Transcriptomic Evidence That Longevity of Acquired Plastids in the Photosynthetic Slugs Elysia timida and Plakobranchus ocellatus Does Not Entail Lateral Transfer of Algal Nuclear Genes

Sacoglossan sea slugs are unique in the animal kingdom in that they sequester and maintain active plastids that they acquire from the siphonaceous algae upon which they feed, making the animals photosynthetic. Although most sacoglossan species digest their freshly ingested plastids within hours, four species from the family Plakobranchidae retain their stolen plastids (kleptoplasts) in a photosynthetically active state on timescales of weeks to months. The molecular basis of plastid maintenance within the cytosol of digestive gland cells in these photosynthetic metazoans is yet unknown but is widely thought to involve gene transfer from the algal food source to the slugs based upon previous investigations of single genes. Indeed, normal plastid development requires hundreds of nuclear-encoded proteins, with protein turnover in photosystem II in particular known to be rapid under various conditions. Moreover, only algal plastids, not the algal nuclei, are sequestered by the animals during feeding. If algal nuclear genes are transferred to the animal either during feeding or in the germ line, and if they are expressed, then they should be readily detectable with deep-sequencing methods. We have sequenced expressed mRNAs from actively photosynthesizing, starved individuals of two photosynthetic sea slug species, Plakobranchus ocellatus Van Hasselt, 1824 and Elysia timida Risso, 1818. We find that nuclear-encoded, algal-derived genes specific to photosynthetic function are expressed neither in P. ocellatus nor in E. timida. Despite their dramatic plastid longevity, these photosynthetic sacoglossan slugs do not express genes acquired from algal nuclei in order to maintain plastid function

Mass Spectrometric Back Reaction Screening of Quasi-Enantiomeric Products as a Mechanistic Tool

In this account, we discuss a mass spectrometric method that enables unambiguous identification of intermediates involved in the enantioselective step of a catalytic cycle. This method, which we originally developed for rapid evaluation of chiral catalysts, is based on monitoring the back reaction of mass-labeled quasi-enantiomeric products by ESI-MS. In this way, the intrinsic enantioselectivity of a chiral catalyst can be determined directly by quantification of catalytically relevant intermediates. By comparing the results from the forward and back reaction, direct evidence for the involvement of a catalytic intermediate in the enantioselective step can be obtained. In addition, insights about the energy profile of the catalytic cycle may be gained. The potential of back reaction screening as a mechanistic tool is demonstrated for organocatalytic aldol reactions, 1,4-additions of aldehydes to nitroolefins, Diels-Alder reactions, Michael additions, and Morita-Baylis-Hillman reactions

Prognostic implications of p16 and HPV discordance in oropharyngeal cancer (HNCIG-EPIC-OPC): a multicentre, multinational, individual patient data analysis

Background p16(INK4a) (p16) immunohistochemistry is the most widely used biomarker assay for inferring HPV causation in oropharyngeal cancer in clinical and trial settings. However, discordance exists between p16 and HPV DNA or RNA status in some patients with oropharyngeal cancer. We aimed to clearly quantify the extent of discordance, and its prognostic implications. Methods In this multicentre, multinational individual patient data analysis, we did a literature search in PubMed and Cochrane database for systematic reviews and original studies published in English between Jan 1, 1970, and Sept 30, 2022. We included retrospective series and prospective cohorts of consecutively recruited patients previously analysed in individual studies with minimum cohort size of 100 patients with primary squamous cell carcinoma of the oropharynx. Patient inclusion criteria were diagnosis with a primary squamous cell carcinoma of oropharyngeal cancer; data on p16 immunohistochemistry and on HPV testing; information on age, sex, tobacco, and alcohol use; staging by TNM 7th edition; information on treatments received; and data on clinical outcomes and follow-up (date of last follow-up if alive, date of recurrence or metastasis, and date and cause of death). There were no limits on age or performance status. The primary outcomes were the proportion of patients of the overall cohort who showed the different p16 and HPV result combinations, as well as 5-year overall survival and 5-year disease-free survival. Patients with recurrent or metastatic disease or who were treated palliatively were excluded from overall survival and disease-free survival analyses. Multivariable analysis models were used to calculate adjusted hazard ratios (aHR) for different p16 and HPV testing methods for overall survival, adjusted for prespecified confounding factors. Findings Our search returned 13 eligible studies that provided individual data for 13 cohorts of patients with oropharyngeal cancer from the UK, Canada, Denmark, Sweden, France, Germany, the Netherlands, Switzerland, and Spain. 7895 patients with oropharyngeal cancer were assessed for eligibility. 241 were excluded before analysis, and 7654 were eligible for p16 and HPV analysis. 5714 (74middot7%) of 7654 patients were male and 1940 (25middot3%) were female. Ethnicity data were not reported. 3805 patients were p16-positive, 415 (10middot9%) of whom were HPV-negative. This proportion differed significantly by geographical region and was highest in the areas with lowest HPV-attributable fractions (r=-0middot744, p=0middot0035). The proportion of patients with p16+/HPV- oropharyngeal cancer was highest in subsites outside the tonsil and base of tongue (29middot7% vs 9middot0%, p<0middot0001). 5-year overall survival was 81middot1% (95% CI 79middot5-82middot7) for p16+/HPV+, 40middot4% (38middot6-42middot4) for p16-/HPV-, 53middot2% (46middot6-60middot8) for p16-/HPV+, and 54middot7% (49middot2-60middot9) for p16+/HPV-. 5-year disease-free survival was 84middot3% (95% CI 82middot9-85middot7) for p16+/HPV+, 60middot8% (58middot8-62middot9) for p16-/HPV-; 71middot1% (64middot7-78middot2) for p16-/HPV+, and 67middot9% (62middot5-73middot7) for p16+/HPV-. Results were similar across all European sub-regions, but there were insufficient numbers of discordant patients from North America to draw conclusions in this cohort. Interpretation Patients with discordant oropharyngeal cancer (p16-/HPV+ or p16+/HPV-) had a significantly worse prognosis than patients with p16+/HPV+ oropharyngeal cancer, and a significantly better prognosis than patients with p16-/HPV- oropharyngeal cancer. Along with routine p16 immunohistochemistry, HPV testing should be mandated for clinical trials for all patients (or at least following a positive p16 test), and is recommended where HPV status might influence patient care, especially in areas with low HPV-attributable fractions. Copyright (c) 2023 The Author(s). Published by Elsevier Ltd

Prognostic implications of p16 and HPV discordance in oropharyngeal cancer (HNCIG-EPIC-OPC): a multicentre, multinational, individual patient data analysis

Background: p16INK4a (p16) immunohistochemistry is the most widely used biomarker assay for inferring HPV causation in oropharyngeal cancer in clinical and trial settings. However, discordance exists between p16 and HPV DNA or RNA status in some patients with oropharyngeal cancer. We aimed to clearly quantify the extent of discordance, and its prognostic implications. Methods: In this multicentre, multinational individual patient data analysis, we did a literature search in PubMed and Cochrane database for systematic reviews and original studies published in English between Jan 1, 1970, and Sept 30, 2022. We included retrospective series and prospective cohorts of consecutively recruited patients previously analysed in individual studies with minimum cohort size of 100 patients with primary squamous cell carcinoma of the oropharynx. Patient inclusion criteria were diagnosis with a primary squamous cell carcinoma of oropharyngeal cancer; data on p16 immunohistochemistry and on HPV testing; information on age, sex, tobacco, and alcohol use; staging by TNM 7th edition; information on treatments received; and data on clinical outcomes and follow-up (date of last follow-up if alive, date of recurrence or metastasis, and date and cause of death). There were no limits on age or performance status. The primary outcomes were the proportion of patients of the overall cohort who showed the different p16 and HPV result combinations, as well as 5-year overall survival and 5-year disease-free survival. Patients with recurrent or metastatic disease or who were treated palliatively were excluded from overall survival and disease-free survival analyses. Multivariable analysis models were used to calculate adjusted hazard ratios (aHR) for different p16 and HPV testing methods for overall survival, adjusted for prespecified confounding factors. Findings: Our search returned 13 eligible studies that provided individual data for 13 cohorts of patients with oropharyngeal cancer from the UK, Canada, Denmark, Sweden, France, Germany, the Netherlands, Switzerland, and Spain. 7895 patients with oropharyngeal cancer were assessed for eligibility. 241 were excluded before analysis, and 7654 were eligible for p16 and HPV analysis. 5714 (74·7%) of 7654 patients were male and 1940 (25·3%) were female. Ethnicity data were not reported. 3805 patients were p16-positive, 415 (10·9%) of whom were HPV-negative. This proportion differed significantly by geographical region and was highest in the areas with lowest HPV-attributable fractions (r=–0·744, p=0·0035). The proportion of patients with p16+/HPV– oropharyngeal cancer was highest in subsites outside the tonsil and base of tongue (29·7% vs 9·0%, p<0·0001). 5-year overall survival was 81·1% (95% CI 79·5–82·7) for p16+/HPV+, 40·4% (38·6–42·4) for p16–/HPV–, 53·2% (46·6–60·8) for p16–/HPV+, and 54·7% (49·2–60·9) for p16+/HPV–. 5-year disease-free survival was 84·3% (95% CI 82·9–85·7) for p16+/HPV+, 60·8% (58·8–62·9) for p16–/HPV–; 71·1% (64·7–78·2) for p16–/HPV+, and 67·9% (62·5–73·7) for p16+/HPV–. Results were similar across all European sub-regions, but there were insufficient numbers of discordant patients from North America to draw conclusions in this cohort. Interpretation: Patients with discordant oropharyngeal cancer (p16–/HPV+ or p16+/HPV–) had a significantly worse prognosis than patients with p16+/HPV+ oropharyngeal cancer, and a significantly better prognosis than patients with p16–/HPV– oropharyngeal cancer. Along with routine p16 immunohistochemistry, HPV testing should be mandated for clinical trials for all patients (or at least following a positive p16 test), and is recommended where HPV status might influence patient care, especially in areas with low HPV-attributable fractions

Co-expression patterns of cancer associated fibroblast markers reveal distinct subgroups related to patient survival in oropharyngeal squamous cell carcinoma

Background: The incidence of oropharyngeal squamous cell carcinoma (OPSCC) is rapidly increasing in high income countries due to its association with persistent high-risk human papilloma virus (HPV) infection. Recent scientific advances have highlighted the importance of the tumor microenvironment in OPSCC. In this study, including 216 OPSCC patients, we analyze the composition of four established markers of cancer associated fibroblasts (CAFs) in the context of intratumoral CD8 T-cell infiltration.Methods: Immunohistochemical staining for fibroblast activation protein (FAP), platelet-derived growth factor receptor beta (PDGFRb), periostin, alpha smooth muscle actin (α-SMA) and CD8 were analyzed digitally and their association with survival, tumor- and patient characteristics was assessed.Results: Co-expression of CAF markers was frequent but not associated with HPV status. FAPhigh and PDGFRbhigh expression were associated with increased CD8 T-cell infiltration. Low expression of PDGFRb improved patient survival in female patients but not in male patients. We identified PDGFRblow periostinlow α-SMAlow status as an independent predictor of improved survival (hazard ratio 0.377, p = 0.006).Conclusion: These findings elucidate the co-expression of four established CAF markers in OPSCC and underscore their association with T-cell infiltration and patient survival. Future analyses of CAF subgroups in OPSCC may enable the development of individualized therapies

Prebiotically plausible mechanisms increase compositional diversity of nucleic acid sequences

During the origin of life, the biological information of nucleic acid polymers must have increased to encode functional molecules (the RNA world). Ribozymes tend to be compositionally unbiased, as is the vast majority of possible sequence space. However, ribonucleotides vary greatly in synthetic yield, reactivity and degradation rate, and their non-enzymatic polymerization results in compositionally biased sequences. While natural selection could lead to complex sequences, molecules with some activity are required to begin this process. Was the emergence of compositionally diverse sequences a matter of chance, or could prebiotically plausible reactions counter chemical biases to increase the probability of finding a ribozyme? Our in silico simulations using a two-letter alphabet show that template-directed ligation and high concatenation rates counter compositional bias and shift the pool toward longer sequences, permitting greater exploration of sequence space and stable folding. We verified experimentally that unbiased DNA sequences are more efficient templates for ligation, thus increasing the compositional diversity of the pool. Our work suggests that prebiotically plausible chemical mechanisms of nucleic acid polymerization and ligation could predispose toward a diverse pool of longer, potentially structured molecules. Such mechanisms could have set the stage for the appearance of functional activity very early in the emergence of life

ANO1 amplification and expression in HNSCC with a high propensity for future distant metastasis and its functions in HNSCC cell lines

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) is associated with poor survival. To identify prognostic and diagnostic markers and therapeutic targets, we studied ANO1, a recently identified calcium-activated chloride channel (CaCC). METHODS: High-resolution genomic and transcriptomic microarray analysis and functional studies using HNSCC cell line and CaCC inhibitors. RESULTS: Amplification and overexpression of genes within the 11q13 amplicon are associated with the propensity for future distance metastasis of HPV-negative HNSCC. ANO1 was selected for functional studies based on high correlations, cell surface expression and CaCC activity. ANO1 overexpression in cells that express low endogenous levels stimulates cell movement, whereas downregulation in cells with high endogenous levels has the opposite effect. ANO1 overexpression also stimulates attachment, spreading, detachment and invasion, which could account for its effects on migration. CaCC inhibitors decrease movement, suggesting that channel activity is required for the effects of ANO1. In contrast, ANO1 overexpression does not affect cell proliferation. INTERPRETATION: ANO1 amplification and expression could be markers for distant metastasis in HNSCC. ANO1 overexpression affects cell properties linked to metastasis. Inhibitors of CaCCs could be used to inhibit the tumourigenic properties of ANO1, whereas activators developed to increase CaCC activity could have adverse effects

Epithelial maturation and molecular biology of oral HPV

Human papillomavirus (HPV) is widespread and can cause latent infection in basal cells, with low HPV DNA copy-number insufficient for transmission of infection; can cause subclinical infection that is active but without clinical signs; or can cause clinical infection leading to benign, potentially malignant or malignant lesions. The HPV cycle is influenced by the stage of maturation of the infected keratinocytes, and the production of virions is restricted to the post-mitotic suprabasal epithelial cells where all the virus genes are expressed

Aptamers for pharmaceuticals and their application in environmental analytics

Aptamers are single-stranded DNA or RNA oligonucleotides, which are able to bind with high affinity and specificity to their target. This property is used for a multitude of applications, for instance as molecular recognition elements in biosensors and other assays. Biosensor application of aptamers offers the possibility for fast and easy detection of environmental relevant substances. Pharmaceutical residues, deriving from human or animal medical treatment, are found in surface, ground, and drinking water. At least the whole range of frequently administered drugs can be detected in noticeable concentrations. Biosensors and assays based on aptamers as specific recognition elements are very convenient for this application because aptamer development is possible for toxic targets. Commonly used biological receptors for biosensors like enzymes or antibodies are mostly unavailable for the detection of pharmaceuticals. This review describes the research activities of aptamer and sensor developments for pharmaceutical detection, with focus on environmental applications