bluuebunny/crossref_metadata_2025_split huggingface dataset:
Analytical kinetic model of native tandem promoters in E. coli
Authors: Chauhan Vatsala , Bahrudeen Mohamed N.M. , Palma Cristina S.D. , Baptista Ines S.C. , Almeida Bilena L.B. , Dash Suchintak , Kandavalli Vinodh , Ribeiro Andre S. 
Published: November 2021

AbstractClosely spaced promoters in tandem formation are abundant in bacteria. We investigated the evolutionary conservation, biological functions, and the RNA and single-cell protein expression of genes regulated by tandem promoters in E. coli. We also studied the sequence (distance between transcription start sites ‘dTSS’, pause sequences, and distances from oriC) and potential influence of the input transcription factors of these promoters. From this, we propose an analytical model of gene expression based on measured expression dynamics, where RNAP-promoter occupancy times and dTSS are the key regulators of transcription interference due to TSS occlusion by RNAP at one of the promoters (when dTSS ≤ 35 bp) and RNAP occupancy of the downstream promoter (when dTSS > 35 bp). Occlusion and downstream promoter occupancy are modeled as linear functions of occupancy time, while the influence of dTSS is implemented by a continuous step function, fit to in vivo data on mean single-cell protein numbers of 30 natural genes controlled by tandem promoters. The best-fitting step is at 35 bp, matching the length of DNA occupied by RNAP in the open complex formation. This model accurately predicts the squared coefficient of variation and skewness of the natural single-cell protein numbers as a function of dTSS. Additional predictions suggest that promoters in tandem formation can cover a wide range of transcription dynamics within realistic intervals of parameter values. By accurately capturing the dynamics of these promoters, this model can be helpful to predict the dynamics of new promoters and contribute to the expansion of the repertoire of expression dynamics available to synthetic genetic constructs.Author SummaryTandem promoters are common in nature, but investigations on their dynamics have so far largely relied on synthetic constructs. Thus, their regulation and potentially unique dynamics remain unexplored. We first performed a comprehensive exploration of the conservation of genes regulated by these promoters in E. coli and the properties of their input transcription factors. We then measured protein and RNA levels expressed by 30 Escherichia coli tandem promoters, to establish an analytical model of the expression dynamics of genes controlled by such promoters. We show that start site occlusion and downstream RNAP occupancy can be realistically captured by a model with RNAP binding affinity, the time length of open complex formation, and the nucleotide distance between transcription start sites. This study contributes to a better understanding of the unique dynamics tandem promoters can bring to the dynamics of gene networks and will assist in their use in synthetic genetic circuits.
Progressive sub-MIC Exposure ofKlebsiella pneumoniae43816 to Cephalothin Induces the Evolution of beta-lactam Resistance without Acquisition of beta-lactamase Genes
Authors: Anderson Jasmine R. , Lam Nghi B. , Jackson Jazmyne L. , Dorenkott Sean M. , Ticer Taylor , Maldosevic Emir , Velez Amanda , Camden Megan R. , Ellis Terri N. 
Published: November 2021

AbstractBacterial exposure to antibiotic concentrations below the minimum inhibitory concentration (MIC) may result in a selection window allowing for the rapid evolution of resistance. These sub-MIC concentrations are commonly found in the greater environment. This study aimed to evaluate the adaptive genetic changes inKlebsiella pneumoniae43816 after prolonged but increasing sub-MIC levels of the common antibiotic cephalothin over a fourteen-day period. Over the course of the experiment, antibiotic concentrations increased from 0.5 μg/mL to 7.5 μg/mL. At the end of this extended exposure, the final adapted bacterial culture exhibited clinical resistance to both cephalothin and tetracycline, altered cellular and colony morphology, and a highly mucoid phenotype. Cephalothin resistance exceeded 125 μg/mL without the acquisition of beta-lactamase genes. Whole genome sequencing identified a series of genetic changes that could be mapped over the fourteen-day exposure period to the onset of antibiotic resistance. Specifically, mutations in therpoBsubunit of RNA Polymerase, thetetR/acrRregulator, and thewcaJsugar transferase each fix at specific timepoints in the exposure regimen where the MIC susceptibility dramatically increases. These mutations indicate that alterations in the secretion of colanic acid and attachment of colonic acid to LPS, may contribute to the resistant phenotype. These data demonstrate that very low, sub-MIC concentrations of antibiotics can have dramatic impacts on the bacterial evolution of resistance. Additionally, this study demonstrates that beta-lactam resistance can be achieved through sequential accumulation of specific mutations without the acquisition of a beta-lactamase gene.ImportanceBacteria are constantly exposed to low levels of antibiotics in the environment. The impact of this low-level exposure on bacterial evolution is not well understood. In this work, we developed a model to exposeKlebsiella pneumoniaeto progressive, low doses of the antibiotic cephalothin. After a fourteen-day exposure regimen, our culture exhibited full clinical resistance to this antibiotic without the traditional acquisition of inactivating genes. This culture also exhibited resistance to tetracycline, had a highly mucoid appearance, and exhibited altered, elongated cellular morphology. Whole genome sequencing identified a collection of mutations to the bacterial genome that could be mapped to the emergence of the resistant phenotype. This study demonstrates that antibiotic resistance can be achieved in response to low level antibiotic exposure and without the traditional acquisition of resistance genes. Further, this study identifies new genes that may play a role in the evolution of antibiotic resistant bacteria.
PDFGRα+ Stromal Cells Promote Salivary Gland Proacinar Differentiation Through FGF2-dependent BMP7 Signaling
Authors: Moskwa Nicholas , Mahmood Ayma , Nelson Deirdre A. , Altrieth Amber L. , Forni Paolo , Larsen Melinda 
Published: November 2021

3.0AbstractStromal cells can direct epithelial differentiation during organ development; however, these pathways remain poorly defined. FGF signaling is essential for submandibular salivary gland development, and FGF2 can regulate proacinar cell differentiation in organoids through autocrine signaling in stromal cells. We performed scRNA Seq and identified stromal cell subsets expressing Fgf2 and Fgf10 that also express Pdgfrα. When combined with epithelial cells in organoids, MACS-sorted PDGFRα+ cells sufficiently promoted proacinar differentiation. Gene expression analysis revealed FGF2 activates the gene Bmp7 in the stroma. BMP7 could replace stromalsignaling and stimulate epithelial acinar differentiation but not branching. However, in the absence of FGF2, pathway analysis revealed that the stromal cells differentiated into myofibroblasts. Myofibroblast differentiation was induced when we treated organoids with TGFβ1, which also prevented proacinar differentiation. Conversely, FGF2 reversed TGFβ’s effects. Dissecting pathways driving acinar differentiation will facilitate development of regenerative therapies.2.0Summary StatementEmbryonic salivary glands contain multiple stromal cell populations. FGF2 maintains the stromal Pdgfrα+ population in-vitro. The PDGFRα+ stromal cells drive early epithelial secretory cell differentiation using BMP7.
ARL3 Mediates BBSome Ciliary Turnover by Promoting Its Outward Diffusion through the Transition Zone
Authors: Liu Yan-Xia , Sun Wei-Yue , Xue Bin , Zhang Rui-Kai , Li Wen-Juan , Xie Xixian , Fan Zhen-Chuan 
Published: November 2021

AbstractCiliary receptors and their certain downstream signaling components undergo intraflagellar transport (IFT) as BBSome cargoes to maintain their ciliary dynamics for sensing and transducing extracellular stimuli inside the cell. Cargo laden BBSomes shed from retrograde IFT at the proximal ciliary region above the transition zone (TZ) followed by diffusing through the TZ for ciliary retrieval, while how the BBSome barrier passage is controlled remains elusive. Here, we show that the BBSome is a major effector of the Arf-like 3 (ARL3) GTPase inChlamydomonas. Under physiological condition, ARL3GDPbinds the membrane for diffusing into and residing in cilia. Following a nucleotide conversion, ARL3GTPdissociates with the ciliary membrane and binds and recruits the IFT-detached and cargo (phospholipase D, PLD)-laden BBSome at the proximal ciliary region to diffuse through the TZ and out of cilia. ARL3 deficiency impairs ciliary signaling, e.g. phototaxis ofChlamydomonascells, by disrupting BBSome ciliary retrieval, providing a mechanistic understanding behind BBSome ciliary turnover required for ciliary signaling.
Inactivation effect and damage of multi-irradiance by UVCLED on Acinetobacter baumannii
Authors: Li Mengmeng , Zhao Baiqin , Hanlei  , Paste Wangzhen 
Published: November 2021

AbstractIt is acknowledged that the inactivation of ultraviolet has been widely used in various fields. Much literature has been reported that ultraviolet C caused DNA damage to achieve inactivation of microorganisms. There is a lack of unified dose calibration and related parameters in this field. In this study, we used a device consisted of the LED of 272 nm to conduct sterilization experiments against A. baumanii. We confirmed the effectiveness of ultraviolet C sterilization for both sensitive and drug resistance strains and explored the relationship between bactericidal rate and ultraviolet doses under various irradiance. Dose requirements of various irradiance were clarified. High irradiance improved sterilization efficiency greatly. The overall damage to the total genome was observed though gel electrophoresis. Ultrastructure of damaged bacteria were investigated by transmission electron microscope in detail. The study revealed that damage to DNA and to the cytoplasm matrix and ribosomes. The study has yielded the possible effects of ultraviolet light on cells by amplifying the energy. The radiation significantly promoted the production of cell wall and cellular membrane.Significance StatementThe statistical results of bactericidal efficiency are influenced by the quantity of bacteria on the medium. Irradiance on the target surface affects the sterilization efficiency directly. And doses required in low irradiance are much more than high. A high irradiance reduced dosage efficiently to achieve the sterilization which improves the sterilization efficiency. There is a difference between low and high UVC dosage damage to the structure of bacteria. Less energy can make DNA coagulation solidified or be dispersed to the edge. Meanwhile the cytoplasm matrix is ruined. When the energy was enough, there is a boost of cell wall and cellular membrane production. The invisible light causes comprehensive damage to bacteria.
Histidine-rich protein 2: a new pathogenic factor of Plasmodium falciparum malaria
Authors: Iwasaki Takashi , Shimoda Mayu , Kanayama Haru , Kawano Tsuyoshi 
Published: November 2021

AbstractPlasmodium falciparum causes serious malaria symptoms; when this protozoan parasite infects human erythrocytes, it produces and secretes large amounts of histidine-rich protein 2 (PfHRP2) into human blood. Thus, PfHRP2 is a well-known diagnostic marker for malaria infection. Here, however, we also identified PfHRP2 as a pathogenic factor produced by P. falciparum. PfHRP2 showed cell penetration and cytotoxicity against various human cells. In particular, PfHRP2 showed significant cytotoxicity over 5 days at the same concentration as in P. falciparum-infected patients’ blood (90–100 nM). This result is consistent with the mortality rate of P. falciparum malaria, which increases rapidly in untreated cases for 3–7 days. In addition, the cell penetration and cytotoxicity of PfHRP2 increased 2.5- and 2.6-fold, respectively, in the absence of serum, which suggests that low serum protein concentrations (occurring during malnutrition, for example) increase the risk of adverse effects from PfHRP2 (consistent with malnutrition increasing the lethality of malaria infection). We also showed that PfHRP2 bound to Ca2+ ions, localized to intracellular lysosomes, increased lysosomal Ca2+ levels, and inhibited the basal level of autophagy by inhibiting autolysosome formation. Furthermore, the Ca2+-dependent cytotoxicity of PfHRP2 was suppressed by the metal ion chelator ethylenediaminetetraacetic acid (EDTA). In summary, our findings suggest that PfHRP2 acts as a pathogenic factor in P. falciparum-infected patients and is associated with the exacerbation of malaria. Furthermore, EDTA is a promising candidate as a therapeutic agent for the suppression of PfHRP2 pathogenicity. Overall, this study provides new insights into P. falciparum malaria pathogenesis and treatment.
Substrate stiffness controls the cell cycle of human mesenchymal stem cells via cellular traction
Authors: Kureel Sanjay Kumar , Sinha Shatarupa , Purkayastha Purboja , Barretto Sarah , Majumder Abhijit 
Published: November 2021

AbstractThe microenvironment of human mesenchymal stem cells (hMSCs) regulates their self-renewal and differentiation properties. Previously it was shown that hMSCs remained quiescent on soft (0.25 kPa) polyacrylamide (PA) gels but re-entered into cell cycle on a stiff (7.5 kPa) gel. However, how cells behave on intermediate stiffness and what intracellular factors transmit mechanical changes to cell interior thereby regulating cell cycle remained unknown. In this work we demonstrated that PA gels between 1 and 5 kPa act as a mechanical switch in regulating cell cycle of hMSCs. By experiments on cell-cycle exit and re-entry, we found that hMSCs demonstrated a sharp transition from quiescence to proliferation between 1 and 5 kPa. Further studies with ROCK inhibitor Y-27632 revealed that contractile proteins, but not cell spread area, accounts for the sensitivity of hMSCs towards substrate stiffness and hence correlates with their changes in cell cycle. These observations therefore suggest that substrate stiffness regulates hMSC proliferation through contractile forces as generated by cellular contractile proteins in a unique pattern which is distinct from other cell types as studied.Graphical abstract
Label-free imaging of collagen fibers in tissue slices using phase imaging with computational specificity
Authors: Sakakura Masayoshi , Macias Virgilia , Borhani Sohelia , Kajdacsy-Balla André , Popescu Gabriel 
Published: November 2021

ABSTRACTEvaluating the tissue collagen content in addition to the epithelial morphology has been proven to offer complementary information in histopathology, especially in oncology tumor staging and prediction of survival in cancer patients. One imaging modality widely used for this purpose is second harmonic generation microscopy (SHGM), which reports on the nonlinear susceptibility associated with the collagen fibers. Another method is polarization light microscopy (PLM) combined with picrosirius-red (PSR) tissue staining. However, SHGM requires expensive equipment and provides limited throughput, while PLM and PSR staining are not part of the routine surgical pathology workflow. Here, we utilize phase imaging with computational specificity (PICS) to computationally infer the collagen distribution ofunlabeledtissue, with high specificity. PICS utilizes deep learning to translate quantitative phase images (QPI) into corresponding PSR images with high accuracy and inference speed of 200 milisecond per forwardpass through the model once trained. We developed a multimodal imaging instrument that yields both Spatial light Inference Microscopy (SLIM) and polarized light microscopy (PLM) images from the same field of view. Our results indicate that the distributions of collagen fiber orientation, length, and straightness reported by PICS closely match the ones from ground truth as defined by KL-divergence.
Cleavage of PGAM5 by the intramembrane protease PARL is governed by transmembrane helix dynamics and oligomeric state
Authors: Siebert Verena , Silber Mara , Heuten Elena , Muhle-Goll Claudia , Lemberg Marius K. 
Published: November 2021

AbstractThe intramembrane protease PARL is a crucial mitochondrial safeguard by cleaving the mitophagy regulators PINK1 and PGAM5. PGAM5 substrate determinates have not been rigorously investigated and it is unclear how uncoupling the mitochondrial membrane potential regulates its processing inversely to PINK1. Here we show that in PGAM5 several hydrophilic residues distant from the cleavage site serve as key determinant for PARL- catalyzed cleavage. NMR analysis indicates that a short N-terminal amphipathic helix, followed by a kink and a C-terminal helix harboring the scissile peptide bond, is key for a productive interaction with PARL. In difference to PINK1, PGAM5 is stably inserted into the inner mitochondrial membrane until uncoupling the membrane potential triggers its disassembly into monomers that are vulnerable to PARL-catalyzed processing. We suggest a model in which PGAM5 is a slowly processed substrate with PARL-catalyzed cleavage that is influenced by multiple hierarchical substrate features including a membrane-potential- dependent oligomeric switch.
Loss of zinc transporters ZIP1 and ZIP3 augments platelet reactivity in response to G protein-coupled receptor agonists and accelerates thrombus formation in vivo
Authors: Elgheznawy A , Öftering P , Englert M , Kaiser F , Kusch C , Gbureck U , Bösl MR , Nieswandt B , Vögtle T , Hermanns HM 
Published: November 2021

AbstractZinc (Zn2+) is considered as an important mediator for thrombosis and haemostasis. However, our understanding of the transport mechanisms that regulate Zn2+ homeostasis in platelets is limited. Zn2+ transporters, ZIPs and ZnTs, are widely expressed in eukaryotic cells. Using mice globally lacking ZIP1 and ZIP3 (ZIP1/3 DKO), our aim was to explore the potential role of these well-known Zn2+ transporters in maintaining platelet Zn2+ homeostasis and in the regulation of platelet function. While ICP-MS measurements indicated unaltered overall Zn2+ concentrations in platelets of ZIP1/3 DKO mice, we observed a significantly delayed and less efficient Zn2+ release upon thrombin-stimulated platelet activation. This resulted in a hyperactive platelet response not only in response to thrombin, but also towards other G protein-coupled receptor (GPCR) agonists. Immunoreceptor tyrosine-based activation (ITAM)-coupled receptor agonist signalling, however, was unaffected. Augmented GPCR responses were accompanied by enhanced Ca2+ signalling and PKC activation. Further functional analysis of ZIP1/3 double deficient mice revealed enhanced platelet aggregation, bigger thrombus volume under flow ex vivo and faster in vivo thrombus formation. The current study thereby identifies ZIP1 and ZIP3 as important regulators for the maintenance of platelet Zn2+ homeostasis and function.
Fusobacterium nucleatum induces pancreatic cancer cell proliferation and migration by regulating host autocrine and paracrine signaling
Authors: Udayasuryan Barath , Nguyen Tam T. D. , Umaña Ariana , Ahmad Raffae N. , Roberts LaDeidra Monét , Sobol Polina , Jones Stephen D. , Munson Jennifer M. , Slade Daniel J. , Verbridge Scott S. 
Published: November 2021

AbstractPancreatic ductal adenocarcinoma (PDAC) harbors a complex tumor microbiome that has been implicated in cancer progression and resistance to chemotherapy. Recent clinical investigations uncovered a correlation between high loads of intratumor Fusobacterium nucleatum and decreased patient survival. Here we show that healthy and cancerous pancreatic cell lines harboring intracellular F. nucleatum secrete increased levels of cancer-associated cytokines including GM-CSF, CXCL1, IL-8, and MIP-3α. We report that GM-CSF (granulocyte-macrophage colony stimulating factor) secretion directly increases the proliferation and migration of pancreatic cancer cells via an autocrine mechanism, notably in the absence of immune cell participation. Furthermore, we show that non-cancerous pancreatic epithelial cells do not exhibit increased proliferation or migration in response to these cytokines, but nevertheless, their secreted cytokines stimulate these responses in cancerous cell lines through paracrine signaling. Our results provide evidence that intratumor F. nucleatum in the pancreas elicits an infection-specific cytokine secretion profile from both normal and cancerous cells that adversely contributes to cancer progression through autocrine and paracrine mechanisms. Therefore, these results support the importance of investigating the contributions of both microbiome and host driven processes in pancreatic cancer to guide future therapeutic interventions.
Preclinical and Toxicology Studies of BRD5529, a Selective Inhibitor of CARD9
Authors: Kottom Theodore J. , Schaefbauer Kyle , Carmona Eva M. , Yi Eunhee S. , Limper Andrew H. 
Published: November 2021

ABSTRACTBackgroundExuberant inflammation during Pneumocystis pneumonia leads to lung injury. CARD9 is a central mediator of inflammatory signaling mediated by C-type lectin receptors. CARD9 inhibitor BRD5529 has been shown to be an effective in vitro inhibitor of Pneumocystis β-glucan-induced proinflammatory signaling and downstream TNF-alpha production, suggesting its viability as a candidate for preliminary drug testing as an anti- inflammatory agent in the rodent Pneumocystis pneumonia model (PCP).MethodsTo assess for potential toxicity, mice were injected intraperitoneally (IP) daily either with vehicle or BRD5529 at 0.1 mg/kg or 1.0 mg/kg for two weeks. Mouse weights were taken daily. At day 14, mice were euthanized, weighed, and analyzed by flexiVent™ for lung stiffness. Lungs, liver, and kidney were then harvested for H&E staining and pathology scoring. Lung samples were further analyzed for proinflammatory cytokines via ELISA and extracellular matrix generation via quantitative PCR (q-PCR). Blood collection postmortem was performed for blood chemistry analysis.ResultsBRD5529 at both doses of IP administration resulted in no significant changes in daily or final weight gain. Analysis of lung stiffness by flexiVent™ showed no significant differences between the control or treated groups. Furthermore, ELISA results for proinflammatory IL-1 Beta, IL-6, and TNF-alpha showed no major differences in the respective groups. qPCR analysis of extracellular matrix transcripts collagen type I, alpha 1 (Col1a1) and fibronectin (Fn) were statically similar as well in the treated and control groups. Examination and pathology scoring of H&E slides from lung, liver, and kidney from the each of the mice in all groups and subsequent pathology scoring showed no significant change. Blood chemistry analysis revealed similar, non-significant patterns.ConclusionsBRD5529 in our initial general safety and toxicology assessments displayed no inherent safety concerns in the analyzed parameters. These data support broader in vivo testing of the inhibitor as a timed adjunct therapy to the deleterious proinflammatory host immune response often associated with anti-Pneumocystis therapy.