bluuebunny/crossref_metadata_2025 huggingface dataset:
Elimination of human papillomavirus 16-induced tumors by a mucosal rAd5 therapeutic vaccination in a pre-clinical study
Authors: Braun Molly R. , Lindbloom Jonathan D. , Moore Anne C. , Hodgson Katherine A. , Dora Emery G. , Tucker Sean N.
Published: March 2024

Therapeutic vaccination can harness the body’s cellular immune system to target and destroy cancerous cells. Several invasive treatments are currently used to eliminate cancerous lesions caused by human papillomaviruses (HPV), however therapeutic vaccination may offer and effective and minimally intrusive alternative. We have developed recombinant, non-replicating human adenovirus type 5 (rAd5) vaccines that encode the HPV16 oncogenic proteins E6 and E7 alongside a molecular dsRNA adjuvant. The potency of these vaccines were examined in a mouse model of HPV tumorigenesis where E6E7-expressing and transformed cells were implanted subcutaneously into C57BL/6 mice. After tumor growth, mice were treated via intranasal administration with E6E7-encoding rAd5 vaccines expressing either a mutant form of E6E7 (rAd5-16/E6E7m), or predicted T cell epitopes of E6E7 (rAd5-16/E6E7epitopes). Animals receiving therapeutic treatments of rAd5-16/E6E7mand rAd5-16/E6E7epitopeshad significant reductions in tumor volume and increased survival compared to animals treated with an empty rAd5 or left untreated. Further, antigen-specific CD8+ T effector memory cells (TEM) were observed in the animals treated with E6E7-encoding rAd5, but not in rAd5-empty group. The work described here demonstrates that mucosal rAd5 can be used in a therapeutic capacity to elicit antigen-specific cellular immunity and further identifies a clinical candidate with immense potential for the treatment and prevention of human cervical cancer.
AUTO-TUNE: SELECTING THE DISTANCE THRESHOLD FOR INFERRING HIV TRANSMISSION CLUSTERS
Authors: Weaver Steven , Dávila-Conn Vanessa , Ji Daniel , Verdonk Hannah , Ávila-Ríos Santiago , Brown Andrew J. Leigh , Wertheim Joel O. , Pond Sergei L. Kosakovsky
Published: March 2024

Molecular surveillance of viral pathogens and inference of transmission networks from genomic data play an increasingly important role in public health efforts, especially for HIV-1. For many methods, the genetic distance threshold used to connect sequences in the transmission network is a key parameter informing the properties of inferred networks. Using a distance threshold that is too high can result in a network with many spurious links, making it difficult to interpret. Conversely, a distance threshold that is too low can result in a network with too few links, which may not capture key insights into clusters of public health concern. Published research using the HIV-TRACE software package frequently uses the default threshold of 0.015 substitutions/site for HIV pol gene sequences, but in many cases, investigators heuristically select other threshold parameters to better capture the underlying dynamics of the epidemic they are studying.

Here, we present a general heuristic scoring approach for tuning a distance threshold adaptively, which seeks to prevent the formation of giant clusters. We prioritize the ratio of the sizes of the largest and the second largest cluster, maximizing the number of clusters present in the network.

We apply our scoring heuristic to outbreaks with different characteristics, such as regional or temporal variability, and demonstrate the utility of using the scoring mechanism’s suggested distance threshold to identify clusters exhibiting risk factors that would have otherwise been more difficult to identify. For example, while we found that a 0.015 substitutions/site distance threshold is typical for US-like epidemics, recent outbreaks like the CRF07_BC subtype among men who have sex with men (MSM) in China have been found to have a lower optimal threshold of 0.005 to better capture the transition from injected drug use (IDU) to MSM as the primary risk factor. Alternatively, in communities surrounding Lake Victoria in Uganda, where there has been sustained hetero-sexual transmission for many years, we found that a larger distance threshold is necessary to capture a more risk factor-diverse population with sparse sampling over a longer period of time. Such identification may allow for more informed intervention action by respective public health officials.
SLRfinder: a method to detect candidate sex-linked regions with linkage disequilibrium clustering
Authors: Yi Xueling , Kemppainen Petri , Merilä Juha
Published: March 2024

Despite their critical roles in genetic sex determination, sex chromosomes remain unknown in many non-model organisms. In contrast to conserved sex chromosomes in mammals and birds, studies of fish, amphibians, and reptiles have found highly labile sex chromosomes with newly evolved sex-linked regions (SLRs). These labile sex chromosomes are important for understanding early sex chromosome evolution but are difficult to identify due to the lack of Y/W degeneration and SLRs limited to small genomic regions. Here we present SLRfinder, a method to identify candidate SLRs and labile sex chromosomes using linkage disequilibrium (LD) clustering, patterns of heterozygosity, and genetic divergence. SLRfinder does not rely on specific sequencing methods or reference genomes and does not require phenotypic sexes which may be unknown from population sampling, although sex information can be incorporated to provide additional inference on candidate SLRs. We tested SLRfinder using various published datasets and compared it to SATC, a method that identifies sex chromosomes based on the depth of coverage and also does not require phenotypic sex. Results show that SATC works better on conserved sex chromosomes (e.g., in African leopards), whereas SLRfinder outperforms SATC in analyzing labile sex chromosomes (e.g., in nine-spined sticklebacks and chum salmon). Since SLRfinder primarily relies on LD clusters, it is expected to be most sensitive to the SLRs harboring structural variants (e.g., inversion) due to strongly reduced recombination rates in heterozygotes. SLRfinder provides a novel and complementary approach for identifying SLRs and uncovering additional sex chromosome diversity in nature.
The association between delirium and falls in older adults in the community: a systematic review
Authors: Eost-Telling Charlotte , McNally Lucy , Yang Yang , Shi Chunhu , Norman Gill , Ahmed Saima , Poku Brenda , Money Annemarie , Hawley-Hague Helen , Todd Chris , Shenkin Susan D. , Vardy Emma R.L.C.
Published: March 2024

Objective

Systematically review and critically appraise evidence for the association between delirium and falls in community-dwelling adults aged 60 years and above

Methods

We searched EMBASE, MEDLINE, PsycINFO, Cochrane Database of Systematic Reviews, CINAHL and Evidence-Based Medicine Reviews (EBMR) databases in April 2023. Standard methods were used to screen, extract data, assess risk of bias (using Newcastle Ottawa scale), provide a narrative synthesis and where appropriate conduct meta-analysis.

Results

We included eight studies, with at least 3505 unique participants. Five found limited evidence for an association between delirium and subsequent falls: one adjusted study showed an increase in falls (RR 6.66;95% CI 2.16-20.53) but the evidence was low certainty. Four non-adjusted studies found no clear effect. Three studies (one with two subgroups treated separately) found some evidence for an association between falls and subsequent delirium: meta-analysis of three adjusted studies showed an increase in delirium (pooled OR 2.01; 95%CI 1.52-2.66), one subgroup of non-adjusted data found no clear effect. Number of falls and fallers were reported in the studies. Four studies and one subgroup were at high risk of bias and one study had some concerns.

Conclusions

We found limited evidence for the association between delirium and falls. More methodologically rigorous research is needed to understand the complex relationship, establish how and why this operates bi-directionally and identify potential modifying factors involved. We recommend the use of standardised assessment measures for delirium and falls. Clinicians should be aware of the potential relationship between these common presentations.

Key points

This is the first systematic review of the association between delirium and falls in the wider community population.

There is relatively limited but consistent evidence on the direction of effect for both delirium preceding falls and falls preceding delirium.

More high-quality longitudinal work is needed to explore the nature of this potentially complex and bidirectional relationship.

History of falls and delirium should be considered when assessing patients with incidence/suspected incidence of falls or delirium.
Unlocking the Treatment of Facioscapulohumeral Muscular Dystrophy Type 2: The Bisphenol Connection
Authors: Sayad Saed , Hiatt Mark , Mustafa Hazem
Published: March 2024

Background

Facioscapulohumeral muscular dystrophy type 2 (FSHD2) poses a significant challenge within the domain of neuromuscular disorders, marked by a progressive decline in muscle strength accompanied by tissue wasting. FSHD2 results from chromosomal deletions triggering the activation of a dormant gene known as DUX4. While DUX4 typically regulates early embryonic development, its activation in adult muscle cells leads to premature cell death. Despite this understanding, the exact pathology of FSHD2 remains unclear. To date, no effective treatment for FSHD2 exists.

Method

We acquired single-cell RNA sequencing (RNA-Seq) data (GSE143452) from primary myoblasts for FSHD2 from the United States National Institutes of Health (NIH) portal website. Our analysis encompassed a comprehensive examination of differentially expressed genes, alongside associated compounds sourced from the Chemical Entities of Biological Interest (ChEBI) database. Employing rigorous statistical methods, we pinpointed the most prominently upregulated and downregulated genes. Subsequently, we determined the compounds capable of modulating the expression of these top genes, either enhancing or reducing their activity.

Results

Bisphenol S (BPS) can upregulate 52 of 100 top downregulated genes in FSHD2 without downregulating any other genes and Bisphenol F (BPF) can upregulate 45 of 100 downregulated genes with downregulating only one other gene. The enrichment analysis of both sets of 52 genes related to BPS and 45 genes corresponding to BPF highlights their significant involvement in various aspects of muscle biology, particularly as pertaining to the function and dysfunction of cardiac and skeletal muscle.

Conclusions

Leveraging single-cell RNA-Seq data and computational analysis, we identified key dysregulated genes in FSHD2 and elucidated their modulation by compounds such as BPS and BPF. While effective treatments for FSHD2 remain elusive, our study provides valuable insights into potential therapeutic targets and pathways for further investigation in the pursuit of effective interventions for this debilitating condition. However, more research is needed to understand whether the roles of BPS and F are constructive or destructive.
Developing multifactorial dementia prediction models using clinical variables from cohorts in the US and Australia
Authors: Finney Caitlin A. , Brown David A. , Shvetcov Artur ,   the Alzheimer’s Disease Neuroimaging Initiative,   the Australian Imaging Biomarkers and Lifestyle Flagship Study of Ageing
Published: March 2024

INTRODUCTION

Existing dementia prediction models using non-neuroimaging clinical measures have been limited in their ability to identify disease. This study used machine learning to re-examine the diagnostic potential of clinical measures for dementia.

METHODS

Data was sourced from the Australian Imaging, Biomarkers, and Lifestyle Flagship Study of Ageing (AIBL) and the Alzheimer’s Disease Neuroimaging Initiative (ADNI). Clinical variables included 21 measures across medical history, hematological and other blood tests, and APOE genotype. Tree-based machine learning algorithms and artificial neural networks were used.

RESULTS

APOE genotype was the best predictor of dementia cases and healthy controls. Our results, however, demonstrated that there are limitations when using publicly accessible cohort data that may limit the generalizability and interpretability of such predictive models.

DISCUSSION

Future research should examine the use of routine APOE genetic testing for dementia diagnostics. It should also focus on clearly unifying data across clinical cohorts.
Neural Networks Estimate Muscle Force in Dynamic Conditions Better than Hill-type Muscle Models
Authors: Athanasiadou Maria Eleni , Daley Monica A. , Koelewijn Anne D.
Published: March 2024

Hill-type muscle models are widely used, even though they do not accurately represent certain muscle mechanics. We explored neural networks to develop new muscle models. We trained neural networks to estimate muscle force from activation, muscle length, and muscle velocity. Training data was recorded using sonomicrometry, electromyography, and a tendon buckle on two muscles of guinea fowl. First, we compared the neural network to a Hill-type muscle model, using the same data for network training and model optimization. Second, we trained neural networks on large datasets, in a more realistic machine learning scenario. We found that neural networks generally yielded higher coefficients of determination and lower errors than Hill-type muscle models. Our neural networks performed better when estimating forces on the muscle used for training, but on another bird, than on a different muscle of the same bird, which could be explained by inaccuracies in activation and force scaling. We extracted forcelength and force-velocity relationships from the trained neural networks and found that both effects were underestimated and that both relationships were not replicated well outside of the training data distribution. We discuss suggested experimental designs to collect suitable training data and conclude that neural networks could provide an accurate alternative to Hill-type muscle models, particularly for modeling dynamic muscle behavior that is prevalent in faster movements, given a suitable training dataset, while scaling of the training data should be comparable between muscles and animals.

Neural networks predict muscle forces more accurately than Hill-type muscle models, particularly under dynamic conditions. However, they struggle to replicate the force-length and force-velocity relationships well.
Circular RNA circASH1L(4,5) protects microRNA-129-5p from target-directed microRNA degradation in human skin wound healing
Authors: Wang Qizhang , Niu Guanglin , Liu Zhuang , Toma Maria A. , Geara Jennifer , Bian Xiaowei , Zhang Letian , Piipponen Minna , Li Dongqing , Sommar Pehr , Landén Ning Xu
Published: March 2024

Both circular RNAs (circRNA) and microRNAs (miRNA) have emerged to play important roles in health and disease. To understand their function in tissue repair, we profiled circRNA, linear RNA, and miRNA expression dynamics in human wound-edge keratinocytes across the wound healing process. Our investigation spotlighted circASH1L(4,5) and its engagement with miR-129-5p, both of which levels were increased with wound repair. Unlike conventional miRNA sponging, circASH1L enhanced miR-129 stability and silencing activity by protecting this miRNA from target-directed miRNA degradation (TDMD) triggered by NR6A1 mRNA. TGF-β signaling, pivotal in wound healing, fostered circASH1L expression while suppressing NR6A1, thus enhancing the miR-129 abundance at the post-transcriptional level. Functionally, circASH1L and miR-129 enhanced keratinocyte migration and proliferation, crucial for re-epithelialization of human wounds. Collectively, our study uncovers circRNAs’ novel role as shields for miRNAs and sheds light on the physiological importance of regulated miRNA degradation in human skin wound healing.
Frontal engagement in perceptual integration under low subjective visibility
Authors: Bae Jisub , Jung Koeun , James Oliver , Suzuki Satoru , Kim Yee Joon
Published: March 2024

We investigated how spatiotemporal neural dynamics underlying perceptual integration changed with the degree of conscious access to a set of backward-masked pacman-shaped inducers that generated the percept of an illusory triangle. We kept the stimulus parameters at a fixed near-threshold level throughout the experiment and recorded electroencephalography from participants who reported the orientation and subjective visibility of the illusory triangle on each trial. Our multivariate pattern analysis revealed that posterior and central regions initially used dynamic neural code and later switched to stable neural code. The transition from dynamic to stable neural code in posterior region occurred increasingly later and eventually disappeared with decreasing conscious access. Anterior region primarily used stable neural code which waned with decreasing conscious access, but increased at below-median visibility and remained even when stimulus awareness was minimal. These results demonstrate differential spatiotemporal neural dynamics underlying perceptual integration depending on conscious access and emphasize a unique role of anterior region in processing integrated shape information especially under low subjective visibility.
Deciphering molecular mechanisms of synergistic growth reduction in kinase inhibitor combinations
Authors: Tsirvouli Eirini , Martinez del Val Ana , Thommesen Liv , Lægreid Astrid , Kuiper Martin , Olsen Jesper , Flobak Åsmund
Published: March 2024

In cancer treatment, the persistent challenge of unresponsiveness of certain patients to drugs or the development of resistance post-treatment remains a significant concern. Drug combinations that synergistically reduce tumor growth emerge as a promising avenue to address this issue. Here, we aimed to characterize the mechanism of action of two synergistic drug combinations that target PI3K together with MEK1 or with TAK1 and used time course measurements of phosphoproteomics and transcriptomics in response to single inhibitors and their combinations. Our analysis untangled those responses driven by single drugs and responses that were unique to the combinations. We observed a high overlap between single-drug responses and their combinations, suggesting that single-drug mechanisms dominate the mechanism of action of the combinations of the kinase inhibitors. Despite a high overlap, both drug combinations exhibited a synergistic modulation of several cell fate regulators found at the convergence points of the targeted pathways, including the key regulator of intrinsic apoptosis BCL2L11. Interestingly, the responses in both combinations were largely limited to the targeted pathways, namely PI3K/AKT and MAPKs, with very limited change of any other additional cell fate decision pathways. In addition, we observed a strong downregulation of nucleotide metabolism and tRNA biosynthesis uniquely in the combinations, which could be attributed to the reduced activity of mTOR and ATF4. Our approach provides insights into the molecular mechanisms affected by the PI3Ki-TAK1i and PI3Ki-MEKi combinations and can serve as a flexible framework for dissecting drug combination responses based on multi-omics measurements.
What makes clocks tick? Characterizing developmental dynamics of adult epigenetic clock sites
Authors: Mulder Rosa H. , Neumann Alexander , Felix Janine F. , Suderman Matthew , Cecil Charlotte A. M.
Published: March 2024

DNA methylation (DNAm) at specific sites can be used to calculate ‘epigenetic clocks’, which in adulthood are used as indicators of age(ing). However, little is known about how these clock sites ‘behave’ during development and what factors influence their variability in early life. This knowledge could be used to optimize healthy aging well before the onset of age-related conditions. Here, we leveraged results from two longitudinal population-based cohorts (N=5,019 samples from 2,348 individuals) to characterize trajectories of adult clock sites from birth to early adulthood. We find that clock sites (i) diverge widely in their developmental trajectories, often showing non-linear change over time; (ii) are substantially more likely than non-clock sites to vary between individuals already from birth, differences that are predictive of DNAm variation at later ages; and (iii) show enrichment for genetic and prenatal environmental exposures, supporting an early-origins perspective to epigenetic aging.
Harnessing Genome Representation Learning for Decoding Phage-Host Interactions
Authors: Badam Sumanth , Rao Shrisha
Published: March 2024

Accurate prediction of the phages that target a bacterial host plays an important role in combating anti-microbial resistance. Our work explores the power of deep neural networks, convolutional neural networks, and pre-trained large DNA/protein language models to predict the host for a given phage. This work mainly uses the data provided by Gonzales et al. that contains receptor-binding protein sequences of the phages and the target host genus. We used pre-trained language models to obtain the dense representations of protein/nucleotide sequences to train a deep neural network to predict the target host genus. Additionally, convolutional neural networks were trained on one-hot encoding of nucleotide sequences to predict the target host genus. We achieved a weighted F1-score of 73.76% outperforming state-of-the-art models with an improvement of around 11% by using the protein language model ESM-1b.

The data and the source code are available athttps://github.com/sumanth2002629/Bacteriophage-Research.
CDK4 and CDK6 upregulation promotes DNA replication stress, genomic instability and resistance to EGFR targeted therapy in lung cancer
Authors: Gini Beatrice , Gui Philippe , Wu Wei , Kerr D. Lucas , Tan Lisa , Barbosa Dora , Olivas Victor , Gomez Carlos , Elmes Sarah , Steri Veronica , Chakrabarti Turja , Bivona Trever G. , Blakely Collin M.
Published: March 2024

Genetic interactions impact both normal human physiology and human diseases, such as cancer. Here, we study genetic interactions through the lens of human lung cancers driven by oncogenic forms of the epidermal growth factor receptor (EGFR), which we and others previously showed harbor a rich landscape of genetic co-alterations and potential genetic interactions. Among the most common genetic co-alterations with oncogenicEGFRare genomic amplifications of cell cycle regulatorsCDK4orCDK6, which have been implicated in EGFR inhibitor clinical resistance, although the mechanism underlying this effect is not well characterized. We show that CDK4/6 upregulation overcomes EGFR inhibitor-induced G1/S cell cycle arrest in association with increased replication stress, DNA damage and genomic instability. These biological effects arising in CDK4/6 upregulated tumors help to enable resistance to EGFR targeted therapies through established genetic resistance mechanisms. Combinatorial EGFR and CDK4/6 inhibitor treatment alleviated genomic instability and EGFR inhibitor resistance in patient-derived preclinical models. This study reveals mechanistic and clinical impacts of the genetic interaction between oncogenicEGFRandCDK4/6co-alterations in human lung cancer.
Inhibition of HDAC7 reprograms the histone H3.3 landscape to induce heterochromatin spreading and DNA replication defects in cancer cells
Authors: Hassan Ola , Pizzagalli Mattia , Leary Owen , Zepecki John P. , Corseri Adrianne , Wu Laura Jinxuan , Sasipalli Shiven , Lee Daniel , Hayward Lindsey , Tran Lily , Fajardo Eduardo , Fiser Andras , Karambizi David , Tapinos Nikos
Published: March 2024

Class IIa histone deacetylases (HDACs) are a family of enzymes with minimal histone deacetylase activity but can function as multi-protein interaction hubs. Here we demonstrate the expression of HDAC7, a Class IIa HDAC family member, in glioblastoma tumor tissue from 84 patients, patient-derived glioma stem cells (GSCs) from six patients, and pediatric diffuse pontine glioma (DIPG) cells from three patients. HDAC7 binds to Histone H3.3 and interacts with H3.3 and HIRA on chromatin. Targeted downregulation of HDAC7 expression with a subtype-specific siRNA inhibits the interaction of H3.3 with HIRA while increasing the association of H3.3 with DAXX and H3K9me3. This results in H3.3 being deposited on H3K9me3+/DAPI+ heterochromatin nuclear foci. Inhibition of HDAC7 triggers H3K9me3+ heterochromatin spreading, increased H3K9me3 binding in the cancer genome, and significant alterations in gene expression. Using single molecule DNA fiber approach, we show that HDAC7 inhibition results in a significant increase in replication fork speed without affecting fork symmetry. This altered replication fork speed leads to replication stress, evidenced by phosphorylation of RPA2 and impact on global DNA synthesis, resulting in reduced EdU incorporation. Finally, HDAC7 depletion leads to reduced BRCA2 expression and increased sensitivity of cancer cells to DNA damaging agents. Taken together, these studies uncover the involvement of HDAC7 in the euchromatic H3.3 chaperone network and the effect of HDAC7 depletion on chromatin dynamics, inducing epigenetic restriction and DNA damage in cancer cells.
Protection againstAPOE4-associated aging phenotypes with the longevity-promoting intervention 17α-estradiol in male mice
Authors: McGill Cassandra J. , Christensen Amy , Qian Wenjie , Thorwald Max A. , Lugo Jose Godoy , Namvari Sara , White Olivia S. , Finch Caleb E. , Benayoun Bérénice A. , Pike Christian J.
Published: March 2024

The apolipoprotein ε4 allele (APOE4) is associated with decreased longevity, increased vulnerability to age-related declines, and disorders across multiple systems. Interventions that promote healthspan and lifespan represent a promising strategy to attenuate the development ofAPOE4-associated aging phenotypes. Here we studied the ability of the longevity-promoting intervention 17α-estradiol (17αE2) to protect against age-related impairments inAPOE4versus the predominantAPOE3genotype using early middle-aged mice with knock-in of humanAPOEalleles. Beginning at age 10 months, maleAPOE3orAPOE4mice were treated for 20 weeks with 17αE2 or vehicle then compared for indices of aging phenotypes body-wide. Across peripheral and neural measures,APOE4was associated with poorer outcomes. Notably, 17αE2 treatment improved outcomes in a genotype-dependent manner favoringAPOE4mice. These data demonstrate a positiveAPOE4bias in 17αE2-mediated healthspan actions, suggesting that longevity-promoting interventions may be useful in mitigating deleterious age-related risks associated withAPOE4genotype.
A brainstem circuit for gravity-guided vertical navigation
Authors: Zhu Yunlu , Gelnaw Hannah , Auer Franziska , Hamling Kyla R. , Ehrlich David E. , Schoppik David
Published: March 2024

The sensation of gravity anchors our perception of the environment and is crucial for navigation. However, the neural circuits that transform gravity into commands for navigation are undefined. We first determined that larval zebrafish (Danio rerio) navigate vertically by maintaining a consistent heading across a series of upward climb or downward dive bouts. Gravity-blind mutant fish swim with more variable heading and excessive veering, leading to inefficient vertical navigation. After targeted photoablation of ascending vestibular neurons and spinal projecting midbrain neurons, but not vestibulospinal neurons, vertical navigation was impaired. These data define a sensorimotor circuit that uses evolutionarily-conserved brainstem architecture to transform gravitational signals into persistent heading for vertical navigation. The work lays a foundation to understand how vestibular inputs allow animals to move efficiently through their environment.
PURIFICATION METHOD FOR ADENO-ASSOCIATED VIRAL VECTOR SEROTYPE 9 USING CERAMIC HYDROXYAPATITE CHROMATOGRAPHY AND ITS QUALIFICATION
Authors: Kurosawa Yae , Tsunekawa Yuji , Wada Mikako , Aizen Yuko , Nitahara-Kasahara Yuko , Okada Takashi
Published: March 2024

Adeno-associated virus (AAV) vectors can efficiently transduce exogenous genes into various tissuesin vivo. Owing to their convenience, high efficiency, long-term stable gene expression, and minimal side effects, AAV vectors have become one of the gold standards for investigating gene functionsin vivo, especially in non-clinical studies. However, challenges persist in efficiently preparing a substantial quantity of high-quality AAV vectors that comprise only genome-packaged full particles. Commercial AAV vectors are typically associated with high costs. Further, in-laboratory production is hindered by the lack of specific laboratory equipment, such as ultracentrifuges. Therefore, a simple, quick, and scalable preparation method for AAV vectors is needed for proof-of-concept experiments. Herein, we present an optimized method for producing and purifying high-quality AAV serotype 9 (AAV9) vectors using standard laboratory equipment and chromatography. Using ceramic hydroxyapatite as a mixed-mode chromatography medium can markedly increase the quality of purified AAV vectors. Basic protocols and optional methods for evaluating purified AAV vectors are also described.
Diverse origins of near-identical antifreeze proteins in unrelated fish lineages provide insights into evolutionary mechanisms of new gene birth and protein sequence convergence
Authors: Rives Nathan , Lamba Vinita , Cheng C.-H. Christina , Zhuang Xuan
Published: March 2024

Determining the origins of novel genes and the genetic mechanisms underlying the emergence of new functions is challenging yet crucial for understanding evolutionary innovations. The convergently evolved fish antifreeze proteins provide excellent opportunities to investigate evolutionary origins and pathways of new genes. Particularly notable is the near-identical type I antifreeze proteins (AFPI) in four phylogenetically divergent fish taxa. This study tested the hypothesis of protein sequence convergence beyond functional convergence in three unrelated AFPI-bearing fish lineages, revealing different paths by which a similar protein arose from diverse genomic resources. Comprehensive comparative analyses ofde novosequenced genome of the winter flounder and grubby sculpin, available high-quality genome of the cunner and 14 other relevant species found that the near-identical AFPI originated from a distinct genetic precursor in each lineage. Each independently evolved a coding region for the novel ice-binding protein while retaining sequence identity in the regulatory regions with their respective ancestor. The deduced evolutionary processes and molecular mechanisms are consistent with the Innovation-Amplification-Divergence (IAD) model applicable to AFPI formation in all three lineages, a new Duplication-Degeneration-Divergence (DDD) model we propose for the sculpin lineage, and a DDD model with gene fission for the cunner lineage. This investigation illustrates the multiple ways by which a novel functional gene with sequence convergence at the protein level could evolve across divergent species, advancing our understanding of the mechanistic intricacies in new gene formation.
Tracking inflammation resolution signatures in lungs after SARS-CoV-2 omicron BA.1 infection of K18-hACE2 mice
Authors: Carolin Agnes , Yan Kexin , Bishop Cameron R. , Tang Bing , Nguyen Wilson , Rawle Daniel J. , Suhrbier Andreas
Published: March 2024

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes Coronavirus Disease 2019 (COVID-19), which can result in severe disease, often characterised by a ‘cytokine storm’ and the associated acute respiratory distress syndrome. However, many infections with SARS-CoV-2 are mild or asymptomatic throughout the course of infection. Although blood biomarkers of severe disease are well studied, less well understood are the inflammatory signatures in lung tissues associated with mild disease or silent infections, wherein infection and inflammation are rapidly resolved leading to sequelae-free recovery. Herein we described RNA-Seq and histological analyses of lungs over time in an omicron BA.1/K18-hACE2 mouse infection model, which displays these latter features. Although robust infection was evident at 2 days post infection (dpi), viral RNA was largely cleared by 10 dpi. Acute inflammatory signatures showed a slightly different pattern of cytokine signatures compared with severe infection models, and where much diminished 30 dpi and absent by 66 dpi. Cellular deconvolution identified significantly increased abundance scores for a number of anti-inflammatory pro-resolution cell types at 5/10 dpi. These included type II innate lymphoid cells, T regulatory cells, and interstitial macrophages. Genes whose expression trended downwards over 2 – 66 dpi included biomarkers of severe disease and were associated with ‘cytokine storm’ pathways. Genes whose expression trended upward during this period were associated with recovery of ciliated cells, AT2 to AT1 transition, reticular fibroblasts and innate lymphoid cells, indicating a return to homeostasis. Very few differentially expressed host genes were identified at 66 dpi, suggesting near complete recovery. The parallels between mild or subclinical infections in humans and those observed in this BA.1/K18-hACE2 mouse model are discussed with reference to the concept of “protective inflammation”.
Host evolution improves genetic circuit function in complex growth environments
Authors: Zhang Joanna T. , Lezia Andrew , Emmanuele Philip , Wu Muyao , Olson Connor A. , Feist Adam M. , Hasty Jeff
Published: March 2024

Genetically engineered bacteria have become an attractive platform for numerous biomedical and industrial applications. Despite genetic circuitry functioning predictably under favorable growth conditions in the lab, the same cannot be said when placed in more complex environments for eventual deployment. Here, we used a combination of evolutionary and rational engineering approaches to enhanceE. colifor robust genetic circuit behavior in non-traditional growth environments. We utilized adaptive laboratory evolution (ALE) onE. coliMG1655 in a minimal media with a sole carbon source and saw improved dynamics of a population-lysis-based circuit after host evolution. Additionally, we improved lysis circuit tolerance of a more clinically relevant strain, the probioticE. coliNissle, using ALE of the host strain in a more complex media environment with added reactive oxygen species (ROS) stress. We observed improved recovery from circuit-induced lysis in the evolved Nissle strain, and in combination with directed mutagenesis, recovered circuit function in the complex media. These findings serve as a proof-of-concept that relevant strains of bacteria can be optimized for improved growth and performance in complex environments using ALE and that these changes can modify and improve synthetic gene circuit function for real-world applications.
Assessing Long-Term Stored Tissues for Multi-Omics Data Quality and Proteogenomics Suitability
Authors: Song Kyu Jin , Kim Minsuh , Heo Yong Jin , Cho Kyung-Cho , Oh Jae-Won , Kim Dae Ho , Hwa Chanwoong , Do Yeji , Choi Seunghyuk , Hwang Hee Sang , Kim Kwoneel , Kim Kyunggon , Na Seungjin , Paek Eunok , An Joon-Yong , Jang Se Jin , Kim Min-Sik , Kim Kwang Pyo
Published: March 2024

As research into the complexities of cancer biology deepens, the integration of multi-omics analyses has emerged as a powerful approach to unravel the complex molecular basis of cancers. However, challenges related to sample availability, including size, collection procedures, and storage duration, hinder the broad application of this methodology. Despite these limitations, there is a growing interest in exploring the potential of archived samples to expand the scope of multi-omics research. Our study aims to investigate the impact of storage duration on the measurment in genomic, transcriptomic, and proteomic profiles of archived samples, demonstrating their viability for advancing our understanding of cancer biology. To comprehensively address these trends and limitations, we systematically examined archived samples collected over a decade, focusing on their transcriptomic, proteomic, and phosphoproteomic attributes. Analysis revealed intricate patterns and dynamic shifts, especially in long-term transcriptomic data, with observed declines in read counts related to protein coding and gene coverage. However, these changes did not compromise the fundamental gene expression landscape. Proteomic result also demonstrated that storage period did not significantly influence proteomic measurement. Comparisons of housekeeping gene (HKG) and housekeeping protein (HKP) expressions unveiled consistent transcriptomic levels across samples, while distinctive proteomic disparities between tumor and normal tissues. In conclusion, the challenges posed by limited sample availability in multi-omics studies can be partially overcome through the strategic integration of archived samples. While technical shifts were evident in certain aspects of transcriptomic data, core gene expression patterns remained robust, and the functionality of essential transcription factors (TFs) and kinases remained unaffected. These findings underscore the potential of archived samples as valuable resources for multi-omics research, providing a broader landscape for investigating cancer biology and paving the way for more comprehensive insights into this intricate field.
Single Cell MALDI-MSI Analysis of Lipids and Proteins within a Replicative Senescence Fibroblast Model
Authors: Sekera Emily R. , Rosas Lorena , Holbrook Joseph H. , Angeles-Lopez Quetzalli D. , Khaliullin Timur , Rojas Mauricio , Mora Ana L. , Hummon Amanda B.
Published: March 2024

In this study, we evaluate lipids and select proteins in human lung fibroblasts (hLFs) to interrogate changes occurring due to aging and senescence. To study single cell populations, a comparison of cells adhered onto slides using poly-D-lysine versus centrifugal force deposition was first analyzed to determine whether specific alterations were observed between preparations. The poly-D-lysine approach was than utilized to interrogate the lipidome of the cell populations and further evaluate potential applications of the MALDI-immunohistochemistry (IHC) platform for single-cell level analyses. Two protein markers of senescence, vimentin and p21, were both observed within the fibroblast populations and quantified. Lipidomic analysis of the fibroblasts found twelve lipids significantly altered because of replicative senescence, including fatty acids, such as stearic acid, and ceramide phosphoethanolamine species (CerPE). Similar to previous reports, alterations were detected in putative fatty acid building blocks, ceramides, among other lipid species. Altogether, our results reveal the ability to detect lipids implicated in senescence and show alterations to protein expression between normal and senescent fibroblast populations, including differences between young and aged cells. This report is the first time that the MALDI-IHC system has been utilized at a single-cell level to analyze both protein expression and lipid profiles in cultured cells, with a particular focus on changes associated with aging and senescence.
Comprehensive analysis of RNA-chromatin, RNA- and DNA-protein interactions
Authors: Khlebnikov Daniil A. , Nikolskaya Arina A. , Zharikova Anastasia A. , Mironov Andrey A.
Published: March 2024

RNA-chromatin interactome data is considered to be one of the noisiest types of data in biology. This is due to protein-coding RNA contacts and non-specific interactions between RNA and chromatin caused by protocol specifics. Therefore, finding regulatory interactions between certain transcripts and genome loci requires a wide range of filtering techniques to obtain significant results. Using data on pairwise interactions between these molecules, we propose a concept of triad interaction involving RNA, protein and a DNA locus. The constructed triads show significantly less noise contacts and are more significant when compared to a background model for generating pairwise interactions. RNA-chromatin contacts data can be used to validate the proposed triad object as positive (Red-ChIP experiment) or negative (RADICL-Seq NPM) controls. Our approach also filters RNA-chromatin contacts in chromatin regions associated with protein functions based on ChromHMM annotation.
Bacterial RNA promotes proteostasis through inter-tissue communication inC. elegans
Authors: Kyriakakis Emmanouil , Medde Chiara , Ritz Danilo , Fucile Geoffrey , Schmidt Alexander , Spang Anne
Published: March 2024

Life expectancy has been increasing over the last decades, which is not matched by an increase in healthspan. Besides genetic composition, environmental and nutritional factors influence both health- and lifespan. Diet is thought to be a major factor for healthy ageing. Here, we show that dietary RNA species extend healthspan inC. elegans. Inherent bacterial-derived double stranded RNA reduces protein aggregation in aC. elegansmuscle proteostasis model. This beneficial effect depends on low levels of systemic selective autophagy, the RNAi machinery in the germline, even when the RNA is delivered through ingestion in the intestine and the integrity of muscle cells. Our data suggest a requirement of inter-organ communication between the intestine, the germline and muscles. Our results demonstrate that bacterial-derived RNAs elicit a systemic response inC. elegans, which protects the animal from protein aggregation during ageing. We provide evidence that low stress levels are beneficial for healthspan.

One-Sentence Summary

Bacteria-derived dietary cues and inter-tissue communication promote proteostasis and fitness inC. elegans
Mutability and hypermutation antagonize immunoglobulin codon optimality
Authors: McGrath Joshua J.C. , Park Juyeon , Troxell Chloe A. , Chervin Jordan C. , Li Lei , Kent Johnathan R. , Changrob Siriruk , Fu Yanbin , Huang Min , Zheng Nai-Ying , Wilbanks G. Dewey , Nelson Sean A. , Sun Jiayi , Inghirami Giorgio , Madariaga Maria Lucia L. , Georgiou George , Wilson Patrick C.
Published: March 2024

The efficacy of polyclonal antibody responses is inherently linked to paratope diversity, as generated through V(D)J recombination and somatic hypermutation (SHM). These processes arose in early jawed vertebrates; however, little is known about how immunoglobulin diversity, mutability, and hypermutation have evolved in tandem with another more ubiquitous feature of protein-coding DNA – codon optimality. Here, we explore these relationships through analysis of germlineIGgenes, natural V(D)J repertoires, serum VH usage, and monoclonal antibody (mAb) expression, each through the lens of multiple optimality metrics. Strikingly, proteomic serum IgG sequencing showed that germlineIGHVcodon optimality positively correlated with VH representation after influenza vaccination, andin vitro, codon deoptimization of mAbs with synonymous amino acid sequences caused consistent expression loss. Germline V genes exhibit a range of codon optimality that is maintained by functionality, and inversely related to mutability. SHM caused a load-dependent deoptimization ofIGHVDJ repertoires within human tonsils, bone marrow, and lymph nodes (including SARS-CoV-2-specific clones from mRNA vaccinees), influenza-infected mice, and zebrafish. Comparison of natural mutation profiles to true random suggests the presence of selective pressures that constrain deoptimization. These findings shed light on immunoglobulin evolution, providing unanticipated insights into the antagonistic relationship between variable region diversification, codon optimality, and antibody secretion; ultimately, the need for diversity takes precedence over that for the most efficient expression of the antibody repertoire.
Temporal and spatial variability in availability bias has consequences for marine bird abundance estimates during the non-breeding season
Authors: Dunn Ruth E , Duckworth James , O’Brien Susan , Furness Robert W , Buckingham Lila , Daunt Francis , Bogdanova Maria , Green Jonathan A.
Published: March 2024

To effectively monitor how marine ecosystems are being reshaped by anthropogenic pressures, we require understanding of species abundances and distributions. Due to their socio-economic and ecological value, predatory species are often at the forefront of survey efforts. However, survey data are only valuable if they can reliably be converted into estimates of underlying distributions.

We consider at-sea surveys of marine predators that often inform ecological impact assessments of offshore windfarms. These surveys are subject to a form of detection bias called ‘availability bias’ whereby individuals which are submerged below the surface are consequently ‘unavailable’ for detection. Although correction factors are commonly used in these surveys, they are currently based on limited data that may not be species-, time-, or area-specific. Here, we use time-depth-recorder data to investigate variation in marine bird availability bias.

We found that the proportion of diving marine birds submerged below the sea surface during daylight hours, and therefore unavailable to be counted during surveys, varied by species, month, and area. For three of our focal species wintering around northwest Europe (Atlantic puffin, common guillemot, razorbill) our results were different to comparable values previously used to correct for the availability bias, whereas no correction factors are regularly used for the fourth species (red-throated diver). We now present availability bias correction factors that are species- and month-specific to the areas the study populations use during their non-breeding seasons: the North Sea, the north and west coasts of the UK, the Baltic Sea, and Icelandic coastal waters.

Synthesis and applications:Variation in the proportion of daylight hours that marine birds spent submerged lead to differences in availability bias correction factors, thereby impacting estimations of their abundances. We encourage use of correction factors that use data from the species, marine area, and month during which surveys are conducted to provide more accurate abundance estimates. Using more relevant correction factors will result in increasingly accurate abundance and distribution estimates of diving marine birds, with relevance for a range of applications including planning for offshore windfarm developments, the designation and monitoring of protected areas, and understanding environmental change.