Secondary structure prediction of long noncoding RNA: review and experimental comparison of existing approaches.

MOTIVATION: In contrast to messenger RNAs, the function of the wide range of existing long noncoding RNAs (lncRNAs) largely depends on their structure, which determines interactions with partner molecules. Thus, the determination or prediction of the secondary structure of lncRNAs is critical to uncover their function. Classical approaches for predicting RNA secondary structure have been based on dynamic programming and thermodynamic calculations. In the last 4 years, a growing number of machine learning (ML)-based models, including deep learning (DL), have achieved breakthrough performance in structure prediction of biomolecules such as proteins and have outperformed classical methods in short transcripts folding. Nevertheless, the accurate prediction for lncRNA still remains far from being effectively solved. Notably, the myriad of new proposals has not been systematically and experimentally evaluated. RESULTS: In this work, we compare the performance of the classical methods as well as the most recently proposed approaches for secondary structure prediction of RNA sequences using a unified and consistent experimental setup. We use the publicly available structural profiles for 3023 yeast RNA sequences, and a novel benchmark of well-characterized lncRNA structures from different species. Moreover, we propose a novel metric to assess the predictive performance of methods, exclusively based on the chemical probing data commonly used for profiling RNA structures, avoiding any potential bias incorporated by computational predictions when using dot-bracket references. Our results provide a comprehensive comparative assessment of existing methodologies, and a novel and public benchmark resource to aid in the development and comparison of future approaches. AVAILABILITY: Full source code and benchmark datasets are available at: https://github.com/sinc-lab/lncRNA-folding. CONTACT: lbugnon@sinc.unl.edu.ar.

Loss-of-Function Variants in PPP1R12A: From Isolated Sex Reversal to Holoprosencephaly Spectrum and Urogenital Malformations.

In two independent ongoing next-generation sequencing projects for individuals with holoprosencephaly and individuals with disorders of sex development, and through international research collaboration, we identified twelve individuals with de novo loss-of-function (LoF) variants in protein phosphatase 1, regulatory subunit 12a (PPP1R12A), an important developmental gene involved in cell migration, adhesion, and morphogenesis. This gene has not been previously reported in association with human disease, and it has intolerance to LoF as illustrated by a very low observed-to-expected ratio of LoF variants in gnomAD. Of the twelve individuals, midline brain malformations were found in five, urogenital anomalies in nine, and a combination of both phenotypes in two. Other congenital anomalies identified included omphalocele, jejunal, and ileal atresia with aberrant mesenteric blood supply, and syndactyly. Six individuals had stop gain variants, five had a deletion or duplication resulting in a frameshift, and one had a canonical splice acceptor site loss. Murine and human in situ hybridization and immunostaining revealed PPP1R12A expression in the prosencephalic neural folds and protein localization in the lower urinary tract at critical periods for forebrain division and urogenital development. Based on these clinical and molecular findings, we propose the association of PPP1R12A pathogenic variants with a congenital malformations syndrome affecting the embryogenesis of the brain and genitourinary systems and including disorders of sex development.

Subtle hydrogen bond preference and dual Franck-Condon activity - the interesting pairing of 2-naphthol with anisole.

The hydrogen-bonded complexes between 2-naphthol (or ß-naphthol) and anisole are explored by detecting their IR absorption in the OH stretching range as well as their UV absorption by means of laser-induced fluorescence and resonance-enhanced two-photon UV ionisation. For the more stable cis and the metastable trans conformations of the OH group in 2-naphthol, hydrogen bonding to the oxygen atom of anisole is consistently detected in different supersonic jet expansions. Alternative hydrogen bonding to the aromatic ring of anisole remains elusive, although the majority of state-of-the-art hybrid DFT functionals with London dispersion correction and - less surprisingly - MP2 wavefunction theory predict it to be slightly more stable at zero-point level, unless three-body dispersion correction is added to the B3LYP-D3(BJ) approach. This changes at the CCSD(T) level, which forecasts an energy advantage of 1-3 kJ mol-1 for the classical hydrogen bond arrangement even after including (DFT) zero-point energy contributions. The UV and IR spectra of the cis complex exhibit clear evidence for intensity redistribution of the primary OH stretch oscillator to combination states with the same low-frequency intermolecular bending mode by Franck-Condon-type vertical excitation mechanisms. This rare case of dual (vibronic and vibrational) Franck-Condon activity of a low-frequency mode invites future studies of homologues where aromatic ring docking of the OH group may be further stabilised, e.g. through anisole ring methylation.

Unveiling the Configurational Landscape of Carbamate: Paving the Way for Designing Functional Sequence-Defined Polymers.

Carbamate is an emerging class of a polymer backbone for constructing sequence-defined, abiotic polymers. It is expected that new functional materials can be de novo designed by controlling the primary polycarbamate sequence. While amino acids have been actively studied as building blocks for protein folding and peptide self-assembly, carbamates have not been widely investigated from this perspective. Here, we combined infrared (IR), vibrational circular dichroism (VCD), and nuclear magnetic resonance (NMR) spectroscopy with density functional theory (DFT) calculations to understand the conformation of carbamate monomer units in a nonpolar, aprotic environment (chloroform). Compared with amino acid building blocks, carbamates are more rigid, presumably due to the extended delocalization of π-electrons on the backbones. Cis configurations of the amide bond can be energetically stable in carbamates, whereas peptides often assume trans configurations at low energies. This study lays an essential foundation for future developments of carbamate-based sequence-defined polymer material design.

A CCR4-NOT Transcription Complex, Subunit 1, CNOT1, Variant Associated with Holoprosencephaly.

Holoprosencephaly is the incomplete separation of the forebrain during embryogenesis. Both genetic and environmental etiologies have been determined for holoprosencephaly; however, a genetic etiology is not found in most cases. In this report, we present two unrelated individuals with semilobar holoprosencephaly who have the identical de novo missense variant in the gene CCR4-NOT transcription complex, subunit 1 (CNOT1). The variant (c.1603C>T [p.Arg535Cys]) is predicted to be deleterious and is not present in public databases. CNOT1 has not been previously associated with holoprosencephaly or other brain malformations. In situ hybridization analyses of mouse embryos show that Cnot1 is expressed in the prosencephalic neural folds at gestational day 8.25 during the critical period for subsequent forebrain division. Combining human and mouse data, we show that CNOT1 is associated with incomplete forebrain division.

Propagule size and sex ratio influence colonisation dynamics after introduction of a non-native lizard.

The composition of founding populations plays an important role in colonisation dynamics and can influence population growth during early stages of biological invasion. Specifically, founding populations with small propagules (i.e. low number of founders) are vulnerable to the Allee effect and have reduced likelihood of establishment compared to those with large propagules. The founding sex ratio can also impact establishment via its influence on mating success and offspring production. Our goal was to test the effects of propagule size and sex ratio on offspring production and annual population growth following introductions of a non-native lizard species (Anolis sagrei). We manipulated propagule composition on nine small islands, then examined offspring production, population growth and survival rate of founders and their descendants encompassing three generations. By the third reproductive season, per capita offspring production was higher on islands seeded with a relatively large propagule size, but population growth was not associated with propagule size. Propagule sex ratio did not affect offspring production, but populations with a female-biased propagule had positive growth, whereas those with a male-biased propagule had negative growth in the first year. Populations were not affected by propagule sex ratio in subsequent years, possibly due to rapid shifts towards balanced (or slightly female biased) population sex ratios. Overall, we show that different components of population fitness have different responses to propagule size and sex ratio in ways that could affect early stages of biological invasion. Despite these effects, the short life span and high fecundity of A. sagrei likely helped small populations to overcome Allee effects and enabled all populations to successfully establish. Our rare experimental manipulation of propagule size and sex ratio can inform predictions of colonisation dynamics in response to different compositions of founding populations, which is critical in the context of population ecology and invasion dynamics.

Detection by multiplex PCR of Mycoplasma species associated with dairy cattle in Argentina.

There is scarce information about the frequency and epidemiological and clinical features associated with the presence of Mycoplasma spp. in Argentine dairy herds. The objectives of this study were to develop a multiplex PCR for identifying M.bovis and M.canadense and to describe the frequency of Mycoplasma spp. isolated from clinical samples submitted to a diagnostic laboratory. Of a total of 1548 samples from intramammary infections, bulk tank milk and biological fluids, 38 Mycoplasma isolates were obtained. M. bovis, M. canadense, M.californicum and M.leachii were detected by using two multiplex PCRs, confirming their presence in clinical conditions in dairy cattle. The techniques used in the present study can be useful to broaden the knowledge about Mycoplasma infections in cattle, since the search for these organisms is not usually included in routine diagnoses.

Eliciting the educational priorities of home care workers caring for adults with heart failure.

BACKGROUND: Although home care workers (HCWs) are increasingly caring for adults with heart failure (HF), many feel unprepared and lack HF training. To serve as the foundation for a future educational intervention, we aimed to elicit HCWs' HF educational needs. METHODS: We partnered with the largest healthcare union in the US to recruit 41 HCWs employed by 17 home care agencies. Using the nominal group technique, we asked HCWs to respond to three questions: When caring for an HF patient: (1) What information do you want? (2) What symptoms worry you? (3) What situations do you struggle with? Participants ranked their responses by priority. Data were consolidated by question. RESULTS: For question 1, participants ranked HF signs and symptoms most highly, followed by HF treatment and medications. For question 2, chest pain was most worrisome, followed by neurologic changes and shortness of breath. For question 3, participants struggled with encouraging patients to follow a heart-specific diet. CONCLUSIONS: HCWs expressed a need to learn more about signs and symptoms of HF and ways to assist patients with HF self-care. These findings can inform the development of an HF training program for HCWs that specifically addresses their expressed needs.

An integrative model of cancer cell differentiation with immunotherapy.

In order to improve cancer treatments, cancer cell differentiation and immunotherapy are the subjects of several studies in different branches of interdisciplinary sciences. In this work, we develop a new population model that integrates other complementary ones, thus emphasizing the relationship between cancer cells at different differentiation stages and the main immune system cells. For this new system, specific ranges were found where transdifferentiation of differentiated cancer cells can occur. In addition, a specific therapy against cancer stem cells was analysed by simulating cytotoxic cell vaccines. In reference to the latter, the different combinations of parameters that optimize it were studied.

Determination of kinetic properties in unimolecular dissociation of complex systems from graph theory based analysis of an ensemble of reactive trajectories.

In this paper, we report how graph theory can be used to analyze an ensemble of independent molecular trajectories, which can react during the simulation time-length, and obtain structural and kinetic information. This method is totally general and here is applied to the prototypical case of gas phase fragmentation of protonated cyclo-di-glycine. This methodology allows us to analyze the whole set of trajectories in an automatic computer-based way without the need of visual inspection but by getting all the needed information. In particular, we not only determine the appearance of different products and intermediates but also characterize the corresponding kinetics. The use of colored graph and canonical labeling allows for the correct characterization of the chemical species involved. In the present case, the simulations consist of an ensemble of unimolecular fragmentation trajectories at constant energy such that from the rate constants at different energies, the threshold energy can also be obtained for both global and specific pathways. This approach allows for the characterization of ion-molecule complexes, likely through a roaming mechanism, by properly taking into account the elusive nature of such species. Finally, it is possible to directly obtain the theoretical mass spectrum of the fragmenting species if the reacting system is an ion as in the specific example.

Eliciting the educational needs and priorities of home care workers on end-of-life care for patients with heart failure using nominal group technique.

BACKGROUND: Home care workers, as paid caregivers, assist with many aspects of home-based heart failure care. However, most home care workers do not receive systematic training on end-of-life care for heart failure patients. AIM: To elicit the educational needs and priorities of home care workers caring for community- dwelling adults with heart failure at the end-of-life. DESIGN: Nominal group technique involving a semi-quantitative structured group process and point rating system was used to designate the importance of priorities elicited from home care workers. Individual responses to the question, "If you have ever cared for a heart failure patient who was dying (or receiving end-of-life care on hospice), what are some of the challenges you faced?", were aggregated into categories using directed content analysis methods. SETTING/PARTICIPANTS: Forty-one home care workers were recruited from a non-profit training and education organization in New York City. RESULTS: Individual responses to the question were aggregated into five categories: (1) how to cope and grieve; (2) assisting patients with behavior changes, (3) supporting patients to improve their quality of life, (4) assisting patients with physical symptom management, and (5) symptom recognition and assessment. CONCLUSIONS: Our findings confirm the need for the formal development and evaluation of an educational program for home care workers to improve the care of heart failure patients at the end-of-life. There is also a need for research on integrating home care workers into the interprofessional healthcare team to support optimal health outcomes for patients with heart failure.

The CHD4-related syndrome: a comprehensive investigation of the clinical spectrum, genotype-phenotype correlations, and molecular basis.

PURPOSE: Sifrim-Hitz-Weiss syndrome (SIHIWES) is a recently described multisystemic neurodevelopmental disorder caused by de novo variants inCHD4. In this study, we investigated the clinical spectrum of the disorder, genotype-phenotype correlations, and the effect of different missense variants on CHD4 function. METHODS: We collected clinical and molecular data from 32 individuals with mostly de novo variants in CHD4, identified through next-generation sequencing. We performed adenosine triphosphate (ATP) hydrolysis and nucleosome remodeling assays on variants from five different CHD4 domains. RESULTS: The majority of participants had global developmental delay, mild to moderate intellectual disability, brain anomalies, congenital heart defects, and dysmorphic features. Macrocephaly was a frequent but not universal finding. Additional common abnormalities included hypogonadism in males, skeletal and limb anomalies, hearing impairment, and ophthalmic abnormalities. The majority of variants were nontruncating and affected the SNF2-like region of the protein. We did not identify genotype-phenotype correlations based on the type or location of variants. Alterations in ATP hydrolysis and chromatin remodeling activities were observed in variants from different domains. CONCLUSION: The CHD4-related syndrome is a multisystemic neurodevelopmental disorder. Missense substitutions in different protein domains alter CHD4 function in a variant-specific manner, but result in a similar phenotype in humans.

Correction: The CHD4-related syndrome: a comprehensive investigation of the clinical spectrum, genotype-phenotype correlations, and molecular basis.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Understanding the Workflow of Home Health Care for Patients with Heart Failure: Challenges and Opportunities.

BACKGROUND: Readmission rates are high among heart failure (HF) patients who require home health care (HHC) after hospitalization. Although HF patients who require HHC are often sicker than those who do not, HHC delivery itself may also be suboptimal. OBJECTIVE: We aimed to describe the workflow of HHC among adults discharged home after a HF hospitalization, including the roles of various stakeholders, and to determine where along these workflow challenges and opportunities for improvement exist. DESIGN AND PARTICIPANTS: In this qualitative study, we used purposeful sampling to approach and recruit a variety of key stakeholders including home health aides, nurses, HF patients, family caregivers, physicians, social workers, home care agency leaders, and policy experts. The study took place in New York, NY, from March to October 2018. APPROACH: Using a semi-structured topic guide, we elicited participants' experiences with HHC in HF through a combination of one-on-one interviews and focus groups. Data were recorded, transcribed, and analyzed thematically. We also asked selected participants to depict in a drawing their understanding of HHC workflow after hospitalization for HF patients. We synthesized the drawings into a final image. KEY RESULTS: Study participants (N = 80) described HHC for HF patients occurring in 6 steps, with home health aides playing a main role: (1) transitioning from hospital to home; (2) recognizing clinical changes; (3) making decisions; (4) managing symptoms; (5) asking for help; and (6) calling 911. Participants identified challenges and opportunities for improvement for each step. CONCLUSIONS: Our findings suggest that HHC for HF patients occurs in discrete steps, each with different challenges. Rather than a one-size-fits-all approach, various interventions may be required to optimize HHC delivery for HF patients in the post-discharge period.

Cohesin complex-associated holoprosencephaly.

Marked by incomplete division of the embryonic forebrain, holoprosencephaly is one of the most common human developmental disorders. Despite decades of phenotype-driven research, 80-90% of aneuploidy-negative holoprosencephaly individuals with a probable genetic aetiology do not have a genetic diagnosis. Here we report holoprosencephaly associated with variants in the two X-linked cohesin complex genes, STAG2 and SMC1A, with loss-of-function variants in 10 individuals and a missense variant in one. Additionally, we report four individuals with variants in the cohesin complex genes that are not X-linked, SMC3 and RAD21. Using whole mount in situ hybridization, we show that STAG2 and SMC1A are expressed in the prosencephalic neural folds during primary neurulation in the mouse, consistent with forebrain morphogenesis and holoprosencephaly pathogenesis. Finally, we found that shRNA knockdown of STAG2 and SMC1A causes aberrant expression of HPE-associated genes ZIC2, GLI2, SMAD3 and FGFR1 in human neural stem cells. These findings show the cohesin complex as an important regulator of median forebrain development and X-linked inheritance patterns in holoprosencephaly.

Identification of a nuclear localization signal and importin beta members mediating NUAK1 nuclear import inhibited by oxidative stress.

NUAK1 is a serine/threonine kinase member of the AMPK-α family. NUAK1 regulates several processes in tumorigenesis; however, its regulation and molecular targets are still poorly understood. Bioinformatics analysis predicted that the majority of NUAK1 localizes in the nucleus. However, there are no studies about the regulation of NUAK1 subcellular distribution. Here, we analyzed NUAK1 localization in several human cell lines, mouse embryo fibroblasts, and normal mouse tissues. We found that NUAK1 is located in the nucleus and also in the cytoplasm. Through bioinformatics analysis and studies comparing subcellular localization of wild type and NUAK1 mutants, we identified a conserved bipartite nuclear localization signal at the N-terminal domain of NUAK1. Based on mass spectrometry analysis, we found that NUAK1 interacts with importin-ß members including importin-ß1 (KPNB1), importin-7 (IPO7), and importin-9 (IPO9). We confirmed that importin-ß members are responsible for NUAK1 nuclear import through the inhibition of importin-ß by Importazole and the knockdown of either IPO7 or IPO9. In addition, we found that oxidative stress induces NUAK1 cytoplasmic accumulation, indicating that oxidative stress affects NUAK1 nuclear transport. Thus, our study is the first evidence of an active nuclear transport mechanism regulating NUAK1 subcellular localization. These data will lead to investigations of the molecular targets of NUAK1 according to its subcellular distribution, which could be new biomarkers or targets for cancer therapies.

ß-catenin aggregation in models of ALS motor neurons: GSK3ß inhibition effect and neuronal differentiation.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by motor neuron death. A 20% of familial ALS cases are associated with mutations in the gene coding for superoxide dismutase 1 (SOD1). The accumulation of abnormal aggregates of different proteins is a common feature in motor neurons of patients and transgenic ALS mice models, which are thought to contribute to disease pathogenesis. Developmental morphogens, such as the Wnt family, regulate numerous features of neuronal physiology in the adult brain and have been implicated in neurodegeneration. ß-catenin is a central mediator of both, Wnt signaling activity and cell-cell interactions. We previously reported that the expression of mutant SOD1 in the NSC34 motor neuron cell line decreases basal Wnt pathway activity, which correlates with cytosolic ß-catenin accumulation and impaired neuronal differentiation. In this work, we aimed a deeper characterization of ß-catenin distribution in models of ALS motor neurons. We observed extensive accumulation of ß-catenin supramolecular structures in motor neuron somas of pre-symptomatic mutant SOD1 mice. In cell-cell appositional zones of NSC34 cells expressing mutant SOD1, ß-catenin displays a reduced co-distribution with E-cadherin accompanied by an increased association with the gap junction protein Connexin-43; these findings correlate with impaired intercellular adhesion and exacerbated cell coupling. Remarkably, pharmacological inhibition of the glycogen synthase kinase-3ß (GSK3ß) in both NSC34 cell lines reverted both, ß-catenin aggregation and the adverse effects of mutant SOD1 expression on neuronal differentiation. Our findings suggest that early defects in ß-catenin distribution could be an underlying factor affecting the onset of neurodegeneration in familial ALS.

De Novo Mutations in CHD4, an ATP-Dependent Chromatin Remodeler Gene, Cause an Intellectual Disability Syndrome with Distinctive Dysmorphisms.

Chromodomain helicase DNA-binding protein 4 (CHD4) is an ATP-dependent chromatin remodeler involved in epigenetic regulation of gene transcription, DNA repair, and cell cycle progression. Also known as Mi2ß, CHD4 is an integral subunit of a well-characterized histone deacetylase complex. Here we report five individuals with de novo missense substitutions in CHD4 identified through whole-exome sequencing and web-based gene matching. These individuals have overlapping phenotypes including developmental delay, intellectual disability, hearing loss, macrocephaly, distinct facial dysmorphisms, palatal abnormalities, ventriculomegaly, and hypogonadism as well as additional findings such as bone fusions. The variants, c.3380G>A (p.Arg1127Gln), c.3443G>T (p.Trp1148Leu), c.3518G>T (p.Arg1173Leu), and c.3008G>A, (p.Gly1003Asp) (GenBank: NM_001273.3), affect evolutionarily highly conserved residues and are predicted to be deleterious. Previous studies in yeast showed the equivalent Arg1127 and Trp1148 residues to be crucial for SNF2 function. Furthermore, mutations in the same positions were reported in malignant tumors, and a de novo missense substitution in an equivalent arginine residue in the C-terminal helicase domain of SMARCA4 is associated with Coffin Siris syndrome. Cell-based studies of the p.Arg1127Gln and p.Arg1173Leu mutants demonstrate normal localization to the nucleus and HDAC1 interaction. Based on these findings, the mutations potentially alter the complex activity but not its formation. This report provides evidence for the role of CHD4 in human development and expands an increasingly recognized group of Mendelian disorders involving chromatin remodeling and modification.

Low-level parental mosaicism affects the recurrence risk of holoprosencephaly.

PURPOSE: De novo variants (DNVs) represent an important fraction of the pathogenic variant burden in holoprosencephaly (HPE). However, unexpected recurrences can occur, as evidenced by multiple affected children harboring the same apparently DNV. This study was performed to estimate the rate of parental mosaicism in a cohort of patients with HPE. METHODS: We developed a targeted capture next-generation sequencing (NGS) panel of 153 genes with potential implication in HPE. Sequencing data from a cohort of 136 HPE family trios were analyzed to identify probands with apparently DNVs. DNVs were examined in the proband and their parents to detect any deviations from the expected ~50/50 allele ratio of true heterozygosity. Selected variants were confirmed by Droplet Digital™ polymerase chain reaction (ddPCR). RESULTS: We identified 28 high-confidence DNVs, 20 of which occurred in known HPE genes. Nineteen of the 20 variants (95%) were pathogenic or likely pathogenic. Sequence data analysis showed evidence of parental mosaicism in five cases, for an overall mosaicism rate of 26%. In addition, we found evidence for likely postzygotic events in four cases (50%). CONCLUSIONS: High sensitivity methods, such as high-depth NGS and ddPCR, are essential to providing an accurate assessment of recurrence risk in HPE families with apparently DNVs.