Building a complex for destruction.

Ranes et al. (2021) report on an in vitro reconstituted ß-catenin destruction complex and elucidate the contributions of full-length and cancer-related mutated core components to ß-catenin turnover, thereby advancing our understanding of the inner workings of this tumor suppressor complex.

AXIN1 bi-allelic variants disrupting the C-terminal DIX domain cause craniometadiaphyseal osteosclerosis with hip dysplasia.

Sclerosing skeletal dysplasias result from an imbalance between bone formation and resorption. We identified three homozygous, C-terminally truncating AXIN1 variants in seven individuals from four families affected by macrocephaly, cranial hyperostosis, and vertebral endplate sclerosis. Other frequent findings included hip dysplasia, heart malformations, variable developmental delay, and hematological anomalies. In line with AXIN1 being a central component of the ß-catenin destruction complex, analyses of primary and genome-edited cells harboring the truncating variants revealed enhanced basal canonical Wnt pathway activity. All three AXIN1-truncating variants resulted in reduced protein levels and impaired AXIN1 polymerization mediated by its C-terminal DIX domain but partially retained Wnt-inhibitory function upon overexpression. Addition of a tankyrase inhibitor attenuated Wnt overactivity in the AXIN1-mutant model systems. Our data suggest that AXIN1 coordinates the action of osteoblasts and osteoclasts and that tankyrase inhibitors can attenuate the effects of AXIN1 hypomorphic variants.

Photoinduced Electron Transfer in Inclusion Complexes of Carbon Nanohoops.

ConspectusPhotoinduced electron transfer (PET) in carbon materials is a process of great importance in light energy conversion. Carbon materials, such as fullerenes, graphene flakes, carbon nanotubes, and cycloparaphenylenes (CPPs), have unusual electronic properties that make them interesting objects for PET research. These materials can be used as electron-hole transport layers, electrode materials, or passivation additives in photovoltaic devices. Moreover, their appropriate combination opens up new possibilities for constructing photoactive supramolecular systems with efficient charge transfer between the donor and acceptor parts. CPPs build a class of molecules consisting of para-linked phenylene rings. CPPs and their numerous derivatives are appealing building blocks in supramolecular chemistry, acting as suitable concave receptors with strong host-guest interactions for the convex surfaces of fullerenes. Efficient PET in donor-acceptor systems can be observed when charge separation occurs faster than charge recombination. This Account focuses on selected inclusion complexes of carbon nanohoops studied by our group. We modeled charge separation and charge recombination in both previously synthesized and computationally designed complexes to identify how various modifications of host and guest molecules affect the PET efficiency in these systems. A consistent computational protocol we used includes a time-dependent density-functional theory (TD-DFT) formalism with the Tamm-Dancoff approximation (TDA) and CAM-B3LYP functional to carry out excited state calculations and the nonadiabatic electron transfer theory to estimate electron-transfer rates. We show how the photophysical properties of carbon nanohoops can be modified by incorporating additional π-conjugated fragments and antiaromatic units, multiple fluorine substitutions, and extending the overall π-electron system. Incorporating π-conjugated groups or linkers is accompanied by the appearance of new charge transfer states. Perfluorination of the nanohoops radically changes their role in charge separation from an electron donor to an electron acceptor. Vacancy defects in π-extended nanohoops are shown to hinder PET between host and guest molecules, while large fully conjugated π-systems improve the electron-donor properties of nanohoops. We also highlight the role of antiaromatic structural units in tuning the electronic properties of nanohoops. Depending on the aromaticity degree of monomeric units in nanohoops, the direction of electron transfer in their complexes with C60 fullerene can be altered. Nanohoops with aromatic units usually act as electron donors, while those with antiaromatic monomers serve as electron acceptors. Finally, we discuss why charged fullerenes are better electron acceptors than neutral C60 and how the charge location allows for the design of more efficient donor-acceptor systems with an unusual hypsochromic shift of the charge transfer band in polar solvents.

Genomic encoding of transcriptional burst kinetics.

Mammalian gene expression is inherently stochastic1,2, and results in discrete bursts of RNA molecules that are synthesized from each allele3-7. Although transcription is known to be regulated by promoters and enhancers, it is unclear how cis-regulatory sequences encode transcriptional burst kinetics. Characterization of transcriptional bursting, including the burst size and frequency, has mainly relied on live-cell4,6,8 or single-molecule RNA fluorescence in situ hybridization3,5,8,9 recordings of selected loci. Here we determine transcriptome-wide burst frequencies and sizes for endogenous mouse and human genes using allele-sensitive single-cell RNA sequencing. We show that core promoter elements affect burst size and uncover synergistic effects between TATA and initiator elements, which were masked at mean expression levels. Notably, we provide transcriptome-wide evidence that enhancers control burst frequencies, and demonstrate that cell-type-specific gene expression is primarily shaped by changes in burst frequencies. Together, our data show that burst frequency is primarily encoded in enhancers and burst size in core promoters, and that allelic single-cell RNA sequencing is a powerful model for investigating transcriptional kinetics.

The RNA m6A Reader YTHDF2 Is Essential for the Post-transcriptional Regulation of the Maternal Transcriptome and Oocyte Competence.

YTHDF2 binds and destabilizes N6-methyladenosine (m6A)-modified mRNA. The extent to which this branch of m6A RNA-regulatory pathway functions in vivo and contributes to mammalian development remains unknown. Here we find that YTHDF2 deficiency is partially permissive in mice and results in female-specific infertility. Using conditional mutagenesis, we demonstrate that YTHDF2 is autonomously required within the germline to produce MII oocytes that are competent to sustain early zygotic development. Oocyte maturation is associated with a wave of maternal RNA degradation, and the resulting relative changes to the MII transcriptome are integral to oocyte quality. The loss of YTHDF2 results in the failure to regulate transcript dosage of a cohort of genes during oocyte maturation, with enrichment observed for the YTHDF2-binding consensus and evidence of m6A in these upregulated genes. In summary, the m6A-reader YTHDF2 is an intrinsic determinant of mammalian oocyte competence and early zygotic development.

Pathogenic Aß production by heterozygous PSEN1 mutations is intrinsic to the mutant protein and not mediated by conformational hindrance of wild-type PSEN1.

Presenilin-1 (PSEN1) is the catalytic subunit of the intramembrane protease γ-secretase and undergoes endoproteolysis during its maturation. Heterozygous mutations in the PSEN1 gene cause early-onset familial Alzheimer's disease (eFAD) and increase the proportion of longer aggregation-prone amyloid-ß peptides (Aß42 and/or Aß43). Previous studies had suggested that PSEN1 mutants might act in a dominant-negative fashion by functional impediment of wild-type PSEN1, but the exact mechanism by which PSEN1 mutants promote pathogenic Aß production remains controversial. Using dual recombinase-mediated cassette exchange (dRMCE), here we generated a panel of isogenic embryonic and neural stem cell lines with heterozygous, endogenous expression of PSEN1 mutations. When catalytically inactive PSEN1 was expressed alongside the wild-type protein, we found the mutant accumulated as a full-length protein, indicating that endoproteolytic cleavage occurred strictly as an intramolecular event. Heterozygous expression of eFAD-causing PSEN1 mutants increased the Aß42/Aß40 ratio. In contrast, catalytically inactive PSEN1 mutants were still incorporated into the γ-secretase complex but failed to change the Aß42/Aß40 ratio. Finally, interaction and enzyme activity assays demonstrated the binding of mutant PSEN1 to other γ-secretase subunits, but no interaction between mutant and wild-type PSEN1 was observed. These results establish that pathogenic Aß production is an intrinsic property of PSEN1 mutants and strongly argue against a dominant-negative effect in which PSEN1 mutants would compromise the catalytic activity of wild-type PSEN1 through conformational effects.

Genetic and clinical determinants of abdominal aortic diameter: genome-wide association studies, exome array data and Mendelian randomization study.

Progressive dilation of the infrarenal aortic diameter is a consequence of the ageing process and is considered the main determinant of abdominal aortic aneurysm (AAA). We aimed to investigate the genetic and clinical determinants of abdominal aortic diameter (AAD). We conducted a meta-analysis of genome-wide association studies in 10 cohorts (n = 13 542) imputed to the 1000 Genome Project reference panel including 12 815 subjects in the discovery phase and 727 subjects [Partners Biobank cohort 1 (PBIO)] as replication. Maximum anterior-posterior diameter of the infrarenal aorta was used as AAD. We also included exome array data (n = 14 480) from seven epidemiologic studies. Single-variant and gene-based associations were done using SeqMeta package. A Mendelian randomization analysis was applied to investigate the causal effect of a number of clinical risk factors on AAD. In genome-wide association study (GWAS) on AAD, rs74448815 in the intronic region of LDLRAD4 reached genome-wide significance (beta = -0.02, SE = 0.004, P-value = 2.10 × 10-8). The association replicated in the PBIO1 cohort (P-value = 8.19 × 10-4). In exome-array single-variant analysis (P-value threshold = 9 × 10-7), the lowest P-value was found for rs239259 located in SLC22A20 (beta = 0.007, P-value = 1.2 × 10-5). In the gene-based analysis (P-value threshold = 1.85 × 10-6), PCSK5 showed an association with AAD (P-value = 8.03 × 10-7). Furthermore, in Mendelian randomization analyses, we found evidence for genetic association of pulse pressure (beta = -0.003, P-value = 0.02), triglycerides (beta = -0.16, P-value = 0.008) and height (beta = 0.03, P-value < 0.0001), known risk factors for AAA, consistent with a causal association with AAD. Our findings point to new biology as well as highlighting gene regions in mechanisms that have previously been implicated in the genetics of other vascular diseases.

Effects and interaction of single nucleotide polymorphisms at the pharmacokinetic/pharmacodynamic site: insights from the Rotterdam study into metformin clinical response and dose titration.

Our study investigated the impact of genetic variations on metformin glycemic response in a cohort from the Rotterdam Study, comprising 14,926 individuals followed for up to 27 years. Among 1285 metformin users of European ancestry, using linear mixed models, we analyzed the association of single nucleotide polymorphisms (SNPs) and a Polygenic Risk Score (PRS) with glycemic response, measured by changes in metformin dosage or HbA1c levels. While individual genetic variants showed no significant association, rs622342 on SLC2A1 correlated with increased glycemic response only in metformin monotherapy patients (ß = -2.09, P-value < 0.001). The collective effect of variants, as represented by PRS, weakly correlated with changes in metformin dosage (ß = 0.023, P-value = 0.027). Synergistic interaction was observed between rs7124355 and rs8192675. Our findings suggest that while higher PRS correlates with increased metformin dosage, its modest effect size limits clinical utility, emphasizing the need for future research in diverse populations to refine genetic risk models.

Smallpox vaccination induces a substantial increase in commensal skin bacteria that promote pathology and influence the host response.

Interactions between pathogens, host microbiota and the immune system influence many physiological and pathological processes. In the 20th century, widespread dermal vaccination with vaccinia virus (VACV) led to the eradication of smallpox but how VACV interacts with the microbiota and whether this influences the efficacy of vaccination are largely unknown. Here we report that intradermal vaccination with VACV induces a large increase in the number of commensal bacteria in infected tissue, which enhance recruitment of inflammatory cells, promote tissue damage and influence the host response. Treatment of vaccinated specific-pathogen-free (SPF) mice with antibiotic, or infection of genetically-matched germ-free (GF) animals caused smaller lesions without alteration in virus titre. Tissue damage correlated with enhanced neutrophil and T cell infiltration and levels of pro-inflammatory tissue cytokines and chemokines. One month after vaccination, GF and both groups of SPF mice had equal numbers of VACV-specific CD8+ T cells and were protected from disease induced by VACV challenge, despite lower levels of VACV-neutralising antibodies observed in GF animals. Thus, skin microbiota may provide an adjuvant-like stimulus during vaccination with VACV and influence the host response to vaccination.

Microsampling Techniques Suitable for Therapeutic Drug Monitoring of Antipsychotics.

BACKGROUND: Therapeutic drug monitoring (TDM) of antipsychotics for dose titration or detection of noncompliance is not uncommon in daily practice. Normally, TDM implies measuring a drug concentration in venous blood samples. This technique is invasive and requires trained assistants and patients normally need to go to an outpatient clinic. Over the past decades, sensitivity of analytical equipment has improved leading to a growing interest in microsampling techniques. These techniques are minimally invasive, require a small volume (<100 µL), usually result in stable samples, and can be collected by the patient or a caregiver at home. Before a microsampling technique can be used in daily routine, proper method development and a clinical validation study should be performed. METHOD: For this review, the databases of PubMed and Embase were systematically searched. Currently available microsampling techniques for antipsychotics in blood, serum, or plasma are summarized. Subsequently, it has also been assessed whether these techniques are sufficiently validated for TDM monitoring in daily practice. RESULTS: Several microsampling techniques are available today, for example, dried blood spot sampling, dried plasma extraction cards, and volumetric absorptive microsampling. Eighteen studies were identified in which a microsampling technique for 1 or a few antipsychotics was chemically analytically and clinically validated. However, the majority of these studies have relevant shortcomings that mean its usefulness for different antipsychotics is not yet well established. CONCLUSIONS: Microsampling for TDM can be recommended for patients using clozapine. For TDM of other antipsychotics, it is a very promising development.

Unveiling the Reaction Mechanisms of the Synthesis and the Excited State Intramolecular Proton Transfer of 2-(2'-hydroxyphenyl)imidazo[1,2-a]pyridine.

Given its wide variety of applications in the pharmaceutical industry, the synthesis of imidazo[1,2-a]pyridines has been extensively studied since the beginning of the last century. Here, we disclose the mechanism for the synthesis of imidazo[1,2-a]pyridines by means of the Ortoleva-King reaction. We also reveal the reaction pathway leading to the formation of a iodinated byproduct, demonstrating the challenge of preventing the formation of such a byproduct because of the low energy barrier to access it. Moreover, quantum chemistry tools were employed to investigate the mechanism of intramolecular proton transfer in the excited state, and connections with aromaticity were explored.

CGG toolkit: Software components for computational genomics.

Public-domain availability for bioinformatics software resources is a key requirement that ensures long-term permanence and methodological reproducibility for research and development across the life sciences. These issues are particularly critical for widely used, efficient, and well-proven methods, especially those developed in research settings that often face funding discontinuities. We re-launch a range of established software components for computational genomics, as legacy version 1.0.1, suitable for sequence matching, masking, searching, clustering and visualization for protein family discovery, annotation and functional characterization on a genome scale. These applications are made available online as open source and include MagicMatch, GeneCAST, support scripts for CoGenT-like sequence collections, GeneRAGE and DifFuse, supported by centrally administered bioinformatics infrastructure funding. The toolkit may also be conceived as a flexible genome comparison software pipeline that supports research in this domain. We illustrate basic use by examples and pictorial representations of the registered tools, which are further described with appropriate documentation files in the corresponding GitHub release.

PARP1 Links CHD2-Mediated Chromatin Expansion and H3.3 Deposition to DNA Repair by Non-homologous End-Joining.

The response to DNA double-strand breaks (DSBs) requires alterations in chromatin structure to promote the assembly of repair complexes on broken chromosomes. Non-homologous end-joining (NHEJ) is the dominant DSB repair pathway in human cells, but our understanding of how it operates in chromatin is limited. Here, we define a mechanism that plays a crucial role in regulating NHEJ in chromatin. This mechanism is initiated by DNA damage-associated poly(ADP-ribose) polymerase 1 (PARP1), which recruits the chromatin remodeler CHD2 through a poly(ADP-ribose)-binding domain. CHD2 in turn triggers rapid chromatin expansion and the deposition of histone variant H3.3 at sites of DNA damage. Importantly, we find that PARP1, CHD2, and H3.3 regulate the assembly of NHEJ complexes at broken chromosomes to promote efficient DNA repair. Together, these findings reveal a PARP1-dependent process that couples ATP-dependent chromatin remodeling with histone variant deposition at DSBs to facilitate NHEJ and safeguard genomic stability.

Cognitive behavioural therapy and third-wave approaches for anxiety and related disorders in older people.

BACKGROUND: Cognitive behavioural therapy (CBT) is the most researched psychological therapy for anxiety disorders in adults, and known to be effective in this population. However, it remains unclear whether these results apply to older adults, as most studies include participants between 18 and 55 years of age. This systematic review aims to provide a comprehensive and up-to-date synthesis of the available evidence on CBT and third wave approaches for older adults with anxiety and related disorders. OBJECTIVES: To assess the effects of Cognitive Behavioural Therapy (CT, BT, CBT and third-wave CBT interventions) on severity of anxiety symptoms compared with minimal management (not providing therapy) for anxiety and related disorders in older adults, aged 55 years or over. To assess the effects of CBT and related therapies on severity of anxiety symptoms compared with other psychological therapies for anxiety and related disorders in older adults, aged 55 years or over. SEARCH METHODS: We searched the Cochrane Common Mental Disorders Controlled studies Register (CCMDCTR), CENTRAL, Ovid MEDLINE, Ovid Embase and Ovid PsycINFO to 21 July 2022. These searches were updated on 2 February 2024. We also searched the international studies registries, including Clinicalstudies.gov and the WHO International Clinical Trials Registry Platform (ICTRP), to identify additional ongoing and unpublished studies. These sources were manually searched for studies up to 12 February 2024. SELECTION CRITERIA: We included randomised controlled trials (RCTs) in older adults (≥ 55 years) with an anxiety disorder, or a related disorder, including obsessive compulsive disorder (OCD), acute stress disorder and post-traumatic stress disorder (PTSD), that compared CBT to either minimal management or an active (non-CBT) psychological therapy. Eligible studies had to have an anxiety-related outcome. DATA COLLECTION AND ANALYSIS: Several authors independently screened all titles identified by the searches. All full texts were screened for eligibility according to our prespecified selection criteria. Data were extracted and the risk of bias was assessed using the Cochrane tool for RCTs. The certainty of evidence was evaluated using GRADE. Meta-analyses were performed for outcomes with quantitative data from more than one study. MAIN RESULTS: We included 21 RCTs on 1234 older people allocated to either CBT or control conditions. Ten studies focused on generalised anxiety disorder; others mostly included a mix of clinical diagnoses. Nineteen studies focused on the comparison between CBT and minimal management. Key issues relating to risk of bias were lack of blinding of participants and personnel, and participants dropping out of studies, potentially due to treatment preference and allocation. CBT may result in a small-to-moderate reduction of anxiety post-treatment (SMD -0.51, 95% CI -0.66 to -0.36, low-certainty evidence). However, compared to this benefit with CBT immediately after treatment, at three to six months post-treatment, there was little to no difference between CBT and minimal management (SMD -0.29, 95% CI -0.59 to 0.01, low-certainty evidence). CBT may have little or no effect on clinical recovery/ improvement post-treatment compared to minimal management, but the evidence is very uncertain (RR 1.56, 95% CI 1.20 to 2.03, very low-certainty evidence). Results indicate that five people would need to receive treatment for one additional person to benefit (NNTB = 5). Compared to minimal management, CBT may result in a reduction of comorbid depression symptoms post-treatment (SMD -0.57, 95% CI -0.74 to -0.40, low-certainty evidence). There was no difference in dropout rates post-treatment, although the certainty of the evidence was low (RR 1.19, 95% CI 0.80 to 1.78). Two studies reported adverse events, both of which related to medication in the control groups (very low-certainty evidence, no quantitative estimate). Only two studies compared CBT to other psychological therapies, both of which only included participants with post-traumatic stress disorder. Low-certainty evidence showed no difference in anxiety severity post-treatment and at four to six months post-treatment, symptoms of depression post-treatment, and dropout rates post-treatment. Other outcomes and time points are reported in the results section of the manuscript. AUTHORS' CONCLUSIONS: CBT may be more effective than minimal management in reducing anxiety and symptoms of worry and depression post-treatment in older adults with anxiety disorders. The evidence is less certain longer-term and for other outcomes including clinical recovery/improvement. There is not enough evidence to determine whether CBT is more effective than alternative psychological therapies for anxiety in older adults.

Remote physical activity intervention to promote physical activity and health in adolescent girls (the HERizon project): a multi-arm, pilot randomised trial.

BACKGROUND: Engaging in physical activity (PA) during adolescence is beneficial for health and positive development. However, most adolescent girls have low PA levels, and there is a need for interventions outside of school hours. This pilot randomised controlled trial aimed to explore the preliminary effectiveness of three different remote PA interventions in increasing adolescent girls' moderate-to- vigorous PA (MVPA), fitness and psychosocial outcomes. METHODS: Girls living in the UK or Ireland, aged between 13 and 16 years old, who wished to increase their activity levels, were eligible for the study. Using a random number generator, participants (n = 153; 14.8y ± 1.4) were randomised into one of three 12-week intervention groups (i) PA programme, (ii) Behaviour change support, or (iii) Combined PA programme and Behaviour change support, or (iv) a Comparison group. Outcome measures included accelerometer and self-reported PA, physical fitness (cardiorespiratory fitness; 20 m shuttle run, muscular endurance; push up, muscular strength; long jump), and psychosocial assessments (perceived competence; body appreciation; self-esteem; behavioural regulation). Linear mixed models were used to analyse differences between each intervention arm and the comparison group immediately postintervention (12 weeks) and at follow up (3-months post-intervention), while adjusting for potential confounders. RESULTS: Participation in the PA programme group was associated with higher perceived competence (0.6, 95% CI 0.1 to 1.2), identified regulation (0.7, 95% CI 0.2 to 1.1) and intrinsic motivation (0.9, 95% CI 0.2 to 1.6) at post-intervention. Participation in the Behaviour change group was associated with higher perceived competence at post-intervention (0.6, 95% CI 0.1 to 1.2), and higher push-up scores at the 3-month follow-up (4.0, 95% CI 0.0 to 7.0). Participation in the Combined group was also associated with higher perceived competence at post-intervention (0.8, 95% CI 0.2 to 1.4), and higher push-up scores at the 3-month follow-up (5.0, 95% CI 1.0 to 8.0). No other significant differences were found between the intervention arms and the comparison group. CONCLUSION: Results suggest perceived competence increased across all intervention arms, while the PA programme group enhanced autonomous motivation in the short term. Intervention arms with behaviour change support appear most promising in improving muscular endurance. However, a larger scale trial is needed for a better understanding of between-group differences and the impact of intervention arms on MVPA and fitness, given the small sample size and short-term follow-up.

Does a lack of emotions make chatbots unfit to be psychotherapists?

Mental health chatbots (MHCBs) designed to support individuals in coping with mental health issues are rapidly advancing. Currently, these MHCBs are predominantly used in commercial rather than clinical contexts, but this might change soon. The question is whether this use is ethically desirable. This paper addresses a critical yet understudied concern: assuming that MHCBs cannot have genuine emotions, how this assumption may affect psychotherapy, and consequently the quality of treatment outcomes. We argue that if MHCBs lack emotions, they cannot have genuine (affective) empathy or utilise countertransference. Consequently, this gives reason to worry that MHCBs are (a) more liable to harm and (b) less likely to benefit patients than human therapists. We discuss some responses to this worry and conclude that further empirical research is necessary to determine whether these worries are valid. We conclude that, even if these worries are valid, it does not mean that we should never use MHCBs. By discussing the broader ethical debate on the clinical use of chatbots, we point towards how further research can help us establish ethical boundaries for how we should use mental health chatbots.

Patient Perceptions of Illness Causes and Treatment Preferences for Obsessive-Compulsive Disorder: A Mixed-Methods Study.

Obsessive-compulsive disorder (OCD) is a condition with high patient morbidity and mortality. Research shows that eliciting patient explanations about illness causes and treatment preferences promotes cross-cultural work and engagement in health services. These topics are in the Cultural Formulation Interview (CFI), a semi-structured interview first published in DSM-5 that applies anthropological approaches within mental health services to promote person-centered care. This study focuses on the New York City site of an international multi-site study that used qualitative-quantitative mixed methods to: (1) analyze CFI transcripts with 55 adults with OCD to explore perceived illness causes and treatment preferences, and (2) explore whether past treatment experiences are related to perceptions about causes of current symptoms. The most commonly named causes were circumstantial stressors (n = 16), genetics (n = 12), personal psychological traits (n = 9), an interaction between circumstantial stressors and participants' brains (n = 6), and a non-specific brain problem (n = 6). The most common treatment preferences were psychotherapy (n = 42), anything (n = 4), nothing (n = 4), and medications (n = 2). Those with a prior medication history had twice the odds of reporting a biological cause, though this was not a statistically significant difference. Our findings suggest that providers should ask patients about illness causes and treatment preferences to guide treatment choice.

Targeting oncogenic microRNAs from the miR-371~373 and miR-302/367 clusters in malignant germ cell tumours causes growth inhibition through cell cycle disruption.

BACKGROUND: MiR-371~373 and miR-302/367 cluster over-expression occurs in all malignant germ cell tumours (GCTs), regardless of age (paediatric/adult), site (gonadal/extragonadal), or subtype [seminoma, yolk sac tumour (YST), embryonal carcinoma (EC)]. Six of eight microRNAs from these clusters contain the seed sequence 'AAGUGC', determining mRNA targeting. Here we sought to identify the significance of these observations by targeting these microRNAs functionally. METHODS: We targeted miR-371~373 and/or miR-302/367 clusters in malignant GCT cell lines, using CRISPR-Cas9, gapmer primary miR-302/367 transcript inhibition, and peptide nucleic acid (PNA) or locked nucleic acid (LNA)-DNA inhibition targeting miR-302a-d-3p, and undertook relevant functional assays. RESULTS: MiR-302/367 cluster microRNAs made the largest contribution to AAGUGC seed abundance in malignant GCT cells, regardless of subtype (seminoma/YST/EC). Following the unsuccessful use of CRISPR-Cas9, gapmer, and PNA systems, LNA-DNA-based targeting resulted in growth inhibition in seminoma and YST cells. This was associated with the de-repression of multiple mRNAs targeted by AAGUGC seed-containing microRNAs, with pathway analysis confirming predominant disruption of Rho-GTPase signalling, vesicle organisation/transport, and cell cycle regulation, findings corroborated in clinical samples. Further LNA-DNA inhibitor studies confirmed direct cell cycle effects, with an increase of cells in G0/G1-phase and a decrease in S-phase. CONCLUSION: Targeting of specific miR-371~373 and miR-302/367 microRNAs in malignant GCTs demonstrated their functional significance, with growth inhibition mediated through cell cycle disruption.

Short- and long-range cis interactions between integrated HPV genomes and cellular chromatin dysregulate host gene expression in early cervical carcinogenesis.

Development of cervical cancer is directly associated with integration of human papillomavirus (HPV) genomes into host chromosomes and subsequent modulation of HPV oncogene expression, which correlates with multi-layered epigenetic changes at the integrated HPV genomes. However, the process of integration itself and dysregulation of host gene expression at sites of integration in our model of HPV16 integrant clone natural selection has remained enigmatic. We now show, using a state-of-the-art 'HPV integrated site capture' (HISC) technique, that integration likely occurs through microhomology-mediated repair (MHMR) mechanisms via either a direct process, resulting in host sequence deletion (in our case, partially homozygously) or via a 'looping' mechanism by which flanking host regions become amplified. Furthermore, using our 'HPV16-specific Region Capture Hi-C' technique, we have determined that chromatin interactions between the integrated virus genome and host chromosomes, both at short- (<500 kbp) and long-range (>500 kbp), appear to drive local host gene dysregulation through the disruption of host:host interactions within (but not exceeding) host structures known as topologically associating domains (TADs). This mechanism of HPV-induced host gene expression modulation indicates that integration of virus genomes near to or within a 'cancer-causing gene' is not essential to influence their expression and that these modifications to genome interactions could have a major role in selection of HPV integrants at the early stage of cervical neoplastic progression.

Excited State Processes in Supramolecular Complexes of Cyclic Dibenzopyrrolopyrrole Isomers with C60 Fullerene.

An approach to modulating the properties of carbon nanorings by incorporating pyrrolo[3,2-b]pyrrole units is of particular interest due to the combined effect of heteroatom and antiaromatic character on electronic properties. The inclusion of units other than phenylene leads to the formation of stereoisomers. In this work, we computationally study how the spatial orientation of monomeric units in the ring affects the properties of cyclic dibenzopyrrolo[3,2-b]pyrroles and their complexes with C60 fullerene. For [4]PP and [4]DHPP, the most symmetrical AAAA isomer is the most stable and forms stronger interactions with fullerene than the isomers where one or two monomeric units are flipped, mostly due to less Pauli repulsion. π-Electron delocalization in the monomeric unit is crucial for directing the electron transfer (from or to nanoring). The energy of excited states with charge transfer depends on the HOMO-LUMO gap, which varies from one stereoisomer to another only for [4]DHPP⊃C60 with aromatic 1,4-dihydropyrrolo[3,2-b]pyrrole units. The rates of electron transfer and charge recombination reactions are relatively weakly dependent of the spatial isomerism of nanorings.