The landscape of human SVA retrotransposons.

SINE-VNTR-Alu (SVA) retrotransposons are evolutionarily young and still-active transposable elements (TEs) in the human genome. Several pathogenic SVA insertions have been identified that directly mutate host genes to cause neurodegenerative and other types of diseases. However, due to their sequence heterogeneity and complex structures as well as limitations in sequencing techniques and analysis, SVA insertions have been less well studied compared to other mobile element insertions. Here, we identified polymorphic SVA insertions from 3646 whole-genome sequencing (WGS) samples of >150 diverse populations and constructed a polymorphic SVA insertion reference catalog. Using 20 long-read samples, we also assembled reference and polymorphic SVA sequences and characterized the internal hexamer/variable-number-tandem-repeat (VNTR) expansions as well as differing SVA activity for SVA subfamilies and human populations. In addition, we developed a module to annotate both reference and polymorphic SVA copies. By characterizing the landscape of both reference and polymorphic SVA retrotransposons, our study enables more accurate genotyping of these elements and facilitate the discovery of pathogenic SVA insertions.

Advancing immunomodulatory functions in mesenchymal stem/stromal cells through targeting the GATA6-mediated pathway.

BACKGROUND AIMS: The immunomodulatory capacity of mesenchymal stem/stromal cells (MSCs) is a key feature that makes them particularly valuable for regenerative medicine. However, this potential is affected by the chronological aging of the donors and the cell expansion procedures in culture. We have demonstrated that GATA binding protein 6 (GATA6) plays a pivotal role in the aging of MSCs and inhibiting GATA6 rejuvenates the characteristics of MSCs. METHODS: In this study, we compared the immunomodulatory capabilities of young and old MSC models, using induced pluripotent stem cells-derived rejuvenated MSCs (rMSCs) and their parental MSCs (pMSCs), respectively, to identify a key mechanism involved in the differential regulation of these capabilities. Additionally, we explored the role of GATA6 in mediating the mechanism. RESULTS: Our results demonstrated that rMSCs exhibited downregulated aging-associated regulators, including p53, p21 and GATA6, and showed enhanced suppression of T cell proliferation compared to pMSCs. Through analyzing our previous RNA-seq data and employing target gene knockdown, we determined both suppressors of cytokine signaling 3 (SOCS3) and interleukin 6 were involved in GATA6-induced regulation, collectively affecting the expression of programmed death ligand 1 (PDL1) in both pMSCs and rMSCs. CONCLUSIONS: Our findings underline the significance of the GATA6/SOCS3/PDL1 pathway in regulating aging-associated changes in MSC immunomodulatory activity, providing valuable insights into the potential use of rMSCs in the treatment of immune diseases and regenerative medicine.

Studies with Direct Industry Affiliation are More Likely to Report Positive Results in Randomized Controlled Trials for Platelet-Rich Plasma Use in Rotator Cuff Pathology: A Systematic Review.

PURPOSE: To perform a systematic review to evaluate the effect of industry affiliation on the outcomes of randomized controlled trials (RCTs) for platelet-rich plasma (PRP) injections in rotator cuff tears. METHODS: PubMed, SPORTdiscus, and Scopus databases were searched from 2010 to the present for terms "rotator cuff" and "platelet-rich plasma." Inclusion criteria were RCTs comparing PRP to controls for treatment of rotator cuff tears and exclusion criteria were systematic reviews, meta-analyses, case reports, cohort studies, basic science studies, other level 3 and below studies, and studies not in English. Degree of industry affiliation was categorized into three groups: direct, indirect, and not affiliated. Direct affiliation required the study or its authors to receive financial support from the company manufacturing the devices used in the study to prepare or administer PRP. Indirect affiliation required financial association with a different company that produces or administers PRP than the one used in the study. Studies were classified as favorable if study outcomes achieved significance (p < 0.05) of PRP over the control, or analogous if there was no statistical significance between PRP and control. Data was analyzed using chi-squared and fisher's exact tests. RESULTS: Of the 47 studies selected for analysis, 8 (17.0%) had no direct industry affiliation, 9 (19.1%) indirect affiliation and 30 (63.8%) no industry affiliation. 22 (46.8%) studies reported favorable results with PRP compared to the control and 25 (53.2%) showed analogous results between PRP and control. Degree of industry affiliation was significantly associated with increased likelihood of reporting favorable study outcomes (p = 0.041). Directly affiliated studies had a significantly increased likelihood of reporting favorable results (p = 0.024) compared to indirectly affiliated. CONCLUSIONS: Studies that used PRP produced by companies that directly fund the study or were financially affiliated with the authors were significantly more likely to report favorable results. LEVEL OF EVIDENCE: Systematic review of Level I and II studies.

Difference of Cerebrospinal Fluid Biomarkers and Neuropsychiatric Symptoms Profiles among Normal Cognition, Mild Cognitive Impairment, and Dementia Patient.

The delineation of biomarkers and neuropsychiatric symptoms across normal cognition, mild cognitive impairment (MCI), and dementia stages holds significant promise for early diagnosis and intervention strategies. This research investigates the association of neuropsychiatric symptoms, evaluated via the Neuropsychiatric Inventory (NPI), with cerebrospinal fluid (CSF) biomarkers (Amyloid-ß42, P-tau, T-tau) across a spectrum of cognitive states to enhance diagnostic accuracy and treatment approaches. Drawing from the National Alzheimer's Coordinating Center's Uniform Data Set Version 3, comprising 977 individuals with normal cognition, 270 with MCI, and 649 with dementia. To assess neuropsychiatric symptoms, we employed the NPI to understand the behavioral and psychological symptoms associated with each cognitive category. For the analysis of CSF biomarkers, we measured levels of Amyloid-ß42, P-tau, and T-tau using the enzyme-linked immunosorbent assay (ELISA) and Luminex multiplex xMAP assay protocols. These biomarkers are critical in understanding the pathophysiological underpinnings of Alzheimer's disease and its progression, with specific patterns indicative of disease stage and severity. This study cohort consists of 1896 participants, which is composed of 977 individuals with normal cognition, 270 with MCI, and 649 with dementia. Dementia is characterized by significantly higher NPI scores, which are largely reflective of mood-related symptoms (p < 0.001). In terms of biomarkers, normal cognition shows median Amyloid-ß at 656.0 pg/mL, MCI at 300.6 pg/mL, and dementia at 298.8 pg/mL (p < 0.001). Median P-tau levels are 36.00 pg/mL in normal cognition, 49.12 pg/mL in MCI, and 58.29 pg/mL in dementia (p < 0.001). Median T-tau levels are 241.0 pg/mL in normal cognition, 140.6 pg/mL in MCI, and 298.3 pg/mL in dementia (p < 0.001). Furthermore, the T-tau/Aß-42 ratio increases progressively from 0.058 in the normal cognition group to 0.144 in the MCI group, and to 0.209 in the dementia group (p < 0.001). Similarly, the P-tau/Aß-42 ratio also escalates from 0.305 in individuals with normal cognition to 0.560 in MCI, and to 0.941 in dementia (p < 0.001). The notable disparities in NPI and CSF biomarkers among normal, MCI and Alzheimer's patients underscore their diagnostic potential. Their combined assessment could greatly improve early detection and precise diagnosis of MCI and dementia, facilitating more effective and timely treatment strategies.

Investigating inherited arrhythmias using hiPSC-derived cardiomyocytes.

Fundamental to the functional behavior of cardiac muscle is that the cardiomyocytes are integrated as a functional syncytium. Disrupted electrical activity in the cardiac tissue can lead to serious complications including cardiac arrhythmias. Therefore, it is important to study electrophysiological properties of the cardiac tissue. With advancements in stem cell research, protocols for the production of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have been established, providing great potential in modelling cardiac arrhythmias and drug testing. The hiPSC-CM model can be used in conjunction with electrophysiology-based platforms to examine the electrical activity of the cardiac tissue. Techniques for determining the myocardial electrical activity include multielectrode arrays (MEAs), optical mapping (OM), and patch clamping. These techniques provide critical approaches to investigate cardiac electrical abnormalities that underlie arrhythmias.

Shortwave Infrared Fluorofluorophores for Multicolor In Vivo Imaging.

Developing chemical tools to detect and influence biological processes is a cornerstone of chemical biology. Here we combine two tools which rely on orthogonality- perfluorocarbons and multiplexed shortwave infrared (SWIR) fluorescence imaging- to visualize nanoemulsions in real time in living mice. Drawing inspiration from fluorous and SWIR fluorophore development, we prepared two SWIR-emissive, fluorous-soluble chromenylium polymethine dyes. These are the most red-shifted fluorous fluorophores- "fluorofluorophores"-to date. After characterizing the dyes, their utility was demonstrated by tracking perfluorocarbon nanoemulsion biodistribution in vivo. Using an excitation-multiplexed approach to image two variables simultaneously, we gained insight into the importance of size and surfactant identity on biodistribution.

Fog Harvesting with Highly Wetting and Nonwetting Vertical Strips.

Highly wetting and nonwetting substrates have been widely used in fogwater collection systems for enhanced water harvesting. In this work, fog harvesting substrates comprising PVC strips of different wetting properties and widths ranging from 1-5 mm were vertically aligned and spaced apart at regular intervals to give the same solid area fraction of 0.8. Evaluation of the water collection efficiencies of the tested configurations revealed that 1 mm wide superhydrophilic strips was the most efficient, achieving double the amount of water harvested compared with 2.8 mm wide strips. This finding was attributed to the low Stokes numbers of the aerosol particle distribution of the fog which tended to result in them being brought by the flow streamlines toward the air gaps between the strips. Stagnant flow regions at the edges of each strip, revealed through potential flow calculations, then caused higher liquid imbibition and impaction there for water harvesting. It was also found that the Cassie nonwetting substrates that originally exhibited contact angles of 161° transformed to Wenzel wetting with zero contact angle within 60 min of fog interception. Optical profilometry revealed no obvious difference in surface roughness between the central region and edges of the strips, indicating that surface morphology was unlikely to be a contributing factor for enhanced water collection at the edges. The findings here indicated that highly wetting vertical strip architectures with narrow widths (1 mm) were favorable over wider strips for water harvesting provided that clogging and re-entrainment were not significant factors.

Low-cost Imaging of Fluorescent DNA in Agarose Gel Electrophoresis using Raspberry Pi cameras.

Low-cost analytical solutions built around microcomputers like the Raspberry Pi help to facilitate laboratory investigations in resource limited venues. Here, three camera modules (V1.3 with and without filter, as well as NoIR) that work with this microcomputer were assessed for their suitability in imaging fluorescent DNA following agarose gel electrophoresis. Evaluation of their utility was based on signal-to-noise (SNR) and noise variance metrics that were developed. Experiments conducted with samples were subjected to Polymerase Chain Reaction (PCR), and the amplified products were separated using gel electrophoresis and stained with Midori green. Image analysis revealed the NoIR camera performed the best with SNR and noise variance values of 21.7 and 0.222 respectively. In experiments conducted using UV LED lighting to simulate ethidium bromide (EtBr) excitation, the NoIR and V1.3 with filter removed cameras showed comparable SNR values.

Cryoprotectant-free preservation of bacteria using semi-spherical drops.

Cryopreservation is a widely used long-term preservation method to ensure the quality and vitality of microbes in laboratories and biological resource facilities. However, freeze-thaw damage and osmotic pressure changes during cryopreservation adversely impacts microbial survival. Significant expenditure of resources and expertise are required to select the right cryoprotectant and optimize its concentration for maximum survival of diverse microorganisms. This work describes a cryopreservation method that obviates the need for cryoprotectants by exploiting the unique thermal characteristics of semi-spherical drops. Here, a plurality of these drops, each 10 µl in volume, created on a highly non-wetting flat-sheet substrate with holes and frozen at -70 °C. Deriving an f (x) metric as a measure of relative viability, storage in drops in the absence of cryoprotectants was found to improve the survivability of Staphylococcus epidermidis by 1.91 times compared with the same sample stored in larger 50-µl volumes in standard 1.5-ml tubes. This also compares well with a value of 2.33 obtained with standard preservation with cryoprotectant. The drop method allows high throughput aliquoting of the bacterial culture into multiple discrete drops using multichannel pipettes or automated liquid handlers and the edges of the holes provides a pinning action that holds the drop stably against gravitational roll-offs. It also allows samples to be removed in discrete small volumes, thus, reducing the number of freeze thaw cycles and associated cell damage. The flat-sheet architecture of the substrate reduces the amount of plastic waste generated and augments green laboratory practices.

In vitro analyses of suspected arrhythmogenic thin filament variants as a cause of sudden cardiac death in infants.

Sudden unexpected death of an infant (SUDI) is a devastating occurrence for families. To investigate the genetic pathogenesis of SUDI, we sequenced >70 genes from 191 autopsy-negative SUDI victims. Ten infants sharing a previously unknown variant in troponin I (TnI) were identified. The mutation (TNNI1 R37C+/-) is in the fetal/neonatal paralog of TnI, a gene thought to be expressed in the heart up to the first 24 months of life. Using phylogenetic analysis and molecular dynamics simulations, it was determined that arginine at residue 37 in TNNI1 may play a critical functional role, suggesting that the variant may be pathogenic. We investigated the biophysical properties of the TNNI1 R37C mutation in human reconstituted thin filaments (RTFs) using fluorometry. RTFs reconstituted with the mutant R37C TnI exhibited reduced Ca2+-binding sensitivity due to an increased Ca2+ off-rate constant. Furthermore, we generated TNNI1 R37C+/- mutants in human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) using CRISPR-Cas9. In monolayers of hiPSC-CMs, we simultaneously monitored voltage and Ca2+ transients through optical mapping and compared them to their isogenic controls. We observed normal intrinsic beating patterns under control conditions in TNNI1 R37C+/- at stimulation frequencies of 55 beats/min (bpm), but these cells showed no restitution with increased stimulation frequency to 65 bpm and exhibited alternans at >75 bpm. The WT hiPSC-CMs did not exhibit any sign of arrhythmogenicity even at stimulation frequencies of 120 bpm. The approach used in this study provides critical physiological and mechanistic bases to investigate sarcomeric mutations in the pathogenesis of SUDI.