Clinical trials from the patient perspective: survey in an online patient community.

BACKGROUND: Developing new medicines relies on the successful conduct of clinical trials. As trial protocols become more arduous, it becomes harder to recruit and retain patient volunteers, although recent efforts such as OMERACT and I-SPY2 show that partnering with patients can be beneficial. We sought to describe drivers and barriers to trial participation, as well as condition-specific trial preferences. METHODS: An online survey was fielded via the patient-powered research network PatientsLikeMe to 1,621 members living with nine selected chronic health conditions. Questions included demographics, trial experience, reasons for non-participation, questions relating to aspects of trial design, and an adaptation of the Net Promoter Score (NPS) for trial satisfaction. RESULTS: Mean age of respondents was 55 years; most patients were white (93%), female (67%), and living in the United States (72%). Primary conditions were MS (21%), Parkinson's (20%), fibromyalgia (15%), ALS (10%), type 2 diabetes (10%), rheumatoid arthritis (RA, 8%), epilepsy (8%), major depressive disorder (MDD, 5%) and systemic lupus erythematosus (SLE, 3%). Most patients had not discussed a trial with their physician and only 21% had ever enrolled, with rates highest in ALS (36%), Parkinson's disease (36%) and MS (20%) and lowest among SLE (9%), MDD (11%) and Fibromyalgia (11%). Common reasons for non-participation were eligibility criteria, inconvenience of travel and concerns about side effects. NPS suggested that many patients were unsatisfied; patients with lupus, epilepsy, RA, and fibromyalgia reported negative scores, i.e. they would dissuade other patients like them from taking part in trials. The most important considerations in trial participation were the opportunity to improve one's own health and that of others, the reputation of the institution, and having medical bills covered in case of injury. Least important were remuneration and possibility of receiving a placebo. ALS patients were more willing to tolerate undesirable aspects of trials. CONCLUSIONS: Most patients are willing to enroll yet very few are invited. When they do, trial participation is often burdensome, but patients are willing to help improve their design. Researchers should let patients help design better trials to overcome recruitment and retention issues and hasten the development of new medicines.

Arabidopsis double-stranded RNA binding protein DRB3 participates in methylation-mediated defense against geminiviruses.

UNLABELLED: Arabidopsis encodes five double-stranded RNA binding (DRB) proteins. DRB1 and DRB2 are involved in microRNA (miRNA) biogenesis, while DRB4 functions in cytoplasmic posttranscriptional small interfering RNA (siRNA) pathways. DRB3 and DRB5 are not involved in double-stranded RNA (dsRNA) processing but assist in silencing transcripts targeted by DRB2-associated miRNAs. The goal of this study was to determine which, if any, of the DRB proteins might also participate in a nuclear siRNA pathway that leads to geminivirus genome methylation. Here, we demonstrate that DRB3 functions with Dicer-like 3 (DCL3) and Argonaute 4 (AGO4) in methylation-mediated antiviral defense. Plants employ repressive viral genome methylation as an epigenetic defense against geminiviruses, using an RNA-directed DNA methylation (RdDM) pathway similar to that used to suppress endogenous invasive DNAs such as transposons. Chromatin methylation inhibits virus replication and transcription, and methylation-deficient host plants are hypersusceptible to geminivirus infection. Using a panel of drb mutants, we found that drb3 plants uniquely exhibit a similar hypersensitivity and that viral genome methylation is substantially reduced in drb3 compared to wild-type plants. In addition, like dcl3 and ago4 mutants, drb3 plants fail to recover from infection and cannot accomplish the viral genome hypermethylation that is invariably observed in asymptomatic, recovered tissues. Small RNA analysis, bimolecular fluorescence complementation, and coimmunoprecipitation experiments show that DRB3 acts downstream of siRNA biogenesis and suggest that it associates with DCL3 and AGO4 in distinct subnuclear compartments. These studies reveal that in addition to its previously established role in the miRNA pathway, DRB3 also functions in antiviral RdDM. IMPORTANCE: Plants use RNA-directed DNA methylation (RdDM) as an epigenetic defense against geminiviruses. RNA silencing pathways in Arabidopsis include five double-stranded RNA binding proteins (DRBs) related to Drosophila R2D2 and mammalian TRBP and PACT. While DRB proteins have defined roles in miRNA and cytoplasmic siRNA pathways, a role in nuclear RdDM was elusive. Here, we used the geminivirus system to show that DRB3 is involved in methylation-mediated antiviral defense. Beginning with a panel of Arabidopsis drb mutants, we demonstrated that drb3 plants uniquely show enhanced susceptibility to geminiviruses. Further, like dcl3 and ago4 mutants, drb3 plants fail to hypermethylate the viral genome, a requirement for host recovery. We also show that DRB3 physically interacts with the RdDM pathway components DCL3 and AGO4 in the nucleus. This work highlights the utility of geminiviruses as models for de novo RdDM and places DRB3 protein in this fundamental epigenetic pathway.

Suppression of methylation-mediated transcriptional gene silencing by ßC1-SAHH protein interaction during geminivirus-betasatellite infection.

DNA methylation is a fundamental epigenetic modification that regulates gene expression and represses endogenous transposons and invading DNA viruses. As a counter-defense, the geminiviruses encode proteins that inhibit methylation and transcriptional gene silencing (TGS). Some geminiviruses have acquired a betasatellite called DNA ß. This study presents evidence that suppression of methylation-mediated TGS by the sole betasatellite-encoded protein, ßC1, is crucial to the association of Tomato yellow leaf curl China virus (TYLCCNV) with its betasatellite (TYLCCNB). We show that TYLCCNB complements Beet curly top virus (BCTV) L2⁻ mutants deficient for methylation inhibition and TGS suppression, and that cytosine methylation levels in BCTV and TYLCCNV genomes, as well as the host genome, are substantially reduced by TYLCCNB or ßC1 expression. We also demonstrate that while TYLCCNB or ßC1 expression can reverse TGS, TYLCCNV by itself is ineffective. Thus its AC2/AL2 protein, known to have suppression activity in other geminiviruses, is likely a natural mutant in this respect. A yeast two-hybrid screen of candidate proteins, followed by bimolecular fluorescence complementation analysis, revealed that ßC1 interacts with S-adenosyl homocysteine hydrolase (SAHH), a methyl cycle enzyme required for TGS. We further demonstrate that ßC1 protein inhibits SAHH activity in vitro. That ßC1 and other geminivirus proteins target the methyl cycle suggests that limiting its product, S-adenosyl methionine, may be a common viral strategy for methylation interference. We propose that inhibition of methylation and TGS by ßC1 stabilizes geminivirus/betasatellite complexes.

Acacia catechu (L.f.) Willd.: A Review on Bioactive Compounds and Their Health Promoting Functionalities.

With the advent of pandemics and infectious diseases, numerous research activities on natural products have been carried out to combat them. Researchers are investigating natural products for the treatment and/or management of various infectious diseases and/or disorders. Acacia catechu (L.f.) Willd. belongs to the family Fabaceae (subfamily Mimosoideae) known as Khair or Cutch tree, possesses diverse pharmacological actions, and has been widely used in Asia and different parts of the world. The purpose of the present study is to highlight the phytochemical profile of different parts of A. catechu, the different biological activities of A. catechu extract, and the utilization of A. catechu as food and beverage. The present work constitutes a review of A. catechu; we performed searches (books, Google, Google Scholar, and Scopus publications) to compile the work/investigations made on A. catechu to the present. From our survey, it was concluded that the main phytochemicals compounds in A. catechu are protocatechuic acid, taxifolin, epicatechin, epigallocatechin, catechin, epicatechin gallate, procyanidin, phloroglucin, aldobiuronic acid, gallic acid, D-galactose, afzelchin gum, L-arabinose, D-rhamnose, and quercetin. The whole plant of A. catechu possesses a comprehensive variety of medicinal potentials such as antimicrobial, antidiarrheal, antinociceptive, antihyperlipidemic, antiulcer, antioxidant, antidiabetic, antiproliferative, haemolytic, and anti-inflammatory properties due to the presence of bioactive compounds like flavonoids, alkaloids, and tannins. However, even though the plant's metabolites were reported to have many different pharmacological uses, there is limited information about their toxicity or clinical trials. Further research on diverse metabolites of A. catechu should be carried out to ensure the safety or utilization of this plant in the pharma or food industries and in the development of potent plant-based drugs.

RNA silencing directed against geminiviruses: post-transcriptional and epigenetic components.

It is well-established that plants use cytoplasmic, post-transcriptional gene silencing (PTGS) as a defense against RNA viruses and DNA virus transcripts. More recently, it has become clear that small RNA-directed methylation leading to transcriptional gene silencing (TGS) is also used as a defense against DNA virus chromatin. Here we use the DNA-containing geminiviruses as models to discuss what is currently known about both types of antiviral silencing, and viral suppression of PTGS and TGS as a counterdefense.

Regulation of host and virus genes by neuronal miR-138 favours herpes simplex virus 1 latency.

MicroRNA miR-138, which is highly expressed in neurons, represses herpes simplex virus 1 (HSV-1) lytic cycle genes by targeting viral ICP0 messenger RNA, thereby promoting viral latency in mice. We found that overexpressed miR-138 also represses lytic processes independently of ICP0 in murine and human neuronal cells; therefore, we investigated whether miR-138 has targets besides ICP0. Using genome-wide RNA sequencing/photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation followed by short interfering RNA knockdown of candidate targets, we identified the host Oct-1 and Foxc1 messenger mRNAs as miR-138's targets, whose gene products are transcription factors important for HSV-1 replication in neuronal cells. OCT-1 has a known role in the initiation of HSV transcription. Overexpression of FOXC1, which was not known to affect HSV-1, promoted HSV-1 replication in murine neurons and ganglia. CRISPR-Cas9 knockout of FOXC1 reduced viral replication, lytic gene expression and miR-138 repression in murine neuronal cells. FOXC1 also collaborated with ICP0 to decrease heterochromatin on viral genes and compensated for the defect of an ICP0-null virus. In summary, miR-138 targets ICP0, Oct-1 and Foxc1 to repress HSV-1 lytic cycle genes and promote epigenetic gene silencing, which together enable favourable conditions for latent infection.

Viral genome methylation as an epigenetic defense against geminiviruses.

Geminiviruses encapsidate single-stranded DNA genomes that replicate in plant cell nuclei through double-stranded DNA intermediates that associate with cellular histone proteins to form minichromosomes. Like most plant viruses, geminiviruses are targeted by RNA silencing and encode suppressor proteins such as AL2 and L2 to counter this defense. These related proteins can suppress silencing by multiple mechanisms, one of which involves interacting with and inhibiting adenosine kinase (ADK), a cellular enzyme associated with the methyl cycle that generates S-adenosyl-methionine, an essential methyltransferase cofactor. Thus, we hypothesized that the viral genome is targeted by small-RNA-directed methylation. Here, we show that Arabidopsis plants with mutations in genes encoding cytosine or histone H3 lysine 9 (H3K9) methyltransferases, RNA-directed methylation pathway components, or ADK are hypersensitive to geminivirus infection. We also demonstrate that viral DNA and associated histone H3 are methylated in infected plants and that cytosine methylation levels are significantly reduced in viral DNA isolated from methylation-deficient mutants. Finally, we demonstrate that Beet curly top virus L2- mutant DNA present in tissues that have recovered from infection is hypermethylated and that host recovery requires AGO4, a component of the RNA-directed methylation pathway. We propose that plants use chromatin methylation as a defense against DNA viruses, which geminiviruses counter by inhibiting global methylation. In addition, our results establish that geminiviruses can be useful models for genome methylation in plants and suggest that there are redundant pathways leading to cytosine methylation.

CCCTC-Binding Factor Acts as a Heterochromatin Barrier on Herpes Simplex Viral Latent Chromatin and Contributes to Poised Latent Infection.

Herpes simplex virus 1 (HSV-1) establishes latent infection in neurons via a variety of epigenetic mechanisms that silence its genome. The cellular CCCTC-binding factor (CTCF) functions as a mediator of transcriptional control and chromatin organization and has binding sites in the HSV-1 genome. We constructed an HSV-1 deletion mutant that lacked a pair of CTCF-binding sites (CTRL2) within the latency-associated transcript (LAT) coding sequences and found that loss of these CTCF-binding sites did not alter lytic replication or levels of establishment of latent infection, but their deletion reduced the ability of the virus to reactivate from latent infection. We also observed increased heterochromatin modifications on viral chromatin over the LAT promoter and intron. We therefore propose that CTCF binding at the CTRL2 sites acts as a chromatin insulator to keep viral chromatin in a form that is poised for reactivation, a state which we call poised latency.IMPORTANCE Herpes simplex virus 1 (HSV-1) is a human pathogen that persists for the lifetime of the host as a result of its ability to establish latent infection within sensory neurons. The mechanism by which HSV-1 transitions from the lytic to latent infection program is largely unknown; however, HSV-1 is able to coopt cellular silencing mechanisms to facilitate the suppression of lytic gene expression. Here, we demonstrate that the cellular CCCTC-binding factor (CTCF)-binding site within the latency associated transcript (LAT) region is critical for the maintenance of a specific local chromatin structure. Additionally, loss of CTCF binding has detrimental effects on the ability to reactivate from latent infection. These results argue that CTCF plays a critical role in epigenetic regulation of viral gene expression to establish and/or maintain a form of latent infection that can reactivate efficiently.

Viral gene products actively promote latent infection by epigenetic silencing mechanisms.

Many viruses undergo an acute infection in the host organism and then are cleared by the ensuing host immune response, but other viruses establish a persistent infection involving a latent infection or a chronic infection. Latent infection by the herpesviruses or human immunodeficiency virus involves epigenetic silencing of the DNA genome or proviral genome, respectively. Latent infection was previously thought to be a default pathway resulting from infection of a nonpermissive cell, but recent studies have shown that viral gene products can promote epigenetic silencing and latent infection. This review will summarize the viral gene products that have been shown to promote epigenetic silencing of the genomes and their potential for therapeutics to target these viral gene products and disrupt or lock in latent infection.

What Do Ovarian Cancer Patients Expect From Treatment?: Perspectives From an Online Patient Community.

BACKGROUND: Advances in ovarian cancer treatment have improved outcomes. However, the gap between patients' hopes and expectations and their actual outcomes remains an understudied aspect of treatment decision making. This gap has been noted to be a predictor of poorer health-related quality of life. Incorporating patient hopes and expectations for treatment into clinical care may improve patient experiences. OBJECTIVES: The aim of this study is to describe the hopes and expectations of ovarian cancer patients at diagnosis and throughout treatment transitions. METHODS: This study sampled from an online community of patients with ovarian cancer. Thirty members completed an online qualitative survey about their diagnostic and treatment journey, including hopes and expectations around treatment. RESULTS: Initially, ovarian cancer patients hoped for a complete cure or removal of cancer. As they progressed through treatment, hopes and expectations centered on issues related to living with ovarian cancer. A subset of patients emphasized a lack of information about treatment side effects. CONCLUSIONS: The shift in expectations from survival to living with ovarian cancer may demonstrate an enhanced understanding of ovarian cancer prognosis as patients learn more about their condition. Patients underscored that a more involved discussion of side effect profiles with clinicians would enhance their treatment decision making and expectation setting process. IMPLICATIONS FOR PRACTICE: Expectation setting may be improved by contextualizing treatment in terms of the transitions ovarian cancer patients experience. Providing relevant information that aligns with patient needs, desires, and concerns at critical transitions may improve clinical care and decision making.

Herpesviral ICP0 Protein Promotes Two Waves of Heterochromatin Removal on an Early Viral Promoter during Lytic Infection.

UNLABELLED: Herpesviruses must contend with host cell epigenetic silencing responses acting on their genomes upon entry into the host cell nucleus. In this study, we confirmed that unchromatinized herpes simplex virus 1 (HSV-1) genomes enter primary human foreskin fibroblasts and are rapidly subjected to assembly of nucleosomes and association with repressive heterochromatin modifications such as histone 3 (H3) lysine 9-trimethylation (H3K9me3) and lysine 27-trimethylation (H3K27me3) during the first 1 to 2 h postinfection. Kinetic analysis of the modulation of nucleosomes and heterochromatin modifications over the course of lytic infection demonstrates a progressive removal that coincided with initiation of viral gene expression. We obtained evidence for three phases of heterochromatin removal from an early gene promoter: an initial removal of histones and heterochromatin not dependent on ICP0, a second ICP0-dependent round of removal of H3K9me3 that is independent of viral DNA synthesis, and a third phase of H3K27me3 removal that is dependent on ICP0 and viral DNA synthesis. The presence of ICP0 in transfected cells is also sufficient to promote removal of histones and H3K9me3 modifications of cotransfected genes. Overall, these results show that ICP0 promotes histone removal, a reduction of H3K9me3 modifications, and a later indirect reduction of H3K27me3 modifications following viral early gene expression and DNA synthesis. Therefore, HSV ICP0 promotes the reversal of host epigenetic silencing mechanisms by several mechanisms. IMPORTANCE: The human pathogen herpes simplex virus (HSV) has evolved multiple strategies to counteract host-mediated epigenetic silencing during productive infection. However, the mechanisms by which viral and cellular effectors contribute to these processes are not well defined. The results from this study demonstrate that HSV counteracts host epigenetic repression in a dynamic stepwise process to remove histone 3 (H3) and subsequently target specific heterochromatin modifications in two distinct waves. This provides the first evidence of a stepwise reversal of host epigenetic silencing by viral proteins. This work also suggests that targets capable of disrupting the kinetics of epigenetic regulation could serve as potential antiviral therapeutic agents.

A Herpesviral Lytic Protein Regulates the Structure of Latent Viral Chromatin.

UNLABELLED: Latent infections by viruses usually involve minimizing viral protein expression so that the host immune system cannot recognize the infected cell through the viral peptides presented on its cell surface. Herpes simplex virus (HSV), for example, is thought to express noncoding RNAs such as latency-associated transcripts (LATs) and microRNAs (miRNAs) as the only abundant viral gene products during latent infection. Here we describe analysis of HSV-1 mutant viruses, providing strong genetic evidence that HSV-infected cell protein 0 (ICP0) is expressed during establishment and/or maintenance of latent infection in murine sensory neurons in vivo Studies of an ICP0 nonsense mutant virus showed that ICP0 promotes heterochromatin and latent and lytic transcription, arguing that ICP0 is expressed and functional. We propose that ICP0 promotes transcription of LATs during establishment or maintenance of HSV latent infection, much as it promotes lytic gene transcription. This report introduces the new concept that a lytic viral protein can be expressed during latent infection and can serve dual roles to regulate viral chromatin to optimize latent infection in addition to its role in epigenetic regulation during lytic infection. An additional implication of the results is that ICP0 might serve as a target for an antiviral therapeutic acting on lytic and latent infections. IMPORTANCE: Latent infection by viruses usually involves minimizing viral protein synthesis so that the host immune system cannot recognize the infected cells and eliminate them. Herpes simplex virus has been thought to express only noncoding RNAs as abundant gene products during latency. In this study, we found genetic evidence that an HSV lytic protein is functional during latent infection, and this protein may provide a new target for antivirals that target both lytic and latent infections.

History and genomic sequence analysis of the herpes simplex virus 1 KOS and KOS1.1 sub-strains.

A collection of genomic DNA sequences of herpes simplex virus (HSV) strains has been defined and analyzed, and some information is available about genomic stability upon limited passage of viruses in culture. The nature of genomic change upon extensive laboratory passage remains to be determined. In this report we review the history of the HSV-1 KOS laboratory strain and the related KOS1.1 laboratory sub-strain, also called KOS (M), and determine the complete genomic sequence of an early passage stock of the KOS laboratory sub-strain and a laboratory stock of the KOS1.1 sub-strain. The genomes of the two sub-strains are highly similar with only five coding changes, 20 non-coding changes, and about twenty non-ORF sequence changes. The coding changes could potentially explain the KOS1.1 phenotypic properties of increased replication at high temperature and reduced neuroinvasiveness. The study also provides sequence markers to define the provenance of specific laboratory KOS virus stocks.

Inhibition of LSD1 reduces herpesvirus infection, shedding, and recurrence by promoting epigenetic suppression of viral genomes.

Herpesviruses are highly prevalent and maintain lifelong latent reservoirs, thus posing challenges to the control of herpetic disease despite the availability of antiviral pharmaceuticals that target viral DNA replication. The initiation of herpes simplex virus infection and reactivation from latency is dependent on a transcriptional coactivator complex that contains two required histone demethylases, LSD1 (lysine-specific demethylase 1) and a member of the JMJD2 family (Jumonji C domain-containing protein 2). Inhibition of either of these enzymes results in heterochromatic suppression of the viral genome and blocks infection and reactivation in vitro. We demonstrate that viral infection can be epigenetically suppressed in three animal models of herpes simplex virus infection and disease. Treating animals with the monoamine oxidase inhibitor tranylcypromine to inhibit LSD1 suppressed viral lytic infection, subclinical shedding, and reactivation from latency in vivo. This phenotypic suppression was correlated with enhanced epigenetic suppression of the viral genome and suggests that, even during latency, the chromatin state of the virus is dynamic. Therefore, epi-pharmaceuticals may represent a promising approach to treat herpetic diseases.

Setting the stage for Cancer Advocacy in Africa: how?

BACKGROUND: Oftentimes, cancer advocates in Africa look at the developed nations in North America and Europe for guidance on cancer advocacy. However, lessons learnt from developed nations do not necessarily apply to the situational context of Africa. Without a doubt, successful cancer advocates in Africa can best serve as learning sources and role models for advocacy in Africa. This paper describes the results of an environmental scan of advocacy organizations in Africa. METHODS: A cross-sectional study design was employed for this project. Using a structured survey data collection form, participants submitted their responses either by online submission (Google docs) or by electronic mail to admin@aortic-africa.org. RESULTS: A total of 39 African advocates representing 17 countries participated in the project. The majority of participants have been advocates for more than five years; and mostly advocate for both males and females and individuals between the ages of 30 and 39. The most common cancers focused on by the advocacy organizations include breast, prostate, liver, cervix, stomach, bladder, pediatric, colorectal and neck. The information provided by participants offer clear guidelines on establishing and maintaining an advocacy program in Africa despite the various challenges faced by these organizations. CONCLUSION: Whilst this paper only highlights a subset of advocacy initiatives on the Continent, there is an opportunity for a more inclusive dialogue for advocates to share ideas with each other, connect with other advocates, learn about other innovative advocacy programs, and join the global war against cancer. To this end, the biennial International Workshop on Cancer Advocacy for African Countries (CAAC) during the next AORTIC International Cancer conference, offers an opportunity to further Africa's cancer advocacy initiatives.