bluuebunny/crossref_metadata_2025_split huggingface dataset:
Epigenetic-focused CRISPR/Cas9 screen identifies ASH2L as a regulator of glioblastoma cell survival
Authors: Ozyerli-Goknar Ezgi , Kala Ezgi Yagmur , Aksu Ali Cenk , Bulut Ipek , Cingöz Ahmet , Nizamuddin Sheikh , Biniossek Martin , Seker-Polat Fidan , Morova Tunc , Aztekin Can , Kung Sonia H.A. , Syed Hamzah , Tuncbag Nurcan , Gonen Mehmet , Philpott Martin , Cribbs Adam P. , Acilan Ceyda , Lack Nathan A. , Onder Tamer T. , Timmers H.T. Marc , Bagci-Onder Tugba 
Published: August 2022

ABSTRACTGlioblastoma is the most common and aggressive primary brain tumor with poor prognosis, highlighting an urgent need for novel treatment strategies. In this study, we investigated epigenetic regulators of glioblastoma cell survival through CRISPR/Cas9 based genetic ablation screens using a customized sgRNA library EpiDoKOL, which targets critical functional domains of chromatin modifiers. Screens conducted in multiple cell lines revealed ASH2L, a histone lysine methyltransferase complex subunit, as a major regulator of glioblastoma cell viability. ASH2L depletion led to cell cycle arrest and apoptosis. RNA sequencing and greenCUT&RUN together identified a set of cell cycle regulatory genes, such as TRA2B, BARD1, KIF20B, ARID4A and SMARCC1 that were downregulated upon ASH2L depletion. Mass spectrometry analysis revealed the interaction partners of ASH2L in glioblastoma cell lines as SET1/MLL family members including SETD1A, SETD1B, MLL1 and MLL2. We further showed that glioblastoma cells had a differential dependency on expression of SET1/MLL family members for survival. The growth of ASH2L-depleted glioblastoma cells was markedly slower than controls in orthotopic in vivo models. TCGA analysis showed high ASH2L expression in glioblastoma compared to low grade gliomas and immunohistochemical analysis revealed significant ASH2L expression in glioblastoma tissues, attesting to its clinical relevance. Therefore, high throughput, robust and affordable screens with focused libraries, such as EpiDoKOL, holds great promise to enable rapid discovery of novel epigenetic regulators of cancer cell survival, such as ASH2L. Together, we suggest that targeting ASH2L could serve as a new therapeutic opportunity for glioblastoma.
Oxygen kinetics during CXL using symmetrically and asymmetrically pulsed UV-irradiation
Authors: Komninou Maria A. , Nambiar Malavika H. , Frueh Beatrice E. , Enzmann Volker , Büchler Philippe , Seiler Theo G. 
Published: August 2022

AbstractPurposeTo investigate oxygen kinetics during symmetrically pulsed and asymmetrically pulsed crosslinking (p-CXL) with and without supplementary oxygen at different irradiances and corneal depths.DesignExperimental, laboratory studyMethodsIn de-epithelialized porcine eyes, a femtosecond-laser generated tunnel was used to place a fibre-probe in corneal depths of 200 and 300 µm to measure the local oxygen concentration. After riboflavin imbibition, the corneas were irradiated at 9, 18 and 30 mW/cm2 for 10 seconds On and 10 seconds Off; while the oxygen concentration was continuously measured until oxygen levels depleted below the oxygen sensor’s threshold (1%) or until stabilized. All experiments were performed under normoxic (21%) and hyperoxic (>95%) conditions and the obtained data were used to identify parameters of a numerical algorithm for oxygen consumption and diffusion. Following the algorithm’s development, the suggested asymmetrical pulsing values were experimentally tested. For 9, 18 and 30 mW/cm2 the suggested tested pulsing schemes were 3 seconds On : 9 seconds Off, 2 seconds On : 9 seconds Off and 1 second On : 9 seconds Off respectively.ResultsThe minimum, available stromal oxygen for p-CXL in normoxic environment was decreasing <1% for 9, 18 and 30 mW/cm2 in 200 and 300 μm. Using optimized p-CXL, the minimum available oxygen increased to 3.8, 1.8 and 2.8 % at 200 μm, for irradiances of 9, 18 and 30 mW/cm2, respectively, where the periods exhibited an equilibrium state. At 300 μm, 1.1 % of oxygen was available for 30 mW/cm2. Using a hyperoxic environment, the oxygen concentration was 19.2% using 9 mW/cm2 in 200 μm, dropping to 17.0% in 300 μm. At 18 mW/cm2, the concentrations were 3.9% and 1% in 200 and 300 μm, respectively. Using 30 mW/cm2, all oxygen was depleted below the threshold limit (1% O2) for both depths. Using optimized pulsing in combination with hyperoxic environment, the oxygen concentration was 42.0% using 9 mW/cm2 in 200 μm and 43.3% in 300 μm. At 18 mW/cm2, the concentrations were 24.7% and 16.1% in 200 and 300 μm, respectively. Using 30 mW/cm2, the minimum oxygen availability was 25.7% and 13.7% in 200 and 300 μm, respectively.ConclusionSupplementary oxygen during symmetrical and asymmetrical p-CXL increased the oxygen availability during corneal cross-linking. The pulsed irradiance and the hyperoxic environment potentially increased the efficacy of corneal cross-linking in deeper corneal layers and higher irradiances. The numerical algorithm for asymmetrical pulsing led to the quantification of “On” and “Off” times related to different scenarios such as irradiances.
The nanomechanical fingerprint of colorectal cancer -derived peritoneal metastasis
Authors: Lorenc Ewelina , Varinelli Luca , Chighizola Matteo , Brich Silvia , Pisati Federica , Guaglio Marcello , Baratti Dario , Deraco Marcello , Gariboldi Manuela , Podesta Alessandro 
Published: August 2022

AbstractPeritoneal metastases (PM) are one of the most common routes of dissemination for colorectal cancer (CRC) and remain a lethal disease with a poor prognosis. The compositional, mechanical and structural properties of the extracellular matrix (ECM) play an important role in cancer development; studying how these properties change during the progression of the disease is crucial to understand CRC-PM development.The elastic properties of ECMs derived from human samples of normal and neoplastic PM in different pathological conditions were studied by atomic force microscopy (AFM); results were correlated to patients’ clinical data and to the expression of ECM components related to metastatic spread.Our results show that PM progression is accompanied by stiffening of ECM as a common feature; spatially resolved mechanical analysis highlighted significant spatial heterogeneity of the elastic properties of both normal and neoplastic ECMs, which show significant overlap in the two conditions. On the micrometre scale, ECMs that are considered normal according to the pathological classification possess stiffer spatial domains, which are typically associated with cancer associated fibroblasts (CAF) activity and tumour development in neoplastic matrices; on the other hand, softer regions are found in neoplastic ECMs on the same scales. Our results support the hypothesis that local changes (stiffening) in the normal ECM can create the ground for growth and spread from the tumour of invading metastatic cells.Mechanical changes correlate well with the presence of CAF and an increase in collagen deposition, which are well known markers of cancer progression. Furthermore, we have found correlations between the mechanical properties of the ECM and patients’ clinical data like age, sex, presence of mutations inBRAFandKRASgenes and tumour grade.Overall, our findings suggest that the mechanical phenotyping of the PM-ECM has the potential for predicting tumour development.
A balance between pro-inflammatory and pro-reparative macrophages is observed in regenerative D-MAPS
Authors: Liu Yining , Suarez-Arnedo Alejandra , Shetty Shamitha , Wu Yaoying , Schneider Michelle , Collier Joel H. , Segura Tatiana 
Published: August 2022

AbstractMicroporous annealed particle scaffolds (MAPS) are a new class of granular materials generated through the interlinking of tunable microgels, which produce an interconnected network of void space. These microgel building blocks can be designed with different mechanical or bio-active parameters to facilitate cell infiltration and modulate host response. Previously, changing the chirality of the microgel crosslinking peptides from L- to D-amino acids led to significant tissue regeneration and functional recovery in D-MAPS-treated cutaneous wounds. In this study, we investigated the immunomodulatory effect of D-MAPS in a subcutaneous implantation model. We uncovered how macrophages are the key antigen-presenting cells to uptake and present these biomaterials to the adaptive immune system. A robust linker-specific IgG2b/IgG1 response to D-MAPS was detected as early as 14 days post-implantation. The fine balance between pro-regenerative and pro-inflammatory macrophage phenotypes was observed in D-MAPS as an indicator for regenerative scaffolds. Our work offers valuable insights into the temporal cellular response to synthetic porous scaffolds and establishes a foundation for further optimization of immunomodulatory pro-regenerative outcomes.
Genetic diversity and spread dynamics of SARS-CoV-2 variants present in African populations
Authors: Mtetwa Desire , Mutetwa Tapiwa Fidelity , Manjengwa Tafadzwa , Mazadza Allen , Chikwambi Zedias 
Published: August 2022

ABSTRACTThe dynamics of COVID-19 disease have been extensively researched in many settings around the world, but little is known about these patterns in Africa. 6139 complete nucleotide genomes from 51 African nations were obtained and analyzed from the National Center for Biotechnology Information (NCBI) and Global Initiative on Sharing Influenza Data (GISAID) databases to examine genetic diversity and spread dynamics of SARS-CoV-2 lineages circulating in Africa. We investigated their diversity using several clade and lineage nomenclature systems, and used maximum parsimony inference methods to recreate their evolutionary divergence and history. According to this study, only 193 of the 2050 Pango lineages discovered worldwide circulated in Africa after two years of the COVID-19 pandemic outbreak, with five different lineages dominating at various points during the outbreak. We identified South Africa, Kenya, and Nigeria as key sources of viral transmissions between Sub-Saharan African nations because they had the most SARS-CoV-2 genomes sampled and sequenced. These results shed light on the evolutionary dynamics of the circulating viral strains in Africa. Genomic surveillance is one of the important techniques in the pandemic preparedness toolbox and to better understand the molecular, evolutionary, epidemiological, and spatiotemporal dynamics of the COVID-19 pandemic in Africa, genomic surveillance activities across the continent must be expanded. The effectiveness of molecular surveillance as a method for tracking pandemics strongly depends on continuous and reliable sampling, speedy virus genome sequencing, and prompt reporting and we have to improve in all these aspects in Africa. Additionally, the pandemic breakout revealed that current land-border regulations aimed at limiting virus’s international transmission are ineffective and a lot needs to be done to implement and improve our African land-borders as far as epidemiology is concerned in order to contain such outbreaks in the future.
A Simplified Function-First Method for the Discovery and Optimization of Bispecific Immune Engaging Antibodies
Authors: Shepherd Alex , Bennychen Bigitha , Marcil Anne , Bloemberg Darin , Pon Rob , Weeratna Risini , McComb Scott 
Published: August 2022

AbstractBi-specific T-cell engager antibodies (BITEs) are synthetic soluble molecules derived from antibodies that induce active contact between T-cells and other target cells in the body. BITE therapeutics have shown great promise for the treatment of various forms of cancer; however, the current development process for BITEs is time consuming and costly. BITE development requires empirical testing and characterization of the individual antigen binding domains, followed by extensive engineering and optimization in bi-specific molecular format to generate a molecule with strong biological activity and appropriate characteristics for clinical development. Here, we sought to create a cost efficient high-throughput method for creating and evaluating BITEs using a simplified function first approach to identify bioactive molecules without purification. Using a plasmid with a modular structure to allow high efficiency exchange of either binder arm, we established a simple method to combine many novel tumour-targeting single chain variable (scFv) domains with the well-characterized OKT3 scFv CD3-targeting domain. After generating these novel plasmids, we demonstrate two systems for high throughput functional screening of BITE molecules based on Jurkat T cells (referred to as BITE-J). Using BITE-J we evaluate four EGFRvIII BITEs, identifying two constructs with superior activity. We then confirmed this activity in primary T cells, where novel EGFRvIII-BITEs induced T cell activation and antigen selective tumor killing. We also demonstrate that we can similarly exchange the CD3-interacting element of our bi-modular plasmid. By testing several novel CD3-targeting scFv elements for activity in EGFRvIII-targeted BITEs, we were able to identify highly active BITE molecules with desirable properties for downstream development. In summary, BITE-J presents a low cost, high-throughput method for the rapid assessment of novel BITE molecules without the need for purification and quantification.
Prevalence and heterogeneity of antibiotic-resistant genes inOrientia tsutsugamushiand other rickettsial genomes
Authors: Rao R. Shyama Prasad , Ghate Sudeep D. , Shastry Rajesh P. , Kurthkoti Krishna , Suravajhala Prashanth , Patil Prakash , Shetty Praveenkumar 
Published: August 2022

AbstractDespite a million infections every year and an estimated one billion people at risk, scrub typhus is regarded as a neglected tropical disease. The causative bacteriumOrientia tsutsugamushi, a member of rickettsiae, seems to be intrinsically resistant to several classes of antibiotics. The emergence of antibiotic-resistant scrub typhus is likely to become a global public health concern. Yet, it is unknown as to how common antibiotic-resistant genes are inO. tsutsugamushi, and how variable these loci are among the genomes of rickettsiae. By using the comprehensive antibiotic resistance database, we explored 79 complete genomes from 24 species of rickettsiae for putative antibiotic-resistant loci. There were 244 unique antibiotic-resistant genes in rickettsiae. Both the total and unique antibiotic-resistant genes inO. tsutsugamushiwere significantly less compared to other members of rickettsiae. However, antibiotic-resistant genes inO. tsutsugamushigenomes were more unique and highly variable. Many genes such as resistant versions ofevgS, andvanS A/Gwere present in numerous copies. These results will have important implications in the context of antibiotic-resistant scrub typhus.
Mesenchymal stromal cell encapsulation in uniform chitosan beads using microchannel emulsification
Authors: Shin Dongjin S , Touani Francesco K , Aboud Damon G.K , Kietzig Anne-Marie , Lerouge Sophie , Hoesli Corinne A 
Published: August 2022

ABSTRACTMesenchymal stromal/stem cells hold potential in repairing damaged tissue through paracrine effects. Their delivery though injectable biodegradable microbeads can improve cell retention and survival at the infusion site. A stirred emulsion process was previously implemented to immobilize these cells in injectable chitosan microbeads for cell therapy applications, but this process leads to broad bead size distribution (coefficient of variation > 40 %). Polydisperse beads may negatively affect the viability of the entrapped cells through oxygen limitations, damage to larger beads during injection, and reduced control over the cell payload and treatment reproducibility. The objective of this work was to modify a microchannel emulsification system initially designed for alginate-based encapsulation to immobilize mesenchymal stromal/stem cells in monodisperse chitosan microbeads. The main factors (e.g., microchannel geometry, chitosan solution viscosity, interfacial tension and flow rate) affecting droplet generation and diameter were investigated. The adapted process enabled the production of monodisperse chitosan microbeads with controlled sizes ranging from 600 µm – 1500 µm in diameter at a coefficient of variation less than 10 %. In a single pass through a 21 G syringe needle (ID: 513 µm), the fraction of ruptured beads was significantly reduced for microchannel-generated vs stirred emulsion-generated beads with matching volume-weighed bead diameter (D[4,3]). The viability of the immobilized cells immediately after the process was 95 % ± 2 % and no significant difference in cell survival and growth factor secretion was observed between microchannel and stirred emulsion-generated beads over 3 days of culture. Future directions include channel multiplexing to increase throughput for clinical applications. Although the device was developed for cell encapsulation, this process could be implemented for encapsulation of other biomolecules, bioactive or living cell agents for applications in the food and drug industry.
Macrophage-Based iNos Reporter Reveals Polarization and Reprogramming in the Context of Breast Cancer
Authors: Mas-Rosario Javier A. , Medor Josue D. , Jeffway Mary I. , Martínez-Montes José M. , Farkas Michelle E. 
Published: August 2022

AbstractAs part of the first line of defense against pathogens, macrophages possess the ability to differentiate into divergent phenotypes with varying functions. The process by which these cells change their characteristics, commonly referred to as macrophage polarization, allows them to change into broadly pro-inflammatory (M1) or anti-inflammatory (M2) subtypes, and depends on the polarizing stimuli. Deregulation of macrophage phenotypes can result in different pathologies or affect the nature of some diseases, such as cancer and atherosclerosis. For this reason, it is necessary to better understand macrophage phenotype conversion in relevant models. However, there are few existing probes to track macrophage changes in multicellular environments. In this study, we generated an eGFP reporter cell line based on inducible nitric oxide synthase (iNos) promoter activity in RAW264.7 cells (RAW:iNos-eGFP). iNos is associated with macrophage activation to pro-inflammatory states, and decreases in immune-suppressing ones. We validated the fidelity of the reporter for iNos, including following cytokine-mediated polarization, and confirmed that reporter and parental cells behaved similarly. RAW:iNos-eGFP cells were then used to track macrophage responses in different in vitro breast cancer models, and their re-education from anti- to pro-inflammatory phenotypes via a previously reported pyrimido(5,4-b)indole small molecule, PBI1. Using two mouse mammary carcinoma cell lines, 4T1 and EMT6, effects on macrophages were assessed via conditioned media, two-dimensional/monolayer co-culture, and three-dimensional spheroid models. While conditioned media derived from 4T1 or EMT6 cells and monolayer co-cultures of each with RAW:iNos-eGFP cells all resulted in decreased fluorescence, the trends and extents of effects differed. We also observed decreases in iNos-eGFP signal in the macrophages in co-culture assays with 4T1- or EMT6-based spheroids. We then showed that we are able to enhance iNos production in the context of these cancer models using PBI1, tracking increased fluorescence. Taken together, we demonstrate that this reporter-based approach provides a facile means to study macrophage responses in complex, multicomponent environments. Beyond the initial studies presented here, this platform can be used with a variety of in vitro models and extended to in vivo applications with intravital imaging.
A Double Dissociation between Savings and Long-Term Memory in Motor Learning
Authors: Hadjiosif Alkis M. , Morehead J. Ryan , Smith Maurice A. 
Published: August 2022

AbstractBoth declarative and procedural memories are easier to reacquire than learn from scratch. This advantage, known as savings, has been widely assumed to result from the reemergence of stable long-term memories. In fact, the presence of savings has often been used as a marker for whether a memory had been consolidated. However, recent findings have demonstrated that motor learning rates can be systematically controlled, providing a mechanistic alternative to the reemergence of a stable long-term memory, and recent work has reported conflicting results about whether implicit contributions to savings in motor learning are present, absent, or inverted, suggesting a limited understanding of the underlying mechanisms. In order to elucidate the mechanism responsible for savings in motor learning, we investigate the relationship between savings and long-term memory by determining how they depend on different components of motor learning. To accomplish this, we experimentally dissect motor adaptation based on short-term (1-minute) temporal persistence. Surprisingly, we find that a temporally-volatile component of implicit learning leads to savings whereas temporally-persistent learning does not, but that temporally-persistent learning leads to long-term memory at 24 hours whereas temporally-volatile learning does not. Moreover, we find that temporally-persistent implicit learning not only fails to contribute to savings, but that it produces an anti-savings which acts to reduce the net savings, and we show that the balance between temporally-volatile and temporally-persistent components can explain seemingly inconsistent reports about implicit savings. The clear double dissociation between the mechanisms for savings and long-term memory formation challenges widespread assumptions about the connection between savings and memory consolidation, and provides new insight into the mechanisms for motor learning.