Short Tandem Repeat DNA Profiling Using Perylene-Oligonucleotide Fluorescence Assay.

We report an amplification-free genotyping method to determine the number of human short tandem repeats (STRs). DNA-based STR profiling is a robust method for genetic identification purposes such as forensics and biobanking and for identifying specific molecular subtypes of cancer. STR detection requires polymerase amplification, which introduces errors that obscure the correct genotype. We developed a new method that requires no polymerase. First, we synthesized perylene-nucleoside reagents and incorporated them into oligonucleotide probes that recognize five common human STRs. Using these probes and a bead-based hybridization approach, accurate STR detection was achieved in only 1.5 h, including DNA preparation steps, with up to a 1000-fold target DNA enrichment. This method was comparable to PCR-based assays. Using standard fluorometry, the limit of detection was 2.00 ± 0.07 pM for a given target. We used this assay to accurately identify STRs from 50 human subjects, achieving >98% consensus with sequencing data for STR genotyping.

A 9-year-old boy with a nonmalignant forehead tumor - a rare case of pediatric Pott's puffy tumor.

BACKGROUND: Pott's puffy tumor (PPT) is a rare and potentially deadly complication of frontal sinusitis consisting of subperiosteal abscess and osteomyelitis of the frontal bone. CASE PRESENTATION: We report the case of a 9-year-old boy who presented with fever and soft tissue swelling of the forehead. Magnetic resonance imaging (MRI) depicted an abscess in the subcutaneous tissue frontally and an epidural empyema, while a cranial computed tomography (CT) scan revealed bone erosion as a sign of osteomyelitis. The patient was treated accordingly. CONCLUSIONS: This rare condition is essential to keep in mind as it needs a multidisciplinary approach and relevant imaging to start proper treatment and thus decrease the risk of intracranial complications.

Cerebral Palsy - Early Diagnosis and Intervention Trial: protocol for the prospective multicentre CP-EDIT study with focus on diagnosis, prognostic factors, and intervention.

BACKGROUND: Early diagnosis of cerebral palsy (CP) is important to enable intervention at a time when neuroplasticity is at its highest. Current mean age at diagnosis is 13 months in Denmark. Recent research has documented that an early-diagnosis set-up can lower diagnostic age in high-risk infants. The aim of the current study is to lower diagnostic age of CP regardless of neonatal risk factors. Additionally, we want to investigate if an early intervention program added to standard care is superior to standard care alone. METHODS: The current multicentre study CP-EDIT (Early Diagnosis and Intervention Trial) with the GO-PLAY intervention included (Goal Oriented ParentaL supported home ActivitY program), aims at testing the feasibility of an early diagnosis set-up and the GO-PLAY early intervention. CP-EDIT is a prospective cohort study, consecutively assessing approximately 500 infants at risk of CP. We will systematically collect data at inclusion (age 3-11 months) and follow a subset of participants (n = 300) with CP or at high risk of CP until the age of two years. The GO-PLAY early intervention will be tested in 80 infants with CP or high risk of CP. Focus is on eight areas related to implementation and perspectives of the families: early cerebral magnetic resonance imaging (MRI), early genetic testing, implementation of the General Movements Assessment method, analysis of the GO-PLAY early intervention, parental perspective of early intervention and early diagnosis, early prediction of CP, and comparative analysis of the Hand Assessment for Infants, Hammersmith Infant Neurological Examination, MRI, and the General Movements method. DISCUSSION: Early screening for CP is increasingly possible and an interim diagnosis of "high risk of CP" is recommended but not currently used in clinical care in Denmark. Additionally, there is a need to accelerate identification in mild or ambiguous cases to facilitate appropriate therapy early. Most studies on early diagnosis focus on identifying CP in infants below five months corrected age. Little is known about early diagnosis in the 50% of all CP cases that are discernible later in infancy. The current study aims at improving care of patients with CP even before they have an established diagnosis. TRIAL REGISTRATION: ClinicalTrials.gov ID 22013292 (reg. date 31/MAR/2023) for the CP-EDIT cohort and ID 22041835 (reg. date 31/MAR/2023) for the GO-PLAY trial.

Cyclic Citrullinated Peptide Aptamer Treatment Attenuates Collagen-Induced Arthritis.

We describe the study of a novel aptamer-based candidate for treatment of seropositive rheumatoid arthritis. The candidate is a nanoparticle-formulated cyclic citrullinated peptide aptamer, which targets autoantibodies and/or the immune reactions leading to antibody production. Due to its specificity, the peptide aptamer nanoparticles might not interfere with normal immune functions as seen with other disease-modifying antirheumatic drugs. Over a 3-week course of treatment, joint swelling and arthritis score in collagen-induced rats were significantly decreased compared with animals treated with phosphate-buffered saline, unloaded nanoparticles, or nanoparticles with a noncitrullinated control peptide. The reduction in joint swelling was associated with decreased anticitrullinated peptide autoantibody levels in the blood. Treatment with aptamer nanoparticles also increased interleukin-10 levels. The effect seen with the proposed treatment candidate could be mediated by upregulation of anti-inflammatory mediators and decreased levels of anticitrullinated peptide antibodies.

Enhanced target cell specificity and uptake of lipid nanoparticles using RNA aptamers and peptides.

Lipid nanoparticles (LNPs) constitute a facile and scalable approach for delivery of payloads to human cells. LNPs are relatively immunologically inert and can be produced in a cost effective and scalable manner. However, targeting and delivery of LNPs across the blood-brain barrier (BBB) has proven challenging. In an effort to target LNPs composed of an ionizable cationic lipid (DLin-MC3-DMA), cholesterol, the phospholipid 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG 2000) to particular cell types, as well as to generate LNPs that can cross the BBB, we developed and assessed two approaches. The first was centered on the BBB-penetrating trans-activator of transcription (Tat) peptide or the peptide T7, and the other on RNA aptamers targeted to glycoprotein gp160 from human immunodeficiency virus (HIV) or C-C chemokine receptor type 5 (CCR5), a HIV-1 coreceptor. We report herein a CCR5-selective RNA aptamer that acts to facilitate entry through a simplified BBB model and that drives the uptake of LNPs into CCR5-expressing cells, while the gp160 aptamer did not. We further observed that the addition of cell-penetrating peptides, Tat and T7, did not increase BBB penetration above the aptamer-loaded LNPs alone. Moreover, we found that these targeted LNPs exhibit low immunogenic and low toxic profiles and that targeted LNPs can traverse the BBB to potentially deliver drugs into the target tissue. This approach highlights the usefulness of aptamer-loaded LNPs to increase target cell specificity and potentially deliverability of central-nervous-system-active RNAi therapeutics across the BBB.

Ultra-fast detection and quantification of nucleic acids by amplification-free fluorescence assay.

Two types of clinically important nucleic acid biomarkers, microRNA (miRNA) and circulating tumor DNA (ctDNA) were detected and quantified from human serum using an amplification-free fluorescence hybridization assay. Specifically, miRNAs hsa-miR-223-3p and hsa-miR-486-5p with relevance for rheumatoid arthritis and cancer related mutations BRAF and KRAS of ctDNA were directly measured. The required oligonucleotide probes for the assay were rationally designed and synthesized through a novel "clickable" approach which is time and cost-effective. With no need for isolating nucleic acid components from serum, the fluoresence-based assay took only 1 hour. Detection and absolute quantification of targets was successfully achieved despite their notoriously low abundance, with a precision down to individual nucleotides. Obtained miRNA and ctDNA amounts showed overall a good correlation with current techniques. With appropriate probes, our novel assay and signal boosting approach could become a useful tool for point-of-care measuring other low abundance nucleic acid biomarkers.

Citrullinated Peptide Epitope Targets Therapeutic Nanoparticles to Human Neutrophils.

Multiple drugs have been proposed for reducing harsh symptoms of human rheumatic diseases. However, a targeted therapy with mild to no side effects is still missing. In this study, we have prepared and tested a series of therapeutic nanoparticles for specific targeting of human neutrophils associated with rheumatoid arthritis. In doing this, a series of citrullinated peptide epitopes derived from human proteins, fibrinogen, vimentin, and histone 3, were screened with regard to specific recognition of neutrophils. The most potent epitope proved to be a mutated fragment of an alpha chain in human fibrinogen. Next, a straightforward synthetic strategy was developed for nanoparticles decorated with this citrullinated peptide epitope and an antisense oligonucleotide targeting disease associated microRNA miR-125b-5p. Our study shows that the nanoparticles specifically recognize neutrophils and knock down miR-125b-5p, with no apparent toxicity to human cells. In contrast to organic dendrimers, chitosan-hyaluronic acid formulations do not activate human innate immune response. Our data proves that the strategy we report herein is effective in developing peptide epitopes for decorating delivery vehicles bearing biological drugs, targeted to a specific cell type.

Is Tadpole Pupil in an Adolescent Girl Caused by Denervation Hypersensitivity?

Tadpole pupil is a rarely encountered phenomenon caused by episodic, segmental iris dilator muscle spasm of short duration (2-15 minutes), occurring in clusters without a known precipitating factor. It has most commonly been described in women aged 28 to 48 years. A few hypotheses on pathogenesis have been discussed but none has been proved. Here, we present an adolescent girl with bilateral tadpole pupil that appeared during physical exercise. This is the first pediatric case of tadpole pupil, not caused by preceding surgery, to be published. Based on (1) this case in which tadpole pupil developed when the norepinephrine level rose during exercise, (2) the high ratio of patients with tadpole pupil who concurrently have or later develop Horner syndrome, in which denervation hypersensitivity is well described, (3) a previous report of a patient with both tadpole pupil and Horner syndrome who had denervation hypersensitivity on pharmacological testing, (4) a 29-year-old man with unilateral tadpole pupil induced by exercise, and (5) a 19-year-old man with bilateral tadpole pupil and possible autonomic neuropathy, we suggest denervation hypersensitivity as a probable pathogenic mechanism causing tadpole pupil. In addition, a suggestion for investigations to be performed in future pediatric cases is provided.

Three new double-headed nucleotides with additional nucleobases connected to C-5 of pyrimidines; synthesis, duplex and triplex studies.

In the search for double-coding DNA-systems, three new pyrimidine nucleosides, each coded with an additional nucleobase anchored to the major groove face, are synthesized. Two of these building blocks carry a thymine at the 5-position of 2'-deoxyuridine through a methylene linker and a triazolomethylene linker, respectively. The third building block carries an adenine at the 6-position of pyrrolo-2'-deoxycytidine through a methylene linker. These double-headed nucleosides are introduced into oligonucleotides and their effects on the thermal stabilities of duplexes are studied. All studied double-headed nucleotide monomers reduce the thermal stability of the modified duplexes, which is partially compensated by using consecutive incorporations of the modified monomers or by flanking the new double-headed analogs with members of our former series containing propyne linkers. Also their potential in triplex-forming oligonucleotides is studied for two of the new double-headed nucleotides as well as the series of analogs with propyne linkers. The most stable triplexes are obtained with single incorporations of additional pyrimidine nucleobases connected via the propyne linker.

Deciphering biased-agonism complexity reveals a new active AT1 receptor entity.

Functional selectivity of G protein-coupled receptor (GPCR) ligands toward different downstream signals has recently emerged as a general hallmark of this receptor class. However, pleiotropic and crosstalk signaling of GPCRs makes functional selectivity difficult to decode. To look from the initial active receptor point of view, we developed new, highly sensitive and direct bioluminescence resonance energy transfer-based G protein activation probes specific for all G protein isoforms, and we used them to evaluate the G protein-coupling activity of [(1)Sar(4)Ile(8)Ile]-angiotensin II (SII), previously described as an angiotensin II type 1 (AT(1)) receptor-biased agonist that is G protein independent but ß-arrestin selective. By multiplexing assays sensing sequential signaling events, from receptor conformations to downstream signaling, we decoded SII as an agonist stabilizing a G protein-dependent AT(1A) receptor signaling module different from that of the physiological agonist angiotensin II, both in recombinant and primary cells. Thus, a biased agonist does not necessarily select effects from the physiological agonist but may instead stabilize and create a new distinct active pharmacological receptor entity.

Creatine kinase response to high-intensity aerobic exercise in adult-onset muscular dystrophy.

INTRODUCTION: We investigated the effect of high-intensity exercise on plasma creatine kinase (CK) in patients with muscular dystrophies. METHODS: Fourteen patients with Becker (BMD), facioscapulohumeral (FSHD), or limb-girdle type 2 (LGMD2) muscular dystrophy, and 8 healthy subjects performed 5 cycling tests: an incremental max test, and tests at 65%, 75%, 85%, and 95% of maximal oxygen uptake (VO2max ). Heart rate and oxygen consumption were measured during the tests, and plasma CK was measured before, immediately after, and 24 hours after exercise. RESULTS: All subjects were able to perform high-intensity exercise at the different levels. In patients with LGMD2 and FSHD, CK normalized 24 hours after exercise compared with the pre-exercise value, whereas those with BMD and healthy controls had elevated CK values 24 hours after exercise. CONCLUSIONS: The findings suggest that high-intensity exercise is generally well tolerated in patients with LGMD2 and FSHD, whereas those with BMD may be more prone to exercise-induced damage.

Mixed-field orientation of molecules without rotational symmetry.

The mixed-field orientation of an asymmetric-rotor molecule with its permanent dipole moment nonparallel to the principal axes of polarizability is investigated experimentally and theoretically. We find that for the typical case of a strong, nonresonant laser field and a weak static electric field complete 3D orientation is induced if the laser field is elliptically polarized and if its major and minor polarization axes are not parallel to the static field. For a linearly polarized laser field solely the dipole moment component along the most polarizable axis of the molecule is relevant resulting in 1D orientation even when the laser polarization and the static field are nonparallel. Simulations show that the dipole moment component perpendicular to the most-polarizable axis becomes relevant in a strong dc electric field combined with the laser field. This offers an alternative approach to 3D orientation by combining a linearly polarized laser field and a strong dc electric field arranged at an angle equal to the angle between the most polarizable axis of the molecule and its permanent dipole moment.

FindZebra online search delving into rare disease case reports using natural language processing.

Early diagnosis is crucial for well-being and life quality of the rare disease patient. Access to the most complete knowledge about diseases through intelligent user interfaces can play an important role in supporting the physician reaching the correct diagnosis. Case reports may offer information about heterogeneous phenotypes which often further complicate rare disease diagnosis. The rare disease search engine FindZebra.com is extended to also access case report abstracts extracted from PubMed for several diseases. A search index for each disease is built in Apache Solr adding age, sex and clinical features extracted using text segmentation to enhance the specificity of search. Clinical experts performed retrospective validation of the search engine, utilising real-world Outcomes Survey data on Gaucher and Fabry patients. Medical experts evaluated the search results as being clinically relevant for the Fabry patients and less clinically relevant for the Gaucher patients. The shortcomings for Gaucher patients mainly reflect a mismatch between the current understanding and treatment of the disease and how it is reported in PubMed, notably in the older case reports. In response to this observation, a filter for the publication date was added in the final version of the tool available from deep.findzebra.com/ with = gaucher, fabry, hae (Hereditary angioedema).

Control and femtosecond time-resolved imaging of torsion in a chiral molecule.

We study how the combination of long and short laser pulses can be used to induce torsion in an axially chiral biphenyl derivative (3,5-difluoro-3',5'-dibromo-4'-cyanobiphenyl). A long, with respect to the molecular rotational periods, elliptically polarized laser pulse produces 3D alignment of the molecules, and a linearly polarized short pulse initiates torsion about the stereogenic axis. The torsional motion is monitored in real-time by measuring the dihedral angle using femtosecond time-resolved Coulomb explosion imaging. Within the first 4 picoseconds (ps), torsion occurs with a period of 1.25 ps and an amplitude of 3° in excellent agreement with theoretical calculations. At larger times, the quantum states of the molecules describing the torsional motion dephase and an almost isotropic distribution of the dihedral angle is measured. We demonstrate an original application of covariance analysis of two-dimensional ion images to reveal strong correlations between specific ejected ionic fragments from Coulomb explosion. This technique strengthens our interpretation of the experimental data.

Validating the automation of different measures of high temperature tolerance of small terrestrial insects.

Accurately phenotyping numerous test subjects is essential for most experimental research. Collecting such data can be tedious or time-consuming, and it can be biased or limited using manual observations. The thermal tolerance of small ectotherms is a good example of this type of phenotypic data, and it is widely used to investigate thermal adaptation, acclimation capacity and climate change resilience of small ectotherms. Here, we present the results of automatically generated thermal tolerance data using motion-tracking software on video recordings. The automatization was applied to two different heat tolerance assays, in two Drosophila species and used temperature acclimation to create variation in thermal tolerances. We find similar effect sizes of acclimation and hardening responses between manual and automated approaches, but different absolute tolerance estimates. This discrepancy likely reflects both technical differences in the assay conditions as well as the measured end-points of the assays. We conclude that both methods generate biological meaningful results, which reflect different aspects of the thermal biology, find no evidence of inflated variance in the manually scored assays, but find that automation can increase throughput several times without compromising quality. Further we show that the method can be applied to a wide range of arthropod taxa. We suggest that this automated method is a useful example of high throughput phenotyping. Further, we suggest this approach might be applied to other tedious laboratory traits, such as desiccation or starvation tolerance, with similar benefits to throughput but caution that the interpretation and potential comparison to results using different methodology rely on thorough validation of the assay and the involved biological mechanism.

Central Serotonin/Noradrenaline Transporter Availability and Treatment Success in Patients with Obesity.

Serotonin (5-hydroxytryptamine, 5-HT) as well as noradrenaline (NA) are key modulators of various fundamental brain functions including the control of appetite. While manipulations that alter brain serotoninergic signaling clearly affect body weight, studies implicating 5-HT transporters and NA transporters (5-HTT and NAT, respectively) as a main drug treatment target for human obesity have not been conclusive. The aim of this positron emission tomography (PET) study was to investigate how these central transporters are associated with changes of body weight after 6 months of dietary intervention or Roux-en-Y gastric bypass (RYGB) surgery in order to assess whether 5-HTT as well as NAT availability can predict weight loss and consequently treatment success. The study population consisted of two study cohorts using either the 5-HTT-selective radiotracer [11C]DASB to measure 5-HTT availability or the NAT-selective radiotracer [11C]MRB to assess NAT availability. Each group included non-obesity healthy participants, patients with severe obesity (body mass index, BMI, >35 kg/m2) following a conservative dietary program (diet) and patients undergoing RYGB surgery within a 6-month follow-up. Overall, changes in BMI were not associated with changes of both 5-HTT and NAT availability, while 5-HTT availability in the dorsal raphe nucleus (DRN) prior to intervention was associated with substantial BMI reduction after RYGB surgery and inversely related with modest BMI reduction after diet. Taken together, the data of our study indicate that 5-HTT and NAT are involved in the pathomechanism of obesity and have the potential to serve as predictors of treatment outcomes.

The importance of bulk Ti3+ defects in the oxygen chemistry on titania surfaces.

The role of bulk defects in the oxygen chemistry on reduced rutile TiO(2)(110)-(1 × 1) has been studied by means of temperature-programmed desorption spectroscopy and scanning tunneling microscopy measurements. Following O(2) adsorption at 130 K, the amount of O(2) desorbing at ∼410 K initially increased with increasing density of surface oxygen vacancies but decreased after further reduction of the TiO(2)(110) crystal. We explain these results by withdrawal of excess charge (Ti(3+)) from the TiO(2)(110) lattice to oxygen species on the surface and by a reaction of Ti interstitials with O adatoms upon heating. Important consequences for the understanding of the O(2)-TiO(2) interaction are discussed.

Time-resolved photoelectron angular distributions from strong-field ionization of rotating naphthalene molecules.

A nanosecond laser pulse confines the spatial orientation of naphthalene in 1D or 3D while a femtosecond kick pulse initiates rotation of the molecular plane around the fixed long axis. Time-dependent photoelectron angular distributions (PADs), resulting from ionization by an intense femtosecond probe pulse, exhibit pronounced changes as the molecular plane rotates. Enhanced 3D alignment, occurring shortly after the kick pulse, provides strongly improved contrast in molecular-frame PADs. Calculations in the strong-field approximation show that the striking structures observed in the PADs originate from nodal planes in occupied valence orbitals.

Carbon Nanotubes-Potent Carriers for Targeted Drug Delivery in Rheumatoid Arthritis.

Two types of single-walled carbon nanotubes (SWCNTs), HiPco- and carboxyl-SWCNT, are evaluated as drug carriers for the traditional anti-inflammatory drug methotrexate (MTX) and a small interfering RNA (siRNA) targeting NOTCH1 gene. The nanotubes are solubilized by PEGylation and covalently loaded with MTX. The coupling efficiency (CE%) of MTX is 77-79% for HiPco-SWCNT and 71-83% for carboxyl-SWCNT. siRNA is noncovalently attached to the nanotubes with efficiency of 90-97% for HiPco-SWCNT and 87-98% for carboxyl-SWCNT. Through whole body imaging in the second near-infrared window (NIR-II window, 1000-1700 nm), SWCNTs were found to be selectively accumulated in inflamed joints in a serum transfer mouse model. We further investigated the interactions of the siRNA/MTX loaded nanotubes with human blood and mice bone marrow cells. In human blood, both types of unloaded SWCNTs were associated with B cells, monocytes and neutrophils. Interestingly, loading with MTX suppressed SWCNTs targeting specificity to immune cells, especially B cells; in contrast, loading siRNA alone enhanced the targeting specificity. Loading both MTX and siRNA to carboxyl-SWCNT enhanced targeting specificity to neutrophils and monocytes but not B cells. The targeting specificity of SWCNTs can potentially be adjusted by altering the ratio of MTX and siRNA loaded. The combined results show that carbon nanotubes have the potential for delivery of cargo drugs specifically to immune cells involved in rheumatoid arthritis.

Elp2 mutations perturb the epitranscriptome and lead to a complex neurodevelopmental phenotype.

Intellectual disability (ID) and autism spectrum disorder (ASD) are the most common neurodevelopmental disorders and are characterized by substantial impairment in intellectual and adaptive functioning, with their genetic and molecular basis remaining largely unknown. Here, we identify biallelic variants in the gene encoding one of the Elongator complex subunits, ELP2, in patients with ID and ASD. Modelling the variants in mice recapitulates the patient features, with brain imaging and tractography analysis revealing microcephaly, loss of white matter tract integrity and an aberrant functional connectome. We show that the Elp2 mutations negatively impact the activity of the complex and its function in translation via tRNA modification. Further, we elucidate that the mutations perturb protein homeostasis leading to impaired neurogenesis, myelin loss and neurodegeneration. Collectively, our data demonstrate an unexpected role for tRNA modification in the pathogenesis of monogenic ID and ASD and define Elp2 as a key regulator of brain development.