Sci Rep. 2024 Feb 26;14(1):4631. doi: 10.1038/s41598-024-54463-5.

ABSTRACT

Of all methods exercised in modern molecular biology, modification of cellular properties through the introduction or removal of nucleic acids is one of the most fundamental. As such, several methods have arisen to promote this process; these include the condensation of nucleic acids with calcium, polyethylenimine or modified lipids, electroporation, viral production, biolistics, and microinjection. An ideal transfection method would be (1) low cost, (2) exhibit high levels of biological safety, (3) offer improved efficacy over existing methods, (4) lack requirements for ongoing consumables, (5) work efficiently at any scale, (6) work efficiently on cells that are difficult to transfect by other methods, and (7) be capable of utilizing the widest array of existing genetic resources to facilitate its utility in research, biotechnical and clinical settings. To address such issues, we describe here Pressure-jump-poration (PJP), a method using rapid depressurization to transfect even difficult to modify primary cell types such as embryonic stem cells. The results demonstrate that PJP can be used to introduce an array of genetic modifiers in a safe, sterile manner. Finally, PJP-induced transfection in primary versus transformed cells reveals a surprising dichotomy between these classes which may provide further insight into the process of cellular transformation.

PMID:38409237 | DOI:10.1038/s41598-024-54463-5

Nat Commun. 2024 Feb 26;15(1):1764. doi: 10.1038/s41467-024-45827-6.

ABSTRACT

Analyzing immune cell interactions in the bone marrow is vital for understanding hematopoiesis and bone homeostasis. Three-dimensional analysis of the complete, intact bone marrow within the cortex of whole long bones remains a challenge, especially at subcellular resolution. We present a method that stabilizes the marrow and provides subcellular resolution of fluorescent signals throughout the murine femur, enabling identification and spatial characterization of hematopoietic and stromal cell subsets. By combining a pre-processing algorithm for stripe artifact removal with a machine-learning approach, we demonstrate reliable cell segmentation down to the deepest bone marrow regions. This reveals age-related changes in the marrow. It highlights the interaction between CX3CR1+ cells and the vascular system in homeostasis, in contrast to other myeloid cell types, and reveals their spatial characteristics after injury. The broad applicability of this method will contribute to a better understanding of bone marrow biology.

PMID:38409121 | DOI:10.1038/s41467-024-45827-6

Light Sci Appl. 2024 Feb 27;13(1):59. doi: 10.1038/s41377-024-01406-4.

ABSTRACT

Techniques for high-definition micromanipulations, such as optical tweezers, hold substantial interest across a wide range of disciplines. However, their applicability remains constrained by material properties and laser exposure. And while microfluidic manipulations have been suggested as an alternative, their inherent capabilities are limited and further hindered by practical challenges of implementation and control. Here we show that the iterative application of laser-induced, localized flow fields can be used for the relative positioning of multiple micro-particles, irrespectively of their material properties. Compared to the standing theoretical proposal, our method keeps particles mobile, and we show that their precision manipulation is non-linearly accelerated via the multiplexing of temperature stimuli below the heat diffusion limit. The resulting flow fields are topologically rich and mathematically predictable. They represent unprecedented microfluidic control capabilities that are illustrated by the actuation of humanoid micro-robots with up to 30 degrees of freedom, whose motions are sufficiently well-defined to reliably communicate personal characteristics such as gender, happiness and nervousness. Our results constitute high-definition micro-fluidic manipulations with transformative potential for assembly, micro-manufacturing, the life sciences, robotics and opto-hydraulically actuated micro-factories.

PMID:38409110 | DOI:10.1038/s41377-024-01406-4

Behav Brain Res. 2024 Feb 24:114919. doi: 10.1016/j.bbr.2024.114919. Online ahead of print.

ABSTRACT

Alzheimer's disease (AD) is a severe neurodegenerative disorder and the most common form of dementia in elderly individuals, characterized by memory deficits, cognitive decline, and neuropathology. The identification of preclinical markers for AD remains elusive. We employed an ultrasound-evoked spatial memory assay to investigate path integration (PI) in wild type C57BL/6J and 5xFAD mice. We observed significant recruitment of the mammillary bodies (MB) and subiculum (Sub) - core regions of the Papez circuit during PI, as indicated by increased expression of the immediate early gene c-Fos in C57BL/6J mice. In 5xFAD mice, amyloid-beta (Aβ) vulnerability in the MB and Sub was evident at 3-months of age, preceding widespread pathology at 5-months of age. In parallel, we detected significant behavioral deficits in PI in the 5XFAD mice at 5- but not 3-months of age. Sex based analysis revealed a more profound deficit in males compared to females at 5-months of age. Our data suggest PI may be as an early indicator of AD, potentially associated with dysfunction within the Papez circuit.

PMID:38408521 | DOI:10.1016/j.bbr.2024.114919

Elife. 2024 Feb 26;12:RP87651. doi: 10.7554/eLife.87651.

ABSTRACT

The muscle synergy is a guiding concept in motor control research that relies on the general notion of muscles 'working together' towards task performance. However, although the synergy concept has provided valuable insights into motor coordination, muscle interactions have not been fully characterised with respect to task performance. Here, we address this research gap by proposing a novel perspective to the muscle synergy that assigns specific functional roles to muscle couplings by characterising their task-relevance. Our novel perspective provides nuance to the muscle synergy concept, demonstrating how muscular interactions can 'work together' in different ways: (1) irrespective of the task at hand but also (2) redundantly or (3) complementarily towards common task-goals. To establish this perspective, we leverage information- and network-theory and dimensionality reduction methods to include discrete and continuous task parameters directly during muscle synergy extraction. Specifically, we introduce co-information as a measure of the task-relevance of muscle interactions and use it to categorise such interactions as task-irrelevant (present across tasks), redundant (shared task information), or synergistic (different task information). To demonstrate these types of interactions in real data, we firstly apply the framework in a simple way, revealing its added functional and physiological relevance with respect to current approaches. We then apply the framework to large-scale datasets and extract generalizable and scale-invariant representations consisting of subnetworks of synchronised muscle couplings and distinct temporal patterns. The representations effectively capture the functional interplay between task end-goals and biomechanical affordances and the concurrent processing of functionally similar and complementary task information. The proposed framework unifies the capabilities of current approaches in capturing distinct motor features while providing novel insights and research opportunities through a nuanced perspective to the muscle synergy.

PMID:38407224 | DOI:10.7554/eLife.87651

Elife. 2024 Feb 26;12:RP89321. doi: 10.7554/eLife.89321.

ABSTRACT

Cell-free DNA (cfDNA) tests use small amounts of DNA in the bloodstream as biomarkers. While it is thought that cfDNA is largely released by dying cells, the proportion of dying cells' DNA that reaches the bloodstream is unknown. Here, we integrate estimates of cellular turnover rates to calculate the expected amount of cfDNA. By comparing this to the actual amount of cell type-specific cfDNA, we estimate the proportion of DNA reaching plasma as cfDNA. We demonstrate that <10% of the DNA from dying cells is detectable in plasma, and the ratios of measured to expected cfDNA levels vary a thousand-fold among cell types, often reaching well below 0.1%. The analysis suggests that local clearance, presumably via phagocytosis, takes up most of the dying cells' DNA. Insights into the underlying mechanism may help to understand the physiological significance of cfDNA and improve the sensitivity of liquid biopsies.

PMID:38407214 | DOI:10.7554/eLife.89321

NAR Genom Bioinform. 2024 Feb 24;6(1):lqae022. doi: 10.1093/nargab/lqae022. eCollection 2024 Mar.

ABSTRACT

Breast cancer (BC) is one of the most commonly diagnosed cancers worldwide. As key regulatory molecules in several biological processes, microRNAs (miRNAs) are potential biomarkers for cancer. Understanding the miRNA markers that can detect BC may improve survival rates and develop new targeted therapeutic strategies. To identify a circulating miRNA signature for diagnostic prediction in patients with BC, we developed an evolutionary learning-based method called BSig. BSig established a compact set of miRNAs as potential markers from 1280 patients with BC and 2686 healthy controls retrieved from the serum miRNA expression profiles for the diagnostic prediction. BSig demonstrated outstanding prediction performance, with an independent test accuracy and area under the receiver operating characteristic curve were 99.90% and 0.99, respectively. We identified 12 miRNAs, including hsa-miR-3185, hsa-miR-3648, hsa-miR-4530, hsa-miR-4763-5p, hsa-miR-5100, hsa-miR-5698, hsa-miR-6124, hsa-miR-6768-5p, hsa-miR-6800-5p, hsa-miR-6807-5p, hsa-miR-642a-3p, and hsa-miR-6836-3p, which significantly contributed towards diagnostic prediction in BC. Moreover, through bioinformatics analysis, this study identified 65 miRNA-target genes specific to BC cell lines. A comprehensive gene-set enrichment analysis was also performed to understand the underlying mechanisms of these target genes. BSig, a tool capable of BC detection and facilitating therapeutic selection, is publicly available at https://github.com/mingjutsai/BSig.

PMID:38406797 | PMC:PMC10894035 | DOI:10.1093/nargab/lqae022

Front Pharmacol. 2024 Feb 9;15:1376058. doi: 10.3389/fphar.2024.1376058. eCollection 2024.

ABSTRACT

[This corrects the article DOI: 10.3389/fphar.2023.1243505.].

PMID:38405670 | PMC:PMC10884821 | DOI:10.3389/fphar.2024.1376058

Cell Surf. 2024 Feb 13;11:100121. doi: 10.1016/j.tcsw.2024.100121. eCollection 2024 Jun.

ABSTRACT

Plant cell wall researchers were asked their view on what the major unanswered questions are in their field. This article summarises the feedback that was received from them in five questions. In this issue you can find equivalent syntheses for researchers working on bacterial, unicellular parasite and fungal systems.

PMID:38405175 | PMC:PMC10885547 | DOI:10.1016/j.tcsw.2024.100121

Comput Struct Biotechnol J. 2024 Feb 12;23:972-981. doi: 10.1016/j.csbj.2024.02.008. eCollection 2024 Dec.

ABSTRACT

Antimicrobial peptides (AMPs) are molecules found in most organisms, playing a vital role in innate immune defense against pathogens. Their mechanism of action involves the disruption of bacterial cell membranes, causing leakage of cellular contents and ultimately leading to cell death. While AMPs typically lack a defined structure in solution, they often assume a defined conformation when interacting with bacterial membranes. Given this structural flexibility, we investigated whether intrinsically disordered regions (IDRs) with AMP-like properties could exhibit antimicrobial activity. We tested 14 peptides from different IDRs predicted to have antimicrobial activity and found that nearly all of them did not display the anticipated effects. These peptides failed to adopt a defined secondary structure and had compromised membrane interactions, resulting in a lack of antimicrobial activity. We hypothesize that evolutionary constraints may prevent IDRs from folding, even in membrane-like environments, limiting their antimicrobial potential. Moreover, our research reveals that current antimicrobial predictors fail to accurately capture the structural features of peptides when dealing with intrinsically unstructured sequences. Hence, the results presented here may have far-reaching implications for designing and improving antimicrobial strategies and therapies against infectious diseases.

PMID:38404711 | PMC:PMC10884422 | DOI:10.1016/j.csbj.2024.02.008

Front Genet. 2024 Feb 9;15:1346707. doi: 10.3389/fgene.2024.1346707. eCollection 2024.

ABSTRACT

Mesoplasma florum is an emerging model organism for systems and synthetic biology due to its small genome (∼800 kb) and fast growth rate. While M. florum was isolated and first described almost 40 years ago, many important aspects of its biology have long remained uncharacterized due to technological limitations, the absence of dedicated molecular tools, and since this bacterial species has not been associated with any disease. However, the publication of the first M. florum genome in 2004 paved the way for a new era of research fueled by the rise of systems and synthetic biology. Some of the most important studies included the characterization and heterologous use of M. florum regulatory elements, the development of the first replicable plasmids, comparative genomics and transposon mutagenesis, whole-genome cloning in yeast, genome transplantation, in-depth characterization of the M. florum cell, as well as the development of a high-quality genome-scale metabolic model. The acquired data, knowledge, and tools will greatly facilitate future genome engineering efforts in M. florum, which could next be exploited to rationally design and create synthetic cells to advance fundamental knowledge or for specific applications.

PMID:38404664 | PMC:PMC10884336 | DOI:10.3389/fgene.2024.1346707

Front Microbiol. 2024 Feb 7;15:1351297. doi: 10.3389/fmicb.2024.1351297. eCollection 2024.

ABSTRACT

In the marine α-proteobacterium Dinoroseobacter shibae more than 40 genes of the aerobic anoxygenic photosynthesis are regulated in a light-dependent manner. A genome-wide screen of 5,605 clones from a D. shibae transposon library for loss of pigmentation and changes in bacteriochlorophyll absorbance identified 179 mutant clones. The gene encoding the LOV-domain containing protein Dshi_1135 was identified by its colorless phenotype. The mutant phenotype was complemented by the expression of a Dshi_1135-strep fusion protein in trans. The recombinantly produced and chromatographically purified Dshi_1135 protein was able to undergo a blue light-induced photocycle mediated by bound FMN. Transcriptome analyses revealed an essential role for Dshi_1135 in the light-dependent expression of the photosynthetic gene cluster. Interactomic studies identified the repressor protein PpsR as an interaction partner of Dshi_1135. The physical contact between PpsR and the Dshi_1135 protein was verified in vivo using the bacterial adenylate cyclase-based two-hybrid system. In addition, the antirepressor function of the Dshi_1135 protein was demonstrated in vivo testing of a bchF-lacZ reporter gene fusion in a heterologous Escherichia coli-based host system. We therefore propose to rename the Dshi_1135 protein to LdaP (light-dependent antirepressor of PpsR). Using the bacterial two-hybrid system, it was also shown that cobalamin (B12) is essential for the interaction of the antirepressor PpaA with PpsR. A regulatory model for the photosynthetic gene cluster in D. shibae was derived, including the repressor PpsR, the light-dependent antirepressor LdaP and the B12-dependent antirepressor PpaA.

PMID:38404597 | PMC:PMC10890935 | DOI:10.3389/fmicb.2024.1351297

Neurobiol Learn Mem. 2024 Feb 23:107903. doi: 10.1016/j.nlm.2024.107903. Online ahead of print.

ABSTRACT

Formation of long-term memories requires learning-induced changes in both transcription and translation. Epitranscriptomic modifications of RNA recently emerged as critical regulators of RNA dynamics, whereby adenosine methylation (m6A) regulates translation, mRNA stability, mRNA localization, and memory formation. Prior work demonstrated a pro-memory phenotype of m6A, as loss of m6A impairs and loss of the m6A/m demethylase FTO improves memory formation. Critically, these experiments focused exclusively on aversive memory tasks and were only performed in male mice. Here we show that the task type and sex of the animal alter effects of m6A on memory, whereby FTO-depletion impaired object location memory in male mice, in contrast to the previously reported beneficial effects of FTO depletion on aversive memory. Additionally, we show that female mice have no change in performance after FTO depletion, demonstrating that sex of the mouse is a critical variable for understanding how m6A contributes to memory formation. Our study provides the first evidence for FTO regulation of non-aversive spatial memory and sexspecific effects of m6A, suggesting that identification of differentially methylated targets in each sex and task will be critical for understanding how epitranscriptomic modifications regulate memory.

PMID:38403011 | DOI:10.1016/j.nlm.2024.107903

Biochem Pharmacol. 2024 Feb 23:116078. doi: 10.1016/j.bcp.2024.116078. Online ahead of print.

ABSTRACT

A drug Mechanism of Action (MoA) is a complex biological phenomenon that describes how a bioactive compound produces a pharmacological effect. The complete knowledge of MoA is fundamental to fully understanding the drug activity. Over the years, many experimental methods have been developed and a huge quantity of data has been produced. Nowadays, considering the increasing omics data availability and the improvement of the accessible computational resources, the study of a drug MoA is conducted by integrating experimental and bioinformatics approaches. The development of new in silico solutions for this type of analysis is continuously ongoing; herein, an updating review on such bioinformatic methods is presented. The methodologies cited are based on multi-omics data integration in biochemical networks and Machine Learning (ML). The multiple types of usable input data and the advantages and disadvantages of each method have been analyzed, with a focus on their applications. Three specific research areas (i.e. cancer drug development, antibiotics discovery, and drug repurposing) have been chosen for their importance in the drug discovery fields in which the study of drug MoA, through novel bioinformatics approaches, is particularly productive.

PMID:38402909 | DOI:10.1016/j.bcp.2024.116078

J Environ Manage. 2024 Feb 24;354:120412. doi: 10.1016/j.jenvman.2024.120412. Online ahead of print.

ABSTRACT

Effluents of wastewater treatment plants can abundantly spread endocrine disrupting chemicals in the environment. To improve water quality monitoring, the use of effect-based tools that measure estrogenic activity has been suggested, however their results could be influenced by different factors. This study compared the estrogenic activity of wastewater samples extracted with two stationary phases and tested with two in vitro effect-based assays to investigate whether and how stationary phases and assays could influence biomonitoring data. During four seasonal periods, the effluents of six WWTPs located in northern Italy were sampled. After the extraction using two different stationary phases (HLB, C18), the samples (n = 72) were tested using two effect-based assays: a gene reporter luciferase assay on mammalian cells (MELN) and yeast estrogen screen assay (YES). The results showed that estrogenic activity of HLB extracts was significantly different from the activity of C18 extracts, suggesting that extraction phase can influence biomonitoring data. Moreover, the estrogenic activity was overall higher using gene reporter MELN assay than using YES assay, suggesting that, due to difference in cell membrane permeability and metabolic activation, the applied cell model can affect the biomonitoring results. Finally, from the comparison between the activity of the final effluent and the environmentally safe estrogenic levels in surface waters, MELN data suggested that the activity of this effluent may pose an environmental risk, while YES data showed that it should not be considered a threat to the receiving surface waters. This study pointed out that a standardized approach is needed to assess the estrogenic activity of waters; it reported important data to select the most suitable stationary phase for samples extraction (samples extracted with C18 sorbent showed higher estradiol equivalent concentration values) and the most appropriate bioassay (gene reporter luciferase MELN assay was more sensitive than YES assay) to assess the environmental risk, thus protecting human health.

PMID:38402785 | DOI:10.1016/j.jenvman.2024.120412

Carbohydr Res. 2024 Feb 22;537:109069. doi: 10.1016/j.carres.2024.109069. Online ahead of print.

ABSTRACT

Milk oligosaccharides, complex carbohydrates unique to mammalian milk, play crucial roles in infant nutrition and immune development. This review explores their biochemical diversity, tracing the evolutionary paths that have led to their variation across different species. We highlight the intersection of nutrition, biology, and chemistry in understanding these compounds. Additionally, we discuss the latest computational methods and analytical techniques that have revolutionized the study of milk oligosaccharides, offering insights into their structural complexity and functional roles. This brief but essential review not only aims to provide a deeper understanding of milk oligosaccharides but also discuss the road toward their potential applications.

PMID:38402731 | DOI:10.1016/j.carres.2024.109069

Stem Cell Reports. 2024 Feb 12:S2213-6711(24)00012-2. doi: 10.1016/j.stemcr.2024.01.009. Online ahead of print.

ABSTRACT

Quality control of human induced pluripotent stem cells (iPSCs) is critical to ensure reproducibility of research. Recently, KOLF2.1J was characterized and published as a male iPSC reference line to study neurological disorders. Emerging evidence suggests potential negative effects of mtDNA mutations, but its integrity was not analyzed in the original publication. To assess mtDNA integrity, we conducted a targeted mtDNA analysis followed by untargeted metabolomics analysis. We found that KOLF2.1J mtDNA integrity was intact at the time of publication and is still preserved in the commercially distributed cell line. In addition, the basal KOLF2.1J metabolome profile was similar to that of the two commercially available iPSC lines IMR90 and iPSC12, but clearly distinct from an in-house-generated ERCC6R683X/R683X iPSC line modeling Cockayne syndrome. Conclusively, we validate KOLF2.1J as a reference iPSC line, and encourage scientists to conduct mtDNA analysis and unbiased metabolomics whenever feasible.

PMID:38402620 | DOI:10.1016/j.stemcr.2024.01.009

Cancer Cell. 2024 Feb 20:S1535-6108(24)00036-9. doi: 10.1016/j.ccell.2024.01.012. Online ahead of print.

ABSTRACT

KRASG12C inhibitors (adagrasib and sotorasib) have shown clinical promise in targeting KRASG12C-mutated lung cancers; however, most patients eventually develop resistance. In lung patients with adenocarcinoma with KRASG12C and STK11/LKB1 co-mutations, we find an enrichment of the squamous cell carcinoma gene signature in pre-treatment biopsies correlates with a poor response to adagrasib. Studies of Lkb1-deficient KRASG12C and KrasG12D lung cancer mouse models and organoids treated with KRAS inhibitors reveal tumors invoke a lineage plasticity program, adeno-to-squamous transition (AST), that enables resistance to KRAS inhibition. Transcriptomic and epigenomic analyses reveal ΔNp63 drives AST and modulates response to KRAS inhibition. We identify an intermediate high-plastic cell state marked by expression of an AST plasticity signature and Krt6a. Notably, expression of the AST plasticity signature and KRT6A at baseline correlates with poor adagrasib responses. These data indicate the role of AST in KRAS inhibitor resistance and provide predictive biomarkers for KRAS-targeted therapies in lung cancer.

PMID:38402609 | DOI:10.1016/j.ccell.2024.01.012

Bioinformatics. 2024 Feb 24:btae088. doi: 10.1093/bioinformatics/btae088. Online ahead of print.

ABSTRACT

MOTIVATION: Genomic intervals are one of the most prevalent data structures in computational genome biology, and used to represent features ranging from genes, to DNA binding sites, to disease variants. Operations on genomic intervals provide a language for asking questions about relationships between features. While there are excellent interval arithmetic tools for the command line, they are not smoothly integrated into Python, one of the most popular general-purpose computational and visualization environments.

RESULTS: Bioframe is a library to enable flexible and performant operations on genomic interval dataframes in Python. Bioframe extends the Python data science stack to use cases for computational genome biology by building directly on top of two of the most commonly-used Python libraries, NumPy and Pandas. The bioframe API enables flexible name and column orders, and decouples operations from data formats to avoid unnecessary conversions, a common scourge for bioinformaticians. Bioframe achieves these goals while maintaining high performance and a rich set of features.

AVAILABILITY: Bioframe is open-source under MIT license, cross-platform, and can be installed from the Python Package Index. The source code is maintained by Open2C on GitHub at https://github.com/open2c/bioframe.

SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

PMID:38402507 | DOI:10.1093/bioinformatics/btae088

Metab Eng. 2024 Feb 22:S1096-7176(24)00026-0. doi: 10.1016/j.ymben.2024.02.010. Online ahead of print.

ABSTRACT

Ectoine, a crucial osmoprotectant for salt adaptation in halophiles, has gained growing interest in cosmetics and medical industries. However, its production remains challenged by stringent fermentation process in model microorganisms and low production level in its native producers. Here, we systematically engineered the native ectoine producer Halomonas bluephagenesis for ectoine production by overexpressing ectABC operon, increasing precursors availability, enhancing product transport system and optimizing its growth medium. The final engineered H. bluephagenesis produced 85 g/L ectoine in 52 h under open unsterile incubation in a 7 L bioreactor in the absence of plasmid, antibiotic or inducer. Furthermore, it was successfully demonstrated the feasibility of decoupling salt concentration with ectoine synthesis and co-production with bioplastic P(3HB-co-4HB) by the engineered H. bluephagenesis. The unsterile fermentation process and significantly increased ectoine titer indicate that H. bluephagenesis as the chassis of Next-Generation Industrial Biotechnology (NGIB), is promising for biomanufacturing of not only intracellular bioplastic PHA but also of small molecular compound such as ectoine.

PMID:38401747 | DOI:10.1016/j.ymben.2024.02.010