bluuebunny/crossref_metadata_2025 huggingface dataset:
D4-Chip Reveals Impaired T Cell Function in Sepsis: Insights from Plasma Microenvironment Analysis and Mitochondrial Targeted Therapy
Authors: Liu Lijuan , Wang Ruoyu , Pu Xuexue , Zha Yutao , Yang Xiao , Fang Xiao , Liu Yong , Shao Min , Zhu Ling , Ren Xiaoou , Deng Guoqing , Yang Ke
Published: August 2024

Background

Sepsis, a systemic inflammation syndrome initiated by infection, poses significant challenges due to its intricate pathophysiology. T cells play a crucial role in combating infections during sepsis. Despite previous observations indicating T cell dysfunction in sepsis, reliable in-vitro detection methods were lacking, and the factors influencing these impairments remained unclear.

Methods

We developed a novel method using the D4-Chip to assess sepsis T cell migration function. This microfluidic platform enabled precise analysis of migration function under controlled conditions. Additionally, We explored the impact of the plasma microenvironment on T cell behavior, along with the redox environment in sepsis, and assessed the potential efficacy of Mitoquinone mesylate (MitoQ), a mitochondrial-targeted drug.

Results

Our findings revealed impaired migration function in sepsis T cells compared to healthy controls. Interestingly, sepsis plasma enhanced the migration of healthy T cells, yet incubation with healthy plasma did not fully restore migration impairments in sepsis T cells. Subsequent investigations uncovered a significant increase in NADH/NAD+ levels in sepsis T cells, with healthy T cells exposed to various sepsis plasma conditions also showing elevated NADH/NAD+ levels. Importantly, MitoQ normalized abnormal intracellular NADH/NAD+ levels and enhanced the migration ability of T cells.

Conclusions

Short-term incubation with sepsis plasma does not directly inhibit T cell migration but instead affects T cell function by disrupting the intracellular redox environment. Improving the intracellular redox environment of sepsis patients contributes to restoring impaired migration and proliferation, with MitoQ demonstrating therapeutic potential.
Optimization of TMS target engagement: novel evidence based on combined TMS–EEG and dMRI tractography of brain circuitry
Authors: Lioumis Pantelis , Roine Timo , Granö Ida , Aydogan Dogu Baran , Ukharova Elena , Souza Victor H. , Kičić Dubravko , Ilmoniemi Risto J. , Makris Nikos
Published: August 2024

Neuromodulation is based on the principle that brain stimulation produces plastic changes in cerebral circuitry. Given the intersubject structural and functional variability, neuromodulation has a personalized effect in the brain. Moreover, because of cerebral dominance and interhemispheric functional and structural differences in the same individual, the characterization of specific brain circuitries involved is currently not feasible. This notion is extremely important for neuromodulation treatments applied in neuropsychiatry. Specifically, the efficacy of the neuromodulation treatments is critically dependent on the anatomical precision of the brain target and the circuitry which has been affected. However, a complete understanding of how the brain behaves under stimulation needs a combined characterization of its neurophysiological response. This can be achieved by TMS–EEG guided by current multimodal neuroimaging techniques in real time. Herein, we present novel data based on dMRI tractography-guided TMS–EEG on one healthy young adult volunteer.
Physical activity modifies the metabolic profile of CD4+ and CD8+ T cell subtypes at rest and upon activation in older adults
Authors: Withnall Edward , Hazeldine Jon , Llibre Alba , Duggal Niharika A. , Lord Janet M. , Sardeli Amanda V.
Published: August 2024

Aging reduces the functional competence of T cells. T cell metabolism regulates their function, with mitochondrial defects in mice resulting in aged phenotypes, including accelerated senescence. Physical activity (PA) maintains T cell function in older adults, although the mechanisms underlying this effect are poorly understood. This study examined the effects of aging on the metabolic profile of T cell subsets and investigated whether PA could improve metabolic function in T cells from older donors. We recruited nine young adults (23 ± 3y) and 19 healthy older adults who had high PA (HPA, N=9, 75.5 ± 4.7y) or low PA levels (LPA, N=10, 76.4 ± 2.1y), based on their moderate-to-vigorous PA scores. We investigated the metabolic profiles of CD4+ and CD8+ T cells at rest and post-activation (PMA and ionomycin), via SCENITH flow cytometry. Compared to young adults, older adults had higher mitochondrial dependence (MD) in unstimulated CD4+ and CD8+ naive, effector memory (EM) and central memory (CM); and higher protein synthesis in CD4+ EM, CD4+ naïve, CD8+ EM, suggesting higher energetic demand in T cells with aging. Upon activation there was a lower reduction in MD of CD4+ EMRA and CD8+ EMRA; and a greater increase in IL-6 and TNFα expression in CD8+ cells of older than young adults, indicative of impaired metabolic flexibility with aging. PA effects were more prominent in unstimulated CD8+ cells, where HPA had lower glucose dependence (GD) for overall CD8+, CD8+EM and a trend to higher MD in CD8+ CM than LPA. Upon activation, HPA had a lower increase in CD4+ TNFα expression and trended to have a higher reduction in MD of overall CD4+ and a higher reduction in GD of CD4+ EMRA, than LPA. This suggests a lower metabolic demand in CD4+ T cells of HPA. We concluded that PA could modify T cell metabolic profile at rest, and following activation, in older adults, which may explain the better T cell function in physically active older individuals.
Stacked MXene/PEDOT-PSS Electrode Fiber for High-Performance Recording and Stimulation
Authors: Gou Shuchun , Li Peixuan , Yang Shu , Bi Guoqiang , Du Zhanhong
Published: August 2024

The development of microelectrodes with high electrical performance is imperative, particularly for invasive interfaces such as deep brain stimulation (DBS) electrodes. MXene, a new family of 2D early transition metal carbides or nitrides, exhibits outstanding electrical properties and has been researched to improve bioelectronic interface. Through a wet spinning process, we fabricate MXene/PEDOT-PSS electrode fibers measuring 30 μm in diameter, exhibiting an electrical conductivity of 2.16 ± 1.46 × 10^5 S/m and notably low interfacial impedance. The excellent cathodic charge storage capacity (CSCc) and charge injection capacity (CIC) lead to their high performance in recording or stimulation. The electrode fibers are electrochemically stable, biocompatible, magnetic resonance imaging (MRI)-compatible, and demonstrate excellent performance in electromyography (EMG), electrocardiograph (ECG), cortical recording and subthalamic nuclei deep brain stimulation (STN-DBS) experiments.
Effects of Release of TSG-6 from Heparin Hydrogels on Supraspinatus Muscle Regeneration
Authors: Pearson Joseph J. , Mao Jiahui , Temenoff Johnna S.
Published: August 2024

Muscle degeneration after rotator cuff tendon tear is a significant clinical problem. In these experiments, we developed a poly(ethylene glycol)-based injectable granular hydrogel containing two heparin derivatives (fully sulfated (Hep) and fully desulfated (Hep-)) as well as a matrix metalloproteinase-sensitive peptide to promote sustained release of Tumor Necrosis Factor Stimulated Gene 6 (TSG-6) over 14+ daysin vivoin a rat model of rotator cuff muscle injury. The hydrogel formulations demonstrated similar release profilesin vivo, thus facilitating comparisons between delivery from heparin derivatives on level of tissue repair in two different areas of muscle (near the myotendious junction (MTJ) and in the muscle belly (MB)) that have been shown previously to have differing responses to rotator cuff tendon injury. We hypothesized that sustained delivery of TSG-6 would enhance the anti-inflammatory response following rotator cuff injury through macrophage polarization, and that release from a fully sulfated heparin derivative (Hep) would potentiate this effect throughout the muscle. Inflammatory/immune cells, satellite cells, and fibroadipogenic progenitor cells, were analyzed by flow cytometery 3 and 7 days after injury and hydrogel injection, while metrics of muscle healing were examined via immunohistochemistry up to Day 14. Results showed controlled delivery of TSG-6 from Hep caused heightened macrophage response (Day 14 macrophages, 4.00 ± 1.85% single cells, M2a, 3.27 ± 1.95% single cells) and increased markers of early muscle regeneration (embryonic heavy chain staining) by Day 7, particularly in the MTJ region of the muscle, compared to release from desulfated heparin hydrogels. This work provides a novel strategy for localized, controlled delivery of TSG-6 to enhance muscle healing after rotator cuff tear.

IMPACT STATEMENT

Rotator cuff tear is a significant problem that can cause muscle degeneration. In this study, a hydrogel particle system was developed for sustained release of an anti-inflammatory protein, Tumor Necrosis Factor Stimulated Gene 6 (TSG-6), to injured muscle. Release of the protein from a fully sulfated heparin hydrogel-based carrier demonstrated greater changes in amount inflammatory cells and more early regenerative effects than a less-sulfated carrier. Thus, this work provides a novel strategy for localized, controlled delivery of an anti-inflammatory protein to enhance muscle healing after rotator cuff tear.
Behavioral and neural correlates of diverse conditioned fear responses in male and female rats
Authors: Mitchell Julia R. , Vincelette Lindsay , Tuberman Samantha , Sheppard Vivika , Bergeron Emmett , Calitri Roberto , Clark Rose , Cody Caitlyn , Kannan Akshara , Keith Jack , Parakoyi Abigail , Pikus MaryClare , Vance Victoria , Ziane Leena , Brenhouse Heather , Laine Mikaela A. , Shansky Rebecca M.
Published: August 2024

Pavlovian fear conditioning is a widely used tool that models associative learning in rodents. For decades the field has used predominantly male rodents and focused on a sole conditioned fear response: freezing. However, recent work from our lab and others has identified darting as a female-biased conditioned response, characterized by an escape-like movement across a fear conditioning chamber. It is also accompanied by a behavioral phenotype: Darters reliably show decreased freezing compared to Non-darters and males and reach higher velocities in response to the foot shock (“shock response”). However, the relationship between shock response and conditioned darting is not known. This study investigated if this link is due to differences in general processing of aversive stimuli between Darters, Non-darters and males. Across a variety of modalities, including corticosterone measures, the acoustic startle test, and sensitivity to thermal pain, Darters were found not to be more reactive or sensitive to aversive stimuli, and, in some cases, they appear less reactive to Non-darters and males. Analyses of cFos activity in regions involved in pain and fear processing following fear conditioning identified discrete patterns of expression among Darters, Non-darters, and males exposed to low and high intensity foot shocks. The results from these studies further our understanding of the differences between Darters, Non-darters and males and highlight the importance of studying individual differences in fear conditioning as indicators of fear state.
A deficiency screen identifies genomic regions critical for sperm head-tail connection
Authors: Galletta Brian J. , Konstantinidou Parthena , Haase Astrid D. , Rusan Nasser M.
Published: August 2024

A stable connection between the sperm head and tail is critical for fertility in species with flagellated sperm. The head-tail coupling apparatus (HTCA) serves as the critical link between the nucleus (head) and the axoneme (tail) via the centriole. To identify regions of theDrosophila melanogastergenome that contain genetic elements that influence HTCA formation, we undertook a two part screen using theDrosophiladeficiency (Df) kit. For this screen, we utilized a sensitized genetic background that overexpresses the pericentriolar material regulatory protein Pericentrin-Like Protein (PLP). We had previously shown that PLP overexpression (PLPOE) disrupts the head-tail connection in some spermatids, but not to a degree sufficient to reduce fertility. In the first step of the screen we tested for Dfs that in combination with PLPOEcause a reduction in fertility. We ultimately identified 11 regions of the genome that showed an enhanced fertility defect when combined with PLP overexpression. In the second step of the screen we tested these Dfs for their ability to enhance the HTCA defect caused by PLPOE, finding six. We then tested smaller Dfs to narrow the region of the genome that contained these enhancers. To further analyze the regions of the genome removed by these Dfs, we examined the expression patterns of the genes within these Dfs in publicly available datasets of RNAseq ofDrosophilatissues and snRNAseq ofDrosophilatestes. In total, our analysis suggests that some of these Dfs may contain a single gene that might influence HTCA formation and / or fertility, while others appear to be regions of the genome especially rich in testis-expressed genes that might affect the HTCA because of complex, multi-gene interactions.

Article Summary

We perform a genetic enhancer deficiency screen to uncover genomic regions required for proper sperm head-tail connection. We identified 6 regions and provide insight into these regions using publicly available RNA sequence data. Our data reveal that these regions are exceptionally rich in testes specific genes. Further analysis using small deficiencies resulted in two classes of enhancers: one class likely enhances head-tail connection by disrupting multiple genes, while the second class might house a single gene responsible for the reduction in fertility.
Molecular Mechanisms ofCoxiella burnetiiFormalin Fixed Cellular Vaccine Reactogenicity
Authors: Fratzke A. P. , Szule J. A. , Butler S. M. , van Schaik E. J. , Samuel J. E.
Published: August 2024

Local and systemic reactogenic responses to Q-VAX® have prevented licensing of this vaccine outside of Australia. These reactogenic responses occur in previously sensitize individuals and have not been well defined at the cellular level, in part because many studies have been done in guinea pigs that have limited molecular tools. We previously characterized a mouse model of reactogenicity where local reactions sites showed an influx of CD8+ and IFNγ-expressing IL17a+ CD4+ T cells consistent with a Th1 delayed-type hypersensitivity. In this study we determined using depletion and adoptive transfer experiments that both anti-Coxiellaantibodies and CD4+ T cells were essential for localized reactions at the site of vaccination. Furthermore, IFNγ depletion showed significant histological changes at the local reaction sites demonstrating the essential nature of this cytokine to reactogenicity. In addition to the cells and cytokines required for this response, we determined WCV material remained at the site of vaccination for at least 26 weeks post-injection. Transmission electron microscopy of these sites demonstrated intact rod-shaped bacteria at 2 weeks post-injection and partially degraded bacteria within macrophages at 26 weeks post-injection. Finally, since SCVs are an environmentally stable form, we determined that local reactions were more severe when the WCV material was prepared with higher levels of SCVs compared to typical WCV or with higher levels of LCV. These studies support the hypothesis that antigen persistence at the site of injection contributes to this reactogenicity and that anti-Coxiellaantibodies, CD4+ T cells, and IFNγ each contribute to this process.
TUSCAN: Tumor segmentation and classification analysis in spatial transcriptomics
Authors: Zang Chenxuan , Guo Charles C. , Wei Peng , Li Ziyi
Published: August 2024

The identification of tumor cells is pivotal to understanding tumor heterogeneity and the tumor microenvironment. Recent advances in spatially resolved transcriptomics (SRT) have revolutionized the way that transcriptomic profiles are characterized and have enabled the simultaneous quantification of transcript locations in intact tissue samples. SRT is a promising alternative method of studying gene expression patterns in spatial domains. Nevertheless, the precise detection of tumor regions within intact tissue remains a great challenge. The common way of identifying tumor cells is via tumor-specific marker gene expression signatures, which is highly dependent on marker accuracy. Another effective approach is through aneuploid copy number events, as most types of cancer exhibit copy number abnormalities. Here, we introduce a novel computational method, called TUSCAN (TUmor Segmentation and Classification ANalysis in spatial transcriptomics), which constructs a spatial copy number variation profile to improve the accuracy of tumor region identification. TUSCAN combines the gene information from SRT data and the hematoxylin-and-eosin-staining image to annotate tumor sections and other benign tissues. We benchmark the performance of TUSCAN and several existing methods through the application to multiple datasets from different SRT platforms. We demonstrate that TUSCAN can effectively delineate tumor regions, with improved accuracy compared to other approaches. Additionally, the output of TUSCAN provides interpretable clonal evolution inferences that may lead to novel insights into disease development and potential druggable targets.
Deep Learning-driven Automatic Nuclei Segmentation of Label-free Live Cell Chromatin-sensitive Partial Wave Spectroscopic Microscopy Imaging
Authors: Alom MD Shahin , Daneshkhah Ali , Acosta Nicolas , Anthony Nick , Liwag Emily Pujadas , Backman Vadim , Gaire Sunil Kumar
Published: August 2024

Chromatin-sensitive Partial Wave Spectroscopic (csPWS) microscopy offers a non-invasive glimpse into the mass density distribution of cellular structures at the nanoscale, leveraging the spectroscopic information. Such capability allows us to analyze the chromatin structure and organization and the global transcriptional state of the cell nuclei for the study of its role in carcinogenesis. Accurate segmentation of the nuclei in csPWS microscopy images is an essential step in isolating them for further analysis. However, manual segmentation is error-prone, biased, time-consuming, and laborious, resulting in disrupted nuclear boundaries with partial or over-segmentation. Here, we present an innovative deep-learning-driven approach to automate the accurate nuclei segmentation of label-free live cell csPWS microscopy imaging data. Our approach, csPWS-seg, harnesses the Convolutional Neural Networks-based U-Net model with an attention mechanism to automate the accurate cell nuclei segmentation of csPWS microscopy images. We leveraged the structural, physical, and biological differences between the cytoplasm, nucleus, and nuclear periphery to construct three distinct csPWS feature images for nucleus segmentation. Using these images of HCT116 cells, csPWS-seg achieved superior performance with a median Intersection over Union (IoU) of 0.80 and a Dice Similarity Coefficient (DSC) score of 0.88. The csPWS-seg overcame the segmentation performance over the baseline U-Net model and another attention-based model, SE-U-Net, marking a significant improvement in segmentation accuracy. Further, we analyzed the performance of our proposed model with four loss functions: binary cross-entropy loss, focal loss, dice loss, and Jaccard loss. The csPWS-seg with focal loss provided the best results compared to other loss functions. The automatic and accurate nuclei segmentation offered by the csPWS-seg not only automates, accelerates, and streamlines csPWS data analysis but also enhances the reliability of subsequent chromatin analysis research, paving the way for more accurate diagnostics, treatment, and understanding of cellular mechanisms for carcinogenesis.
Characterizing the role of exosomal miRNAs in metastasis
Authors: Agrawal Piyush , Olgun Gulden , Singh Arashdeep , Gopalan Vishaka , Hannenhalli idhar
Published: August 2024

Background

Exosomal microRNAs (exomiRs), transported via exosomes, play a pivotal role in intercellular communication. In cancer, exomiRs influence tumor progression by regulating key cellular processes such as proliferation, angiogenesis, and metastasis. Their role in mediating communication between cancer cells and the tumor microenvironment highlights their significance as potential diagnostic and therapeutic targets.

Methodology

In this study, we aimed to characterize the role of exomiRs in influencing the pre-metastatic niche (PMN). Across 7 tumor types, including 4 cell lines and three tumors, we extracted high confidence exomiRs (Log FC >= 2 in exosomes relative to control) and their targets (experimentally identified and targeted by at least 2 exomiRs). Subsequently, we identified enriched pathways and selected the top 100 high-confidence exomiR targets based on the frequency of their appearance in the enriched pathways. These top 100 targets were consistently used throughout the analysis.

Results

Cancer cell line and tumor derived ExomiRs have significantly higher GC content relative to genomic background. Pathway enriched among the top exomiR targets included general cancer-associated processes such as “wound healing” and “regulation of epithelial cell proliferation”, as well as cancer-specific processes, such as “regulation of angiogenesis in kidney” (KIRC), “ossification” in lung (LUAD), and “positive regulation of cytokine production” in pancreatic cancer (PAAD). Similarly, ‘Pathways in cancer’ and ‘MicroRNAs in cancer’ ranked among the top 10 enriched KEGG pathways in all cancer types. ExomiR targets were not only enriched for cancer-specific tumor suppressor genes (TSG) but are also downregulated in pre-metastatic niche formed in lungs compared to normal lung. Motif analysis shows high similarity among motifs identified from exomiRs across cancer types. Our analysis recapitulates exomiRs associated with M2 macrophage differentiation and chemoresistance such as miR-21 and miR-222-3p, regulating signaling pathways such as PTEN/PI3/Akt, NF-κB, etc. Cox regression indicated that exomiR targets are significantly associated with overall survival of patients in TCGA. Lastly, a Support Vector Machine (SVM) model using exomiR target gene expression classified responders and non-responders to neoadjuvant chemotherapy with an AUROC of 0.96 (in LUAD), higher than other previously reported gene signatures.

Conclusion

Our study characterizes the pivotal role of exomiRs in shaping the PMN in diverse cancers, underscoring their diagnostic and therapeutic potential.
Lipid A phosphoethanolamine transferase-mediated site-selective modifications show association with colistin resistance phenotypes and fitness
Authors: Schumann A. , Gaballa A. , Yang H. , Vu D. , Ernst RK. , Wiedmann M.
Published: August 2024

Genes encoding lipid A modifying phosphoethanolamine transferases (PETs) are genetically diverse and can confer resistance to colistin and antimicrobial peptides. To better understand the functional diversity of PETs, we characterized three canonical mobile colistin resistance (mcr) alleles (mcr-1,-3,-9), one intrinsicpet(eptA), and twomcr-like genes (petB,petC). Using an isogenic expression system, we show thatmcr-1andmcr-3are phenotypically similar by conferring colistin resistance with lower fitness costs.mcr-9, which is phylogenetically closely related tomcr-3, andeptAonly provide fitness advantages in the presence of sub-inhibitory concentrations of colistin and significantly reduce fitness in media only. PET-B and PET-C were phenotypically distinct from bonafide PETs; neither conferred colistin resistance or caused considerable fitness cost inEscherichia coli. Strikingly, we found for the first time that different PETs selectively modify different phosphates of lipid A - MCR-1, MCR-3, and PET-C selectively modify the 4’-phosphate, while MCR-9 and EptA modify the 1-phosphate. 4’-phosphate modifications facilitated by MCR-1 and -3 are associated with high levels of colistin resistance and low toxicity. Our results suggest that PETs have a wide phenotypic diversity and that high level colistin resistance is associated with specific lipid A modification patterns that has been largely unexplored thus far.

IMPORTANCE

Rising levels of resistance to increasing numbers of antimicrobials has led to the revival of last resort antibiotic colistin. Unfortunately, resistance to colistin is also spreading in the form ofmcrgenes, making it essential to (i) improve identification of resistant bacteria to allow clinicians to prescribe effective drug regimens and (ii) develop new combination therapies effective at targeting resistant bacteria. Our results demonstrate that PETs, including MCR variants, are site-selective inE. coli, with site-selectivity correlating with the level of resistance and fitness costs conferred by certain PETs. Site-selectivity associated with a given PET may not only help predict colistin resistance phenotypes, but may also provide an avenue to (i) improved drug regimens and (ii) development of new combination therapies to better combat colistin resistant bacteria.
Proliferative events ameliorate DNA damage accumulation without affecting function in hematopoietic stem cells
Authors: Mehatre Shubham Haribhau , Agrawal Harsh , Roy Irene Mariam , Schouteden Sarah , Khurana Satish
Published: August 2024

Upon aging, HSCs show functional decline with increased proliferation, myeloid skewing, and poor engraftment efficiency. Accumulation of DNA damage has been causally linked with this phenomenon, with the debatable role of proliferative events. In this study, we sought to enquire the effect of increased hematopoietic stem cell (HSC) proliferation during the lifetime on the hematopoietic aging in mice. Multiple rounds of blood withdrawals were performed between two to twelve months of adult life to maintain higher proliferation rate in HSC population. Our experiments showed little effect of increased proliferation rate on age-associated functional decline in hematopoietic system. However, we noted a decrease in the double strand breaks (DSBs) accumulated with age in mice that underwent serial bleeding regimen. Analysis of single-cell sequencing data from mouse and human HSPCs showed enrichment of DNA damage response pathways confirmed by increased expression of the genes involved. Importantly, we demonstrate that the induction of HSC proliferation in aged mice is sufficient to decrease the load of DSBs. Hence, our results show that proliferative events during lifetime might aid in clearing age-associated DSBs. While these DNA damages might not be directly linked with the functional decline, proliferation induced clearance can have clinical implications.
Transcriptomic analysis and high throughput functional characterization of human induced pluripotent stem cell derived sensory neurons
Authors: Truong Vincent , Brougher Jackson , Strassmaier Tim , Lu Irene , George Dale , Price Theodore J. , Obergrussberger Alison , Randolph Aaron , Haedo Rodolfo J. , Fertig Niels , Walsh Patrick
Published: August 2024

Peripheral sensory neurons are a primary effector in pain neurotransmission, and have become a useful cellular model for the study of pain. While rodent tissue has historically served as a source of these neurons, it has become increasingly clear that pain mechanisms in rodents and humans are substantially divergent. Sensory neurons harvested from cadaveric human tissue serve as a superior translational model for studying pain mechanisms, however their relative paucity limits their widespread utility. Theoretically, sensory neurons manufactured from human pluripotent stem cells (hPSCs) could help bridge this translational gap given their relative abundance and potential similarity to primary human tissue. However, hPSC-derived sensory neurons manufactured with the most common methodologies correlate poorly to human tissue both transcriptionally and functionally. In the present work, we compare a novel population of hPSC-derived sensory neurons to previously published datasets and find this novel population to more closely resemble human primary dorsal root ganglia transcriptionally. Furthermore, we evaluate the heterogeneity of this novel population via single nucleus RNA sequencing and find it resembles specific nociceptor and mechanoreceptor subsets found in vivo. Finally, we assay the functionality of this population with high throughput automated patch clamp electrophysiology with respect to voltage-gated sodium (Nav) and potassium channels (Kv), and ligand-gated ionotropic GABA and P2X receptors. Overall, we find this population of hPSC-derived sensory neurons to be of relatively high fidelity, and suitable for interrogating numerous potential pain targets on a fully humanized platform.
Gesture encoding in human left precentral gyrus neuronal ensembles
Authors: Vargas-Irwin Carlos E , Hosman Tommy , Gusman Jacob T , Pun Tsam Kiu , Simeral John D , Singer-Clark Tyler , Kapitonava Anastasia , Nicolas Claire , Shah Nishal P , Avansino Donald , Kamdar Foram , Williams Ziv , Henderson Jaimie M , Hochberg Leigh R
Published: August 2024

Understanding the cortical activity patterns driving dexterous upper limb motion has the potential to benefit a broad clinical population living with limited mobility through the development of novel brain-computer interface (BCI) technology. The present study examines the activity of ensembles of motor cortical neurons recorded using microelectrode arrays in the dominant hemisphere of two BrainGate clinical trial participants with cervical spinal cord injury as they attempted to perform a set of 48 different hand gestures. Although each participant displayed a unique organization of their respective neural latent spaces, it was possible to achieve classification accuracies of ~70% for all 48 gestures (and ~90% for sets of 10). Our results show that single unit ensemble activity recorded in a single hemisphere of human precentral gyrus has the potential to generate a wide range of gesture-related signals across both hands, providing an intuitive and diverse set of potential command signals for intracortical BCI use.
Rearrangement of 3D genome organization in breast cancer epithelial - mesenchymal transition and metastasis organotropism
Authors: Das Priyojit , San Martin Rebeca , Hong Tian , McCord Rachel Patton
Published: August 2024

Breast cancer cells exhibit organotropism during metastasis, showing preferential homing to certain organs such as bone, lung, liver, and brain. One potential explanation for this organotropic behavior is that cancer cells gain properties that enable thriving in certain microenvironments. Such specific metastatic traits may arise from gene regulation at the primary tumor site. Spatial genome organization plays a crucial role in oncogenic transformation and progression, but the extent to which chromosome architecture contributes to organ-specific metastatic traits is unclear. This work characterizes chromosome architecture changes associated with organotropic metastatic traits. By comparing a collection of genomic data from different subtypes of localized and lung metastatic breast cancer cells with both normal and cancerous lung cells, we find important trends of genomic reorganization. The most striking differences in 3D genome compartments segregate cell types according to their epithelial vs. mesenchymal status. This EMT compartment signature occurs at genomic regions distinct from transcription-defined EMT signatures, suggesting a separate layer of regulation. Specifically querying organotropism, we find 3D genome changes consistent with adaptations needed to survive in a new microenvironment, with lung metastatic breast cells exhibiting compartment switch signatures that shift the genome architecture to a lung cell-like conformation and brain metastatic prostate cancer cells showing compartment shifts toward a brain-like state. TCGA patient data reveals gene expression changes concordant with these organ-permissive compartment changes. These results suggest that genome architecture provides an additional level of cell fate specification informing organotropism and enabling survival at the metastatic site.

SIGNIFICANCE

Computational analysis of a cohort of cancer cell lines reveals 3D genome spatial compartment changes are associated with transitions in cancer cell state that favor metastasis (EMT) and enable survival in a new organ context.
Proteomic profiling of UV damage repair patches uncovers histone chaperones with central functions in chromatin repair
Authors: Plessier Alexandre , Chansard Audrey , Petit Eliane , Ducassou Julia Novion , Couté Yohann , Polo Sophie E.
Published: August 2024

DNA damage compromises not only genome stability but also the integrity of the chromatin template, which plays a central role in controlling cell identity. Our understanding of chromatin repair mechanisms is very incomplete. To bridge this knowledge gap, here we devise a novel proteomic strategy to characterize dynamic changes in the chromatin landscape during the repair of UV-induced DNA lesions in human cells, in a quantitative, unbiased and time-resolved manner. Thus, we identify the histone chaperones DNAJC9 and MCM2 as central players in chromatin repair. We demonstrate that DNAJC9 and MCM2 are independently recruited to sites of UV damage repair. DNAJC9 provides new H3-H4 histones to CAF-1 and HIRA chaperones for deposition into chromatin and also stimulates old H3-H4 histone recovery. DNAJC9 cooperates with MCM2 to coordinate old and new histone dynamics during UV damage repair. Together, our proteomic dataset provides a molecular framework for further dissecting epigenome maintenance mechanisms.
Understanding the Global Spread of Artemisinin Resistance: Insights from over 100KPlasmodium falciparumSamples
Authors: Balmer Andrew J. , White Nina F. D. , Ünlü Eyyüb S. , Lee Chiyun , Pearson Richard D. , Almagro-Garcia Jacob , Ariani Cristina V.
Published: August 2024

Artemisinin partial resistance (ART-R) inPlasmodium falciparumis one of the most pressing threats to global malaria control. Over the last two decades, ART-R has spread widely across Southeast Asia, compromising public health strategies and hindering elimination efforts. As of 2024, ART-R has now emerged in East Africa, with the potential to dramatically increase human mortality in the region. Mitigating the spread of ART-R requires detailed genomic surveillance of point mutations in thekelch13gene, the primary determinant of resistance to artemisinin derivatives. Although extensive surveillance data on these markers is available, it is distributed across many literature studies and open databases. In this literature review, we aggregate publicly available spatiotemporal data for 112,933P. falciparumsamples between 1980 – 2023 into a single resource, providing the most comprehensive overview ofkelch13markers to date. By synthesising insights from these samples over a global scale, we outline the history and current status ofkelch13mutations associated with ART-R, with particular reference to their emergence in Southeast Asia and recent emergence in East and Northeast Africa. Concerningly, we find their recent increases in frequency in these areas of Africa are comparable to those observed in Southeast Asia 10-15 years ago. We review several factors that may influence the spread of ART-R going forwards, such as fitness costs, treatment strategies, and local epidemiological dynamics, before articulating possible scenarios on how resistance may spread in Africa in coming years. In summary, this review provides a unified, comprehensive account of how the situation of ART-R has unfolded globally so far, highlighting insights both for researchers in the field and public health bodies which aim to reduce its negative effects. More broadly, we highlight the critical role genomic surveillance has had, and will continue to have in combating the spread of ART-R.
Host-Guest binding free energies à la carte: an automated OneOPES protocol
Authors: Febrer Martinez Pedro , Rizzi Valerio , Aureli Simone , Gervasio Francesco Luigi
Published: August 2024

Estimating absolute binding free energies from molecular simulations is a key step in computer-aided drug design pipelines, but agreement between computational results and experiments is still very inconsistent. Both the accuracy of the computational model and the quality of the statistical sampling contribute to this discrepancy, yet disentangling the two remains a challenge. In this study, we present an automated protocol based on OneOPES, an enhanced sampling method that exploits replica exchange and can accelerate several collective variables, to address the sampling problem. We apply this protocol to 37 host-guest systems. The simplicity of setting up the simulations and of producing well-converged binding free energy estimates without the need to optimize simulation parameters provides a reliable solution to the sampling problem. This, in turn, allows for a systematic force field comparison and ranking according to the correlation between simulations and experiments, which can inform the selection of an appropriate model. The protocol can be readily adapted to test more force field combinations and study more complex protein-ligand systems, where the choice of an appropriate physical model is often based on heuristic considerations rather than a systematic optimization.
In vivocontribution ofCyp24a1promoter vitamin D response elements
Authors: Meyer Mark B. , Lee Seong Min , Towne Jordan M. , Cichanski Shannon R. , Kaufmann Martin , Jones Glenville , Pike J. Wesley
Published: August 2024

CYP24A1 is a multifunctional, P450 mitochondrial 24-hydroxylase enzyme that is responsible for catabolism of the most active vitamin D hormone (calcitriol, 1,25(OH)2D3), its precursor (calcifediol, 25(OH)D3), and numerous other vitamin D metabolites at the 23- and 24-carbon positions. In the kidney,Cyp24a1is induced by 1,25(OH)2D3, induced by FGF23, and potently suppressed by PTH to tightly control the circulating blood levels of 1,25(OH)2D3. This gene is believed to be under the control of a pair of classic promoter proximal (PRO) vitamin D response elements (VDREs) that are aided by distal, downstream (DS) containing enhancers that we identified more recently. The DS1 enhancer cluster was found to respond to PTH and FGF23 actions in a kidney-specific manner. The DS2 enhancer cluster was found to assist in the response of 1,25(OH)2D3in kidney, as well as other target tissues. Despite this knowledge, thein vivocontribution of the PRO VDREs to gene expression, what drivesCyp24a1basal expression in the kidney, how FGF23 activatesCyp24a1, and importantly, how PTH suppressesCyp24a1, all remain unknown. Here in this study, we utilize homology directed CRISPR to mutate one or both VDREs in the PRO region of theCyp24a1genein vivoin the mouse to address these questions. We found that the VDRE (VDRE1) more proximal to the to the transcriptional start site (TSS) is the dominant VDRE of the pair and mutation of both VDREs leads to a dramatic loss of VDR, a reduction ofCyp24a1gene expression in the kidney, and a near elimination of 1,25(OH)2D3induction in the intestine. FGF23 induction ofCyp24a1was reduced with mutation of the PRO VDREs, however, co-treatment of 1,25(OH)2D3and FGF23 synergistically increasedCyp24a1expression even with the loss of the PRO VDREs. PTH suppression ofCyp24a1gene expression was unchanged with PRO VDRE mutations, despite a minor reduction in total pCREB occupancy. Finally, VDR occupancy was dramatically reduced across the DS enhancers in theCyp24a1locus after the PRO VDREs mutation. Taken together, our data suggest a cooperative relationship between the DS and PRO enhancers in the regulation ofCyp24a1by 1,25(OH)2D3and FGF23, and despite the overall reduction of CREB on the genome it appeared that suppression either does not rely on CREB or that the PRO VDREs are unconnected to PTH suppression altogether. These studies point to the DS1 region as a basal switch forCyp24a1expression and help further define the interconnected genomic control of these hormones on vitamin D catabolism.
Uncovering methylation-dependent genetic effects on regulatory element function in diverse genomes
Authors: Petersen Rachel M. , Vockley Christopher M. , Lea Amanda J.
Published: August 2024

A major goal in evolutionary biology and biomedicine is to understand the complex interactions between genetic variants, the epigenome, and gene expression. However, the causal relationships between these factors remain poorly understood. mSTARR-seq, a methylation-sensitive massively parallel reporter assay, is capable of identifying methylation-dependent regulatory activity at many thousands of genomic regions simultaneously, and allows for the testing of causal relationships between DNA methylation and gene expression on a region-by-region basis. Here, we developed a multiplexed mSTARR-seq protocol to assay naturally occurring human genetic variation from 25 individuals sampled from 10 localities in Europe and Africa. We identified 6,957 regulatory elements in either the unmethylated or methylated state, and this set was enriched for enhancer and promoter annotations, as expected. The expression of 58% of these regulatory elements was modulated by methylation, which was generally associated with decreased RNA expression. Within our set of regulatory elements, we used allele-specific expression analyses to identify 8,020 sites with genetic effects on gene regulation; further, we found that 42.3% of these genetic effects varied between methylated and unmethylated states. Sites exhibiting methylation-dependent genetic effects were enriched for GWAS and EWAS annotations, implicating them in human disease. Compared to datasets that assay DNA from a single European individual, our multiplexed assay uncovers dramatically more genetic effects and methylation-dependent genetic effects, highlighting the importance of including diverse individuals in assays which aim to understand gene regulatory processes.
State modulation in spatial networks with three interneuron subtypes
Authors: Edwards Madeline M , Rubin Jonathan , Huang Chengcheng
Published: August 2024

Several inhibitory interneuron subtypes have been identified as critical in regulating sensory responses. However, the specific contribution of each interneuron subtype remains uncertain. In this work, we explore the contributions of cell-type specific activity and synaptic connections to dynamics of a spatially organized spiking neuron network. We find that the firing rates of the somatostatin (SOM) interneurons align closely with the level of network synchrony irrespective of the target of modulatory input. Further analysis reveals that inhibition from SOM to parvalbumin (PV) interneurons must be limited to allow gradual transitions from asynchrony to synchrony and that the strength of recurrent excitation onto SOM neurons determines the level of synchrony achievable in the network. Our results are consistent with recent experimental findings on cell-type specific manipulations. Overall, our results highlight common dynamic regimes achieved across modulations of different cell populations and identify SOM cells as the main driver of network synchrony.
MiR-125b differentially impacts mineralization in dexamethasone and calcium-treated human mesenchymal stem cells
Authors: Joris Virginie , Balmayor Elizabeth R. , van Griensven Martijn
Published: August 2024

Bone metabolism is highly regulated, and microRNAs (miR) can contribute to this process. Among them, miR-125b is well-known to enhance osteoporosis and reduce osteogenic differentiation of human mesenchymal stem cells (hMSCs). In this work we aim to evaluate and understand how miR-125b modulates mineralization of hMSCs in two differentin vitromodels. Cells were cultured in dexamethasone or calcium medium and transfected with miR-125b mimic. Exposure to dexamethasone or calcium medium increased mineralization of hMSCs and was associated with decreased miR-125b expression. Transfection of miR-125b mimic in dexamethasone-treated cells increased mineralization while it decreased it in calcium-treated cells. Levels of osteogenic markers presented the same difference. We identified STAT3, p53 and RUNX2 as direct targets of miR-125b in hMSCs. While these targets remained identical in both treatments, their modulation after transfection was different. We showed that miR-125b mimicking differentially modulated the expression of the miR-199a/214 cluster, probably via STAT3/miR-199a/214, and p53/miR-214 pathways. In conclusion, miR-125b affinity for targets implicated in bone remodeling changed depending on thein vitromodels used to induce mineralization and led to opposite physiological effect. This works shows the complexity of drugs such as dexamethasone and opens the door for newin vitromodels of mineralization.
LDB1 establishes multi-enhancer networks to regulate gene expression
Authors: Aboreden Nicholas G. , Lam Jessica C. , Goel Viraat Y. , Wang Siqing , Wang Xiaokang , Midla Susannah C. , Quijano Alma , Keller Cheryl A. , Giardine Belinda M. , Hardison Ross C. , Zhang Haoyue , Hansen Anders S. , Blobel Gerd A.
Published: August 2024

How specific enhancer-promoter pairing is established is still mostly unclear. Besides the CTCF/cohesin machinery, only a few nuclear factors have been studied for a direct role in physically connecting regulatory elements. Here, we show via acute degradation experiments that LDB1 directly and broadly promotes enhancer-promoter loops. Most LDB1-mediated contacts, even those spanning hundreds of kb, can form in the absence of CTCF, cohesin, or YY1 as determined via the use of multiple degron systems. Moreover, an engineered LDB1-driven chromatin loop is cohesin independent. Cohesin-driven loop extrusion does not stall at LDB1 occupied sites but may aid the formation of a subset of LDB1 anchored loops. Leveraging the dynamic reorganization of nuclear architecture during the transition from mitosis to G1-phase, we establish a relationship between LDB1-dependent interactions in the context of TAD organization and gene activation. Lastly, Tri-C and Region Capture Micro-C reveal that LDB1 organizes multi-enhancer networks to activate transcription. This establishes LDB1 as a direct driver of regulatory network inter-connectivity.
Efflux pumps and membrane permeability contribute to intrinsic antibiotic resistance inMycobacterium abscessus
Authors: McGowen Kerry , Funck Tobias , Wang Xin , Zinga Samuel , Wolf Ian D. , Akusobi Chidiebere C. , Denkinger Claudia M. , Rubin Eric J. , Sullivan Mark R.
Published: August 2024

Mycobacterium abscessusis a pulmonary pathogen that exhibits intrinsic resistance to antibiotics, but the factors driving this resistance are incompletely understood. Insufficient intracellular drug accumulation could explain broad-spectrum resistance, but whether antibiotics fail to accumulate inM. abscessusand the mechanisms required for drug exclusion remain poorly understood. We measured antibiotic accumulation inM. abscessususing mass spectrometry and found a wide range of drug accumulation across clinically relevant antibiotics. Of these compounds, linezolid accumulates the least, suggesting that inadequate uptake impacts its efficacy. We utilized transposon mutagenesis screening to identify genes that cause linezolid resistance and found multiple transporters that promote membrane permeability or efflux, including an uncharacterized,M. abscessus-specific protein that effluxes linezolid and several chemically related antibiotics. This demonstrates that membrane permeability and drug efflux are critical mechanisms of antibiotic resistance inM. abscessusand suggests that targeting membrane transporters could potentiate the efficacy of certain antibiotics.