Transcriptome innovations in primates revealed by single-molecule long-read sequencing.

Transcriptomic diversity greatly contributes to the fundamentals of disease, lineage-specific biology, and environmental adaptation. However, much of the actual isoform repertoire contributing to shaping primate evolution remains unknown. Here, we combined deep long- and short-read sequencing complemented with mass spectrometry proteomics in a panel of lymphoblastoid cell lines (LCLs) from human, three other great apes, and rhesus macaque, producing the largest full-length isoform catalog in primates to date. Around half of the captured isoforms are not annotated in their reference genomes, significantly expanding the gene models in primates. Furthermore, our comparative analyses unveil hundreds of transcriptomic innovations and isoform usage changes related to immune function and immunological disorders. The confluence of these evolutionary innovations with signals of positive selection and their limited impact in the proteome points to changes in alternative splicing in genes involved in immune response as an important target of recent regulatory divergence in primates.

A New Chromosome-Assigned Mongolian Gerbil Genome Allows Characterization of Complete Centromeres and a Fully Heterochromatic Chromosome.

Chromosome-scale genome assemblies based on ultralong-read sequencing technologies are able to illuminate previously intractable aspects of genome biology such as fine-scale centromere structure and large-scale variation in genome features such as heterochromatin, GC content, recombination rate, and gene content. We present here a new chromosome-scale genome of the Mongolian gerbil (Meriones unguiculatus), which includes the complete sequence of all centromeres. Gerbils are thus the one of the first vertebrates to have their centromeres completely sequenced. Gerbil centromeres are composed of four different repeats of length 6, 37, 127, or 1,747 bp, which occur in simple alternating arrays and span 1-6 Mb. Gerbil genomes have both an extensive set of GC-rich genes and chromosomes strikingly enriched for constitutive heterochromatin. We sought to determine if there was a link between these two phenomena and found that the two heterochromatic chromosomes of the Mongolian gerbil have distinct underpinnings: Chromosome 5 has a large block of intraarm heterochromatin as the result of a massive expansion of centromeric repeats, while chromosome 13 is comprised of extremely large (>150 kb) repeated sequences. In addition to characterizing centromeres, our results demonstrate the importance of including karyotypic features such as chromosome number and the locations of centromeres in the interpretation of genome sequence data and highlight novel patterns involved in the evolution of chromosomes.

End-to-end approach for the characterization and control of product-related impurities in T cell bispecific antibody preparations.

Antibody-based T cell-activating biologics are promising therapeutic medicines being developed for a number of indications, mainly in the oncology field. Among those, T cell bispecific antibodies are designed to bind one tumor-specific antigen and the T cell receptor at the same time, leading to a robust T cell response against the tumor. Although their unique format and the versatility of the CrossMab technology allows for the generation of safer molecules in an efficient manner, product-related variants cannot be completely avoided. Therefore, it is of extreme importance that both a manufacturing process that limits or depletes product-related impurities, as well as a thorough analytical characterization are in place, starting from the development of the manufacturing cell line until the assessment of potential toxicities. Here, we describe such an end-to-end approach to minimize, quantify and control impurities and -upon their functional characterization- derive specifications that allow for the release of clinical material.

Y chromosome sequence and epigenomic reconstruction across human populations.

Recent advances in long-read sequencing technologies have allowed the generation and curation of more complete genome assemblies, enabling the analysis of traditionally neglected chromosomes, such as the human Y chromosome (chrY). Native DNA was sequenced on a MinION Oxford Nanopore Technologies sequencing device to generate genome assemblies for seven major chrY human haplogroups. We analyzed and compared the chrY enrichment of sequencing data obtained using two different selective sequencing approaches: adaptive sampling and flow cytometry chromosome sorting. We show that adaptive sampling can produce data to create assemblies comparable to chromosome sorting while being a less expensive and time-consuming technique. We also assessed haplogroup-specific structural variants, which would be otherwise difficult to study using short-read sequencing data only. Finally, we took advantage of this technology to detect and profile epigenetic modifications among the considered haplogroups. Altogether, we provide a framework to study complex genomic regions with a simple, fast, and affordable methodology that could be applied to larger population genomics datasets.

Gender-associated differences of perforin polymorphisms in the susceptibility to multiple sclerosis.

The granule-dependent exocytosis pathway is an important mechanism to induce apoptosis by CD8(+) T cells and NK cells and involves lytic molecules such as perforin. In the current study, we investigated the perforin 1 gene (PRF1) as a candidate for multiple sclerosis (MS) susceptibility in the Spanish population. We genotyped three PRF1 single nucleotide polymorphisms (rs885822, rs10999426, and rs3758562) in 420 patients with MS and 512 controls. Associations of PRF1 polymorphisms with the disease were restricted to male patients with MS, and the finding was consistently observed at the allele, genotype, and haplotype levels. Gender-associated differences were validated in an additional replication cohort comprised of 292 MS cases and 300 controls. In addition, we identified minor risk haplotypes strongly associated with male patients having primary progressive MS (PPMS). Further characterization of male patients with PPMS carrying the risk haplotypes by means of gene expression microarrays revealed overrepresentation of the cell killing gene ontology category among downregulated genes in these patients compared with male patients with PPMS carrying protective haplotypes. Moreover, PRF1 mRNA expression levels were significantly lower in patients with risk haplotypes, and changes in perforin protein expression by CD8(+) T cells mirrored those observed in gene expression. These findings suggest a gender dimorphism in the PRF1 association with MS and point to the presence of a generalized defect in the expression of genes that code for proteins involved in cell killing in a subgroup of male patients with PPMS.

Emerging Animal-Associated Fungal Diseases.

The Global Action Fund for Fungal Infections (GAFFI) estimates that fungal diseases kill around 150 people each hour, and yet they are globally overlooked and neglected. Histoplasma and Talaromyces, which are associated with wildlife, cause systemic infections that are often lethal in patients with impaired cellular immunity. Dermatophytes that cause outbreaks in human hosts are often associated with domesticated animals. Changes in human behavior have been identified as a main cause of the emergence of animal-associated fungal diseases in humans, sometimes caused by the disturbance of natural habitats. An understanding of ecology and the transmission modes of causative agents is therefore essential. Here, we focus on fungal diseases contracted from wildlife and domesticated animals, their habitats, feces and carcasses. We discuss some basic fungal lifestyles and the risk of transmission to humans and illustrate these with examples from emerging and established diseases.

A Short Functional Neuroimaging Assay Using Attachment Scenes to Recruit Neural Correlates of Social Cognition-A Replication Study.

Attachment theory provides a conceptual framework to understand the impact of early child-caregiver experiences, such as loss or separation, on adult functioning and psychopathology. In the current study, scenes from the Adult Attachment Projective Picture System (AAP), a validated, commonly used standardized diagnostic instrument to assess adult attachment representations, were used to develop a short fMRI assay eliciting the neural correlates of encoding of potentially hurtful and threatening social situations such as social losses, rejections or loneliness. Data from healthy participants (N = 19) showed activations in brain areas associated with social cognition and semantic knowledge during exposure to attachment-related scenes compared to control scenes. Extensive activation of the temporal poles was observed, suggesting the use of semantic knowledge for generating social concepts and scripts. This knowledge may underlie our ability to explain and predict social interactions, a specific aspect of theory of mind or mentalization. In this replication study, we verified the effectiveness of a modified fMRI assay to assess the external validity of a previously used imaging paradigm to investigate the processing of emotionally negatively valenced and painful social interactions. Our data confirm the recruitment of brain areas associated with social cognition with our very short neuroimaging assay.

Spectroscopically well-characterized RGD optical probe as a prerequisite for lifetime-gated tumor imaging.

Labeling of RGD peptides with near-infrared fluorophores yields optical probes for noninvasive imaging of tumors overexpressing ανß3 integrins. An important prerequisite for optimum detection sensitivity in vivo is strongly absorbing and highly emissive probes with a known fluorescence lifetime. The RGD-Cy5.5 optical probe was derived by coupling Cy5.5 to a cyclic arginine-glycine-aspartic acid-d-phenylalanine-lysine (RGDfK) peptide via an aminohexanoic acid spacer. Spectroscopic properties of the probe were studied in different matrices in comparison to Cy5.5. For in vivo imaging, human glioblastoma cells were subcutaneously implanted into nude mice, and in vivo fluorescence intensity and lifetime were measured. The fluorescence quantum yield and lifetime of Cy5.5 were found to be barely affected on RGD conjugation but dramatically changed in the presence of proteins. By time domain fluorescence imaging, we demonstrated specific binding of RGD-Cy5.5 to glioblastoma xenografts in nude mice. Discrimination of unspecific fluorescence by lifetime-gated analysis further enhanced the detection sensitivity of RGD-Cy5.5-derived signals. We characterized RGD-Cy5.5 as a strongly emissive and stable probe adequate for selective targeting of ανß3 integrins. The specificity and thus the overall detection sensitivity in vivo were optimized with lifetime gating, based on the previous determination of the probes fluorescence lifetime under application-relevant conditions.

Cerebrospinal fluid chitinase 3-like 1 levels are associated with conversion to multiple sclerosis.

In most patients with multiple sclerosis, the disease initiates with a first attack or clinically isolated syndrome. At this phase, magnetic resonance imaging is an important predictor of conversion to multiple sclerosis. With the exception of oligoclonal bands, the role of other biomarkers in patients with clinically isolated syndrome is controversial. In the present study, we aimed to identify proteins associated with conversion to multiple sclerosis in patients with clinically isolated syndrome. We applied a mass spectrometry-based proteomic approach (isobaric labelling) to previously collected pooled cerebrospinal fluid samples from patients with clinically isolated syndrome, who subsequently converted to clinically definite multiple sclerosis (n=30) and patients who remained as having clinically isolated syndrome (n=30). Next, three of the most represented differentially expressed proteins, i.e. ceruloplasmin, vitamin D-binding protein and chitinase 3-like 1 were selected for validation in individual cerebrospinal fluid samples by enzyme-linked immunosorbent assay. Only chitinase 3-like 1 was validated and cerebrospinal fluid levels were increased in patients who converted to clinically definite multiple sclerosis compared with patients who continued as clinically isolated syndrome (P=0.00002) and controls (P=0.012). High cerebrospinal fluid levels of chitinase 3-like 1 significantly correlated with the number of gadolinium enhancing lesions and the number of T2 lesions observed in brain magnetic resonance imaging scans performed at baseline, and were associated with disability progression during follow-up and shorter time to clinically definite multiple sclerosis (log-rank P-value=0.003). Cerebrospinal fluid chitinase 3-like 1 levels were also measured in a second validation clinically isolated syndrome cohort and found to be increased in patients who converted to multiple sclerosis compared with patients who remained as having clinically isolated syndrome (P=0.018). Our results indicate that patients who will convert to clinically definite multiple sclerosis could be distinguished from those patients who will remain as clinically isolated syndrome by proteomic analysis of cerebrospinal fluid samples. Although protein levels are also increased in other disorders characterized by chronic inflammation, chitinase 3-like 1 may serve as a prognostic biomarker for conversion to multiple sclerosis and development of disability which may help to improve the understanding of the aetiopathogenesis in the early stages of multiple sclerosis.

Differential susceptibility to apoptosis of CD4+T cells expressing CCR5 and CXCR3 in patients with MS.

We aimed to evaluate differences in the susceptibility to apoptosis of CD4+CCR5+ and CD4+CXCR3+T cells between MS patients (N=41) and controls (N=15) 6 days after activation of peripheral blood cells with anti-CD3 antibodies and 24 h following stimulation with anti-Fas antibodies. Susceptibility to anti-CD3 induced activation-induced cell death (AICD) and Fas-mediated apoptosis was selectively increased in CD4+CCR5+T cells compared with CD4+CCR5- and CD4+CXCR3-/+T cells. Compared with controls, CD4+CCR5+T cells from patients with primary progressive MS (PPMS) were more resistant to anti-CD3-induced AICD and anti-Fas-induced apoptosis determined with the mitochondrial probe DiOC(6) (3-3'-dihexyloxacarboyanine iodide). Our findings point to a differential regulation in the susceptibility to apoptosis of CD4+T cells expressing CCR5 and CXCR3 and suggest an impairment in the mitochondria-mediated apoptotic deletion of CD4+CCR5+T cells in PPMS patients that may lead to their chronic persistence in peripheral blood from these patients.

Changes in matrix metalloproteinases and their inhibitors during interferon-beta treatment in multiple sclerosis.

Matrix metalloproteinases (MMPs) and tissue inhibitors (TIMPs) play a key role in the pathogenesis of multiple sclerosis (MS) and have been proposed as biomarkers of response to therapy. We investigated serum levels of several MMPs and TIMPs in 43 relapsing-remitting MS (RRMS) patients undergoing interferon-beta (IFN-b) treatment and classified as responders and non-responders based on clinical criteria. Levels of MMP-2, MMP-7, MMP-9, TIMP-1 and TIMP-2 were determined by ELISA before treatment and after 3, 6, 12, and 24 months of therapy. Neutralizing antibodies were determined by the myxovirus A induction bioassay. Treatment with IFN-b induced changes in levels of MMP-9 and TIMP-1. In contrast to non-responders, IFN-b resulted in an early and sustained increase in TIMP-1 levels in MS patients who showed clinical response to IFN-b. The early and sustained increase in TIMP-1 levels could be a marker of the response to IFN-b during the first 2 years of treatment.

Flow Sorting Enrichment and Nanopore Sequencing of Chromosome 1 From a Chinese Individual.

Sorting of individual chromosomes by Flow Cytometry (flow-sorting) is an enrichment method to potentially simplify genome assembly by isolating chromosomes from the context of the genome. We have recently developed a workflow to sequence native, unamplified DNA and applied it to the smallest human chromosome, the Y chromosome. Here, we modify improve upon that workflow to increase DNA recovery from chromosome sorting as well as sequencing yield. We apply it to sequence and assemble the largest human chromosome - chromosome 1 - of a Chinese individual using a single Oxford Nanopore MinION flow cell. We generate a selective and highly continuous assembly whose continuity reaches into the order of magnitude of the human reference GRCh38. We then use this assembly to call candidate structural variants against the reference and find 685 putative novel SV candidates. We propose this workflow as a potential solution to assemble structurally complex chromosomes, or the study of very large plant or animal genomes that might challenge traditional assembly strategies.

Selective single molecule sequencing and assembly of a human Y chromosome of African origin.

Mammalian Y chromosomes are often neglected from genomic analysis. Due to their inherent assembly difficulties, high repeat content, and large ampliconic regions, only a handful of species have their Y chromosome properly characterized. To date, just a single human reference quality Y chromosome, of European ancestry, is available due to a lack of accessible methodology. To facilitate the assembly of such complicated genomic territory, we developed a novel strategy to sequence native, unamplified flow sorted DNA on a MinION nanopore sequencing device. Our approach yields a highly continuous assembly of the first human Y chromosome of African origin. It constitutes a significant improvement over comparable previous methods, increasing continuity by more than 800%. Sequencing native DNA also allows to take advantage of the nanopore signal data to detect epigenetic modifications in situ. This approach is in theory generalizable to any species simplifying the assembly of extremely large and repetitive genomes.

Three-Dimensional Genomic Structure and Cohesin Occupancy Correlate with Transcriptional Activity during Spermatogenesis.

Mammalian gametogenesis involves dramatic and tightly regulated chromatin remodeling, whose regulatory pathways remain largely unexplored. Here, we generate a comprehensive high-resolution structural and functional atlas of mouse spermatogenesis by combining in situ chromosome conformation capture sequencing (Hi-C), RNA sequencing (RNA-seq), and chromatin immunoprecipitation sequencing (ChIP-seq) of CCCTC-binding factor (CTCF) and meiotic cohesins, coupled with confocal and super-resolution microscopy. Spermatogonia presents well-defined compartment patterns and topological domains. However, chromosome occupancy and compartmentalization are highly re-arranged during prophase I, with cohesins bound to active promoters in DNA loops out of the chromosomal axes. Compartment patterns re-emerge in round spermatids, where cohesin occupancy correlates with transcriptional activity of key developmental genes. The compact sperm genome contains compartments with actively transcribed genes but no fine-scale topological domains, concomitant with the presence of protamines. Overall, we demonstrate how genome-wide cohesin occupancy and transcriptional activity is associated with three-dimensional (3D) remodeling during spermatogenesis, ultimately reprogramming the genome for the next generation.

Music Therapy in Parkinson's Disease.

OBJECTIVES: Parkinson's disease (PD) is a chronic progressive neurologic disorder involving degeneration of the dopaminergic system. Its clinical manifestations include motor and nonmotor symptoms. Several nonpharmacologic therapies, such as music therapy (MT), have recently been developed in order to improve the clinical manifestations of this disease. The aim of this narrative literature review is to analyze the scientific evidence for the therapeutic effects of music in PD. DESIGN: We undertook a search in the databases of PubMed, PsycINFO, Scopus, MEDLINE, and Science Direct. SETTINGAND PARTICIPANTS: Inclusion criteria were articles including persons with PD rehabilitated with an MT intervention. MEASURES: Keywords used were music therapy, Parkinson's disease, auditory cueing, non-motor symptoms, motor symptoms, and quality of life. RESULTS: We detected a total of 27 articles, all of which analyzed the therapeutic effects of MT in PD. Of these, 20 studies analyzed the effects in motor symptoms (16 showed beneficial effects and 4, nonbeneficial effects); 9 studies analyzed the effects in nonmotor symptoms, 7 of which demonstrated beneficial effects; and 8 studies analyzed the effects on quality of life, with 6 reporting benefits. None of the articles analyzing nonmotor symptoms and quality of life showed negative effects. CONCLUSIONS/IMPLICATIONS: Most of the studies analyzed demonstrated that MT has beneficial effects for the nonpharmacologic treatment of motor and nonmotor symptoms and quality of life of persons with PD. The use of music as a therapeutic tool combined with conventional therapies should be taken into account.

On the Cost of Introducing Speech-Like Properties to a Stimulus for Auditory Steady-State Response Measurements.

Validating hearing-aid fittings in prelingual infants is challenging because typical measures (aided audiometry, etc.) are impossible with infants. One objective alternative uses an aided auditory steady-state response (ASSR) measurement. To make an appropriate measurement, the hearing aid's signal-processing features must be activated (or deactivated) as if the ASSR stimulus was real speech. Rather than manipulating the hearing-aid settings to achieve this, an ASSR stimulus with speech-like properties was developed. This promotes clinical simplicity and face validity of the validation. The stimulus consists of narrow-band CE-Chirps®, modified to mimic the International Speech Test Signal (ISTS). This study examines the cost of introducing the speech-like features into the ASSR stimulus. Thus, 90 to 100 Hz ASSRs were recorded to the ISTS-modified stimulus as well as an equivalent stimulus without the ISTS modification, presented through insert phones to 10 young normal-hearing subjects. Noise-corrected ASSR magnitudes and clinically relevant detection times were estimated and analyzed with mixed-model analyses of variance. As a supplement, the observed changes to the ASSR magnitudes were compared with an objective characterization of the stimuli based on modulation power. The main findings were a reduction in ASSR magnitude of 4 dB and an increase in detection time by a factor of 1.5 for the ISTS-modified stimulus compared with the standard. Detection rates were unaffected given sufficient recording time. For clinical use of the hearing-aid validation procedure, the key metric is the detection time. While this varied considerably across subjects, the observed 50% mean increase corresponds to less than 1 min of additional recording time.

Single- and two-photon imaging of human micrometastases and disseminated tumour cells with conjugates of nanobodies and quantum dots.

Early detection of malignant tumours and, especially, micrometastases and disseminated tumour cells is still a challenge. In order to implement highly sensitive diagnostic tools we demonstrate the use of nanoprobes engineered from nanobodies (single-domain antibodies, sdAbs) and fluorescent quantum dots (QDs) for single- and two-photon detection and imaging of human micrometastases and disseminated tumour cells in ex vivo biological samples of breast and pancreatic metastatic tumour mouse models expressing human epidermal growth factor receptor 2 (HER2) or carcinoembryonic antigen (CEA). By staining thin (5-10 µm) paraffin and thick (50 µm) agarose tissue sections, we detected HER2- and CEA-positive human tumour cells infiltrating the surrounding tissues or metastasizing to different organs, including the brain, testis, lung, liver, and lymph nodes. Compared to conventional fluorescently labelled antibodies the sdAb-HER2-QD and sdAb-CEA-QD nanoprobes are superior in detecting micrometastases in tissue sections by lower photobleaching and higher brightness of fluorescence signals ensuring much better discrimination of positive signals versus background. Very high two-photon absorption cross-sections of QDs and small size of the nanoprobes ensure efficient imaging of thick tissue sections unattainable with conventional fluorescent probes. The nanobody-QD probes will help to improve early cancer diagnosis and prognosis of progression by assessing metastasis.

Deficient Fas expression by CD4+ CCR5+ T cells in multiple sclerosis.

OBJECTIVE: To evaluate whether T cells expressing CCR5 and CXCR3 from multiple sclerosis (MS) patients are more resistant to apoptosis. METHODS: Expression of CD69, TNF-R1, Fas, FasL, bcl-2, and bax was investigated in 41 MS patients and 12 healthy controls by flow cytometry in CD4+ and CD8+ T cells expressing CCR5 and CXCR3. RESULTS: In MS patients, the percentage of CD69 was increased and Fas expression decreased in CD4+ CCR5+ T cells. INTERPRETATION: The lower Fas expression in activated CD4+ CCR5+ T cells might contribute to disease pathogenesis by prolonging cell survival and favoring their migration into the CNS.

Acciones para la superación de los maestros sobre la postura corporal correcta en los niños de la Educación Primaria / Actions for the overcoming of the teachers on the correct corporal posture in the children of the Primary Education

RESUMEN La postura corporal correcta es un aspecto importante para la salud y la calidad de vida de los niños desde las primeras edades y se incluye como parte del eje temático de Higiene en el Programa Director de Salud que se desarrolla en las escuelas cubanas. Teniendo en cuenta la existencia de esta problemática en la Educación Primaria, se partió de un diagnóstico sobre la situación actual referente a la postura corporal en una muestra de 184 niños de ambos sexos pertenecientes al semi-internado «Isidro de Armas¼ de Pinar del Río. Durante el estudio se empleó la observación científica y el examen somatoscópico, además se aplicó una encuesta a 27 maestros trabajadores en ese centro. A partir de las deficiencias y debilidades encontradas se elaboró un conjunto de acciones encaminadas a la capacitación de los maestros del referido centro, para un desarrollo eficiente de la educación corporal en los niños comprendidos entre 6 a 12 años. La implementación de dichas acciones en la práctica pedagógica permitió a los maestros profundizar en los aspectos teóricos, prácticos y metodológicos indispensables para el logro de un desarrollo adecuado de la postura corporal en los infantes.

ABSTRACT The correct corporal posture is an important aspect for the health and the quality of children's life since the early years and it is included as part of the hygiene thematic axis in the Health Principal Program which is developed in our schools. Taking into account the existence of the problematic situation in the Elementary Education, the authors of this investigation elaborated and validated an up-grading strategic for the primary teachers of the school «Isidoro Garcia¼ from Las Minas de Matahambres municipality, gaining outstanding results after applying the corresponding methods such as the observation, the survey and a Somatoscopical test. Besides, other samples of groups were included in the investigation, like the family, supervisors and principals.

High-sensitivity detection of breast tumors in vivo by use of a pH-sensitive near-infrared fluorescence probe.

We investigated the potential of the pH-sensitive dye, CypHer5E, conjugated to Herceptin (pH-Her) for the sensitive detection of breast tumors in mice using noninvasive time-domain near-infrared fluorescence imaging and different methods of data analysis. First, the fluorescence properties of pH-Her were analyzed as function of pH and/or dye-to-protein ratio, and binding specificity was confirmed in cell-based assays. Subsequently, the performance of pH-Her in nude mice bearing orthotopic HER2-positive (KPL-4) and HER2-negative (MDA-MB-231) breast carcinoma xenografts was compared to that of an always-on fluorescent conjugate Alexa Fluor 647-Herceptin (Alexa-Her). Subtraction of autofluorescence and lifetime (LT)-gated image analyses were performed for background fluorescence suppression. In mice bearing HER2-positive tumors, autofluorescence subtraction together with the selective fluorescence enhancement of pH-Her solely in the tumor's acidic environment provided high contrast-to-noise ratios (CNRs). This led to an improved sensitivity of tumor detection compared to Alexa-Her. In contrast, LT-gated imaging using LTs determined in model systems did not improve tumor-detection sensitivity in vivo for either probe. In conclusion, pH-Her is suitable for sensitive in vivo monitoring of HER2-expressing breast tumors with imaging in the intensity domain and represents a promising tool for detection of weak fluorescent signals deriving from small tumors or metastases.