A highly conserved program of neuronal microexons is misregulated in autistic brains.

Alternative splicing (AS) generates vast transcriptomic and proteomic complexity. However, which of the myriad of detected AS events provide important biological functions is not well understood. Here, we define the largest program of functionally coordinated, neural-regulated AS described to date in mammals. Relative to all other types of AS within this program, 3-15 nucleotide "microexons" display the most striking evolutionary conservation and switch-like regulation. These microexons modulate the function of interaction domains of proteins involved in neurogenesis. Most neural microexons are regulated by the neuronal-specific splicing factor nSR100/SRRM4, through its binding to adjacent intronic enhancer motifs. Neural microexons are frequently misregulated in the brains of individuals with autism spectrum disorder, and this misregulation is associated with reduced levels of nSR100. The results thus reveal a highly conserved program of dynamic microexon regulation associated with the remodeling of protein-interaction networks during neurogenesis, the misregulation of which is linked to autism.

Genome-wide CRISPR-Cas9 Interrogation of Splicing Networks Reveals a Mechanism for Recognition of Autism-Misregulated Neuronal Microexons.

Alternative splicing is crucial for diverse cellular, developmental, and pathological processes. However, the full networks of factors that control individual splicing events are not known. Here, we describe a CRISPR-based strategy for the genome-wide elucidation of pathways that control splicing and apply it to microexons with important functions in nervous system development and that are commonly misregulated in autism. Approximately 200 genes associated with functionally diverse regulatory layers and enriched in genetic links to autism control neuronal microexons. Remarkably, the widely expressed RNA binding proteins Srsf11 and Rnps1 directly, preferentially, and frequently co-activate these microexons. These factors form critical interactions with the neuronal splicing regulator Srrm4 and a bi-partite intronic splicing enhancer element to promote spliceosome formation. Our study thus presents a versatile system for the identification of entire splicing regulatory pathways and further reveals a common mechanism for the definition of neuronal microexons that is disrupted in autism.

Enantioenriched Allylesters via a Copper-Catalyzed Diene Carboesterification with Alkyltrifluoroborates and Carboxylic Acids.

The rapid synthesis of a range of enantioenriched allylic esters is enabled by a new 3-component catalytic enantioselective 1,2-carboesterification of readily available dienes with carboxylic acids and potassium alkyltrifluoroborates. The chiral copper catalyst, formed in situ from Cu(OTf)2 and (4S,4'S)-2,2'-(cyclopentane-1,1-diyl)bis(4-phenyl-4,5-dihydrooxazole), is implicated in both the generation of alkyl radicals from the alkyltrifluoroborates as well as the enantioselective formation of C-O bonds. Potassium salts of primary and secondary alkyltrifluoroborates as well as several benzylic trifluoroborates, tert-butyltrifluoroborate, and phenyltrifluoroborate participate in the reaction. The regioselectivity and enantioselectivity are strongly impacted by variations in all of the reaction components, which in turn are thought to impact the C-O bond-forming reductive elimination from a [Cu(III)] intermediate.

Variable trends in the distribution of Atlantic cod (Gadus morhua) in the Celtic seas.

Despite decades of active fisheries management, many stocks of Atlantic cod in its southern range are in a depleted state and mortality estimates remain high. Recovery of these stocks, as defined by management areas, could be confounded by cod distributions shifting outside of these areas. Here, we assess data from internationally coordinated trawl surveys to investigate the distribution of three cod stocks in the Celtic Seas ecoregion, Irish Sea, Celtic Sea, and West of Scotland, from 1985 to 2021. We mapped cod densities, analyzed trends in mean weighted depth and bottom temperature, and calculated the center of gravity and equivalent area of the stocks. The distribution of the West of Scotland stock shifted north and east, spilling into the North Sea, while the Irish Sea and Celtic Sea stocks shifted west. Each stock showed decreasing trends in equivalent area, but there were no clear trends in the average depth occupied by the fish. There was no apparent relationship between temperature and the distribution of cod, as bottom temperature varied little from 1993 to 2021. Although Irish Sea cod showed a shift into warmer water, this was due to changes in survey distribution. The shift in distribution of the West of Scotland cod stock towards the North Sea whilst impairing local recovery provides further justification for the recent definition of its incorporation into a larger stock unit that includes the northwest of the North Sea. The Irish Sea and Celtic Sea cod stocks are neither shifting northwards, nor into deeper waters, but remained within current boundaries. This suggests that recent temperature conditions did not affect their distribution, but this may change as temperatures increase towards the limit for reproduction.

Aquatic connectivity: challenges and solutions in a changing climate.

The challenge of managing aquatic connectivity in a changing climate is exacerbated in the presence of additional anthropogenic stressors, social factors, and economic drivers. Here we discuss these issues in the context of structural and functional connectivity for aquatic biodiversity, specifically fish, in both the freshwater and marine realms. We posit that adaptive management strategies that consider shifting baselines and the socio-ecological implications of climate change will be required to achieve management objectives. The role of renewable energy expansion, particularly hydropower, is critically examined for its impact on connectivity. We advocate for strategic spatial planning that incorporates nature-positive solutions, ensuring climate mitigation efforts are harmonized with biodiversity conservation. We underscore the urgency of integrating robust scientific modelling with stakeholder values to define clear, adaptive management objectives. Finally, we call for innovative monitoring and predictive decision-making tools to navigate the uncertainties inherent in a changing climate, with the goal of ensuring the resilience and sustainability of aquatic ecosystems.

Prostaglandin E2/EP4 axis is upregulated in Spondyloarthritis and contributes to radiographic progression.

Ankylosing spondylitis (AS) is an inflammatory disease leading to spine ankylosis; however, the mechanisms behind new bone formation are still not fully understood. Single Nucleotide Polymorphisms (SNPs) in PTGER4, encoding for the receptor EP4 of prostaglandin E2 (PGE2), are associated with AS. Since the PGE2-EP4 axis participates in inflammation and bone metabolism, this work aims at investigating the influence of the prostaglandin-E2 axis on radiographic progression in AS. In 185 AS (97 progressors), baseline serum PGE2 predicted progression, and PTGER4 SNP rs6896969 was more frequent in progressors. Increased EP4/PTGER4 expression was observed in AS circulating immune cells, synovial tissue, and bone marrow. CD14highEP4 + cells frequency correlated with disease activity, and when monocytes were cocultured with mesenchymal stem cells, the PGE2/EP4 axis induced bone formation. In conclusion, the Prostaglandin E2 axis is involved in bone remodelling and may contribute to the radiographic progression in AS due to genetic and environmental upregulation.

Analysis of human leukocyte antigen associations in human papillomavirus-positive and -negative head and neck cancer: Comparison with cervical cancer.

BACKGROUND: Although the majority of human papillomavirus (HPV) infections are cleared by the immune system, a small percentage of them progress to develop HPV-driven cancers. Cervical cancer studies highlight that HPV persistence and cancer risk are associated with genetic factors, especially at the human leukocyte antigen (HLA) genes. This study was conducted to investigate such associations in head and neck cancer (HNC). METHODS: In all, 192 patients with HNC and 384 controls were genotyped with the Infinium Global Screening Array (Illumina, Inc). HLA variants were imputed with SNP2HLA, and an association analysis was performed by logistic regression. RESULTS: HPV-positive HNCs were significantly associated with single-nucleotide polymorphisms (SNPs) at DRB1_32660090 (P = 1.728 × 10-6 ) and DRB1_32660116 (P = 1.728 × 10-6 ) and with the amino acid variant DRB1_11_32660115 (P = 1.728 × 10-6 ). None of these associations were observed in the HPV-negative cohort, and this suggested their specificity to convey risk for HPV-associated HNCs. In general, associations observed for HPV-negative HNC were relatively weak, and variants in the HLA-DPA1 region were the strongest among them (P = 4.531 × 10-4 ). Several lead signals reported by previous HNC genome-wide association studies, including SNPs rs3135001 (P = .012), rs1049055 (P = .012), and rs34518860 (P = .029) and allele HLA-DQB1*06 (P = .009), were replicated in the current study. However, these associations were limited to the HPV-positive HNC group. Several cervical cancer-associated HLA variants, including SNPs rs9272143 (P = .002) and rs9271858 (P = .002) and alleles HLA-B-1501 (P = .009) and HLA-B-15 (P = .015), were also exclusively associated with HPV-positive HNC. CONCLUSIONS: HPV-positive HNC risk is associated with distinct HLA variants, and some of them are shared by both cervical cancer and HPV-positive HNC. Human papillomavirus (HPV)-positive head and neck cancer (HNC) risk is associated with distinct human leukocyte antigen variants, and some of them are shared by both cervical cancer and HPV-positive HNC. LAY SUMMARY: Cervical cancer studies highlight that human papillomavirus (HPV)-driven cancer risk is linked with human leukocyte antigen (HLA) polymorphism. Hence, the current study was designed to investigate the HLA associations in HPV-positive and HPV-negative head and neck cancer (HNC) and compare these associations with cervical cancer. Several lead signals reported by previous HNC and cervical genome-wide association studies were replicated in the current study. However, these associations were limited to the HPV-positive HNC group, and this suggests that HPV-positive HNC risk is associated with distinct HLA variants, and some of them are shared by both cervical cancer and HPV-positive HNC.

Pixel MTF response effect on non-null interferometry.

The pixel modulation transfer function response degrades the contrast of non-null interferometric surface figure measurements. We experimentally quantify this effect for spatial frequencies ranging from 0 to 363 lp/mm (≈3.33 times the Nyquist limit). Our results show a low SNR spatial frequency band that behaves like a low-pass filter for sub-Nyquist interferometry and a stop-band filter for multiple-wavelength phase-shifting interferometry. We also introduce a multiple-mode, multiple-wavelength interferometry approach to measure optical surfaces with slope departure angles mapping to spatial frequencies in this low SNR band. The extended measurement range of this approach is achieved without using a sparse-array detector.

Temporal evolution of the biological response to laser-induced refractive index change (LIRIC) in rabbit corneas.

Laser-induced refractive index change (LIRIC) is a new, non-incisional, non-ablative, femtosecond photo-modification technique being developed for vision correction in humans. Prior, exvivo studies showed intra-tissue refractive index change to induce minimal cell death, restricted to the laser focal zone in the corneal stroma, and with no observable damage to the epithelium or endothelium. Here, we used live rabbits to ascertain longer-term consequences of LIRIC in vivo. Specifically, we assessed cell death, fibrosis, corneal nerve distribution, endothelial cell density, and corneal structure for up to 3 months after LIRIC. A +2.5 D gradient-index LIRIC Fresnel lens was inscribed inside 20 applanated corneas of Dutch Belted rabbits, over a circular region of the mid-stroma measuring 4.5 mm in diameter. Twelve additional rabbit eyes were used as applanation-only controls to differentiate the effects of laser treatment and suction applanation on biological and structural parameters. In vivo optical measurements were performed pre-operatively, then immediately, 2, 4, and 12 weeks after the procedure, to measure endothelial cell density and changes in corneal structure. Groups of four rabbits were sacrificed at 4 hours, 2, 4, and 12 weeks after LIRIC for histological determinations; the TUNEL assay was used to evaluate cell death, H&E staining was used to assess inflammatory infiltration, and immunostaining for α-smooth muscle actin (α-SMA) and ßIII tubulin (Tuj-1) was performed to assess myofibroblast differentiation and corneal nerve distribution, respectively. Consistent with prior ex vivo data, only minimal cell death was observed in the laser focal zone, with TUNEL-positive cells restricted to the stromal region of refractive index change 4 h after LIRIC. No TUNEL-positive cells were evident anywhere in the cornea 2, 4, or 12 weeks after LIRIC. Applanation-only corneas were completely TUNEL-negative. Neither LIRIC-treated nor applanation-only eyes exhibited α-SMA-positive staining or altered corneal nerve distributions at any of the time points examined. In vivo confocal imaging revealed normal endothelial cell densities in all eyes (whether LIRIC-treated or applanation-only) at all time points. Optical coherence tomography showed suction applanation to cause a temporary decrease in central corneal thickness, which returned to normal within 4 h. Corneas into which LIRIC Fresnel lenses were written while applanated did not undergo major structural or shape changes beyond the temporary thinning already described for suction applanation. The present findings suggest that LIRIC patterns, which generated a clinically-relevant refractive correction in the mid-stromal region of live rabbit corneas, induced little-to-no disruption to corneal structure and biology for 3 months after the procedure. This affirms the relative safety of LIRIC and predicts that compared to traditional laser vision correction surgeries, common post-operative complications such as dry eye, haze, or patient discomfort may be entirely avoided.

Dynamic Preference for NADP/H Cofactor Binding/Release in E. coli YqhD Oxidoreductase.

YqhD, an E. coli alcohol/aldehyde oxidoreductase, is an enzyme able to produce valuable bio-renewable fuels and fine chemicals from a broad range of starting materials. Herein, we report the first computational solution-phase structure-dynamics analysis of YqhD, shedding light on the effect of oxidized and reduced NADP/H cofactor binding on the conformational dynamics of the biocatalyst using molecular dynamics (MD) simulations. The cofactor oxidation states mainly influence the interdomain cleft region conformations of the YqhD monomers, involved in intricate cofactor binding and release. The ensemble of NADPH-bound monomers has a narrower average interdomain space resulting in more hydrogen bonds and rigid cofactor binding. NADP-bound YqhD fluctuates between open and closed conformations, while it was observed that NADPH-bound YqhD had slower opening/closing dynamics of the cofactor-binding cleft. In the light of enzyme kinetics and structural data, simulation findings have led us to postulate that the frequently sampled open conformation of the cofactor binding cleft with NADP leads to the more facile release of NADP while increased closed conformation sampling during NADPH binding enhances cofactor binding affinity and the aldehyde reductase activity of the enzyme.

A compact high-precision periodic-error-free heterodyne interferometer.

We present the design, bench-top setup, and experimental results of a compact heterodyne interferometer that achieves picometer-level displacement sensitivities in air over frequencies above 100 MHz. The optical configuration with spatially separated beams prevents frequency and polarization mixing, and therefore eliminates periodic errors. The interferometer is designed to maximize common-mode optical laser beam paths to obtain high rejection of environmental disturbances, such as temperature fluctuations and acoustics. The results of our experiments demonstrate the short- and long-term stabilities of the system during stationary and dynamic measurements. In addition, we provide measurements that compare our interferometer prototype with a commercial system, verifying our higher sensitivity of 3 pm, higher thermal stability by a factor of two, and periodic-error-free performance.

Advances in optical metrology and instrumentation: introduction.

Optical measurement and characterization are two of the pillars of metrology. The ability to measure precisely with high dynamic range and accuracy betters our understanding of nature and the universe. In this feature issue, we present a collection of articles that delves into the fundamental techniques used to advance the field.

Tissue-specific alternative splicing remodels protein-protein interaction networks.

Alternative splicing plays a key role in the expansion of proteomic and regulatory complexity, yet the functions of the vast majority of differentially spliced exons are not known. In this study, we observe that brain and other tissue-regulated exons are significantly enriched in flexible regions of proteins that likely form conserved interaction surfaces. These proteins participate in significantly more interactions in protein-protein interaction (PPI) networks than other proteins. Using LUMIER, an automated PPI assay, we observe that approximately one-third of analyzed neural-regulated exons affect PPIs. Inclusion of these exons stimulated and repressed different partner interactions at comparable frequencies. This assay further revealed functions of individual exons, including a role for a neural-specific exon in promoting an interaction between Bridging Integrator 1 (Bin1)/Amphiphysin II and Dynamin 2 (Dnm2) that facilitates endocytosis. Collectively, our results provide evidence that regulated alternative exons frequently remodel interactions to establish tissue-dependent PPI networks.

A scoping review of initiatives to reduce inappropriate or non-beneficial hospital admissions and bed days in people nearing the end of their life: much innovation, but limited supporting evidence.

BACKGROUND: Hospitalisation during the last weeks of life when there is no medical need or desire to be there is distressing and expensive. This study sought palliative care initiatives which may avoid or shorten hospital stay at the end of life and analysed their success in terms reducing bed days. METHODS: Part 1 included a search of literature in PubMed and Google Scholar between 2013 and 2018, an examination of governmental and organisational publications plus discussions with external and co-author experts regarding other sources. This initial sweep sought to identify and categorise relevant palliative care initiatives. In Part 2, we looked for publications providing data on hospital admissions and bed days for each category. RESULTS: A total of 1252 abstracts were reviewed, resulting in ten broad classes being identified. Further screening revealed 50 relevant publications describing a range of multi-component initiatives. Studies were generally small and retrospective. Most researchers claim their service delivered benefits. In descending frequency, benefits identified were support in the community, integrated care, out-of-hours telephone advice, care home education and telemedicine. Nurses and hospices were central to many initiatives. Barriers and factors underpinning success were rarely addressed. CONCLUSIONS: A wide range of initiatives have been introduced to improve end-of-life experiences. Formal evidence supporting their effectiveness in reducing inappropriate/non-beneficial hospital bed days was generally limited or absent. TRIAL REGISTRATION: N/A.

Prospective longitudinal investigation shows correlation of event-related potential to mild traumatic brain injury in adolescents.

STUDY DESIGN: Prospective longitudinal cohort study. BACKGROUND: Adolescent athletes may be more susceptible to the long-term effects of mild traumatic brain injury (mTBI). A diagnostic and prognostic neuromarker may optimize management and return-to-activity decision-making in athletes who experience mTBI. OBJECTIVE: Measure an event-related potential (ERP) component captured with electroencephalography (EEG), called processing negativity (PN), at baseline and post-injury in adolescents who suffered mTBI and determine their longitudinal response relative to healthy controls. METHODS: Thirty adolescents had EEG recorded during an auditory oddball task at a pre-mTBI baseline session and subsequent post-mTBI sessions. Longitudinal EEG data from patients and healthy controls (n= 77) were obtained from up to four sessions in total and processed using Brain Network Analysis algorithms. RESULTS: The average PN amplitude in healthy controls significantly decreased over sessions 2 and 3; however, it remained steady in the mTBI group's 2nd (post-mTBI) session and decreased only in sessions 3 and 4. Pre- to post-mTBI amplitude changes correlated with the time interval between sessions. CONCLUSION: These results demonstrate that PN amplitude changes may be associated with mTBI exposure and subsequent recovery in adolescent athletes. Further study of PN may lead to it becoming a neuromarker for mTBI prognosis and return-to-activity decision-making in adolescents.

Rationalization in the pejorative sense: Cushman's account overlooks the scope and costs of rationalization.

According to Cushman, rationalization occurs when a person has performed an action and then concocts beliefs and desires that would have made it rational. We argue that this isn't the paradigmatic form of rationalization. Consequently, Cushman's explanation of the function and usefulness of rationalization is less broad-reaching than he intends. Cushman's account also obscures some of rationalization's pernicious consequences.

Whole-genome sequencing of human malignant mesothelioma tumours and cell lines.

Pleural mesothelioma is a cancer of serosal surfaces caused by environmental exposure to asbestos. Clinical outcome remains poor and while trials of new treatments are ongoing it remains an understudied cancer. Mesothelioma cell lines can readily be grown from primary tumour and from tumour cells shed into pleural effusion with the latter representing a particularly valuable source of DNA in clinical settings, procurable without the need for additional invasive procedures. However, it is not well understood how accurately patient-derived cultured tumour cells represent the molecular characteristics of their primary tumour. We used whole-genome sequencing of primary tumour and matched cultured cells to comprehensively characterize mutations and structural alterations. Most cases had complex rearranged genomes with evidence of chromoanagenesis and rearrangements reminiscent of chromoplexy. Many of the identified driver mutations were structural, indicating that mesothelioma is often caused by structural alterations and catastrophic genomic events, rather than point mutations. Because the majority of genomic changes detected in tumours were also displayed by the genomes of cultured tumour cells, we conclude that low-passage cultured tumour cells are generally suitable for molecular characterization of mesothelioma and may be particularly useful where tissue samples with high tumour cell content are not available. However, the subclonal compositions of the cell lines did not fully recapitulate the subclonal diversity of the primary tumours. Furthermore, longitudinal acquisition of major alterations in subclonal cell populations was observed after long-term passaging. These two factors define limitations of tumour-derived cell lines as genomic substrate for clinical purposes.

RNA-Seq analysis of chikungunya virus infection and identification of granzyme A as a major promoter of arthritic inflammation.

Chikungunya virus (CHIKV) is an arthritogenic alphavirus causing epidemics of acute and chronic arthritic disease. Herein we describe a comprehensive RNA-Seq analysis of feet and lymph nodes at peak viraemia (day 2 post infection), acute arthritis (day 7) and chronic disease (day 30) in the CHIKV adult wild-type mouse model. Genes previously shown to be up-regulated in CHIKV patients were also up-regulated in the mouse model. CHIKV sequence information was also obtained with up to ≈8% of the reads mapping to the viral genome; however, no adaptive viral genome changes were apparent. Although day 2, 7 and 30 represent distinct stages of infection and disease, there was a pronounced overlap in up-regulated host genes and pathways. Type I interferon response genes (IRGs) represented up to ≈50% of up-regulated genes, even after loss of type I interferon induction on days 7 and 30. Bioinformatic analyses suggested a number of interferon response factors were primarily responsible for maintaining type I IRG induction. A group of genes prominent in the RNA-Seq analysis and hitherto unexplored in viral arthropathies were granzymes A, B and K. Granzyme A-/- and to a lesser extent granzyme K-/-, but not granzyme B-/-, mice showed a pronounced reduction in foot swelling and arthritis, with analysis of granzyme A-/- mice showing no reductions in viral loads but reduced NK and T cell infiltrates post CHIKV infection. Treatment with Serpinb6b, a granzyme A inhibitor, also reduced arthritic inflammation in wild-type mice. In non-human primates circulating granzyme A levels were elevated after CHIKV infection, with the increase correlating with viral load. Elevated granzyme A levels were also seen in a small cohort of human CHIKV patients. Taken together these results suggest granzyme A is an important driver of arthritic inflammation and a potential target for therapy. TRIAL REGISTRATION: ClinicalTrials.gov NCT00281294.

Comprehensive genetic screening: The prevalence of maturity-onset diabetes of the young gene variants in a population-based childhood diabetes cohort.

BACKGROUND: Maturity-onset diabetes of the young (MODY) is caused by autosomal dominant mutations in one of 13 confirmed genes. Estimates of MODY prevalence vary widely, as genetic screening is usually restricted based on clinical features, even in population studies. We aimed to determine prevalence of MODY variants in a large and unselected pediatric diabetes cohort. METHODS: MODY variants were assessed using massively parallel sequencing in the population-based diabetes cohort (n = 1363) of the sole tertiary pediatric diabetes service for Western Australia (population 2.6 million). All individuals were screened, irrespective of clinical features. MODY variants were also assessed in a control cohort (n = 993). RESULTS: DNA and signed consent were available for 821 children. Seventeen children had pathogenic/likely pathogenic variants in MODY genes, two diagnosed with type 2 diabetes, four diagnosed with antibody-negative type 1 diabetes (T1DM), three diagnosed with antibody-positive T1DM, and eight previously diagnosed with MODY. Prevalence of MODY variants in the sequenced cohort was 2.1%, compared to 0.3% of controls. CONCLUSIONS: This is the first comprehensive study of MODY variants in an unselected population-based pediatric diabetes cohort. The observed prevalence, increasing access to rapid and affordable genetic screening, and significant clinical implications suggest that genetic screening for MODY could be considered for all children with diabetes, irrespective of other clinical features.