bluuebunny/crossref_metadata_2025 huggingface dataset:
Orphan cytochrome P450 20A1 CRISPR/Cas 9 mutants and neurobehavioral phenotypes in zebrafish
Authors: Brun Nadja R. , Salanga Matthew C. , Mora-Zamorano Francisco X. , Lamb David C. , Goldstone Jared V. , Stegeman John J.
Published: July 2021

Orphan cytochrome P450 (CYP) enzymes are those for which biological substrates and function(s) are unknown. Cytochrome P450 20A1 (CYP20A1) is the last human orphan P450 enzyme, and orthologs occur as single genes in every vertebrate genome sequenced to date. The occurrence of high levels of CYP20A1 transcripts in human substantia nigra and hippocampus and abundant maternal transcripts in zebrafish eggs strongly suggest roles both in the brain and during early embryonic development. Patients with chromosome 2 microdeletions including CYP20A1 show hyperactivity and bouts of anxiety, among other conditions. Here, we created zebrafish CYP20A1 mutants using CRISPR/Cas9, providing vertebrate models with which to study the role of CYP20A1 in behavior and other neurodevelopmental functions. The homozygous cyp20a1 null mutants exhibited significant behavioral differences from wild-type zebrafish, both in larval and adult animals. Larval cyp20a1−/− mutants exhibited a strong increase in light-simulated movement (i.e., light-dark assay), which was interpreted as hyperactivity. Further, the larvae exhibited mild hypoactivity during the adaptation period of the optomotor assays. Adult cyp20a1 null fish showed a pronounced delay in adapting to new environments, which is consistent with an anxiety paradigm. Taken together with our earlier morpholino cyp20a1 knockdown results, the results described herein suggest that the orphan CYP20A1 has a neurophysiological role.
Intrinsically disordered N-terminal domain (NTD) of p53 interacts with mitochondrial PTP regulator Cyclophilin D
Authors: Zhao Jing , Liu Xinyue , Blayney Alan , Zhang Yumeng , Gandy Lauren , Zhang Fuming , Linhardt Robert J. , Chen Jianhan , Baines Christopher , Loh Stewart N. , Wang Chunyu
Published: July 2021

Mitochondrial permeability transition pore (mPTP) plays crucial roles in cell death in a variety of diseases, including ischemia/reperfusion injury in heart attack and stroke, neurodegenerative conditions, and cancer. To date, cyclophilin D is the only confirmed component of mPTP. Under stress, p53 can translocate into mitochondria and interact with CypD, triggering necrosis and cell growth arrest. However, the molecular details of p53/CypD interaction are still poorly understood. Previously, several studies reported that p53 interacts with CypD through its DNA-binding domain (DBD). However, using surface plasmon resonance (SPR), we found that full-length p53 (FLp53) binds to CypD withKDof ~1 μM, while both NTD-DBD and NTD bind to CypD at ~10 μMKD(Fig. 1C and 1D). Thus, instead of DBD, NTD is the major CypD binding site on p53. NMR titration and MD simulation revealed that NTD binds CypD with broad and dynamic interfaces dominated by electrostatic interactions. NTD 20-70 was further identified as the minimal binding region for CypD interaction, and two NTD fragments, D1 (residues 22-44) and D2 (58-70), can each bind CypD with mM affinity. Our detailed biophysical characterization of the dynamic interface between NTD and CypD provides novel insights on the p53-dependent mPTP opening and drug discovery targeting NTD/CypD interface in diseases.
Nematode Small RNA Pathways in the Absence of piRNAs
Authors: Zagoskin Maxim , Wang Jianbin , Neff Ashley T. , Veronezi Giovana M. B. , Davis Richard E.
Published: July 2021

Small RNA pathways play diverse regulatory roles in the nematodeC. elegans.However, our understanding of small RNA pathways, their conservation, and their roles in other nematodes is limited. Here, we analyzed small RNA pathways in the parasitic nematodeAscaris. Ascarishas ten Argonautes with five worm-specific Argonautes (WAGOs) that are associated with secondary 5’-triphosphate small RNAs (22-24G-RNAs). TheseAscarisWAGOs and their small RNAs target repetitive sequences (WAGO-1, WAGO-2, WAGO-3, and NRDE-3) or mature mRNAs (CSR-1, NRDE-3, and WAGO-3) and are similar to theC. elegansmutator, nuclear, and CSR-1 small RNA pathways.AscarisCSR-1 likely functions to “license” gene expression in the absence of anAscarispiRNA pathway.AscarisALG-4 and its associated 26G-RNAs target and appear to repress specific mRNAs during meiosis in the testes. Notably,AscarisWAGOs (WAGO-3 and NRDE-3) small RNAs change their targets between repetitive sequences and mRNAs during spermatogenesis or in early embryos illustrating target plasticity of these WAGOs. We provide a unique and comprehensive view of mRNA and small RNA expression throughout nematode spermatogenesis that illustrates the dynamics and flexibility of small RNA pathways. Overall, our study provides key insights into the conservation and divergence of nematode small RNA pathways.
Coordination of di-acetylated histone ligands by the ATAD2 bromodomain
Authors: Evans Chiara M. , Phillips Margaret , Malone Kiera L. , Tonelli Marco , Cornilescu Gabriel , Cornilescu Claudia , Holton Simon J. , Gorjánácz Mátyás , Wang Liping , Carlson Samuel , Gay Jamie C. , Nix Jay C. , Demeler Borries , Markley John L. , Glass Karen C.
Published: July 2021

The ATPase Family, AAA domain-containing protein 2 (ATAD2) bromodomain (BRD) has a canonical bromodomain structure consisting of four α-helices. ATAD2 functions as a co-activator of the androgen and estrogen receptors as well as the MYC and E2F transcription factors. ATAD2 also functions during DNA replication, recognizing newly synthesized histones. In addition, ATAD2 is shown to be up regulated in multiple forms of cancer including breast, lung, gastric, endometrial, renal, and prostate. Furthermore, up-regulation of ATAD2 is strongly correlated with poor prognosis in many types of cancer, making the ATAD2 bromodomain an innovative target for cancer therapeutics. In this study, we describe the recognition of histone acetyllysine modifications by the ATAD2 bromodomain. Residue-specific information on the complex formed between the histone tail and the ATAD2 bromodomain, obtained through nuclear magnetic resonance spectroscopy (NMR) and X-ray crystallography, illustrates key residues lining the binding pocket, which are involved in coordination of di-acetylated histone tails. Analytical ultracentrifugation, NMR relaxation data, and isothermal titration calorimetry further confirm the monomeric state of the functionally active ATAD2 bromodomain in complex with di-acetylated histone ligands. Overall, we describe histone tail recognition by ATAD2 BRD and illustrate that one acetyllysine group is primarily engaged by the conserved asparagine (N1064), the “RVF” shelf residues, and the flexible ZA loop. Coordination of a second acetyllysine group also occurs within the same binding pocket, but is essentially governed by unique hydrophobic and electrostatic interactions making the di-acetyllysine histone coordination more specific than previously presumed.
Transfer Learning Compensates Limited Data, Batch-Effects, And Technical Heterogeneity In Single-Cell Sequencing
Authors: Park Youngjun , Hauschild Anne-Christin , Heider Dominik
Published: July 2021

Abstract

Tremendous advances in next-generation sequencing technology have enabled the accumulation of large amounts of omics data in various research areas over the past decade. However, study limitations due to small sample sizes, especially in rare disease clinical research, technological heterogeneity, and batch effects limit the applicability of traditional statistics and machine learning analysis. Here, we present a meta-learning approach to transfer knowledge from big data and reduce the search space in data with small sample sizes. Few-shot learning algorithms integrate meta-learning to overcome data scarcity and data heterogeneity by transferring molecular pattern recognition models from datasets of unrelated domains. We explore few-shot learning models with large scale public dataset, TCGA (The Cancer Genome Atlas) and GTEx dataset, and demonstrate their potential as meta-learning dataset in other molecular pattern recognition tasks. Our results show that transfer learning is very effective for datasets with a limited sample size. Furthermore, we show that our approach can transfer knowledge across technological heterogeneity, e.g., from bulk cell to single-cell data. Our approach can overcome study size constraints, batch effects, and technological limitations in analyzing single-cell data by leveraging existing bulk-cell sequencing data.
Using Deep Learning to Count Monarch Butterflies in Dense Clusters
Authors: Patel Shruti , Kulkarni Amogh , Mukhopadhyay Ayan , Gujar Karuna , de Roode Jaap
Published: July 2021

Monarch butterflies display one of the most fascinating migration patterns of all species, traveling over 3000 miles from their North American breeding grounds to reach overwintering sites in Central Mexico. Recent studies have suggested that monarchs have experienced an alarming decline in population size due to a combination of deforestation, loss of native milkweed and nectaring plants, and climate change. An issue that conservation efforts face is the lack of principled mechanisms to accurately estimate and count the population size of monarchs. This difficulty occurs due to their small size and existence in dense overwintering clusters in forests. We create an open-source tool to aid conservationists estimate the count of monarch butterflies from images automatically. To the best of our knowledge, our approach, based on deep convolutional neural networks, is the first automated application that can count small insects like monarch butterflies in dense clusters. We demonstrate that our approach achieves high accuracy in counting the number of butterflies even in the presence of occlusion. We also release an open-source dataset containing high resolution images of monarch butterflies along with human annotations for each butterfly’s position. Our open-source implementation can be readily used by scientists to estimate monarch numbers in overwintering clusters and could also be adapted for use in other clustering species.
Microtubule-based mitotic spindles contain a micron-sized mixed-nucleotide zone
Authors: Castrogiovanni Cédric , Inchingolo Alessio , Harrison Jonathan U. , Dudka Damian , Sen Onur , Burroughs Nigel , McAinsh Andrew D. , Meraldi Patrick
Published: July 2021

Current models infer that the microtubule-based mitotic spindle is built from GDP-tubulin with small GTP caps at microtubule plus-ends, including those that attach to kinetochores (K-fibres). Here we reveal that K-fibres additionally contain a dynamic mixed-nucleotide zone that reaches several microns in length. This zone becomes visible in cells expressing fluorescently labelled EBs, a known marker for GTP-tubulin, and endogenously-labelled HURP - a protein which we show to preferentially bind the GDP microtubule lattice in vitro. In living cells HURP accumulates on the ends of depolymerising K-fibres, whilst avoiding recruitment to nascent polymerising K-fibres. This gives rise to a growing “HURP-gap” which we can recapitulate in a minimal computational simulation. We therefore postulate that the K-fibre lattice contains a dynamic, micron-sized mixed-nucleotide zone.

One Sentence Summary

We reveal that the microtubules of the mitotic spindle contain a third, uncharacterized domain, a mixed nucleotide zone that resides between the GTP-cap and the GDP-tubulin lattice.
Transcriptome analysis ofPseudomonas aeruginosabiofilm infection in anex vivopig model of the cystic fibrosis lung
Authors: Harrington Niamh E. , Littler Jenny L. , Harrison Freya
Published: July 2021

Pseudomonas aeruginosais the predominant cause of chronic biofilm infections that form in the lungs of people with cystic fibrosis (CF). These infections are highly resistant to antibiotics and persist for years in the respiratory tract. One of the main research challenges is that current laboratory models do not accurately replicate key aspects of a chronicP. aeruginosabiofilm infection, highlighted by previous RNA-sequencing studies. We compared theP. aeruginosaPA14 transcriptome in anex vivopig lung (EVPL) model of CF and a well-studied synthetic cystic fibrosis sputum medium (SCFM).P. aeruginosawas grown in the EVPL model for 1, 2 and 7 days, andin vitroin SCFM for 1 and 2 days. The RNA was extracted and sequenced at each time point. Our findings demonstrate that expression of antimicrobial resistance genes was cued by growth in the EVPL model, highlighting the importance of growth environment in determining accurate resistance profiles. The EVPL model created two distinct growth environments: tissue-associated biofilm and the SCFM surrounding tissue, each of which cued a transcriptome distinct from that seen in SCFMin vitro. The expression of quorum sensing associated genes in the EVPL tissue-associated biofilm at 48 h relative toin vitroSCFM was found to be similar to CF sputum versusin vitroconditions. Hence, the EVPL model can replicate key aspects ofin vivobiofilm infection that are missing from other current models and provides a more accurateP. aeruginosagrowth environment for determining antimicrobial resistance.

Significance Statement

Pseudomonas aeruginosalung infections that affect people with cystic fibrosis are a well-researched area, yet they continue to evade antimicrobial treatments. The lack of a laboratory model that captures all key aspects of these infections hinders not only research progression but also clinical diagnostics. We used transcriptome analysis to demonstrate how a model using pig lungs can more accurately replicate key characteristics ofP. aeruginosalung infection, including mechanisms of antibiotic resistance and infection establishment. Therefore, this model may be used in the future to further understand infection dynamics to develop novel treatments and more accurate treatment plans. This could improve clinical outcomes as well as quality of life for individuals affected by these infections.
Nano-Insecticides Against the Black Cutworm Agrotis ipsilon (Lepidoptera: Noctuidae): Toxicity, Development, Enzyme activity, and DNA Mutagenicity
Authors: Awad Mona , Ibrahim El-Desoky S. , Osman Engy I. , Elmenofy Wael H. , Mahmoud Abdel Wahab M. , Atia Mohamed A. M. , Moustafa Moataz A. M.
Published: July 2021

High-frequency doses of chemical pesticides cause environmental pollution with high pesticide residues. In addition, increasing insecticide resistance in many insect pests requires novel pest control methods. Nanotechnology could be a promising field of modern agriculture, and is receiving considerable attention in the development of novel nano-agrochemicals, such as nanoinsectticides and nanofertilizers. This study assessed the effects of the lethal and sublethal concentrations of chlorantraniliprole, thiocyclam, and their nano-forms on the development, reproductive activity, oxidative stress enzyme activity, and DNA changes at the molecular level of the polyphagous species of black cutworm Agrotis ipsilon. The results revealed that A. ipsilon larvae were more susceptible to the nano-formsthan the regular forms of both nano chlorine and sulfur within the chlorantraniliprole and thiocyclam insecticides, respectively, with higher toxicities than the regular forms (ca. 3.86, and ca.2.06-fold, respectively). Significant differences in biological parameters, including developmental time and reproductive activity (fecundity and hatchability percent) were also observed. Correspondingly, increases in oxidative stress enzyme activities were observed, as were mutagenic effects on the genomic DNA of A. ipsilon after application of the LC50 of the nano-forms of both insecticides compared to the control. The positive results obtained here have led us to apply these nano-forms indifferent insect models in additional studies.
Inhibition of Mitochondrial Fission by Drp-1 Blockade by Short-Term Leptin and Mdvi-1 Treatment Improves White Adipose Tissue Abnormalities in Obesity and Diabetes
Authors: Finocchietto P , Perez H , Blanco G , Miksztowicz V , Marotte C , Morales C , Peralta J , Berg G , Poderoso C , Poderoso JJ , Carreras MC
Published: July 2021

Background

Obesity and type 2 diabetes are chronic diseases characterized by insulin resistance, mitochondrial dysfunction and morphology abnormalities.

Objective

Herein, we investigated if dysregulation of mitochondrial dynamics and biogenesis is involved in an animal model of obesity and diabetes.

Methods

The effect of short-term leptin and mdivi-1 –a selective inhibitor of Drp-1 fission-protein– treatment on mitochondrial dynamics and biogenesis was evaluated in epididymal white adipose tissue (WAT) from male ob/ob mice.

Results

An increase in Drp-1 protein levels and a decrease in Mfn2 and OPA-1 protein expression were observed with enhanced and sustained mitochondrial fragmentation in ob/ob mice compared to wt C57BL/6 animals (p<0.05). The content of mitochondrial DNA and mRNA expression of PGC-1α –both parameters of mitochondrial biogenesis– were reduced in ob/ob mice (p<0.05). Leptin and mdivi-1 treatment significantly increased mitochondrial biogenesis, improved fusion-to-fission balance and attenuated mitochondrial dysfunction, thus inducing white-to-beige adipocyte transdifferentiation. Measurements of glucose and lipid oxidation in adipocytes revealed that both leptin and mdivi-1 increase substrates oxidation while in vivo determination of blood glucose concentration showed decreased levels by 50% in ob/ob mice, almost to the wt level.

Conclusions

Pharmacological targeting of Drp-1 fission protein may be a potential novel therapeutic tool for obesity and type 2 diabetes.
US Dog Importations during the COVID-19 Pandemic: Do we have an erupting problem?
Authors: Pieracci Emily G , Williams Cara , Wallace Ryan M , Kalapura Cheryl , Brown Clive M
Published: July 2021

Dog importation data from 2018-2020 were evaluated to ascertain whether the dog importation patterns in the United States changed during the COVID-19 pandemic, specifically with regard to denial of entry. Dog denial of entry reports from January 1, 2018, to December 31, 2020, stored within the Centers for Disease Control and Prevention (CDC) Quarantine Activity Reporting System, were reviewed. Basic descriptive statistics were used to analyze the data. Reason for denial, country of origin, and month of importation were all examined to determine which countries of origin resulted in the largest number of denials, and whether there was a seasonal change in importations during the COVID-19 pandemic (2020), compared to previous years (2018 and 2019). During 2020, CDC denied entry to 458 dogs. This represents a 52% increase in dogs denied entry compared to the averages in 2018 and 2019. Dogs were primarily denied entry for falsified rabies vaccination certificates (56%). Three countries exported 74% of all dogs denied entry into the United States, suggesting that targeted interventions may be needed for certain countries. Increased attempts to import inadequately vaccinated dogs from countries with canine rabies in 2020 may have been due to the increased demand for domestic pets during the COVID-19 pandemic. Educational messaging should highlight the risk of rabies and the importance of making informed pet purchases from foreign entities to protect pet owners, their families, and the public.
Automated identification of piglet brain tissue from MRI images using Region-based Convolutional Neural Networks
Authors: Stanke Kayla L. , Larsen Ryan J. , Rund Laurie , Leyshon Brian J. , Louie Allison Y. , Steelman Andrew J.
Published: July 2021

Magnetic Resonance Imaging is an important tool for characterizing volumetric changes of the piglet brain during development. Typically, an early step of an imaging analysis pipeline is brain extraction, or skull stripping. Brain extractions are usually performed manually; however, this approach is time-intensive and can lead to variation between brain extractions when multiple raters are used. Automated brain extractions are important for reducing the time required for analyses and improving the uniformity of the extractions. Here we demonstrate the use of Mask R-CNN, a Region-based Convolutional Neural Network (R-CNN), for automated brain extractions of piglet brains. We validate our approach using Nested Cross-Validation on six sets of training/validation data drawn from 32 pigs. Visual inspection of the extractions shows acceptable accuracy, Dice coefficients are in the range of 0.95-0.97, and Hausdorff Distance values in the range of 4.1-8.3 voxels. These results demonstrate that R-CNNs provide a viable tool for skull stripping of piglet brains.
TELSAM polymers accelerate crystallization of fused target proteins by stabilizing minimal crystal contacts and in the absence of direct inter-TELSAM contacts
Authors: Sarath Nawarathnage Supeshala D. , Soleimani Sara , Mathis Moriah H. , Bezzant Braydan D. , Ramírez Diana T , Gajjar Parag , Bunn Derick , Stewart Cameron , Smith Tobin , Pedroza Romo Maria J , Brown Seth , Doukov Tzanko , Moody James D.
Published: July 2021

We extend investigation into the usefulness of genetic fusion to TELSAM polymers as an effective protein crystallization strategy. We tested various numbers of the target protein fused per turn of the TELSAM helical polymer and various TELSAM–target connection strategies. We provide definitive evidence that: 1. A TELSAM–target protein fusion can crystallize more rapidly than the same target protein alone, 2. TELSAM–target protein fusions can form well-ordered, diffracting crystals using either flexible or rigid TELSAM–target linkers, 3. Well-ordered crystals can be obtained when either 2 or 6 copies of the target protein are presented per turn of the TELSAM helical polymer, 4. The TELSAM polymers themselves need not directly contact one another in the crystal lattice, and 5. Fusion to TELSAM polymer confers immense avidity to stabilize exquisitely weak inter-target protein crystal contacts. We report features of TELSAM-target protein crystals and outline future work needed to define the requirements for reliably obtaining optimal crystals of TELSAM–target protein fusions.
Selection of reference genes for quantitative analysis of microRNA expression in three cancers
Authors: Veryaskina Yuliya A. , Titov Sergei E. , Ivanov Mikhail K. , Ruzankin Pavel S. , Tarasenko Anton S. , Shevchenko Sergei P. , Kovynev Igor B. , Stupak Evgenij V. , Pospelova Tatiana I. , Zhimulev Igor F.
Published: July 2021

MicroRNAs (miRNAs) are promising biomarkers in cancer research. Quantitative PCR (qPCR), also known as real-time PCR, is the most frequently used technique for measuring miRNA expression levels. The use of this technique, however, requires that expression data be normalized against reference genes. The problem is that a universal internal control for quantitative analysis of miRNA expression by qPCR has yet to be known. The aim of this work was to find the miRNAs with stable expression in the thyroid gland, brain and bone marrow according to NanoString nCounter miRNA quantification data. As a results, the most stably expressed miRNAs were as follows: miR-361-3p, -151a-3p and -29b-3p in the thyroid gland; miR-15a-5p, -194-5p and -532-5p in the brain; miR-140-5p, -148b-3p and -362-5p in bone marrow; and miR-423-5p, -28-5p and -532-5p, no matter what tissue type. These miRNAs represent promising reference genes for miRNA quantification by qPCR.
Expression of Spred2 in the urothelial tumorigenesis of the urinary bladder
Authors: Oda Shinsuke , Fujisawa Masayoshi , Chunning Li , Ito Toshihiro , Yamaguchi Takahiro , Yoshimura Teizo , Matsukawa Akihiro
Published: July 2021

Aberrant activation of the Ras/Raf/ERK (extracellular-signal-regulated kinase)-MAPK (mitogen-activated protein kinase) pathway is involved in the progression of cancer, including urothelial carcinoma; but the negative regulation remains unclear. In the present study, we investigated pathological expression of Spred2 (Sprouty-related EVH1 domain-containing protein 2), a negative regulator of the Ras/Raf/ERK-MAPK pathway, and the relation to ERK activation and Ki67 index in various categories of 275 urothelial tumors obtained from clinical patients. In situ hybridization demonstrated that Spred2 mRNA was highly expressed in high-grade non-invasive papillary urothelial carcinoma (HGPUC), and the expression was decreased in carcinoma in situ (CIS) and infiltrating urothelial carcinoma (IUC). Immunohistochemically, membranous Spred2 expression, important to interact with Ras/Raf, was preferentially found in HGPUC. Interestingly, membranous Spred2 expression was decreased in CIS and IUC relative to HGPUC, while ERK activation and the expression of the cell proliferation marker Ki67 index were increased. HGPUC with membranous Spred2 expression correlated significantly with lower levels of ERK activation and Ki67 index as compared to those with negative Spred2 expression. Thus, our pathological findings suggest that Spred2 negatively regulates cancer progression in non-invasive papillary carcinoma possibly through inhibiting the Ras/Raf/ERK-MAPK pathway, but this regulatory mechanism is lost in cancers with high malignancy. Spred2 appears to be a key regulator in the progression of non-invasive bladder carcinoma.
CardiOvascular examination in awake Orangutans (Pongo pygmaeus pygmaeus): Low-stress Echocardiography including Speckle Tracking imaging (the COOLEST method)
Authors: Chetboul Valérie , Concordet Didier , Tissier Renaud , Vonfeld Irène , Poissonnier Camille , Alvarado Maria Paz , Passavin Peggy , Gluntz Mathilde , Lefort Solène , Bourgeois Aude , Duby Dylan , Hano Christelle , Chai Norin
Published: July 2021

Introduction

Cardiovascular diseases have been identified as a major cause of mortality and morbidity in Borneo orangutans (Pongo pygmaeus pygmaeus). Transthoracic echocardiography is usually performed under anesthesia in great apes, which may be stressful and risky in cardiac animals. The aim of the present pilot study was hence to develop a quick and non-stressful echocardiographic method (i.e., the COOLEST method) in awake Borneo orangutans (CardiOvascular examination in awake Orangutans: Low-stress Echocardiography including Speckle Tracking imaging) and assess the variability of corresponding variables.

Materials and Methods

Four adult Borneo orangutans trained to present their chest to the trainers were involved. A total of 96 TTE examinations were performed on 4 different days by a trained observer examining each orangutans 6 times per day. Each examination included four two-dimensional views, with offline assessment of 28 variables (i.e., two-dimensional (n=12), M-mode and anatomic M-mode (n=6), Doppler (n=7), and speckle tracking imaging (n=3)), representing a total of 2,688 measurements. A general linear model was used to determine the within-day and between-day coefficients of variation.

Results

Mean±SD (minimum-maximum) images acquisition duration was 3.8±1.6 minutes (1.3-6.3). All within-day and between-day coefficients of variation but one (n=55/56, 98%) were <15%, and most (51/56, 91%) were <10% including those of speckle tracking systolic strain variables (2.7% to 5.4%).

Discussion

Heart morphology as well as global and regional myocardial function can be assessed in awake orangutans with good to excellent repeatability and reproducibility.

Conclusions

This non-stressful method may be used for longitudinal cardiac follow-up in awake orangutans.
Long-term changes in populations of rainforest birds in the Australia Wet Tropics bioregion: a climate-driven biodiversity emergency
Authors: Williams Stephen E. , de la Fuente Alejandro
Published: July 2021

Many authors have suggested that the vulnerability of montane biodiversity to climate change worldwide is significantly higher than in most other ecosystems. Despite the extensive variety of studies predicting severe impacts of climate change globally, few studies have empirically validated the predicted changes in distribution and population density. Here, we used 17 years of bird monitoring across latitudinal/elevational gradients in the rainforest of the Australian Wet Tropics World Heritage Area to assess changes in local abundance and distribution. We used relative abundance in 1977 surveys across 114 sites ranging from 0-1500m above sea level and utilised a trend analysis approach (TRIM) to investigate elevational shifts in abundance of 42 species between 2000 – 2016. The local abundance of most mid and high elevation species has declined at the lower edges of their distribution by >40% while lowland species increased by up to 190% into higher elevation areas. Upland-specialised species and regional endemics have undergone dramatic population declines of almost 50%. The “Outstanding Universal Value” of the Australian Wet Tropics World Heritage Area, one of the most irreplaceable biodiversity hotspots on Earth, is rapidly degrading. These observed impacts are likely to be similar in many tropical montane ecosystems globally.
Comparative analysis of transcriptomic profiles among ascidians, zebrafish, and mice: insights from tissue-specific gene expression
Authors: Matsubara Shin , Osugi Tomohiro , Shiraishi Akira , Wada Azumi , Satake Honoo
Published: July 2021

Tissue/organ-specific genes (TSGs) are important not only for understanding organ development and function, but also for investigating the evolutionary lineages of organs in animals. Here, we investigate the TSGs of 9 adult tissues of an ascidian, Ciona intestinalis Type A (Ciona robusta), which lies in the important position of being the sister group of vertebrates. RNA-seq and qRT-PCR identified the Ciona TSGs in each tissue, and BLAST searches identified their homologs in zebrafish and mice. Tissue distributions of the vertebrate homologs were analyzed and clustered using public RNA-seq data for 12 zebrafish and 30 mouse tissues. Among the vertebrate homologs of the Ciona TSGs in the neural complex, 48% and 63% showed high expression in the zebrafish and mouse brain, respectively, suggesting that the central nervous system is evolutionarily conserved in chordates. In contrast, vertebrate homologs of Ciona TSGs in the ovary, pharynx, and intestine were not consistently highly expressed in the corresponding tissues of vertebrates, suggesting that these organs have evolved in Ciona-specific lineages. Intriguingly, more TSG homologs of the Ciona stomach were highly expressed in the vertebrate liver (17-29%) and intestine (22-33%) than in the mouse stomach (5%). Expression profiles for these gene suggest that the biological roles of the Ciona stomach are distinct from those of their vertebrate counterparts. Collectively, Ciona tissues were categorized into 3 groups: i) high similarity to the corresponding vertebrate tissues (neural complex and heart), ii) low similarity to the corresponding vertebrate tissues (ovary, pharynx, and intestine), and iii) low similarity to the corresponding vertebrate tissues, but high similarity to other vertebrate tissues (stomach, endostyle, and siphons). The present study provides transcriptomic catalogs of adult ascidian tissues and significant insights into the evolutionary lineages of the brain, heart, and digestive tract of chordates.
Improving RT-LAMP Detection of SARS-CoV-2 RNA through Primer Set Selection and Combination
Authors: Zhang Yinhua , Tanner Nathan A.
Published: July 2021

Reverse transcription loop-mediated isothermal amplification (RT-LAMP) has emerged as a viable molecular diagnostic method to expand the breadth and reach of nucleic acid testing, particularly for SARS-CoV-2 detection and surveillance. While rapidly growing in prominence, RT-LAMP remains a relatively new method compared to the standard RT-qPCR, and contribution to our body of knowledge on designing LAMP primer sets and assays can have significant impact on its utility and adoption. Here we evaluate 18 LAMP primer sets for SARS-CoV-2, comparing speed and sensitivity with different LAMP formulations and conditions across more than 5,000 RT-LAMP reactions and identifying several primer sets with similar high sensitivity for different SARS-CoV-2 gene targets. Significantly we observe a consistent sensitivity enhancement by combining primer sets for different targets, confirming and building on earlier work to create a simple, general approach to building better and more sensitive RT-LAMP assays.
Fine-tuned method to extract high purified proteins from the seagrass Halophila stipulacea to be used for proteome analyses
Authors: Piro Amalia , Anagnostopoulou Vasileia , Apostolaki Eugenia T. , Mazzuca Silvia
Published: July 2021

The non-indigenous to the Mediterranean tropical seagrass Halophila stipulacea has the possibility to become more prevalent in the Mediterranean basin, exacerbated by the rapid increase of water temperature. Molecular profiling appears a promising tool to study the traits that render H. stipulacea tolerant and resilient and facilitate its rapid and vast geographical spread. Taking advantage from recent seagrass genomes sequencing, proteomics specialty has been applied to several seagrasses giving new insight on the biology and physiology of this group of angiosperms. Thus, it could be of interest to apply proteomics to H. stipulacea that it could be considered as a possible plant model species to study marine biological invasion. The first step to achieve this goal is to obtain high quality proteins from plant tissue. Tissue fixation and protein extraction protocol are the most challenging steps in proteomics. Here we report a fine-tuned procedure obtained by comparing protein yield from H. stipulacea plants frozen in liquid nitrogen or preserved in RNAlater and processed following two different extraction protocols. Higher protein yield have been extracted from the procedure that use the RNAlater preserved plants, extracted with trichloroacetic acid in water followed by trichloroacetic acid in acetone, compared to those obtained from all other procedures. Protein purity of these samples have been tested by the separation in SDS-PAGE comfirming a better resolved profile of peptide bands suitable for a gel-based proteomics. Then, to assess the quality of proteins the mHPLC-ESI-MS/MS mass spectrometry analyses and bioinformatics have been performed. Hundreds proteins have been identified against several seagrass genomic resources available at UniProt, NCBI, SeagrassDB and transcriptomic datasets, which were merged to form the first customized dataset useful for H. stipulacea proteomic investigations.
Effects of pen enrichment on leg health of fast and slower-growing broiler chickens
Authors: Güz Bahadır Can , de Jong Ingrid C. , Souza Da Silva Carol , Veldkamp Fleur , Kemp Bas , Molenaar Roos , van den Brand Henry
Published: July 2021

Pen enrichment for broiler (meat-type) chickens is one of the potential strategies to stimulate locomotion and consequently contribute to leg health and welfare. This study was designed to evaluate effects of using a plethora of pen enrichments (barrier perches, angular ramps, horizontal platforms, large distance between feed and water and providing live Black Soldier fly larvae in a dustbathing area) on tibia characteristics, locomotion, leg health and home pen behaviour of fast and slower-growing broiler chickens. The experiment was set up as a 2 x 2 factorial arrangement with a total of 840 male broiler chickens in a complete randomized design (7 replicates per treatment and 30 chickens per replicate) with the following treatments: 1) pen enrichment (enriched pen or non-enriched pen); 2) broiler strain (fast-growing Ross 308 or slower-growing Hubbard JA 757). Home pen behaviour and use of enrichment were observed. At approximately 1400 and 2200 gram body weight, two chickens per pen were randomly selected and slaughtered, to investigate tibia morphological, biophysical and mechanical characteristics and leg health. Pen enrichment positively affected tibia biophysical characteristics, e.g., osseous volume (Δ=1.8 cm3,P=0.003), total volume (Δ=1.4 cm3,P=0.03) and volume fraction (Δ=0.02 %,P=0.002), in both fast and slower-growing chickens, suggesting that pen enrichment particularly affects ossification and mineralization mechanisms. Accordingly, locomotion and active behaviours were positively influenced by pen enrichment. However, pen enrichment resulted in lower body weight gain in both strains, which might be due to higher activity or lower feed intake as a result of difficulties of crossing the barrier perches. Regarding the strain, slower-growing chickens showed consistently more advanced tibia characteristics and more active behaviour than fast-growing chickens. It can be concluded that pen enrichment may lead to more activity and better bone development in both fast and slower-growing chickens.
A real-time assay for cell-penetrating peptide-mediated delivery of molecular cargos
Authors: Gentry Schuyler B. , Nowak Scott J. , Ni Xuelei , Hill Stephanie A. , Wade Lydia R. , Clark William R. , Keelaghan Aidan P. , Morris Daniel P. , McMurry Jonathan L.
Published: July 2021

Cell-penetrating peptides (CPPs) are capable of transporting molecules to which they are tethered across cellular membranes. Unsurprisingly, CPPs have attracted attention for their potential drug delivery applications, but several technical hurdles remain to be overcome. Chief among them is the so-called ‘endosomal escape problem,’ i.e. the propensity of CPP-cargo molecules to be endocytosed but remain entrapped in endosomes rather than reaching the cytosol. Previously, a CPP fused to calmodulin that bound calmodulin binding site-containing cargos was shown to efficiently deliver cargos to the cytoplasm, effectively overcoming the endosomal escape problem. The CPP-adaptor, “TAT-CaM,” evinces delivery at nM concentrations and more rapidly than we had previously been able to measure. To better understand the kinetics and mechanism of CPP-adaptor-mediated cargo delivery, a real-time cell penetrating assay was developed in which a flow chamber containing cultured cells was installed on the stage of a confocal microscope to allow for observationab initio. Also examined in this study was an improved CPP-adaptor that utilizes naked mole rat (Heterocephalus glaber) calmodulin in place of human and results in superior internalization, likely due to its lesser net negative charge. Adaptor-cargo complexes were delivered into the flow chamber and fluorescence intensity in the midpoint of baby hamster kidney cells was measured as a function of time. Delivery of 400 nM cargo was observed within seven minutes and fluorescence continued to increase linearly as a function of time. Cargo-only control experiments showed that the minimal uptake which occurred independently of the CPP-adaptor resulted in punctate localization consistent with endosomal entrapment. A distance analysis was performed for cell-penetration experiments in which CPP-adaptor-delivered cargo showing wider dispersions throughout cells as compared to an analogous covalently-bound CPP-cargo. Small molecule endocytosis inhibitors did not have significant effects upon delivery. The real-time assay is an improvement upon static endpoint assays and should be informative in a broad array of applications.
Deficiency inLystfunction leads to accumulation of secreted proteases and predisposition to mechanic stress-induced retinal detachment
Authors: Ji Xiaojie , Zhao Lihong , Umapathy Ankita , Fitzmaurice Bernard , Wang Jieping , Williams David S. , Chang Bo , Naggert Jürgen K. , Nishina Patsy M.
Published: July 2021

Chediak–Higashi syndrome, caused by mutations in theLysosomeTrafficking Regulator (Lyst) gene, is a recessive hypopigmentation disorder characterized by albinism, neuropathies, neurodegeneration, and defective immune responses, with enlargement of lysosomes and lysosome-related organelles. Although recent studies have suggested thatLystmutations impair the regulation of sizes of lysosome and lysosome-related organelle, the underlying pathogenic mechanism of Chediak–Higashi syndrome is still unclear. Here we show striking evidence that deficiency in LYST protein function leads to accumulation of photoreceptor outer segment phagosomes in retinal pigment epithelial cells, and reduces adhesion between photoreceptor outer segment and retinal pigment epithelial cells in a mouse model of Chediak–Higashi syndrome. In addition, we observe elevated levels of cathepsins, matrix metallopeptidase (MMP) 3 and oxidative stress markers in the retinal pigment epithelium ofLystmutants. Previous reports showed that impaired degradation of photoreceptor outer segment phagosomes causes elevated oxidative stress, which could consequently lead to increases of cysteine cathepsins and MMPs in the extracellular matrix. Taken together, we conclude that the loss of LYST function causes accumulation of phagosomes in the retinal pigment epithelium and elevation of several extracellular matrix-remodeling proteases through oxidative stress, which may, in turn, reduce retinal adhesion. Our work reveals previously unreported pathogenic events in the retinal pigment epithelium caused byLystdeficiency, which may place Chediak–Higashi syndrome patients at increased risk for retinal detachment. The same pathogenic events may be conserved in other professional phagocytic cells, such as macrophages in the immune system, contributing to overall Chediak–Higashi syndrome pathology.
Highly selective Cooperative Primers for multiplex detection of rare mutations
Authors: Kent Jana Oliveriusova , Christensen Masen Chad
Published: July 2021

We describe a real time PCR-based technique capable of detecting and quantifying rare somatic mutations in circulating tumor DNA reference materials. Our approach utilizes previously described Cooperative Primers, structurally modified to exhibit high allele-specificity and one-copy target sensitivity. Cooperative Primers are bi-functional molecules, consisting of a high affinity probe fragment that guarantees sensitivity, and a covalently attached lower affinity primer providing specificity. Additional optimization of Cooperative Primer structure generated molecules capable of reliable detection of allele changes as small as a single nucleotide. These highly selective Cooperative Primers maintain excellent discrimination properties in rare mutant allele scenarios, in both monoplex and multiplex assays. With synthetic DNA samples, Cooperative Primers can detect as little as 100 copies of mutant template amongst 1 000 000 copies of wild-type template (minor allele fraction of 0.01 %). Multiplex Cooperative Primer assay was validated with cell-free DNA reference materials and consistently detected the lowest minor allele fraction available (0.1 %) for EGFR L858R, G719S and V769-D770insASV mutations, while simultaneously providing qualitative and quantitative assessment of cell-free DNA with integrated β-Actin assay. Easy to design, rapid and inexpensive, Cooperative Primer - based real time PCR assays are a promising tool for evaluation of cancer therapy response, occurrence of resistance mutations and relapse monitoring.
Bioerosion traces and borings in the Upper Devonian vertebrate remains from the Holy Cross Mountains, Poland Bioerosion in fish remains
Authors: Szrek Piotr , Dworczak Patrycja G. , Wilk Olga
Published: July 2021

Among the hundreds of collected Devonian vertebrate macrofossils in the Holy Cross Mountains, placoderms dominate and provide data on their morphology, distribution and taphonomy. So far 17 out of more than 500 studied specimens have revealed bones with surfaces covered by sediment-filled trace fossils. The traces have been made on the vertebrate remains before their final burial. The borings, oval in cross-section, include dendroidal networks of shallow tunnels or short, straight or curved individual scratches and grooves, which frequently create groups on the both sides of the bones. ?Karethraichnusisp. from Kowala and ?Osteocallisisp. from Wietrznia are the oldest record of these ichnogenera. Sedimentological clues indicate a shallow water environment, probably from the slope below the storm wave base.
Waves and currents decrease the available space in a salmon cage
Authors: Johannesen Ása , Patursson Øystein , Kristmundsson Jóhannus , Dam Signar Pætursonur , Mulelid Mats , Klebert Pascal
Published: July 2021

Due to increasing demand for salmon and environmental barriers preventing expansion in established sites, salmon farmers seek to move or expand their production to more exposed sites. In this study we investigate the effects of strong currents and waves on the behaviour of salmon and how they choose to use the space available to them. Observations are carried out in a site with strong tidal currents and well mixed water. Using video cameras and echo sounders, we show that salmon prefer to use the entire water column, narrowing their range only as a response to cage deformation, waves, or daylight. Conversely, salmon show strong horizontal preference, mostly occupying the portions of the cage exposed to currents. Additionally, waves cause salmon to disperse from the exposed side of the cage to the more sheltered side. Even when strong currents decrease the amount of available space, salmon choose to occupy the more exposed part of the cage. This indicates that at least with good water exchange, the high density caused by limited vertical space is not so aversive that salmon choose to move to less desirable areas of the cage. However, the dispersal throughout the entire available water column indicates that ensuring enough vertical space, even in strong currents, would be beneficial to salmon welfare.
Targeting “Immunogenic Hotspots” in Dengue and Zika Virus: A Novel Approach to a Common Vaccine
Authors: Mahata Dhrubajyoti , Mukherjee Debangshu , Malviya Vanshika , Mukherjee Gayatri
Published: July 2021

Diseases caused by Dengue (DENV) and Zika (ZIKV) viruses cause significant mortality and illness globally. Due to the high sequence similarity of the viral proteins and the purported cross-reactive immune responses against the viruses, we envisioned a common multi-epitope vaccine (MEV) against both viruses by adopting a novel approach of identifying “immunogenic hotspots”. These stretches of the structural and non-structural proteins are enriched with MHC class I and class II supertype-restricted T cell epitopes, and B cell epitopes, in addition to being highly conserved between different DENV serotypes and ZIKV. Such an approach ensures inclusion of multiple overlapping T and B cell epitopes common to both viruses, and also warrants high population coverage. Importantly, epitopes known to cause antibody-dependent-enhancement of infection have been excluded. These immunogenic hotspots have then been stitched together with linkers in-silico along with an adjuvant, CTxB to develop the MEV candidate. Four structural models of the MEV were selected on the basis of conformational preservation of CTxB, and their biophysical parameters, which also conserved the immunogenicity of the multiple epitopes. Importantly, each of the MEV candidates were found to interact with TLR4-MD2 complex by molecular docking studies, indicative of their ability to induce TLR-mediated immune responses.