The cardiac-restricted protein ADP-ribosylhydrolase-like 1 is essential for heart chamber outgrowth and acts on muscle actin filament assembly

AbstractAdprhl1, a member of the ADP-ribosylhydrolase protein family, is expressed exclusively in the developing heart of all vertebrates. In the amphibian Xenopus laevis, distribution of its mRNA is biased towards actively growing chamber myocardium. Morpholino oligonucleotide-mediated knockdown of all Adprhl1 variants inhibits striated myofibril assembly and prevents outgrowth of the ventricle. The resulting ventricles retain normal electrical conduction and express markers of chamber muscle differentiation but are functionally inert. Using a cardiac-specific Gal4 binary expression system, we show that the abundance of Adprhl1 protein in tadpole hearts is tightly controlled through a negative regulatory mechanism targeting the 5′-coding sequence of Xenopus adprhl1. Over-expression of full length (40kDa) Adprhl1 variants modified to escape such repression, also disrupts cardiac myofibrillogenesis. Disarrayed myofibrils persist that show extensive branching, with sarcomere division occurring at the actin-Z-disc boundary. Ultimately, Adprhl1-positive cells contain thin actin threads, connected to numerous circular branch points. Recombinant Adprhl1 can localize to stripes adjacent to the Z-disc, suggesting a direct role for Adprhl1 in modifying Z-disc and actin dynamics as heart chambers grow. Modelling the structure of Adprhl1 suggests this cardiac-specific protein is a pseudoenzyme, lacking key residues necessary for ADP-ribosylhydrolase catalytic activity

Human papillomavirus type 16 causes a defined subset of conjunctival in situ squamous cell carcinomas

Funder: CUH | Addenbrooke's Charitable Trust, Cambridge University Hospitals (Addenbrooke's Charitable Trust, Cambridge University Hospitals NHS Foundation Trust); doi: https://doi.org/10.13039/501100002927Abstract: Squamous cell carcinoma of the conjunctiva is associated with a number of risk factors, including HIV infection, iatrogenic immunosuppression and atopy. In addition, several studies have suggested an involvement of HPV, based on the presence of viral DNA, but did not establish whether there was active infection or evidence of causal disease association. In this manuscript, 31 cases of conjunctival in situ squamous cell carcinoma were classified as HPV DNA-positive or -negative, before being analysed by immunohistochemistry to establish the distribution of viral and cellular biomarkers of HPV gene expression. Our panel included p16INK4a, TP53 and MCM, but also the virally encoded E4 gene product, which is abundantly expressed during productive infection. Subsequent in situ detection of HPV mRNA using an RNAscope approach confirmed that early HPV gene expression was occurring in the majority of cases of HPV DNA-positive conjunctival in situ squamous cell carcinoma, with all of these cases occurring in the atopic group. Viral gene expression correlated with TP53 loss, p16INK4a elevation, and extensive MCM expression, in line with our general understanding of E6 and E7’s role during transforming infection at other epithelial sites. A characteristic E4 expression pattern was detected in only one case. HPV mRNA was not detected in lower grades of dysplasia, and was not observed in cases that were HPV DNA-negative. Our study demonstrates an active involvement of HPV in the development of a subset of conjunctival in situ squamous cell carcinoma. No high-risk HPV types were detected other than HPV16. It appears that the conjunctiva is a vulnerable epithelial site for HPV-associated transformation. These cancers are defined by their pattern of viral gene expression, and by the distribution of surrogate markers of HPV infection

Fgf3 and Fgf8 are required together for formation of the otic placode and vesicle

Fgf3 has long been implicated in otic placode induction and early development of the otocyst; however, the results of experiments in mouse and chick embryos to determine its function have proved to be conflicting. In this study, we determined fgf3 expression in relation to otic development in the zebrafish and used antisense morpholino oligonucleotides to inhibit Fgf3 translation. Successful knockdown of Fgf3 protein was demonstrated and this resulted in a reduction of otocyst size together with reduction in expression of early markers of the otic placode.fgf3 is co-expressed with fgf8 in the hindbrain prior to otic induction and, strikingly, when Fgf3 morpholinos were co-injected together with Fgf8 morpholinos, a significant number of embryos failed to form otocysts. These effects were made manifest at early stages of otic development by an absence of early placode markers (pax2.1 and dlx3) but were not accompanied by effects on cell division or death. The temporal requirement for Fgf signalling was established as being between 60% epiboly and tailbud stages using the Fgf receptor inhibitor SU5402. However, the earliest molecular event in induction of the otic territory, pax8 expression, did not require Fgf signalling, indicating an inductive event upstream of signalling by Fgf3 and Fgf8. We propose that Fgf3 and Fgf8 are required together for formation of the otic placode and act during the earliest stages of its induction

Viral genome amplification during the productive life cycle occurs in cell co-expressing both 16E1^E4 and activated cytoplasmic JNK.

<p><b>(A)</b> Raft sections derived from NIKS cells harboring HPV16 WT or E4KO genomes were analysed at day 14 post-differentiation by quadruple staining for E1^E4 (green), p-JNK (red), nuclear DNA (magenta; DAPI, colour modified using imageJ) and HPV16 genomic DNA (blue, <i>in situ</i> hybridization, colour modified using imageJ). Efficient viral genome amplification is confined to the staining for cytoplasmic E4 and cytoplasmic phosphor-JNK-positive upper epithelial layers of WT rafts. In the E4KO rafts a lower level of genome amplification can occur in cells that are negative for cytoplasmic phosphor-JNK. Such cells are typically in the upper p-JNK-staining layers and typically show only nuclear p-JNK staining. The dotted lines indicate the position of the basal layer. Images were captured using a 10x objective. <b>(B)</b> Enlarged areas of the regions boxed in (A) to show the E4/p-JNK patterns in cells known to be supporting HPV16 genome amplification.</p

E1^E4 expression is required for the maintenance of activated pJNK in the cytoplasm during the late stages of the HPV16 life cycle.

<p><b>(A)</b> Raft tissues derived from NIKS cells or NIKS cells harboring HPV16 WT or E4KO genomes were analyzed at day 14 post-differentiation after staining for 16E1^E4 (green), phospho-JNK (p-JNK, red) and DNA (blue; DAPI). Although elevated cytoplasmic JNK MAPK was apparent before 16E1^E4 became abundant, its activity was noticeably elevated and persisted in cells expressing 16E4. Cytoplasmic p-JNK was not apparent in rafts prepared using ‘empty’ NIKS cells. The boxed areas are enlarged in the right panel. The dotted lines indicate the position of the basal layer. Images were captured using a 10x objective. <b>(B)</b> Tissue sections from HPV16-induced cervical lesions or normal cervix were stained for 16E1^E4 (green), p-JNK (red) and DNA (blue; DAPI). The pattern of p-JNK seen in HPV16-induced cervical lesions and in normal cervical tissue was broadly similar to that seen in NIKS-HPV16 rafts and ‘empty’ NIKS rafts respectively. The boxed areas are enlarged in the lower panel. The dotted lines indicate the position of the basal layer. Images were captured using a 10x objective. <b>(C)</b> Raft tissues derived from NIKS cells or NIKS cells harbouring the HPV18 WT or E4KO genomes were analysed at day 14 post-differentiation. In contrast to the situation seen with HPV16 (see <b>(A)</b> above), no obvious E4-mediated cytoplasmic p-JNK association was apparent. <b>(D, E)</b> p-JNK staining in rafts produced from NIKS cells containing either HPV45 (D) or HPV31 (E) show an absence of E4-mediated p-JNK sequestration.</p

Primer and Probe.

<p>Primer and Probe.</p

The G2 arrest function of HPV16 E1^E4 contributes to viral genome amplification and L1 expression.

<p><b>(A)</b> Mitotic index of Cos-7 cells expressing either the HPV16 WT E1^E4 protein, the E1^E4 PIIP mutant that is defective in G2 arrest function, or a control protein (GFP) that does not inhibit cell cycle progression. The E1^E4 PIIP mutant has lost its ability to prevent mitotic entry when compared to WT 16 E1^E4. <b>(B)</b> HPV16 WT or PIIP mutant genome-containing NIKS were suspended in 1.5% methylcellulose (MC) in order to induce differentiation, and were harvested at 0h, 24h, 48h and 72h. Genomic DNA copy numbers were measured by qPCR. The results are presented as fold-increase in HPV16 genome copy number relative to the zero hour time point. A reduction in genome amplification success was noticeable at all time points. <b>(C & D)</b> Viral genome amplification and L1 expression were examined at day 14 post-differentiation by fluorescence <i>in situ</i> hybridization (FISH signal shown in red in C) and indirect immunofluorescence (L1 staining shown in red in D) in raft tissues derived from NIKS cells harboring either the HPV16 WT or E4KO genomes. Viral genome amplification and L1 expression were compromised in the PIIP mutant. The dotted lines indicate the position of the basal layer. Images were captured using a 10x objective.</p

16E1^E4 expression drives nuclear accumulation of wild type but not mutant 16E1 and enhances E1/E2-mediated HPV16 ori-dependent replication.

<p><b>(A)</b> Sequence alignment showing the location of conserved MAPK phosphorylation sites involved in regulating E1 nuclear/cytoplasmic shuttling in HPV11, 31 and 16. Numbers in parentheses indicate the position of the first amino acid in each sequence. The location of the bipartite nuclear localization signal (NLS) is indicated by a double-arrow and highlighted in yellow. The nuclear export signal (NES) is shaded in blue. Aligned MAPK- and CDK-phosphorylation sites [S/T]-P, are colored in pink. <b>(B, C)</b> SiHa-tetON/CSII-TRE-tight-HA16E1 or CSII-TRE-tight-HA16E1(S93A, S107A cells were infected for 48 hours with rAd16E1^E4 or rAdβ-Gal in the presence of 1 μg/ml of Doxycycline. Cells were stained for 16E1^E4 (green), 16E1 (red; HA-tag) and DNA (blue; DAPI) to show the increased nuclear distribution of wild type E1 but not the phosphorylation-deficient E1 mutant (S93A, S107A) following co-expression with E4. <b>(D)</b> The number of cells showing E1-nuclear localization was quantified, and is shown as a percentage of the total number of E1-positive cells counted amongst the E4-positive and E4-negative cells. Wild type E1, but not the phosphorylation-deficient mutant, was enhanced in the presence of E4. The standard deviations of three independent sets of 1000 E1-positive cells are indicated by the error bars. <b>(E)</b> To assess replication competence, plasmids expressing 16E1/E2/E4 or 16E1/E2 were transfected along with a HPV 16 replication origin-containing plasmid (p16Ori) as described in the Materials and Methods (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006282#ppat.1006282.s006" target="_blank">S6 Fig</a>). Amplified p16Ori plasmid was analysed by Southern blotting using extracted total DNA (upper panel). The levels of p16Ori input are shown in the lower panel. <b>(F)</b> Columns show quantitation of the 72h Southern blot signal averaged across triplicate experiments. Data is shown as ‘fold’ change when compared to the level of replication seen in the absence of E4. <b>(G)</b> As in <b>(F)</b> above, columns show the change in replication seen in the presence or absence of E4, but in this case, a luciferase reporter plasmid system was used (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006282#sec002" target="_blank">materials and methods</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006282#ppat.1006282.s006" target="_blank">S6 Fig</a>). Results were generated from the average of six independent experiments.</p

16 E1^E4 contributes to p38 MAPK and ERK1/2 activity during the HPV16 life cycle.

<p><b>(A)</b> Raft tissues from NIKS containing HPV16 WT or E4KO genomes were harvested at day 14 post-differentiation and stained for 16E1^E4 (green), phospho-p38 MAPK (p-p38 MAPK) (red) and DNA (blue; DAPI). An elevation of p-p38 MAPK staining in the upper layers of the raft is apparent in rafts generated using the WT HPV16 genome. The dotted lines indicate the position of the basal layer. Images were captured using a 10x objective. <b>(B)</b> The extent and intensity of p-p38MAPK staining in the HPV16 WT and E4KO raft tissues at the 14 day time-point post differentiation was digitally scanned from the basal layer to the top of the raft tissue as described in the materials and methods. The expression of E4 in the WT raft is shown as the grey shadow. A very different level of p-p38 MAPK activity was apparent in the upper epithelial layers of rafts prepared using the WT HPV16 genome. <b>(C)</b> Raft sections from HPV16 WT or E4KO harvested at 8, 10, 12 or 14 days respectively post-differentiation were stained for 16E1^E4 (green), p-ERK1/2 (red) and DNA (blue; DAPI), to reveal differences in the levels of ERK1/2 activity in the mid epithelial layers. The dotted lines indicate the position of the basal layer. Images were captured using a 10x objective. <b>(D)</b> The extent and intensity of p-ERK1/2 staining in NIKS rafts harboring HPV16 WT or E4KO raft tissues at day 14 was examined by digitally scanning the raft tissue from the basal layer to the top of the raft as described in materials and methods. The distribution and intensity of E1^E4 staining in the WT raft is shown as a grey shadow. Comparison with data shown in <b>(B)</b> reveals 16E1^E4’s effect on ERK1/2 to be largely confined to the mid epithelial layers, which is distinct from the E4-mediated effect on p38 MAPK which is prominent in the upper epithelial layers.</p