced apparently  technique is widely used to study the charge transport behavior at the single-molecule level36. The conductance of a single-molecule junction highly depends on the contact geometry of the target molecule with the electrodes. To decipher the conformational impact on the molecular conductance, STM-BJ technique is applied to characterize the conductance of these molecules in single-molecule junctions. Typical direct tunneling traces with exponential decay and one-dimensional (1D) histograms shown in Supplementary Fig. G0 (G0 = 2e2/h), corresponding to the single Au–Au atomic conductance37.As the regulation of intramolecular rotations endows v/v) are constructed from over 5000 individual conductance–distance traces  in the mixed THF/mesitylene (THF/TMB) solvents  conductance–displacement histograms are generated by aligning each conductance trace after the point contact ruptures and overlaying all the conductance traces. As shown in Fig. o-PP-2SMe constructed from over 5000 individual traces in THF/TMB mixture , 1,2,4-trichlorobenzene (TCB), and n-decane are performed. Different from the two distinct conductance peaks recorded in THF/TMB mixture, the 1D histograms of o-PP-2SMe in TCB and n-decane only exhibit one single conductance peak and a tiny shoulder at around 10−3.0G0  between AA and AB conformers in n-decane (non-polar) and THF (polar) are calculated  are distinctly different than empty junctions  in THF/TMB  mixture46. An additional lower conductance peak at 10−4.89±0.02G0 can be separated from the LC state   Fig. 4–10,46,454 are carried out. All the flicker noise analyses are focused on the LC state stemming from the major AA conformers in molecular junctions. In the light of previous reports55, the noise power scaling as G2.0 is indicative of through-space coupling while G1.0 stands for through-bond coupling  isomers with a sole through-bond pathway49.To explore the charge transport pathways in these folded molecules, flicker noise analysesG2.0, it is possible that these folded molecules hold through-bond channel in addition to through-space channel for charge transport64. In order to validate this hypothesis and shed light on the transmission mechanism of these molecules, density functional theory (DFT) simulation is carried out using the ATK software package with nonequilibrium Green’s functions (NEGF). The zero-bias transmission spectra , whereas the LUMOs dominate the conductance of o-TP-2Py and o-PP-2CN, which is in accordance with other reported polyphenylenes with the same anchoring groups56. The main cause of the different conductance between o-PP-2SMe with electron-donating SMe anchors and the other two molecules with electron-withdrawing CN and Py anchors is the different dominant molecular orbitals. Since the through-space conjugation mainly occurs in LUMOs  than o-TP-2Py. On the other hand, in AB conformers, three molecules show similar transmission spectra with close conductance at zero bias. The calculated conductance of the AB conformers  is the highest value even reported, indicating the great potential as single-molecule potentiometers. The fact that o-PPs have larger continuous conductance ranges than conventional alkanes and para-phenylenes breaks the stereotype that polyphenylenes are not suitable as a molecular potentiometer. Actually, the helical molecules could be more promising as molecular potentiometers than conventional linear molecules. By strengthening the connection between the electrodes and anchoring groups of ortho-phenylenes derivatives with sufficient lengths, high-performance molecular potentiometers with controllable conductance and giant switching factors are expectable.More importantly, these pseudo-elastic folded molecules can be stretched and compressed by the mechanical force of the Au tip along with a dramatically variable conductance by up to two orders of magnitude. To the best of our knowledge, the switching factor of 1H and 13C NMR spectra were measured on a Bruker AV 500 (500 MHz) spectrometer in CD2Cl2 with tetramethylsilane . High-resolution mass spectra (HRMS) were recorded on a GCT premier CAB048 mass spectrometer operating in MALDI-TOF mode. Single-crystal X-ray diffraction intensity data were collected on a Bruker–Nonices Smart Apex CCD diffractometer with graphite monochromated MoKα radiation. Processing of the intensity data was carried out using the SAINT and SADABS routines, and the structure and refinement were conducted using the SHELTL suite of X-ray programs (version 6.10). UV–vis absorption spectrum was measured on a Shimadzu UV-2600 spectrophotometer. PL spectra were recorded on a Horiba Fluoromax-4 spectrofluorometer. Solution fluorescence quantum yields were measured using a Hamamatsu absolute PL quantum yield spectrometer C11347 Quantaurus_QY. High-performance liquid chromatography spectra were measured using Waters alliance e2695 separation module.All the chemicals and reagents were purchased from a commercial source and used as received without further purification. In the STM-BJ experiment, 5 μL of the solutions of target molecules (0.2 mM) was directly dropped on the gold substrate, which was used for further STM-BJ experiment. During the measurement, the gold tip is first controlled by a stepper motor  to get to the approximate position to contact the substrate (<1 μm), thereafter the tip is controlled by a piezo stack  with the voltage applied from 0 to 10 V, with the approach/retract at the speed of 10 nm/s. There is also a feedback system in the setup, when the voltage changing of the piezo stack from 0 to 10 V can not contact gold tips and substrate or break the junction to the detecting limit of our amplifier (~1 pA), the motor would take control of the movement of the tips to offset the distances between tips and substrates. During the repeating breaking and re-connecting operation, the bias is kept at 100 mV, and the real-time conductance is recorded by the home-built I-V converter with a sampling rate of 20 kHz.z = z − zcorr. Snap-back distance zcorr refers to the instantly increased distance caused by surface atoms reorganization between two gold electrodes. According to the tunneling equation G = G0 eβ−Tz, where βT is the tunneling decay constant and z is the absolute distance between two electrodes, the tunneling equation can be transferred to log(G/G0) = − 0.434βT∆z − 0.434βT∆zcorr. Then a plot of log(G/G0) versus ∆z has a slope of −0.434βT and an intercept of −0.434βT∆zcorr. The decay constant log(∆G/G0)/∆z = −5.5 Å−1 is chosen to scale the traces in pure solvent. By calibrating the distance from 10−3.0G0 to 10−5.0G0 to be 0.36 nm, the stretching rate is determined and used for the calibration of the relative stretching distance. The snap-back distance zcorr is determined to be 0.5 nm. Calibration of snap-back distance and the stretching rate leads to the absolute stretching distance of molecular junctions z = ∆z + zcorr, where ∆z is calculated from 10−0.3G0 to one order magnitude lower than the most probable conductance value.Conductance measurement in the pure solvent is used for the calibration of the snap-back distance and stretching rate as reported. Because of the existence of snap-back distance, the distance we measured in experiments is the relative distance ∆For each molecule, over 5000 curves are collected and analyzed to form the one-dimensional histograms and two-dimensional conductance–displacement cloud maps from which information concerning the molecular conductance and plateau length can be extracted. An automated process for finding STM-BJ traces with certain molecular plateaus is carried out using Labview processed programs.−3.0G0 to 10−5.0G0 based on calibration. Only the traces with certain molecular plateaus are overlaid to form one-dimensional and two-dimensional histograms.To rule out the direct tunneling traces with exponentially decayed conductance, the program will not overlay the traces of which junction lengths is shorter than 0.36 nm in the range from 10o-PP-2SMe, o-PP-2CN, and o-TP-2Py, respectively. For the HC state, the formation probabilities are counted as 41.4% (2362/5703), 30.8% (1719/5583), and 63.20% (4469/7071) for o-PP-2SMe, o-PP-2CN, and o-TP-2Py, respectively. In addition, there are 10226 out of 11452 (89.3%) traces and 9114 out of 9979 (91.3%) traces are selected to construct the conductance histograms of o-PP-2SMe in n-decane and TCB, respectively. In the experiment of o-PP-2CN and o-TP-2Py in n-decane, 2285 out of 7130 (35.5%) traces and 5145 out of 12706 (40.5%) traces are selected, respectively.The formation probability is counted after extracting the traces with certain selected peaks (HC/LC). The formation probabilities are counted as 84.6% (4826/5703), 40.6%, and 37.8% for the LC state of G) versus the average conductance. According to the previous reports, the noise power of tunneling junction scales as G2.0 is contributed to complete through-space coupling while G1.0 refers to complete through-bond coupling.Flicker noise analysis can distinguish the pattern of coupling by noise distribution. The molecular elongation is paused for 150 ms if the junction signal is detected at a bias voltage of 0.1 V and a sampling rate of 20 kHz. The conductance plateau is cut out for discrete Fourier transformation, in which the data are squared to get the noise power density spectra. The data integrated from 100 Hz to 1000 Hz then build the 2D histograms of normalized noise power  that using the SMD model. The Gibbs free energy in solvents is calculated using the M06-2X/6-31G(d) method. The electron density clouds are drawn by Multifwn.Geometry optimization calculations were carried out with Gaussian 16 program using density functional theory (DFT) and time-dependent density functional theory (TD-DFT) for the ground-state and long-range corrected functional M06-2X applied with 6-31G basis set, treating crystal structures as the initial configurations. The vibrations of compounds also calculated under the same method. The flexible scan is processed under the M06-2X/def2-TZVP method, the stable values of 14, for the exchange and correlation effects of the electrons. Double-ζ plus polarization (DZP) basis set for the molecule and single-ζ plus polarization (SZP) basis set for the electrodes. For geometry optimization, 1 k-point for x-, y directions, and 50 k-points for z direction, for transmission spectra and transmission pathways calculation, 5 k-point for x-, y directions, and 100 k-points for z direction. We resolve the interatomic transmission pathways at the Fermi energy with ATK software based on the calculated transmission spectra. A minimum threshold of 10% of the total transmission is used for the pathways.All the molecules are optimized based on the procedure described above, and making sure there is no imaginary frequency existed in the finally adopted structures. The compounds then inserted between the Au electrodes to build up model devices by the Atomistic Tool Kit (ATK) software. The molecular junction was formed by the semi-finite left and right electrodes  and a scattering region where the molecule was settled. A scattering region also comprises screening layers of LE and RE. The molecular device was examined using the Au (111) electrodes (both LE and RE). The Au electrodes were settled to a 5 × 6 × 3 supercell. First, isolated organic molecules were optimized. After the optimization, organic molecules were inserted between the LE and RE, and the distance between the LE and RE was relaxed until the total energy reached a minimum. Next, constraining the Au electrodes, organic molecules were relaxed. During the relaxation, force tolerance was set to 0.05 eV/Å. The distance between Au and S is 2.5 ± 0.1 Å. Then, first-principles calculations were carried out to expose the electronic transport properties. In calculations, the exchange-correlation potential was approximated within the generalized gradient approximation (GGA) with Perdew–BurkeErnzerhof (PBE) functional (GGA.PBE)Supplementary Information


A new zebrafish study identifies compounds that shield ears and kidneys against an anticancer drug. Related research article Wertman JN, Melong N, Stoyek MR, Piccolo O, Langley S, Orr B, Steele SL, Razaghi B, Berman JN. 2020. The identification of dual protective agents against cisplatin-induced oto-and nephrotoxicity using the zebrafish model. eLife9:e56235. doi: 10.7554/eLife.56235Cancer treatments have become increasingly effective over the past few decades, but the chemotherapy drugs that kill tumour cells also damage healthy tissues. This can lead to serious side effects that go on to impair the quality of life of patients after recovery. For instance, cisplatin, a drug used to treat testicular cancer, is toxic to kidneys and hair cells in the ear that are necessary for hearing processes . Now, inDanio rerio, a tiny freshwater tropical fish similar to humans at the molecular level, but can be bred cheaply and quickly (To do so, the team enlisted the zebrafish  quickly . It has  quickly . Indeed, quickly .Wertman et al. examined whether 1200 compounds could protect the kidneys and lateral line hair cells of zebrafish larvae against the toxic effects of cisplatin. The screening highlighted 22 molecules, including two that offered the highest levels of protection: dopamine, a compound that nerve cells use to communicate, and L-mimosine, a rare plant non-protein amino acid similar to the amino acid tyrosine . Their pThe next step would be to investigate how dopamine and L-mimosine perform this protective role. Organic cation transporters are a family of proteins that help to carry molecules – including dopamine – into cells. In their absence, cisplatin is less toxic for ears and kidneys . It is tFinally, it is essential to demonstrate that dopamine and L-mimosine do not impair the anticancer activity of cisplatin in vivo, which could also be done in zebrafish larvae. In addition, this animal model could be useful to study neurotoxicity, another potential side effect of the drug. This would allow scientists to investigate whether the two compounds only protect specific organs, or globally interfere with cisplatin activity.Confirming that dopamine and L-mimosine preserve the anticancer properties of cisplatin in vivo, together with fully understanding how they shield ears and kidneys from the drug’s toxicity should help to develop protective therapies. Ultimately, this would allow more aggressive cancer chemotherapy to be performed, and improve the quality of life of cancer survivors.


Despite its hallmark cutaneous presentation, most Kaposi's sarcoma (KS) in Africa is diagnosed too late for effective treatment. Early diagnosis will only be achievable if patients with KS present earlier for care. We hypothesized that public awareness about KS can be enhanced through exposure to common media.  We developed educational messages regarding early detection of KS for the general African public portraying a three-part theme: “Look” (regularly examine one's skin/mouth), “Show” , and “Test” (ask for a biopsy for definitive diagnosis). We packaged the messages in three common media forms  and tested their effect on increasing KS awareness among adults attending markets in Uganda. Participants were randomized to a single exposure to one of the media and evaluated for change in KS-related knowledge and attitudes. p < 0.001). Most participants (96%) found the media culturally appropriate.  Among 420 participants, media exposure resulted in increased ability to identify KS (from 0.95% pretest to 46% posttest); awareness that anyone is at risk for KS (29% to 50%); belief that they may be at risk (63% to 76%); and knowledge that definitive diagnosis requires biopsy (23% to 51%) (all  Exposure to media featuring a theme of “Look,” “Show,” and “Test” resulted in changes in knowledge and attitudes concerning KS among the general public in Uganda. High incidence and poor survival of KS in Africa are an impetus to further evaluate these media, which are freely available online. As a result of the intersection between the HIV epidemic and the endemic presence of Kaposi sarcoma-associated herpesvirus (KSHV) infection, sub-Saharan Africa accounts for over 80% of incident Kaposi's sarcoma (KS) worldwide . In EastBecause HIV-infected individuals are at particular risk for KS, a natural target to consider for interventions to enhance early detection of KS in Africa is the numerous primary care facilities that have arisen to enable the roll-out of ART. Recent work from Zimbabwe, however, which provided training regarding early KS detection to frontline clinicians at HIV-dedicated primary care clinics, demonstrated that training alone may be insufficient. In this research, providing training increased clinician knowledge but had no effect on the stage of patients' KS at diagnosis . In retrTo address the gap in public knowledge about KS in sub-Saharan Africa and ultimately facilitate early detection, we endeavored to create an educational message regarding KS and the importance of early diagnosis that could be disseminated in the community via common media formats. We subsequently tested whether exposure to the media forms was able to change knowledge and attitudes regarding KS among adults in the general community.Using community-engaged research, we developed an educational message regarding KS and its early detection and incorporated this message into three common media forms. To determine whether the message and media forms influenced knowledge and attitudes concerning KS among members of the general public, we then randomly assigned exposure to the three media forms to adults attending rural and urban commercial markets in Uganda. Lastly, we assessed knowledge and attitudes concerning KS with pre- and postmedia tests.Our objective was to develop a scientifically sound and culturally appropriate educational message about KS and its early detection. We, also, aimed to determine if common media forms could be used to disseminate the message. We worked with an international health science communication consultant  and used community-engaged research. Community engagement involved soliciting input from key stakeholders, at several different stages, including (a) patients with KS who had survived the cancer  and persons living with HIV infection; (b) healthcare practitioners including primary healthcare providers, community health workers, and traditional health practitioners; (c) community leaders and members of the general public; and (d) local media groups experienced with health message dissemination in Uganda.https://soundcloud.com/kaposis_sarcoma), and a short (10-minute) video documentary (https://vimeo.com/224920054). All media were created in English and the four most commonly spoken local languages .We developed an educational message with a three-part theme: “Look,” indicating to perform skin and mouth examination, either by one's self or via a partner, to identify any new or unusual skin/mouth lesions; “Show,” meaning to bring any suspicious skin/mouth lesions to the attention of a healthcare provider; and “Test,” denoting to ask healthcare providers to perform biopsies for suspicious lesions. We incorporated this “Look”, “Show”, and “Test” theme into storylines featuring fictional and real-life  characters finding abnormal skin lesions, presenting the lesions to healthcare providers, receiving a skin punch biopsy for diagnosis, and initiating treatment for KS. The message was crafted drawing upon the concepts of the Information–Motivation–Behavioral skills model (IMB model) . To dissThe target population for our evaluation of the three media forms was the general Ugandan adult population aged at least 18 years. As an accessible population, we choose to sample participants from commercial markets. Markets in Uganda are concentrations of multiple open-air structures where food  and everyday household items are traded. Each market serves a given approximate geographic area. We chose markets from one predominantly rural district  and theWe randomly assigned participants, stratified by gender, to one of the three media types. The first was the comics which we printed on newspaper-sized hard stock cards. The second was the video documentary which we showed on handheld electronic tablets. The third, the radio vignettes, were administered through handheld radios. Because we wanted to simulate a single exposure to the media, participants were only allowed to possess the media for a limited amount of time (ten minutes each for the comics and radio vignettes and 15 minutes for the video), following which they were immediately administered a posttest. Illiterate participants assigned to comics had a research team member read each story once while the participant viewed the corresponding pictures. Any questions about the material were deferred until after testing was completed.Our main objective was to describe the change in knowledge and attitudes concerning KS as a result of being exposed to the media. We determined within-person change by comparing responses on an interviewer-administered test completed prior to the exposure to the media to the responses on the same test administered immediately after the exposure. The test questions were informed by the concepts of the IMB model. They covered visual recognition of KS (by showing participants pictures of KS lesions); knowledge of the causative agent and casual behavioral factors; self-risk assessment; and health-seeking attitudes regarding KS.The interviewer-administered pretest also obtained information on age, sex, education and literacy level, residence, income, and self-reported HIV status.Each item on the questionnaire had a single best specific response according to the messages covered by the media. For some of the items, additional correct  responses were also possible. For each item, we determined within-participant change. We determined the percentage of participants whose responses changed from incorrect on the pretest to correct on the posttest, percentage of participants whose responses changed from correct to incorrect, and percentage whose responses did not change. We performed this analysis for the single best response and, separately, for all possible correct responses. Statistical significance of the within-participant change was determined with McNemar's test. We also evaluated the influence of several factors  on change in KS knowledge and attitudes. The outcome was a change from an incorrect response on the pretest to a correct response on the posttest; this was coded as “1.” All those whose responses did not change or changed in the negative direction were coded as “0.” We used a directed acyclic graph , 12 to dn = 237) and rural (n = 183) Uganda. The median age was 30 years (interquartile range: 24 to 39), and by design, 50% were women  and “microorganism.”p=0.002) lower odds of correctly identifying KS from photographs compared to those residing in rural areas, those in urban areas had higher odds of correct responses in the other two questions. Having at least secondary education had higher odds of correctly naming KS compared to having only primary or no formal education. Notably, education level showed no strong evidence of an association with performance on the other two questions.We explored the relationship between various participant characteristics and ability, after media exposure, to provide correct responses on the posttest. We evaluated these relationships in the context of three test questions: ability to identify and cite KS by name in response to photographs; knowledge of who is at risk for KS; and recognition that any suspicious skin or mouth changes should be presented immediately to a biomedical healthcare provider . In adjuWhen asked about their opinions about the suitability of the educational materials, 93% of participants were satisfied with the media content and offered no suggestions for improvement. Of the 31 (7.4%) participants who suggested some adjustments, 19% believed that including more photographs of KS lesions would help reinforce the messages, and 29% requested more information on the causative agent of KS . Only 16Despite the possibility of remission when diagnosed at an early stage, KS in sub-Saharan Africa is too often detected at advanced disease states for which available treatment is ineffective. The irony is exacerbated by most KS presenting on the skin or mouth, areas in which abnormalities should be readily identified and brought to medical attention. In a community-based sample of Ugandan adults, we found—at least in part explaining why advanced stage presentation is so frequent—that knowledge about KS and its early detection was limited. To begin to remedy this, we developed an educational message about KS targeted to the public. The themes intended to educate the public about the importance of examining their skin (“Look”), timely presentation to a healthcare provider if abnormalities are found (“Show”), and asking providers to perform biopsies for definitive diagnosis (“Test”). To disseminate the message, we incorporated it into three common media forms: comics, radio vignettes, and a video documentary. We demonstrated that exposure to these media increased knowledge and changed attitudes about KS and its early detection. The messages and modes of delivery were acceptable to the public, thus providing a promising foundation for future comprehensive educational efforts. To our knowledge, this is the first formal effort to begin to educate the public in Africa about KS.We found very low knowledge regarding KS in our participants prior to exposing them to the media materials. Almost no participant could recognize and cite “Kaposi sarcoma,” and less than 50% thought of it as cancer. There was also no local terminology or name used to refer to KS. This low level of specific knowledge in the population has been documented for other cancers in sub-Saharan Africa –16. AlthAfter a single setting exposure to one of the three media forms, participants exhibited significant increases in their ability to identify and name KS, recognize that everyone is at risk for KS, state that they would seek attention from a biomedical provider if they developed lesions resembling KS, and know that a biopsy is needed to establish a diagnosis. We are unaware of similar educational efforts targeted at the general public to enhance knowledge about KS. Community-targeted educational efforts have, however, been implemented for breast and cervical cancer in Africa with demonstrable increases in knowledge –25. WhilOne fact that our participants did not learn from our media was the name of the causative agent of KS—HHV-8/KSHV. This is unsurprising given the technical nature of this term and the low level of education we found in our participants. We do not, however, feel that this is a shortcoming of our media in that knowledge of the causative organism for KS is unimportant for community residents. Instead, what is essential is, and what we were seeking to accomplish, is for the public to recognize KS, seek immediate biomedical attention, and request a biopsy for diagnosis. We also paradoxically observed some participants changing their answer from a correct to incorrect one. We attribute this to uncertainty and guessing on the pretest followed by incomplete learning and continued guessing on the posttest.KS in Africa is similar to melanoma in other parts of the world . Like KS, melanoma clinically features hyperpigmented skin lesions  and has The magnitude of learning that our participants exhibited was admittedly modest. We attribute this to their receiving only a single exposure of one media type. In addition, even in those participants who did exhibit correct changes in their test performance after media exposure, it is unknown how long knowledge will be retained from this single exposure and, as noted above, whether it will translate into the desired behavior change. It is not our intention, however, to suggest that a single exposure to one media form will be impactful. Instead, we strongly believe that to cause a meaningful change in behavior, people will need to experience multiple exposures to multiple media types over an extended period of time. Directed discussions of the messages, for example, led by Village Health Team members , might Our work does have limitations. While commercial markets are known to attract all types of individuals, we cannot be certain how our participants represent the overall general target population in Uganda. To mitigate this, we sampled markets in both urban and rural locations, and furthermore, our participants' literacy and HIV infection status mirrored national averages. Because we sought to evaluate learning from exposure to the media irrespective of reading literacy, our study staff both read the comics to those who were illiterate and administered the tests to all participants. It is therefore conceivable that this interaction with staff may have spuriously influenced test performance. To prevent this, staff were instructed to only read the text of the comics, point to the accompanying pictures, and not provide additional explanations. Pre- and posttests followed a standardized script with no prompting. Finally, because the study was only conducted in Uganda, we are uncertain how findings will translate to other parts of Africa.In conclusion, the high incidence and poor survival of KS in Africa—mainly due to advanced stage of disease at diagnosis—are a strong motivation to educate the public about KS and early detection. Although some will argue that a decline in KS incidence with growing ART availability , 55 less


A subset of genes involved in tumorigenesis and inflammation/immune responses showed higher magnitude, but not unique dysregulation in EnKS compared to EpKS. While gender and ART had no detectable contribution, the trend toward higher magnitude of gene dysregulation in EnKS coupled with the absence of HIV-1 transcripts in EpKS may suggest an indirect or systemic effect of HIV-1 to promote KS tumorigenesis.In sub-Saharan Africa, endemic Kaposi’s sarcoma (EnKS) is still prevalent despite high incidence of epidemic Kaposi’s sarcoma (EpKS) resulting from the on-going HIV-1 epidemic. While KSHV is clearly the etiologic agent of KS, the mechanisms underlying KS development are not fully understood. For example, HIV-1 co-infection and concomitant immune dysfunction have been associated with EpKS development. However, the direct or indirect role(s) of HIV-1, and therefore of immune suppression, in EpKS remains unclear. How, or whether, EpKS is mechanistically distinct from EnKS is unknown. Thus, the absence of HIV-1 co-infection in EnKS provides a unique control for investigating and deciphering whether HIV-1 plays a direct or indirect role in the EpKS tumor microenvironment. We hypothesized that HIV-1 co-infection would induce transcriptome changes that differentiate EpKS from EnKS, thereby defining the direct intra-tumor role of HIV-1 in KS. Comparison of ART-treated and -naïve patients would further define the impact of ART on the KS transcriptome. We utilized RNA-seq followed by multiparameter bioinformatics analysis to compare transcriptomes from KS lesions to uninvolved control skin. We provide the first transcriptomic comparison of EpKS versus EnKS, ART-treated vs–naïve EpKS and male vs female EpKS to define the roles of HIV-1 co-infection, the impact of ART, and gender on KS gene expression profiles. Our findings suggest that ART-use and gender have minimal impact on transcriptome profiles of KS lesions. Gene expression profiles strongly correlated between EpKS and EnKS patients  coverage, Kaposi’s sarcoma (KS) remains the most common cancer in people living with HIV/AIDS. KSHV is known to cause KS, but there are no preventive or curative vaccines. Our understanding of the interactions between KSHV, host cell and the microenvironment is lacking. HIV-1 co-infection has been implicated in KS pathogenesis, but its mechanistic role is unclear. We analyzed transcriptomes from lesions and uninvolved control skin from HIV-1-positive and -negative KS patients to determine the direct or indirect role of HIV-1 in KS development. Transcriptomes from the uninvolved control skin from KS subjects were indistinguishable from that of non-KS healthy individuals. This validates the use of uninvolved control skin to control for gene expression pattern in KS lesions. Despite high concordance in gene expression profiles between HIV-1-positive and -negative KS patients, a subset of genes involved in tumorigenesis and inflammation/immune responses showed higher magnitude of dysregulation in EnKS than EpKS patients. The trend toward lower magnitude of gene dysregulation in EpKS coupled with the absence of HIV-1-transcripts in EpKS suggests an indirect or systemic effect of HIV-1 to promote KS tumorigenesis and possibly explain the high KS incidence in regions with endemic KSHV and HIV-1 co-infections. Kaposi’s sarcoma-associated herpesvirus (KSHV) is an oncogenic gamma-herpesvirus associated with Kaposi’s sarcoma (KS), primary effusion lymphoma (PEL) and multicentric Castleman’s disease (MCD) –4. KSHV There are four major forms of KS, classical KS, iatrogenic KS, African-endemic KS (EnKS) and epidemic/HIV-1/AIDS-associated KS (EpKS) –20. EnKSOur group and others have previously analyzed viral and cellular gene expression profiles of EpKS patients –30. HoweApart from inducing anti-inflammatory cytokines , KSHV isIn this study, we provide the first transcriptomic comparison of EpKS vs EnKS patients, ART-treated versus -naïve EpKS patients and male versus female EpKS patients to more completely define the roles of HIV-1 co-infection, ART experience, and gender on KS gene expression profiles. We initially hypothesized that HIV-1 co-infection would induce transcriptome changes that differentiate EpKS from EnKS, thereby defining the direct intra-tumor role of HIV-1 in KS. Comparison of ART-treated and–naïve patients would define the impact of ART on tumor transcriptome. These differentials could prove important in the design of therapeutic strategies to control KSHV and KS development. In addition, we have compared transcriptomes from KS lesions to uninvolved control skin from the same patient as well from asymptomatic volunteers. This is to determine whether KSHV has a systematic effect on gene expression profiles, even in non-disease sites, and to ensure that uninvolved control skin in KS patients is indeed ‘normal’.Our results demonstrate that there are no differentials in most of the transcriptomes between EpKS and EnKS, however a small subset of genes exhibited a higher magnitude of change in EnKS compared to EpKS. This coupled with the absence of HIV-1 transcripts in EpKS suggest an indirect or systemic effect of HIV-1 to promote cellular reprogramming of this group of genes which may be involved in KS development. The data also demonstrated that ART-use and gender have minimal impact on transcriptome profiles of KS lesions. Furthermore, uninvolved control skin from KS subjects is indistinguishable from that of asymptomatic healthy controls.RNAlater  overnight at 4°C to prevent RNA degradation and then frozen at -80°C after removal of RNAlater.KS patients of both genders (N = 24), over 18 years of age were recruited from Tanzania and Zambia. Diagnosis of KS was confirmed by histology and PCR detection of KSHV viral DNA in the lesion. For every KS patient recruited in the study, a 4mm skin biopsy was collected from a representative KS lesion. Similarly, another 4mm skin biopsy was collected from an uninvolved control contralateral/ipsilateral cutaneous site from the same individual. However, because it is possible that even uninvolved control skin tissues in KS patients are transcriptomically dysregulated in relation to skin from non-KS subjects, 4mm skin biopsies were collected from three healthy volunteers for comparison of gene expression profiles. Written informed consent was obtained from all study participants. All biopsies were treated with Biopsied tissues were subjected to RNA extraction using a combined cryo-pulverization (mortar and pestle) and miRNeasy Mini Kit  procedure. Briefly, in a metal tray, the DNA/RNA free mortar and pestle were chilled using liquid nitrogen. Biopsied tissue samples were transferred (one at a time) from dry ice into the pre-chilled mortar and then pulverized and homogenized using the pre-chilled pestle. The homogenized sample was then transferred to a 2mL Eppendorf tube using a pre-chilled spatula. The miRNeasy Mini Kit  was used to extract total RNA from homogenized samples using on-column DNase-I treatment, according to the manufacturer’s protocol. RNA concentration was determined using a Qubit fluorimeter  and the quality of the extracted RNA was assessed by RNA fragment analysis using an Agilent Bio-analyzer . The electrophoretic trace of the 18s and 28s rRNA peaks was used to calculate an RNA Integrity Number (RIN) indicative of the level of RNA integrity. All sequenced samples had RIN ranging from 4.8 to 10. An overall schematic representation of biopsy collection and processing is shown in Plasma was separated from whole blood samples by centrifugation at 400xg for 5 minutes, followed by volume reconstitution and preparation of the peripheral blood mononuclear cell (PBMC) fraction by density gradient centrifugation using Lymphoprep . Plasma and cells with cryoprotectant were stored at -80°C. The collected plasma was then used to determine the serology and plasma viral load for both HIV-1 and KSHV. Approval to conduct this study was obtained from the review boards of Tanzania National Institute for Medical Research, Ocean Road Cancer Institute, University of Zambia Biomedical Research Ethics Committee, and the University of Nebraska-Lincoln (UNL).HIV-1 serology was determined by HIV Rapid Test Algorithm  in TanzaPlasma samples (400μL) were centrifuged at 8,000 x g at room temperature for 10 minutes to remove any residual cells. To eliminate any cell-free DNA, 15μL of DNase-I  was added and incubated for 2 hours at room temperature. The DNase-I was then inactivated by incubation at 65°C for 20 minutes. KSHV virion-associated DNA (vDNA) was extracted according to the QIAamp DNA extraction protocol . A negative PCR amplification result for the human β-actin gene using primers of Actin1 [5’-TTCTACAATGAGCTGCGTGT-3’] and Actin2 [5’-GCCAGACAGCACTGTGTTGG-3’] demonstrated the complete elimination of any cell-free DNA. KSHV vDNA was then detected by nested PCR of the KSHV ORF26 gene using the following primers, forward [5’-AGCCGAAAGATTCCACCAT-3’] and reverse [5’-TCCGTGTTGTCTACGTCCAG-3’] in the first round, and forward [5’-CGAATCCAACGGATTTGACCTC-3’] and reverse [5’-CCCATAAATGACACATTGGTGGTA-3’] in the second round reaction. Each amplification was performed in a total volume of 25 μl using 0.4 μM primers and Takara Ex Taq DNA polymerase kit  according to manufacturer’s protocol. 200ng of DNA isolated from plasma was used as a template for both β-actin and KSHV ORF26 in first round amplification. 2 μl of the initial reaction product was used for nested ORF26 second round amplification. All reactions utilized the following thermal-cycling conditions: 95°C for 5 min, 35 cycles of 95°C for 30 secs, 58°C for 30 secs, 72°C for 30 secs, and one cycle of 72°C for 7 min.Biopsy-derived RNA samples with a RIN ranging from 4.8 to 10 were used for library preparation and sequencing. Library preparation was done using the Illumina’s TruSeq RNA Library Prep Kit , and data were collected using Illumina’s NextSeq HighOutput v2 , in single-read 50bp, rapid run mode at the University of Nebraska DNA Sequencing Core.et.al .,66.65,66in vitro ,68. It ain vitro . Genes rTo explore the potential transcriptomic basis for gender in KS development, we compared transcriptomic differentials between male and female EpKS patients. Most of the dysregulated cellular genes were similar between male and female EpKS patients. Despite marginal differential expression in a few genes (n = 34) between male and female, none of these genes were significant at FDR<5% . CompariThe use of ART to control HIV-1 has been associated with a number of cellular changes –47. SincKSHV predominantly adopts a latent gene expression pattern in infected cells where a few viral genes including latency-associate nuclear antigen, LANA (ORF73), viral cyclin D homolog, vCYC ORF72), Kaposin (K12) and viral Fas-associated protein with death domain-like interleukin 1β converting enzyme/caspase-8- inhibitory protein, vFLIP ORF71), are expressed to maintain the viral episome ,71. Apar, are exp, KaposinTo investigate the KSHV gene expression profile in KS lesions, expression of KSHV genes from each lesion were normalized to the respective total KSHV RNA reads and subjected to unsupervised hierarchical clustering. Consistent with previous studies, three gene expression patterns were observed in both EpKS and EnKS tumors   79,80].,80.79,80Furthermore, irrespective of HIV-1 infection status, five clusters of KSHV genes were observed in KS lesions according to known gene functions . AlthougBecause KSHV is a necessary, but insufficient, agent in KS development, we investigated relationships between KSHV RNA load/reads and cellular gene expression in KS lesions and controls. Principle component analysis (PCA) demonstrated the anticipated separation of lesion and control samples within the first two principle components  . Increas−10, In addition to investigating the impact of HIV-1, ART, and gender on transcriptome profiles of KS patients, we determined gene expression patterns associated with KSHV pathogenesis. We compared our previous dataset (4 paired EpKS samples) with the additional sample pairs from the current dataset (21 paired samples). Of the 3589 genes reported to exhibit significantly dysregulated expression versus uninvolved control skin in the previous study at FDR<5%, 96.4% showed the same direction of change in the completely independent and much larger cohort of patients. Overall expression patterns were similar , with on−10) [To our knowledge, this is the first study to analyze the effects of HIV-1 co-infection on the gene expression profiles in KS lesions with appropriate infection, treatment, and uninvolved tissue controls. This was conducted by comparatively analyzing the transcriptome profiles between EpKS and EnKS patients from SSA. The study involved sequencing 51 individual skin tissues to support investigation of multiple comparison groups to better characterize the impact of HIV-1, ART, and gender, on KS tissue gene expression profiles. For example, gene expression in uninvolved control skin from KS patients was demonstrated to be indistinguishable one from another, and from skin from non-diseased healthy subjects. So uninvolved skin in a KS patient is indeed ‘normal’ skin. Overall, gene expression profiles correlated significantly between our current and previous study  . Here we−10) , suggest−10). Our analyses showed that most of the dysregulated genes involved in immune function and tumorigenesis were shared between the EpKS and EnKS datasets. This is consistent with our recent analysis of peripheral immune responses between ART-treated EpKS and EnKS patients, in which HIV-1 co-infection did not differentially impact cytokine and antibody responses [Comparison of control-normalized EpKS and EnKS transcriptomes revealed that up- and down-regulated cellular genes in EpKS and EnKS were highly concordant  has been shown to lack protective efficacy against natural KSHV infection. Rather, nAb correlates with KS pathogenesis ,89. Onlyet al [a priori partitioning decisions that could limit the capacity to detect globally important transcriptome changes in KS. Also, as a limitation of RNA-seq technique and its processing algorithms, for example, it cannot accurately distinguish reads coming from ORF74 versus K14 (vOX-2), as the NCBI KSHV version NC_009333.1 has ORF74 gene (https://www.ncbi.nlm.nih.gov/gene/4961460) sequence being completely within longer sequence of K14 (https://www.ncbi.nlm.nih.gov/gene/4961465). However, the current analysis, in addition to validating our previous findings on altered glucose and lipid metabolism, has addressed the role of HIV-1 and whether a gene expression bias exists based on gender in EpKS patients. Relationships between HLA and risk of KS are still unclear and complicated by lack of functional immune response/repertoire data for haplotypes other than HLA-A*0201. Sequencing 51 individual biopsies from 27 individuals makes this one of the very few if not the only KS study with a large transcriptome dataset. However, distribution of the samples into multiple comparison groups has led to lower sample sizes in some of the groups. This could have limited our ability to detect transcriptome differentials between some groups. Studies with larger sample size particularly with EnKS are warranted to fully characterize KS transcriptome. In conclusion, transcriptome profiling reveals only minor differences between EpKS and EnKS, and no detectable contribution from gender or ART use. The trend toward higher magnitude of gene dysregulation in EnKS coupled with the absence of HIV-1 transcripts in EpKS may suggest an indirect or systemic effect of HIV-1 to promote KS tumorigenesis. Alternatively, KSHV requires greater extent of cellular reprogramming to induce cancer on its own than when in the presence of HIV-1 co-infection.Our study expands significantly upon the previous findings derived from comparisons of 4 EpKS and contralateral uninvolved control skin samples solely from ART treated patients . As repoet al , one conS1 FigEnKS–Endemic Kaposi’s sarcoma, EpKS–Epidemic Kaposi’s sarcoma.(TIF)Click here for additional data file.S2 FigN—normal/healthy, L–Lesion and C–Control.(TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S4 Fig(A) Comparison of absolute normalized expression in controls and lesions between male and female. (B) Comparison of absolute normalized expression in controls and lesions between ART-treated and HIV (ART-naive) patients.(TIF)Click here for additional data file.S5 Fig(A) A scatter plot comparing transcriptome profiles between EpKS patients treated with ART for more one week (N = 10) and those who were ART naïve or treated for less than a week (N = 8). Green dotted line denotes regression line. Grey dots denote genes whose expression was not significantly different between EpKS patients treated with Art for >1 week and those who are ART naïve of treated for <1 week. Black dots denote genes whose expression was significantly different between EpKS patients treated with Art for >1 week and those who are ART naïve of treated for <1 week at P = 0.005 (N = 23) and unfilled circles denote genes whose expression was significantly different between EpKS patients treated with Art for >1 week and those who are ART naïve of treated for <1 week at FDR<5% (N = 0). (B) A scatter plot comparing transcriptome profiles between EpKS patients treated with ART for more one month (N = 9) and those who were ART naïve or treated for less than a month (N = 9). Green dotted line denotes regression line. Grey dots denote genes whose expression was not significantly different between EpKS patients treated with Art for >1 month and those who are ART naïve of treated for <1 month. Black dots denote genes whose expression was significantly different between EpKS patients treated with Art for >1 month and those who are ART naïve of treated for <1 month at P = 0.005 (N = 46) and unfilled circles denote genes whose expression was significantly different between EpKS patients treated with Art for >1 month and those who are ART naïve of treated for <1 month at FDR<5% (N = 2). (C) A scatter plot comparing transcriptome profiles between EpKS patients with detectable plasma HIV-1 viral load (viremic) (N = 11) and undetectable plasma HIV-1 viral load (aviremic) (N = 7). Green dotted line denotes regression line. Grey dots denote genes whose expression was not significantly different between HIV-1 viremic and aviremic EpKS patients. Black dots denote genes whose expression was significantly different between HIV-1 viremic and aviremic EpKS patients P = 0.005 (N = 33) and unfilled circles denote genes whose expression was significantly different between HIV-1 viremic and aviremic EpKS patients at FDR<5% (N = 0). (D) Principal component analysis (PCA) showing relationship of samples on first and second principal components based on transcriptome profile of 33 genes affected by ART in HIV-1 suppressed patients (green circles) vs patients with HIV-1 viremia (red squares) on at nominal p<0.005 in EpKS lesions. ART treated patients with detectable HIV-1 (white triangles) demonstrate similarity to ART naïve, HIV-1 viremic EpKS patients indicating that these genes represent signature of HIV-1 viremia. EnKS–Endemic Kaposi’s sarcoma, EpKS–Epidemic Kaposi’s sarcoma.(TIF)Click here for additional data file.S1 TableEnKS–Endemic Kaposi’s sarcoma, EpKS–Epidemic Kaposi’s sarcoma.(DOCX)Click here for additional data file.S2 TableL–Lesion, C–Control, N–Normal/Healthy, KSHV–Kaposi’s sarcoma-associated herpesvirus, HIV-1 –Human Immunodeficiency virus type 1(DOCX)Click here for additional data file.


Pharmacological therapies that function independently of dystrophin and complement strategies aimed at dystrophin restoration could significantly improve patient outcomes. Previous observations have suggested that serotonin pathway modulation ameliorates dystrophic pathology, and re-application of serotonin modulators already used clinically would potentially hasten availability to DMD patients. In our study, we used dystrophin-deficient sapje and sapje-like zebrafish models of DMD for rapid and easy screening of several classes of serotonin pathway modulators as potential therapeutics. None of the candidate drugs tested significantly decreased the percentage of zebrafish exhibiting the dystrophic muscle phenotype in the short-term birefringence assay or lengthened the lifespan in the long-term survival assay. Although we did not identify an effective drug, we believe our data is of value to the DMD research community for future studies, and there is evidence that suggests serotonin modulation may still be a viable treatment strategy with further investigation. Given the widespread clinical use of selective serotonin reuptake inhibitors, tricyclic antidepressants and reversible inhibitors of monoamine oxidase, their reapplication to DMD is an attractive strategy in the field's pursuit to identify pharmacological therapies to complement dystrophin restoration strategies.Duchenne muscular dystrophy (DMD) is a progressive muscle-wasting disease caused by mutation of the  Summary: Phenotypic screening of serotonin pathway modulators has been performed in DMD zebrafish in order to identify small-molecules that limit the dystrophic muscle phenotype and lengthen the lifespan as potential therapeutics. Absence protein  and caus protein  that lea protein . The con protein .dystrophin, have recently been approved by the FDA  localizes to the muscle cell membrane and functions similarly as in mammals . The higuman DMD . Mutant sapje and sapje-like zebrafish to assess serotonin and 16 serotonin precursors, products and modulating drugs as DMD therapeutics. We performed both short-term birefringence assays to assess the ability of the candidate drugs to prevent manifestation of the dystrophic phenotype as well as long-term survival assays. Unfortunately, our experiments did not recapitulate previous positive results, but should be taken into account in future efforts to assess serotonin modulation as a strategy for ongoing DMD therapy development.In this study, we used sapje/sapje-like homozygous mutant muscle phenotype detected by birefringence assay  inhibitor aminophylline as our positive control, which was discovered as a positive effector in a previous zebrafish drug screen in our lab and also confirmed independently  and tryptophan, the serotonin product melatonin and the serotonin receptor agonist cisapride. Short-term treatment with serotonin (8.25 μM–66 μM), 5-HTP (16.5 μM–132 μM), tryptophan (8.25 μM–66 μM), melatonin (8.25 μM–66 μM), and cisapride (4.12 μM–66 μM) did not significantly decrease the percentage of affected fish A–E. Cisasapje fish , escitalopram (16.5 μM–132 μM), fluoxetine (4.12 μM–66 μM), fluvoxamine (8.25 μM–132 μM), paroxetine (4.12 μM–66 μM) and sertraline (4.12 μM–66 μM) did not significantly decrease the percentage of affected fish A–F. Fluopje fish . In our pje fish C,G.Fig.Our third set of candidate drugs included the tricyclic antidepressants amitriptyline, clomipramine and imipramine, and the RIMAs moclobemide, pirindole, and toloxatone. Short-term treatment with amitriptyline (4.12 μM–66 μM), clomipramine (4.12 μM–66 μM), imipramine (8.25 μM–132 μM), moclobemide (16.5 μM–132 μM), pirlindole (8.25 μM–66 μM) and toloxatone (16.5 μM–132 μM) did not significantly decrease the percentage of affected fish A–F. Amitsapje fish already exhibiting the muscle phenotype. In the long-term assay, affected and unaffected fish are identified and separated by birefringence assay on 4 dpf. Drug treatment is then initiated, and the number of surviving fish in each cohort is counted every other day through 30 dpf  used in this study were handled in accordance with the Guide for the Care of Laboratory Animals of the National Institutes of Health. Humane endpoints were used during all zebrafish experiments. The specific criteria used were whether zebrafish exhibited a swim response to touch. Those that did not were euthanized with the technique appropriate for the given larval stage in accordance with the National Institute of Health Final Report to OLAW on Euthanasia of Zebrafish. Zebrafish that survived through the course of the survival study (30 dpf) were also euthanized appropriately in accordance to the National Institutes of Health Final Report to OLAW on Euthanasia of Zebrafish. Specifically, zebrafish were immobilized by submersion in ice water  for at least 10 min following cessation of opercular (i.e. gill) movement. The protocol used in this study was approved by the Institutional Animal Care and Use Committee (IACUC) at Boston Children's Hospital (Protocol number: 18-08-3749R).Zebrafish (dx.doi.org/10.17504/protocols.io.bb2iiqce). Fertilized eggs were collected and raised in E2 water at 28.5°C  and sapje-like fish: forward primer 5′-TCTGAGTCAGCTGACCACAGCC-3′; reverse primer 5′-ATGTGCCTGACATCAACATGTGG-3′. Sequencing was preformed by the Molecular Genetics Core Facility at Children's Hospital Boston and analyzed using Sequencher.Zebrafish were housed in the Boston Children's Hospital Aquatics Facility and maintained in accordance to IACUC standards , 5-hydroxy -L-tryptophan (Sigma-Aldrich), tryptophan (Sigma-Aldrich), melatonin (Sigma-Aldrich), cisapride (Sigma-Aldrich), citalopram (Sigma-Aldrich), escitalopram (Sigma-Aldrich), fluoxetine (Sigma-Aldrich), fluvoxamine (Selleckchem), paroxetine (Sigma-Aldrich), sertraline (Sigma-Aldrich), clomipramine (Sigma-Aldrich), imipramine (Sigma-Aldrich), amitriptyline (Sigma-Aldrich), moclobemide (Sigma-Aldrich), pirlindole (Santa Cruz Biotechnology), toloxatone (Sigma-Aldrich) and aminophylline (Sigma-Aldrich) . Each caP-values of <0.05 were considered to be statistically significant.All results are shown as means± standard error of the mean (s.e.m.). Statistical analyses of the data were performed using StatPlus to implement one- and two-way ANOVA followed by Bonferroni post-hoc tests.