bluuebunny/crossref_metadata_2025 huggingface dataset:
2-photon laser printing to mechanically stimulate multicellular systems in 3D
Authors: Colombo Federico , Taale Mohammadreza , Taheri Fereydoon , Villiou Maria , Debatin Teresa , Dulatahu Gent , Kollenz Philipp , Schmidt Målin , Schlagheck Christina , Wittbrodt Joachim , Selhuber-Unkel Christine
Published: December 2023

Most biological activities take place in 3D environments, where cells communicate with each other in various directions and are located in a defined, often microstructured, space. To investigate the effect of defined cyclic mechanical forces on a multicellular system, we develop a sub-millimeter sized stretching device for mechanical stimulation of a structurally restricted, soft multicellular microenvironment. For the stretching device, a multimaterial 3D microstructure made of PDMS and gelatine-based hydrogel is printed via 2-photon polymerization (2PP) method. The printed structures are first characterized microscopically and mechanically to study the effect of different printing parameters. With 2PP, organotypic cell cultures are then directly printed into the hydrogel structures to achieve true 3D cell culture systems. These are mechanically stimulated with a cantilever by indenting the stretching device at a defined point. As a most important result, the cells in the 3D organotypic cell culture change morphology and actin orientation when exposed to cyclic mechanical stretch, even within short timescales of just 30 minutes. As a proof of concept, we encapsulated a Medaka retinal organoid in the same structure to demonstrate that even preformed organoids can be stimulated by our method. The results demonstrate the power of 2PP to manufacturing multifunctional soft devices for mechanically controlling multicellular systems at micrometer resolution and thus mimicking mechanical stress situations, as they occurin vivo.
T cell exhaustion dynamics in systemic autoimmune disease
Authors: Turner Christi N. , Camilo Sanchez Arcila Juan , Huerta Noah , Quiguoe Avi Rae , Jensen Kirk D.C. , Hoyer Katrina K.
Published: December 2023

Unlike in infection and cancer, T cell exhaustion in autoimmune disease has not been clearly defined. Here we set out to understand inhibitory protein (PD-1, Tim3, CTLA4, Lag3) expression in CXCR5- and CXCR5+ CD8 and CD4 T cells in systemic lupus erythematosus. CXCR5+ CD8 and CD4 T cells express PD-1 and engage B cells in germinal center reactions, leading to autoantibody formation in autoimmunity. We hypothesized that CXCR5+ CD8 T cells develop an exhausted phenotype as SLE autoimmunity expands from initial to chronic, self-perpetuating disease due to chronic self-antigen exposure. Our results indicate that there is no exhaustion frequency differences between sexes, although disease kinetics vary by sex. CXCR5+ CD8 T cells express primarily IFNγ, known to promote autoimmune disease development, whereas CXCR5-CD8 T cells express TNFα and IFNγ as disease progresses from 2-6 months. Tim3 is the highest expressed inhibitory marker for all CD4 and CD8 T cell populations demonstrating potential for terminally exhausted populations. CTLA4 expression on CD4 T cells suggests potential tolerance induction in these cells. We identified exhaustion phenotypes within autoimmune disease that progress with increasing lupus erythematosus severity and possibly provide a feedback mechanism for immunological tolerance.

Highlights

CXCR5- and CXCR5+ CD8 T cells expand with rate of disease in SLE mouse model.

CXCR5+ CD8 T cells are low contributors to TNFα disease progression unlike CXCR5-CD8 T cells but may increase disease mechanisms through high IFNγ production.

Inhibitory markers upregulate in frequency with the highest amounts seen in Tim3+ populations. Tim3+Lag3+ expression may be an indicator of terminal differentiation for all populations.

Inhibitory marker expression frequency was unrelated to sex.
Aggregation of the Amyloid-β Peptide (Aβ40) within Condensates Generated through Liquid-Liquid Phase Separation
Authors: Morris Owen M. , Toprakcioglu Zenon , Röntgen Alexander , Cali Mariana , Knowles Tuomas P. J. , Vendruscolo Michele
Published: December 2023

The deposition of the Aβ peptide into amyloid fibrils is characteristic of Alzheimer’s disease. As it has been recently observed that the process of amyloid aggregation can take place within an intermediate liquid-like condensed phase, we investigated whether Aβ could undergo liquid-liquid phase separation, and whether Aβ amyloid aggregation could take place within Aβ liquid condensates. By using a microfluidic protocol, we observed that the 40-residue form of Aβ (Aβ40) can undergo liquid-liquid phase separation, and that accessing a liquid intermediate state enhances primary nucleation and enables Aβ40 to readily self-assemble into amyloid fibrils. These results prompt further studies to investigate the possible role of Aβ condensates in the aggregation of this peptide in Alzheimer’s disease.
Rapid body colouration change inOryzias celebensisas a social signal for intraspecific competition
Authors: Ueda Ryutaro , Ansai Satoshi , Takeuchi Hideaki
Published: December 2023

In some species, such as chameleon and cichlid fish, rapid body colouration changes (within seconds or minutes) serve as visual social signals in male-male competition. This study investigated the relationship between aggressive behaviour and body colouration changes inOryzias celebensis, an Indonesian medaka fish. We analysed aggressive behaviours and corresponding body colouration changes during attack events in a controlled laboratory setting using groups of 3 adult fish in a small tank. In a triadic relationship consisting of 2 males and a female, males with blackened markings attacked more frequently than males without blackened markings and females. Additionally, we observed that the males with blackened markings were seldom attacked by males without blackened markings and females. These tendencies persisted even in groups consisting of 3 males. Our results suggest that the blackened markings in maleO. celebensisnot only indicate the level of aggression but also serve as a social signal to suppress attacks by other individuals.
Analysis of Metabolomics and Transcriptomics Data to Assess Interactions in Microalgal Co-culture ofSkeletonema marinoiandPrymnesium parvum
Authors: Zulfiqar Mahnoor , Abel Anne-Susann , Barth Emanuel , Syhapanha Kristy , Poulin Remington Xavier , Dehiwalage Sassrika Nethmini Costa Warnakulasu , Pohnert Georg , Steinbeck Christoph , Peters Kristian , Sorokina Maria
Published: December 2023

In marine ecosystems, microbial communities often interact using specialised metabolites, which play a central role in shaping the dynamics of the ecological networks and maintaining the balance of the ecosystem. With metabolomics and transcriptomics analyses, this study explores the interactions between two marine microalgae,Skeletonema marinoiandPrymnesium parvum, grown in mono-cultures and non-contact co-cultures. As a growth indicator, the photosynthetic potential, measured via fluorescence, suggested chemical interaction betweenS. marinoiandP. parvum. Using Liquid Chromatography-Mass Spectrometry (LC-MS) data, we identified 346 and 521 differentially produced features in the endo- and exometabolome ofS. marinoiandP. parvum, respectively. Despite limited tandem mass spectrometry data (MS2) for these features, we structurally annotated 14 compounds, most of which were previously under-studied specialised metabolites. Differential gene expression analysis was then performed on the transcriptomes of the microalgae, which uncovered differentially expressed genes involved in energy metabolism and cellular repair for both species. These metabolic and transcriptomics changes depict the adaptation of both species in the co-culture. However, further data acquisition and investigation will be necessary to confirm the type of interaction and the underlying mechanisms.

Importance

Marine microalgae have great ecological importance and biochemical potential. Among these microbes are the diatomSkeletonema marinoi, known for its marine biogeochemical cycling, and the haptophytePrymnesium parvum, which poses adverse environmental consequences. Given these opposing roles for the two cosmopolitan microalgae, we designed a study using untargeted metabolomics and transcriptomics to acquire a comprehensive snapshot of their interactions, grown as mono-cultures and co-cultures. The statistical analysis of the chlorophyllafluorescence levels, and the metabolomics and transcriptomics dataset revealed metabolic communication occurring among the two species via specialised metabolites and activated cellular repair mechanisms. These findings reveal the complexity of the interactions within marine microbial ecosystems, offering a foundation for future research to understand and harness marine ecological systems.
IST-editing: Infinite spatial transcriptomic editing in a generated gigapixel mouse pup
Authors: Wu Jiqing , Berg Ingrid , Koelzer Viktor H.
Published: December 2023

Advanced spatial transcriptomics (ST) techniques provide comprehensive insights into complex living systems across multiple scales, while simultaneously posing challenges in bioimage analysis. The spatial co-profiling of biological tissues by gigapixel whole slide images (WSI) and gene expression arrays motivates the development of innovative and efficient algorithmic approaches. Using Generative Adversarial Nets (GAN), we introduceInfiniteSpatialTranscriptomicediting (IST-editing) and establish gene expression-guided editing in a generated gigapixel mouse pup. Trained with patch-wise high-plex gene expression (input) and matched image data (output), IST-editing enables the synthesis of arbitrarily large bioimages at inference,e.g., with a 106496×53248 resolution. After feeding edited gene expressions to the trained network, we model cell-, tissue- and animal-level morphological transitions in the generated mouse pup. Lastly, we discuss and evaluate editing effects on interpretable morphological features. The generated WSIs of the mouse pup and code are publicly released and accessible viahttps://github.com/CTPLab/IST-editing.
Discerning state estimation and sensory gating, two presumptive predictive signals in mouse barrel cortex
Authors: Gupta Kalpana , Chowdhury Ritu Roy , Chakrabarti Shubhodeep , Schwarz Cornelius
Published: December 2023

The brain is assumed to contain distinctive predictive systems ranging from sensorimotor to cognitive functions. Here we report the successful functional delineation of two different presumptive predictive systems on the neuronal level, state estimation (SE) and sensory gating (SG), which both attenuate sensory flow during movement. Studying neuronal sensorimotor responses throughout the depth of primary somatosensory cortex in mice trained on a whisker reach task, SE appeared as a learned attenuation of tactile signal flow, due to a suppressive predictive signal, precisely at the time of an experimental sensory consequence. In contrast, SG was observable during a much longer interval after onset of the motor command. Both phenomena are internal, presumptively predictive signals, as blockade of the reafference did not affect them. We speculate that SG may be related to cognitive signals monitoring goals of movements, while SE likely is the expression of the classical notion of the reafference principle.
Role ofStaphylococcus aureus’s Buoyant Density in the Development of Biofilm Associated Antibiotic Resistance
Authors: Kispert Sarah , Liguori Madison , Valikaneye Cody , Qiu Chong , Wang Shue , Zhang Nan , Gu Huan
Published: December 2023

Bacterial biofilms are clusters of bacterial cells that form at various interfaces, including those between air and liquid or liquid and solid. Due to their roles in enhancing wastewater treatment processes, and their unfortunate propensity to cause persistent human infections through high antibiotic resistance, understanding and managing bacterial biofilms is of paramount importance. A pivotal stage in biofilm development is the initial bacterial attachment to these interfaces. However, the determinants of bacterial cell choice in colonizing an interface first and heterogeneity in bacterial adhesion remain elusive. Recent research has unveiled variations in the buoyant density ofStaphylococcus aureuscells, irrespective of their growth phase. Cells with a low cell buoyant density, characterized by fewer cell contents, exhibited greater resistance to antibiotic treatments (100 μg/mL vancomycin) and favored biofilm formation at air-liquid interfaces. In contrast, cells with higher buoyant cell density, which have richer cell contents, were more vulnerable to antibiotics and predominantly formed biofilms on liquid-solid interfaces when contained upright. In essence,S. aureuscells with higher buoyant cell density are more inclined to adhere to upright substrates.
Transcriptomic analysis of melanoma cells reveals an association of α-synuclein with regulation of the inflammatory response
Authors: Rajasekaran Santhanasabapathy , Cheng Siyuan , Gajendran Nithya , Shekoohi Sahar , Chesnokova Liudmila , Yu Xiuping , Witt Stephan N.
Published: December 2023

The Parkinson’s disease protein, alpha-synuclein (α-syn/SNCA), is highly expressed in neurons and melanomas. The goal of this study was to reveal the mechanism(s) of α-syn’s involvement in melanoma pathogenesis. To decipher the genes and pathways affected by α-syn, we conducted an RNA sequencing analysis of human SK-MEL-28 cells and several SK-MEL-28SNCA-KO clones. We identified 1098 significantly up-regulated genes and 660 significantly down-regulated genes. Several of the upregulated genes are related to the immune system, i.e., the inflammatory response and the matrisome. We validated five upregulated genes (IL-1ý, SAA1, IGFBP5, CXCL8, and CXCL10) by RT-qPCR and detected IGFBP5 and IL-1ý in spent media of control andSNCA-KO cells. The levels of each of these secreted proteins were significantly higher in the spent media of theSNCA-KO clones than control cells. We suggest that the loss of α-syn expression unleashes chemokine/cytokine secretion, which could help melanoma cells evade the immune system.
EnhancerTracker: Comparing cell-type-specific enhancer activity of DNA sequence triplets via an ensemble of deep convolutional neural networks
Authors: Garza Anthony B. , Garcia Rolando , Solis Luis M. , Halfon Marc S. , Girgis Hani Z.
Published: December 2023

Motivation

Transcriptional enhancers — unlike promoters — are unrestrained by distance or strand orientation with respect to their target genes, making their computational identification a challenge. Further, there are insufficient numbers of confirmed enhancers for many cell types, preventing robust training of machine-learning-based models for enhancer prediction for such cell types.

Results

We presentEnhancerTracker, a novel tool that leverages an ensemble of deep separable convolutional neural networks to identify cell-type-specific enhancers with the need of only two confirmed enhancers.EnhancerTrackeris trained, validated, and tested on 52,789 putative enhancers obtained from the FANTOM5 Project and control sequences derived from the human genome. Unlike available tools, which accept one sequence at a time, the input to our tool is three sequences; the first two are enhancers active in the same cell type.EnhancerTrackeroutputs 1 if the third sequence is an enhancer active in the same cell type(s) where the first two enhancers are active. It outputs 0 otherwise. On a held-out set (15%),EnhancerTrackerachieved an accuracy of 64%, a specificity of 93%, a recall of 35%, a precision of 84%, and an F1 score of 49%.

Availability and implementation

https://github.com/BioinformaticsToolsmith/EnhancerTracker

Contact

hani.girgis@tamuk.edu
Senescent cells inhibit muscle differentiation via the SASP-lipid 15d-PGJ2mediated modification and control of HRas
Authors: Pundlik Swarang Sachin , Barik Alok , Venkateshvaran Ashwin , Sahoo Snehasudha Subhadarshini , Jaysingh Mahapatra Anshuman , Math Raviswamy G H , Ramanathan Arvind
Published: December 2023

Senescent cells, which are characterized by multiple features such as increased expression of Senescence-Associated β-galactosidase activity (SA β-gal) and cell cycle inhibitors such as p21 or p16, accumulate with tissue damage and dysregulate tissue homeostasis. In the context of skeletal muscle, it is known that agents used for chemotherapy such as Doxorubicin cause buildup of senescent cells, leading to the inhibition of tissue regeneration. Senescent cells influence the neighboring cells via numerous secreted factors which form the senescence-associated secreted phenotype (SASP). Lipids are emerging as a key component of SASP that can control tissue homeostasis. Arachidonic acid-derived lipids have been shown to accumulate within senescent cells, specifically 15d-PGJ2, which is an electrophilic lipid produced by the non-enzymatic dehydration of the prostaglandin PGD2. In this study, we show that 15d-PGJ2is also released by Doxorubicin-induced senescent cells as a SASP factor. Treatment of skeletal muscle myoblasts with the conditioned medium from these senescent cells inhibits myoblast fusion during differentiation. Inhibition of L-PTGDS, the enzyme that synthesizes PGD2, diminishes the release of 15d-PGJ2by senescent cells and restores muscle differentiation. We further show that this lipid post-translationally modifies Cys184 of HRas in skeletal muscle cells, causing a reduction in the localization of HRas to the Golgi, increased HRas binding to RAF RBD, and activation of cellular MAPK-Erk signaling (but not the Akt signaling). Mutating C184 of HRas prevents the ability of 15d- PGJ2to inhibit the differentiation of muscle cells and control the activity of HRas. This work shows that 15d-PGJ2released from senescent cells could be targeted to restore muscle homeostasis after chemotherapy.
Accelerated simulations of RNA clustering: a systematic study of repeat sequences
Authors: Aierken Dilimulati , Joseph Jerelle A.
Published: December 2023

Under certain conditions, RNA repeat sequences phase separate yielding protein-free biomolecular condensates. Importantly, RNA repeat sequences have also been implicated in neurological disorders, such as Huntington’s Disease. Thus, mapping repeat sequences to their phase behavior, functions, and dysfunctions is an active area of research. However, despite several advances, it remains challenging to characterize the RNA phase behavior at submolecular resolution. Here, we have implemented a residue-resolution coarse-grained model in LAMMPS – that incorporates both RNA sequence and structure – to study the clustering propensities of protein-free RNA systems. Importantly, we achieve multifold speedup in the simulation time compared to previous work. Leveraging this efficiency, we study the clustering propensity of all 20 non-redundant trinucleotide repeat sequences. Our results align with findings from experiments, emphasizing that canonical base pairing and G-U wobble pairs play a dominant role in regulating cluster formation of RNA repeat sequences. Strikingly, we find strong entropic contributions to the stability and composition of RNA clusters, which is demonstrated for single-component RNA systems, as well as binary mixtures of trinucleotide repeats. Additionally, we investigate clustering behaviors of trinucleotide (odd) repeats and their quadranucleotide (even) counterparts. We observe that odd repeats exhibit stronger clustering tendencies, attributed to the presence of consecutive base pairs in their sequences that are disrupted in even repeat sequences. Altogether, our work extends the set of computational tools for probing RNA cluster formation at submolecular resolution and uncovers physicochemical principles that govern the stability and composition of resulting clusters.
Distinct ancient structural polymorphisms control heterodichogamy in walnuts and hickories
Authors: Groh Jeffrey S. , Vik Diane C. , Stevens Kristian A. , Brown Patrick J. , Langley Charles H. , Coop Graham
Published: December 2023

The maintenance of stable mating type polymorphisms is a classic example of balancing selection, underlying the nearly ubiquitous 50/50 sex ratio in species with separate sexes. One lesser known but intriguing example of a balanced mating polymorphism in angiosperms is heterodichogamy – polymorphism for opposing directions of dichogamy (temporal separation of male and female function in hermaphrodites) within a flowering season. This mating system is common throughout Juglandaceae, the family that includes globally important and iconic nut and timber crops – walnuts (Juglans), as well as pecan and other hickories (Carya). In both genera, heterodichogamy is controlled by a single dominant allele. We fine-map the locus in each genus, and find two ancient (>50 Mya) structural variants involving different genes that both segregate as genus-wide trans-species polymorphisms. TheJuglanslocus maps to a ca. 20 kb structural variant adjacent to a probable trehalose phosphate phosphatase (TPPD-1), homologs of which regulate floral development in model systems.TPPD-1is differentially expressed between morphs in developing male flowers, with increased allele-specific expression of the dominant haplotype copy. Across species, the dominant haplotype contains a tandem array of duplicated sequence motifs, part of which is an inverted copy of theTPPD-13’ UTR. These repeats generate various distinct small RNAs matching sequences within the 3’ UTR and further downstream. In contrast to the single-geneJuglanslocus, theCaryaheterodichogamy locus maps to a ca. 200-450 kb cluster of tightly linked polymorphisms across 20 genes, some of which have known roles in flowering and are differentially expressed between morphs in developing flowers. The dominant haplotype in pecan, which is nearly always heterozygous and appears to rarely recombine, shows markedly reduced genetic diversity and is over twice as long as its recessive counterpart due to accumulation of various types of transposable elements. We did not detect either genetic system in other heterodichogamous genera within Juglandaceae, suggesting that additional genetic systems for heterodichogamy may yet remain undiscovered.
OncogenicPIK3CAcorrupts growth factor signaling specificity
Authors: Madsen R.R. , Le Marois A. , Mruk O. , Voliotis M. , Yin S. , Sufi J. , Qin X. , Zhao S.J. , Gorczynska J. , Morelli D. , Davidson L. , Sahai E. , Korolchuk V.I. , Tape C.J. , Vanhaesebroeck B.
Published: December 2023

Pathological activation of the PI3K/AKT pathway is among the most frequent defects in human cancer and is also the cause of rare overgrowth disorders. Yet, there is currently no systematic understanding of the quantitative flow of information within PI3K/AKT signaling and how it is perturbed by disease-causing mutations. Here, we develop scalable, single-cell approaches for systematic analyses of signal processing within the PI3K pathway, enabling precise calculations of its information transfer for different growth factors. Using genetically-engineered human cell models with allele dose-dependent expression ofPIK3CAH1047R, we show that this oncogene is not a simple, constitutive pathway activator but a context-dependent modulator of extracellular signal transfer.PIK3CAH1047Rreduces information transmission downstream of IGF1 while selectively enhancing EGF-induced signaling and transcriptional responses. This leads to a gross reduction in signaling specificity, akin to “blurred” signal perception. The associated increase in signaling heterogeneity promotes phenotypic diversity in a human cervical cancer cell line model and in human induced pluripotent stem cells. Collectively, these findings and the accompanying methodological advances lay the foundations for a systematic mapping of the quantitative mechanisms of PI3K/AKT-dependent signal processing and phenotypic control in health and disease.

One-sentence summary

Single-cell signaling and information theoretic analyses reveal that oncogenic PI3K/AKT activation leads to a gross reduction in signaling specificity, context-dependent EGF response amplification as well as increased phenotypic heterogeneity.
Multilayer Network Analysis across Cortical Depths in Resting-State 7T fMRI
Authors: Kotlarz Parker , Lankinen Kaisu , Hakonen Maria , Turpin Tori , Polimeni Jonathan R. , Ahveninen Jyrki
Published: December 2023

In graph theory, “multilayer networks” represent systems involving several interconnected topological levels. One example in neuroscience is the stratification of connections between different cortical depths or “laminae”, which is becoming non-invasively accessible in humans using ultra-high-resolution functional MRI (fMRI). Here, we applied multilayer graph theory to examine functional connectivity across different cortical depths in humans, using 7T fMRI (1-mm3voxels; 30 participants). Blood oxygenation level dependent (BOLD) signals were derived from five depths between the white matter and pial surface. We compared networks where the inter-regional connections were limited to a single cortical depth only (“layer-by-layer matrices”) to those considering all possible connections between areas and cortical depths (“multilayer matrix”). We utilized global and local graph theory features that quantitatively characterize network attributes including network composition, nodal centrality, path-based measures, and hub segregation. Detecting functional differences between cortical depths was improved using multilayer connectomics compared to the layer-by-layer versions. Superficial depths of the cortex dominated information transfer and deeper depths drove clustering. These differences were largest in frontotemporal and limbic regions. fMRI functional connectivity across different cortical depths may contain neurophysiologically relevant information; thus, multilayer connectomics could provide a methodological framework for studies on how information flows across this stratification.
PEARL-catalyzed peptide bond formation after chain reversal during the biosynthesis of non-ribosomal peptides
Authors: Yu Yue , van der Donk Wilfred A.
Published: December 2023

A subset of nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs) are encoded in their biosynthetic gene clusters (BGCs) with enzymes annotated as lantibiotic dehydratases. The functions of these putative lantibiotic dehydratases remain unknown. Here, we characterize an NRPS-PKS BGC with a putative lantibiotic dehydratase from the bacteriumStackebrandtia nassauensis(sna). Heterologous expression revealed several metabolites produced by the BGC, and the omission of selected biosynthetic enzymes revealed the biosynthetic sequence towards these compounds. The putative lantibiotic dehydratase catalyzes peptide bond formation that extends the peptide scaffold opposite to the NRPS and PKS biosynthetic direction. The condensation domain of the NRPS catalyzes the formation of a ureido group, and bioinformatics analysis revealed distinct active site residues of ureido-generating condensation (UreaC) domains. This work demonstrates that the annotated lantibiotic dehydratase serves as a separate amide bond-forming machinery in addition to the NRPS, and that the lantibiotic dehydratase enzyme family possesses diverse catalytic activities in the biosynthesis of both ribosomal and non-ribosomal natural products.
Automated In Vitro Wound Healing Assay
Authors: Cwycyshyn Jillian , Stansbury Cooper , Meixner Walter , Hoying James B. , Muir Lindsey A. , Rajapakse Indika
Published: December 2023

Restoring the epidermal barrier after injury requires spatial and temporal orchestration of migration, proliferation, and signaling across many cell types. The mechanisms that coordinate this complex process are incompletely understood. In vitro wound assays are common model systems for examining these mechanisms in wound healing. In the scratch assay, a cell-free gap is created by mechanical removal of cells from a monolayer, followed by monitoring cell migration into the gap over time. While simple and low-cost, manual scratch assays are limited by low reproducibility and low throughput. Here, we have designed a robotics-assisted automated wound healing (AWH) assay that increases reproducibility and throughput while integrating automated live-cell imaging and analysis. Wounds are designed as computer-aided design (CAD) models and recreated in confluent cell layers by the BioAssemblyBot (BAB) 3D-bioprinting platform. The dynamics of migration and proliferation in individual cells are evaluated over the course of wound closure using live-cell fluorescence microscopy and our high-performance image processing pipeline. The AWH assay outperforms the standard scratch assay with enhanced consistency in wound geometry. Our ability to create diverse wound shapes in any multi-well plate with the BAB not only allows for multiple experimental conditions to be analyzed in parallel but also offers versatility in the design of wound healing experiments. Our method emerges as a valuable tool for the automated completion and analysis of high-throughput, reproducible, and adaptable in vitro wound healing assays.
Spatial genomics of AAVs reveals mechanism of transcriptional crosstalk that enables targeted delivery of large genetic cargo
Authors: Coughlin Gerard M. , Borsos Máté , Appling Nathan , Barcelona Bre’Anna H. , Mayfield Acacia M. H. , Mackey Elisha D. , Eser Rana A. , Chen Xinhong , Kumar Sripriya Ravindra , Gradinaru Viviana
Published: December 2023

Integrating cell type-specific regulatory elements (e.g. enhancers) with recombinant adeno-associated viruses (AAVs) can provide broad and efficient genetic access to specific cell types. However, the packaging capacity of AAVs restricts the size of both the enhancers and the cargo that can be delivered. Transcriptional crosstalk offers a novel paradigm for cell type-specific expression of large cargo, by separating distally-acting regulatory elements into a second AAV genome. Here, we identify and profile transcriptional crosstalk in AAV genomes carrying 11 different enhancers active in mouse brain. To understand transcriptional crosstalk, we develop spatial genomics methods to identify and localize AAV genomes and their concatemeric forms in cultured cells and in tissue. Using these methods, we construct detailed views of the dynamics of AAV transduction and demonstrate that transcriptional crosstalk is dependent upon concatemer formation. Finally, we leverage transcriptional crosstalk to drive expression of a large Cas9 cargo in a cell type-specific manner with systemically-administered engineered AAVs and demonstrate AAV-delivered, minimally-invasive, cell type-specific gene editing in wildtype animals that recapitulates known disease phenotypes.

Highlights

Transcriptional crosstalk between enhancers and promoters delivered intransby AAVs is a generalized phenomenon.

Spatial genomics techniques, AAV-Zombie and SpECTr, reveal that AAV genome concatemerization facilitates transcriptional crosstalk.

Transcriptional crosstalk can be leveraged for minimally-invasive, targeted AAV delivery of large cargo, including machinery for CRISPR-based gene editing and manipulation.

Transcriptional crosstalk enables cell-type specific gene disruption in wildtype animals, recapitulating behavioural phenotypes of genetic knockouts.
Substrate complexity buffers negative interactions in a synthetic microbial community of leaf litter degraders
Authors: Abdoli Parmis , Vulin Clément , Lepiz Miriam , Chase Alexander B. , Weihe Claudia , Rodríguez-Verdugo Alejandra
Published: December 2023

Microbes associated with leaf litter, the top layer of soil, collectively decompose organic matter such as plant polysaccharides, and respire carbon dioxide, regulating the land-atmosphere fluxes of carbon. Therefore, it is crucial to understand the processes limiting biopolymer degradation and their influences on soil community properties. For example, it is still unclear how substrate complexity – defined as the structure of the saccharide and the amount of external processing by extracellular enzymes – influences species interactions and species coexistence. Here, we tested the hypothesis that growth on monosaccharides (i.e., xylose) promotes negative interactions through resource competition, and growth on polysaccharides (i.e., xylan) promotes neutral or positive interactions through resource partitioning or synergism among extracellular enzymes. We assembled a three-species community of leaf litter-degrading bacteria isolated from a grassland site in Southern California. In the polysaccharide xylan, pairs of species stably coexisted and grew equally in co-culture and in monoculture. Conversely, in the monosaccharide xylose, competitive exclusion and negative interactions prevailed. These pairwise dynamics remained consistent in a three-species community: all three species coexisted in xylan, while only two species coexisted in xylose. A mathematical model parameterized from single-species growth behaviors showed that in xylose these dynamics could be explained by resource competition. Instead, the resource competition model could not predict the coexistence patterns in xylan. Overall, our study shows that substrate complexity influences species interactions and patterns of coexistence in a synthetic microbial community of leaf litter degraders that can serve as a model for studying carbon cycling and climate change.

Graphical Abstract
Concerns on research history overview, distribution patterns, and potentially neglected ecological consequences of the invasive apple snail (Pomacea sp.): A review
Authors: Luo Du , Li Yuefei , Li Jie
Published: December 2023

Invasive species pose a threat to global ecosystems and biodiversity, with the apple snail standing out as one kind of the worst invasive species due to its exclusive expansion. In order to draw a conceptual framework of its invasion pattern from an overview, we endeavor to conduct a bibliometric analysis and systematic review to explore the existing knowledge on research history and subjective categorization ofPomacea canaliculataandP. maculata, and to uncover emerging trends of distribution and possible neglected impact on food webs. The research history reveals that the first international research publication was documented in 1958. However, a substantial increase in international publications occurred only after 2000. The adoption and effective implementation of a universally agreed upon management strategy may greatly enhance the progress of research on apple snail invasion. Notably, co-word analysis revealed that the content of each thematic cluster has a shift from basic biology to the monitoring of occurrences and environmental adaptation. Currently, the global distribution of apple snails has expanded to 39 countries primarily as a result of anthropogenic translocation. All infested countries, with the exception of Israel and Singapore, are major paddy rice producers. The northernmost distribution limit is Tyumen in Russia, while the southernmost limit is the Buenos Aires Province in Argentina. Biological control has emerged as a prominent technology for the prevention of apple snail infestations, leading to the identification of 98 non-target enemy species as potential control agents. A taxonomic diversity analysis revealed that these species encompassed a wide range of phylogenetic classifications, including 6 Phyla, 12 Classes, 33 Orders, 55 Families, and 82 Genera. This observation suggests that there is a substantial pool of animals from diverse phylogenetic levels that can prey upon apple snails. Given the high population density and extensive distribution of apple snails, there is a pressing need for further research to explore their potential ecological ramifications. The current understanding of the upward ecological impacts of apple snails remains inadequate. These findings provide valuable insights for future research endeavors and contribute to the effective management of invasive apple snail populations.
Proton transfer from bisphenol-A is required to activate extranuclear-initiated estrogen receptor signaling
Authors: Medina-Gali Regla M. , Martinez-Pinna Juan , Dos Santos Reinaldo S. , Alonso-Magdalena Paloma , Serrano-Maciá Marina , Sempere-Navarro Roberto , Soriano Sergi , Fuentes-Paez Georgina , Pasquali Lorenzo , Mallavia Ricardo , Ciordia Sergio , Grimaldi Marina , Balaguer Patrick , Marroqui Laura , Nadal Angel
Published: December 2023

Ligand binding to estrogen receptors (ERs), ERα and ERβ, controls the physiology of estrogen-responsive tissues through nuclear and extranuclear initiated pathways. We found that selective ligands activate the extranuclear pathway by a mechanism involving proton transfer. The low affinity ligand and widespread endocrine disruptor Bisphenol-A (BPA) initiated nuclear and extranuclear actions. BPA concentrations similar to the receptor affinity initiated the nuclear pathway, whereas much lower concentrations initiated the extranuclear pathway. Experiments in different cell types using deuterated molecules of BPA (d16-BPA) and the ERβ agonist, diarylpropionitrile (d2-DPN), indicated that a proton transfer from the hydroxyl groups to an amino acid acceptor within the ligand binding domain is required to activate the extranuclear pathway. Activation of the extranuclear pathway by BPA caused a rapid change in the ERβ-associated interactome that did not occur with d16-BPA. To investigate this mechanism in a physiological system, we acutely injected BPA and d16-BPA into male mice. BPA rapidly decreased blood glucose and increased serum C-peptide levels, whereas d16-BPA had no effect. In mouse pancreatic β-cells, acute treatment with BPA closed ATP-dependent K+channels and increased glucose-induced insulin secretion, however d16-BPA was without effect. Contrary to d16-BPA, BPA modified electrical activity and increased apoptosis. RNA sequencing indicated that neither BPA nor d16-BPA significantly altered gene expression. Our findings suggests that the extranuclear actions of ERs are initiated by a mechanism that requires proton transfer from ligands and may have important biological consequences. This mechanism may work for other ligands, nuclear receptors and cell types.

One Sentence Summary

Proton-transfer from low-affinity ligands activates extranuclear Estrogen Receptor-initiated pathway
Structure and Function Relationships of Mucociliary Clearance in Human and Rat Airways
Authors: Roth Doris , Şahin Ayşe Tuğçe , Ling Feng , Tepho Niels , Senger Christiana N. , Quiroz Erik J. , Calvert Ben A. , van der Does Anne M. , Güney Tankut G. , Glasl Sarah , van Schadewijk Annemarie , von Schledorn Laura , Olmer Ruth , Kanso Eva , Nawroth Janna C. , Ryan Amy L.
Published: December 2023

Mucociliary clearance is a vital defense mechanism of the human airways, protecting against harmful particles and infections. When this process fails, it contributes to respiratory diseases like chronic obstructive pulmonary disease (COPD) and asthma. While advances in single-cell transcriptomics have revealed the complexity of airway composition, much of what we know about how airway structure impacts clearance relies on animal studies. This limits our ability to create accurate human-based models of airway diseases. Here we show that the airways in female rats and in humans exhibit species-specific differences in the distribution of ciliated and secretory cells as well as in ciliary beat, resulting in significantly higher clearance effectiveness in humans. We further reveal that standard lab-grown cultures exhibit lower clearance effectiveness compared to human airways, and we identify the underlying structural differences. By combining diverse experiments and physics-based modeling, we establish universal benchmarks to assess human airway function, interpret preclinical models, and better understand disease-specific impairments in mucociliary clearance.
ProHap enables proteomic database generation accounting for population diversity
Authors: Vašíček Jakub , Kuznetsova Ksenia G. , Skiadopoulou Dafni , Njølstad Pål R. , Johansson Stefan , Bruckner Stefan , Käll Lukas , Vaudel Marc
Published: December 2023

Amid the advances in genomics, the availability of large reference panels of human haplotypes is key to account for human diversity within and across populations. However, mass spectrometry-based proteomics does not benefit from this information. To address this gap, we introduce ProHap, a python-based tool that constructs protein sequence databases from phased genotypes of reference panels. ProHap empowers researchers to account for haplotypic diversity in proteomic searches.
Distractors induce space-specific neural biases in visual working memory
Authors: Raya Deepak V , Gupta Sanchit , Sridharan Devarajan
Published: December 2023

Information held in working memory is remarkably resilient to interference. Yet, distractors containing task-relevant features impact working memory systematically. The neural basis of this interference is poorly understood. Combining behavior with multivariate decoding of human electroencephalography recordings, we show that distractors induce space-specific biases in mnemonic representations. Participants performed a visual working memory task involving grating orientation estimation, with cued and uncued memoranda appearing on opposite visual hemifields. A high-contrast grating distractor, flashed briefly during the delay epoch, biased both behavioral reports and neural representations. Mnemonic representations were biased either towards the distractor’s orientation (attractive) or away from it (repulsive) depending, respectively, on whether the distractor appeared in the same hemifield as the corresponding memorandum, or opposite to it. On the other hand, the magnitude of the bias was much stronger for the uncued than the cued, memorandum, and depended critically on the distractor’s timing, encoding strength and gating efficacy. A two-tier ring attractor model incorporating cross-hemifield competitive inhibition, parsimoniously explained each of these experimental observations. Overall, our results show that prioritization in working memory shields mnemonic representations from distractor interference, and provide a testable computational hypothesis for distractor effects in visual working memory.
Decoding contextual influences on auditory perception from primary auditory cortex
Authors: Englitz B. , Akram S. , Elhilali M. , Shamma S.
Published: December 2023

Perception can be highly dependent on stimulus context, but whether and how sensory areas encode the context remains uncertain. We used an ambiguous auditory stimulus - a tritone pair - to investigate the neural activity associated with a preceding contextual stimulus that strongly influenced the tritone pair’s perception: either as an ascending or a descending step in pitch.

We recorded single-unit responses from a population of auditory cortical cells in awake ferrets listening to the tritone pairs preceded by the contextual stimulus. We find that the responses adapt locally to the contextual stimulus, consistent with human MEG recordings from the auditory cortex under the same conditions. Decoding the population responses demonstrates that cells responding to pitch-class-changes are able to predict well the context-sensitive percept of the tritone pairs. Conversely, decoding the individual pitch-class representations and taking their distance in the circular Shepard tone space predicts theoppositeof the percept. The various percepts can be readily captured and explained by a neural model of cortical activity based on populations of adapting, pitch-class and pitch-class-direction cells, aligned with the neurophysiological responses.

Together, these decoding and model results suggest that contextual influences on perception may well be already encoded at the level of the primary sensory cortices, reflecting basic neural response properties commonly found in these areas.
Structure and dynamics of secondary and mature rainforests: insights from South Asian long-term monitoring plots
Authors: Murali Akhil , Kasinathan Srinivasan , Bhat Kshama , Ratnam Jayashree , Sankaran Mahesh , Mudappa Divya , Raman T. R. Shankar , Osuri Anand M.
Published: December 2023

We assessed tree community and carbon dynamics over 5 years in two 1-ha long-term ecosystem monitoring plots, one each in mature tropical rainforests (MR) and 10-year post-agroforestry secondary rainforests (SR) in India’s Western Ghats mountains. Both plots were established in 2017 and monitored annually six times. We expected (1) higher tree diversity, distinct species composition, and greater carbon stock in MR; (2) higher carbon sequestration rates in SR; and (3) carbon dynamics shaped by growth and mortality in SR and MR, respectively. The SR plot had fewer species (67 vs. 84), stored substantially less carbon (76 vs. 193 Mg), and comprised a distinct community with fewer late-successional species than MR. SR gained 5.8 Mg carbon, due to tree growth exceeding losses from mortality, while in MR mortality exceeded growth and recruitment resulting in a 3.3 Mg carbon decline over five years. While MR had higher tree diversity, carbon stocks and relatively intact composition, the high rates of biodiversity and carbon accrual in SR highlight the conservation and climate significance of post-agroforestry secondary forests. Moderate carbon losses noted here in MR, as in other mature South Asian tropical forests, is a cause for concern under ongoing climate change.
Comprehensive characterization of the integrin family across 32 cancer types
Authors: Zou Cheng , Zhu Jinwei , Xiong Jiangling , Tian Yu , Peng Yousong , Cheung Edwin , Zhang Dingxiao
Published: December 2023

Integrin genes widely involve in tumorigenesis. Yet, a comprehensive characterization of integrin family and their interactome on the pan-cancer level is lacking. Here, we systematically dissect integrin family in nearly 10000 tumors across 32 cancer types. Globally, integrins represent a frequently altered and misexpressed pathway, with alteration and dysregulation overall being protumorigenic. Expression dysregulation, better than mutational landscape, of integrin family successfully identifies a subgroup of aggressive tumors demonstrating a high level of proliferation and stemness. We identify that several molecular mechanisms jointly regulate integrin expression in a context-dependent manner. For potential clinical usage, we construct a weighted score, integrinScore, to measure integrin signaling patterns in individual tumors. Remarkably, integrinScore consistently correlates with predefined molecular subtypes in multiple cancers, with integrinScore high tumors being more aggressive. Importantly, integrinScore is cancer-dependently and closely associated with proliferation, stemness, tumor microenvironment, metastasis, and immune signatures. IntegrinScore also predicts patient’s response to immunotherapy. By mining drug databases, we unravel an array of compounds that may serve as integrin signaling modulators. Finally, we build a user-friendly database to facilitate researchers to explore integrin-related knowledge. Collectively, we provide a comprehensive characterization of integrins across cancers, which offers gene- and cancer-specific rationales for developing integrin-targeted therapy.