Biology Mechanisms Research Articles

Displacement Mechanism and Flow Characteristics of Polymer Particle Dispersion System Based on Capillary Bundle Model

During the development of oil reservoirs, a rapid increase in water cut following reservoir flooding leads to inefficient or ineffective circulation of injected water, rendering a significant portion of the remaining oil in the reservoir inaccessible. The displacement method using polymer particle dispersion systems effectively solves the issue of rapid water breakthrough in oil reservoirs. Owing to the particle phase separation phenomenon, polymer particles can selectively penetrate into the larger pores where water circulation is inefficient, enhance their flow resistance, and thereby achieve equilibrium displacement along with an increased swept volume. This paper investigates the heterogeneous distribution of polymer particles within a porous medium, incorporates the red blood cell dendrite concentration distribution theory from biological fluid mechanics, and develops a mathematical model to delineate the viscosity characteristics of polymer particle dispersion systems, taking into account the phase separation phenomenon. Building on this foundation, it formulates a capillary bundle model for the polymer particle dispersion system specifically designed for oil displacement and proceeds to determine its relative permeability curve. Simulation outcomes reveal that at a water saturation level of 0.063, the concentration of polymer particles in fractured large pore capillaries is markedly elevated, yet capillaries with a pore size under 26 μm remain devoid of polymer particles. With the increase of water saturation, the concentration of polymer particles in large pore capillaries reduces, whereas it progressively augments in medium pore capillaries. Upon reaching a peak water saturation of 0.751, capillaries smaller than 18 μm are entirely free of polymer particles. These findings suggest that the heterogeneous distribution of polymer particles markedly inhibits the percolation capabilities of the dispersed system following a water phase breakthrough, facilitating the entry of more dispersion into oil-laden capillaries and thus enhancing the flow capacity of the oil phase.

Polyphenols and Lactation: Molecular Evidence to Support the Use of Botanical Galactagogues.

Botanicals and herbal supplements contain a diverse array of polyphenols that may affect mammary gland function and promote galactagogue activity. This scoping review is conducted to identify scientific literature elucidating how polyphenols affect mammary gland biology and cellular mechanisms critical for lactation. A literature search of PubMed and Medline reviews relevant studies in dairy animals, rodent models, and cultured mammary epithelial cells that are published from January 2010 until July 2023, to ascertain effects of polyphenols on mechanisms regulating milk production and composition. The PRISMA-ScR (Preferred Reporting Items for Systematic Reviews and Meta-Analyses for Scoping Review) strategy is applied and 80 studies on polyphenols and their implications on milk production and composition are included in this review. Limited information delineating effects of polyphenols on the molecular pathways that affect lactation are found, although available information suggests modulation of Stat5 signaling/differentiation, Stat3 signaling/remodeling, mTOR and insulin signaling/energy production, and nuclear factor kappa beta (NFκβ) signaling/oxidative stress and inflammation may play roles. A profound lack of mechanistic information underscores the critical need for further research to understand the impact of botanical supplements and polyphenols on milk production and composition in humans to establish maternal nutritional guidelines to support lactation and breastfeeding goals.

Study of traveling soliton and fronts phenomena in fractional Kolmogorov-Petrovskii-Piskunov equation

The present research work presents the modified Extended Direct Algebraic Method (m-EDAM) to construct and analyze propagating soliton solutions for fractional Kolmogorov-Petrovskii-Piskunov equation (FKPPE) which incorporates Caputo’s fractional derivatives. The FKPPE has significance in various disciplines such as population growth, reaction-diffusion mechanisms, and mathematical biology. By leveraging the series form solution, the proposed m-EDAM determines plethora of travelling soliton solutions through the transformation of FKPPE into Nonlinear Ordinary Differential equation (NODE). These soliton solutions shed light on propagation processes in the framework of the FKPPE model. Our study also offers some graphical representations that facilitate the characterization and investigation of propagation processes of the obtained soliton solutions which include kink, shock soliton solutions. Our work advances our understanding of complicated phenomena across multiple academic disciplines by fusing insights from mathematical biology and reaction-diffusion mechanisms.

Metabolic difference between patient-derived xenograft model of pancreatic ductal adenocarcinoma and corresponding primary tumor

BackgroundPatients-derived xenograft (PDX) model have been widely used for tumor biological and pathological studies. However, the metabolic similarity of PDX tumor to the primary cancer (PC) is still unknown.MethodsIn present study, we established PDX model by engrafting primary tumor of pancreatic ductal adenocarcinoma (PDAC), and then compared the tumor metabolomics of PC, the first generation of PDX tumor (PDXG1), and the third generation of PDX tumor (PDXG3) by using 1H NMR spectroscopy. Then, we assessed the differences in response to chemotherapy between PDXG1 and PDXG3 and corresponding metabolomic differences in drug-resistant tumor tissues. To evaluate the metabolomic similarity of PDX to PC, we also compared the metabolomic difference of cell-derived xenograft (CDX) vs. PC and PDX vs. PC.ResultsAfter engraftment, PDXG1 tumor had a low level of lactate, pyruvate, citrate and multiple amino acids (AAs) compared with PC. Metabolite sets enrichment and metabolic pathway analyses implied that glycolysis metabolisms were suppressed in PDXG1 tumor, and tricarboxylic acid cycle (TCA)-associated anaplerosis pathways, such as amino acids metabolisms, were enhanced. Then, after multiple passages of PDX, the altered glycolysis and TCA-associated anaplerosis pathways were partially recovered. Although no significant difference was observed in the response of PDXG1 and PDXG3 to chemotherapy, the difference in glycolysis and amino acids metabolism between PDXG1 and PDXG3 could still be maintained. In addition, the metabolomic difference between PC and CDX models were much larger than that of PDX model and PC, indicating that PDX model still retain more metabolic characteristics of primary tumor which is more suitable for tumor-associated metabolism research.ConclusionsCompared with primary tumor, PDX models have obvious difference in metabolomic level. These findings can help us design in vivo tumor metabolomics research legitimately and analyze the underlying mechanism of tumor metabolic biology thoughtfully.

Molecular Ratchets and Kinetic Asymmetry: Giving Chemistry Direction

In recent years ratchet mechanisms have transformed the understanding and design of stochastic molecular systems—biological, chemical and physical—in a move away from the mechanical macroscopic analogies that dominated thinking regarding molecular dynamics in the 1990s and early 2000s (e.g. pistons, springs, etc), to the more scale‐relevant concepts that underpin out‐of‐equilibrium research in the molecular sciences today. Ratcheting has established molecular nanotechnology as a research frontier for energy transduction and metabolism, and has enabled the reverse engineering of biomolecular machinery, delivering insights into how molecules ‘walk’ and track‐based synthesizers operate, how the acceleration of chemical reactions enables energy to be transduced by catalysts, and how dynamic (supra)molecular systems can be driven away from equilibrium. The recognition of molecular ratchet mechanisms in biology, and their invention in synthetic systems, is proving significant in areas as diverse as supramolecular chemistry, systems chemistry, dynamic covalent chemistry, DNA nanotechnology, polymer and materials science, molecular biology, heterogeneous catalysis, endergonic synthesis, and many other branches of chemical science. Put simply, ratchet mechanisms give chemistry direction. Kinetic asymmetry, quantified by the outcome of ratcheting, is the dynamic counterpart of structural asymmetry (i.e. chirality). Given the ubiquity of ratchet mechanisms, it is surely just as fundamentally important.

Molecular Ratchets and Kinetic Asymmetry: Giving Chemistry Direction.

In recent years ratchet mechanisms have transformed the understanding and design of stochastic molecular systems-biological, chemical and physical-in a move away from the mechanical macroscopic analogies that dominated thinking regarding molecular dynamics in the 1990s and early 2000s (e.g. pistons, springs, etc), to the more scale-relevant concepts that underpin out-of-equilibrium research in the molecular sciences today. Ratcheting has established molecular nanotechnology as a research frontier for energy transduction and metabolism, and has enabled the reverse engineering of biomolecular machinery, delivering insights into how molecules 'walk' and track-based synthesizers operate, how the acceleration of chemical reactions enables energy to be transduced by catalysts, and how dynamic (supra)molecular systems can be driven away from equilibrium. The recognition of molecular ratchet mechanisms in biology, and their invention in synthetic systems, is proving significant in areas as diverse as supramolecular chemistry, systems chemistry, dynamic covalent chemistry, DNA nanotechnology, polymerand materials science, molecular biology, heterogeneous catalysis, endergonic synthesis, and many other branches of chemical science. Put simply, ratchet mechanisms give chemistry direction. Kinetic asymmetry, quantified by the outcome of ratcheting, is the dynamic counterpart of structural asymmetry (i.e. chirality). Given the ubiquity of ratchet mechanisms, it is surely just as fundamentally important.

A bibliometric analysis of miRNAs in rheumatoid arthritis from 2001 to 2022: Research hotspots and trends.

MicroRNAs (miRNAs) are widely recognized in the pathogenesis of autoimmune disease. As a key regulatory factor, miRNAs have introduced new biomarkers for the early diagnosis of rheumatoid arthritis (RA) and provided a favorable research direction for the development of novel therapeutic targets. This study aimed to explore the hotspots of miRNA research related to RA published from different countries, organizations, and authors. From 2001 to 2022, publications on miRNA related to RA were identified in the Web of Science database. The total and annual number of publishments, citations, impact factor, H-index, productive authors, and involved journals were collected for quantitative and qualitative comparisons. A total of 29 countries/regions in the world have participated in the research of miRNAs and RA over the past two decades, and China (760, 53.18%) and the United States (233, 16.31%) account for the majority of the total publications. China dominated in total citation (17881) and H-index (62). A total of 507 academic journals have published articles in related fields, and Frontiers in Immunology published the most (53, 3.71%). Chih-hsin Tang of the China Medical University has published the most papers (16, 1.2%). Stanczyk (2008) published the most cited article Altered expression of miRNAs in synovial fibroblasts and synovial tissue in rheumatoid arthritis in Arthritis and Rheumatism, with 660 citations. Inflammation is the high-frequency keyword outside of RA and miRNAs, and related researches have mainly focused on miR-146a and miR-155. In the past two decades, extensive and continuous research has been conducted to investigate the role of miRNAs in RA, and miRNAs are widely recognized in the pathogenesis of RA. Related research has mainly focused on miR-146a and miR-155 that have shown promising results as key factors in RA experimental models. Focusing on clinical applications and translational research may be the future research direction and hotspot based on molecular biology basic research and mechanism exploration.

The Molecular Biological Mechanism of Hydrogen Therapy and Its Application in Spinal Cord Injury.

Hydrogen, which is a novel biomedical molecule, is currently the subject of extensive research involving animal experiments and in vitro cell experiments, and it is gradually being applied in clinical settings. Hydrogen has been proven to possess anti-inflammatory, selective antioxidant, and antiapoptotic effects, thus exhibiting considerable protective effects in various diseases. In recent years, several studies have provided preliminary evidence for the protective effects of hydrogen on spinal cord injury (SCI). This paper provides a comprehensive review of the potential molecular biology mechanisms of hydrogen therapy and its application in treating SCI, with an aim to better explore the medical value of hydrogen and provide new avenues for the adjuvant treatment of SCI.

A novel inducible von Willebrand Factor Cre recombinase mouse strain to study microvascular endothelial cell-specific biological processes in vivo

Mouse models are invaluable to understanding fundamental mechanisms in vascular biology during development, in health and different disease states. Several constitutive or inducible models that selectively knockout or knock in genes in vascular endothelial cells exist; however, functional and phenotypic differences exist between microvascular and macrovascular endothelial cells in different organs. In order to study microvascular endothelial cell-specific biological processes, we developed a Tamoxifen-inducible von Willebrand Factor (vWF) Cre recombinase mouse in the SJL background. The transgene consists of the human vWF promoter with the microvascular endothelial cell-selective 734 base pair sequence to drive Cre recombinase fused to a mutant estrogen ligand-binding domain [ERT2] that requires Tamoxifen for activity (CreERT2) followed by a polyadenylation (polyA) signal. We initially observed Tamoxifen-inducible restricted bone marrow megakaryocyte and sciatic nerve microvascular endothelial cell Cre recombinase expression in offspring of a mixed strain hemizygous C57BL/6-SJL founder mouse bred with mT/mG mice, with >90% bone marrow megakaryocyte expression efficiency. Founder mouse offspring were backcrossed to the SJL background by speed congenics, and intercrossed for >10 generations to develop hemizygous Tamoxifen-inducible vWF Cre recombinase (vWF-iCre/+) SJL mice with stable transgene insertion in chromosome 1. Microvascular endothelial cell-specific Cre recombinase expression occurred in the sciatic nerves, brains, spleens, kidneys and gastrocnemius muscles of adult vWF-iCre/+ SJL mice bred with Ai14 mice, with retained low level bone marrow and splenic megakaryocyte expression. This novel mouse strain would support hypothesis-driven mechanistic studies to decipher the role(s) of specific genes transcribed by microvascular endothelial cells during development, as well as in physiologic and pathophysiologic states in an organ- and time-dependent manner.

Abstract 1434: Modeling the 3q26 OncCassette using genetically engineered mice

Abstract Lung squamous cell carcinoma (LUSC) is a devastating disease, accounting for ~30% of all lung cancer diagnoses and ~40,000 deaths annually in the United States alone. LUSC patients suffer from high prevalence of relapse and a dismal 5-year survival of only ~24%. The lack of animal models that reflect the salient features of human disease to assess the safety and efficacy of drugs, and to explore the underlying molecular mechanisms of LUSC tumor biology, is a major impediment for LUSC patients. We have identified concomitant 3q26 copy number gain (CNG) and TP53 loss as a defining genetic alteration present in ~90% of LUSC tumors. Furthermore, we have identified and characterized three 3q26 oncogenes (PRKCI, ECT2 and SOX2) that genetically and biochemically collaborate to drive LUSC tumor formation. Using syngeneic mouse modeling, we have demonstrated that coordinate overexpression of Prkci, Ect2 and Sox2, and loss of Trp53 in ex vivo cultures of lung basal stem cells (LBSCs; a cell of origin for LUSC) drives transformation of these cells into tumors of exclusively LUSC histopathology. These findings lead to the hypothesis that PRKCI, ECT2 and SOX2 represent a multigenic driver of 3q26 CNG-driven LUSC, which we term the 3q26 OncCassette. Based in these findings, we have developed and begun characterizing the first autochthonous genetically engineered mouse model (GEMM) to study 3q26 OncCassette-driven tumorigenesis. Our novel immune competent GEMM will provide critical insight into tumor initiation and progression of preneoplastic lesions to malignant LUSC and allow characterization of the immune landscape in these tumors. Citation Format: Kayleah M. Meneses, Yi Liu, Capella R. Weems, Lee Jamieson, Duy T. Nguyen, Verline Justilien, Nicole R. Murray, Alan P. Fields. Modeling the 3q26 OncCassette using genetically engineered mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1434.

Application of Omics and Bioinformatics Technologies in Response to COVID-19 Pandemic

The medical biotechnology community has undertaken significant endeavors to gain a comprehensive understanding of SARS-CoV-2’s biology and pathogenesis mechanisms. Omics approaches and technologies have been widely employed in the fight against SARS-CoV-2. Since the onset of the virus outbreak, researchers have demonstrated how recent omics and bioinformatics technological advancements have contributed to the diagnosis, vaccine development, treatment, and control of disease transmission. Studies conducted since the outbreak have been collected and summarized, with a focus on bioinformatics approaches and their contribution to controlling this pandemic. Developments and advanced omics technology in connection to the COVID-19 pandemic have been analyzed. The multi-omics technology, which offers various strategies in identifying potential diagnostics, therapeutics, studies of variants of concern, and drug repurposing approaches, has been assessed. Pandemic response has seen the application of multi-omics and pan-genomics approaches, including genomics, metabolomics, transcriptomics, proteomics, epigenomics, clustered regularly interspaced short palindromic repeats (CRISPR) technology, host-pathogen interactions, artificial intelligence, and machine learning in various research areas. Additionally, bioinformatics and mathematical modeling have played a significant role in disease control. The use of smart technologies to control virus transmission and predict patients’ health conditions and treatment outcomes has also been crucial. Transcriptome analysis has emerged as a major application, contributing to the generation of new knowledge on viral sequences and intracellular signaling pathways that regulate viral infection and pathogenesis mechanisms. The sequencing of the virus has paved the way for the use of omics technologies and an integrative technique in combating the pandemic. In general, the advancement of omics technology during this pandemic has been fascinating and has contributed a significant role to the science of health biotechnology in general and omics and bioinformatics in particular.Keywords: bioinformatics, coronavirus, COVID-19, omics, SARS COV-2

Abstract 3937: Investigating the role of fibulin 3 in pancreatic tumorigenesis

Abstract Pancreatic ductal adenocarcinoma (PDA) is a highly lethal malignancy with a five-year survival rate of less than 11%, making it one of the deadliest cancers. Notably, 95% of PDA cases exhibit KRAS mutations, contributing to highly dysregulated cell signaling networks. Despite extensive research into the interplay of various oncogenic pathways in pancreatic tumorigenesis, targeted therapies against these signaling networks have displayed modest efficacy, with most patients rapidly developing therapeutic resistance. Recent studies highlight the significance of the carbohydrate antigen 19-9 (CA19-9) as a functional biomarker linked to disease progression and resistance. However, understanding the detailed functions of CA19-9-modified proteins in PDA has been limited due to the lack of adequate models for synthesizing CA19-9. To address this challenge, we have developed unique mouse and 3D organoid culture models capable of producing CA19-9. CA19-9 elevation in mice with KRAS-mutations results in an aggressive PDA phenotype through increased tumor proliferation and microenvironment (TME) remodeling. Furthermore, our research has identified Fibulin 3 (FBLN3), an extracellular matrix glycoprotein, as a secreted, CA19-9-modified protein that stimulates tumorigenesis. In this study, we found that FBLN3 not only facilitates tumor proliferation but also drives epithelial-mesenchymal transition (EMT) and promotes STAT3 pathways activation in the CA19-9pos, KRAS-mutant PDA organoids. Additionally, in both CA19-9pos, KRAS-mutant PDA mouse models and human PDA, FBLN3 is expressed by cancer-associated fibroblasts (CAFs), suggesting its role in TME remodeling. These findings have significant implications for providing novel insights into PDA biology and paracrine signaling mechanisms, identifying FBLN3 as a potential therapeutic target for the improved treatment of this devastating disease. Citation Format: Hyemin Song, Jasper Hsu, Xiaoxue Lin, Satoshi Ogawa, Vasiliki Pantazopoulou, Kristina Peck, Chelsea Bottomley, Shira Okhovat, McKenna Stamp, Kassidy Curtis, Jeffrey D. Esko, Dannielle D. Engle. Investigating the role of fibulin 3 in pancreatic tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3937.

Siraitia grosvenorii As a Homologue of Food and Medicine: A Review of Biological Activity, Mechanisms of Action, Synthetic Biology, and Applications in Future Food.

Siraitia grosvenorii (SG), also known as Luo Han Guo or Monk fruit, boasts a significant history in food and medicine. This review delves into SG's historical role and varied applications in traditional Chinese culture, examining its phytochemical composition and the health benefits of its bioactive compounds. It further explores SG's biological activities, including antioxidant, anti-inflammatory, and antidiabetic properties and elucidates the mechanisms behind these effects. The review also highlights recent synthetic biology advances in enhancing the production of SG's bioactive compounds, presenting new opportunities for broadening their availability. Ultimately, this review emphasizes SG's value in food and medicine, showcasing its historical and cultural importance, phytochemistry, biological functions, action mechanisms, and the role of synthetic biology in its sustainable use.

Circadian Mechanisms in Brain Fluid Biology.

The brain is a complex organ, fundamentally changing across the day to perform basic functions like sleep, thought, and regulating whole-body physiology. This requires a complex symphony of nutrients, hormones, ions, neurotransmitters and more to be properly distributed across the brain to maintain homeostasis throughout 24 hours. These solutes are distributed both by the blood and by cerebrospinal fluid. Cerebrospinal fluid contents are distinct from the general circulation because of regulation at brain barriers including the choroid plexus, glymphatic system, and blood-brain barrier. In this review, we discuss the overlapping circadian (≈24-hour) rhythms in brain fluid biology and at the brain barriers. Our goal is for the reader to gain both a fundamental understanding of brain barriers alongside an understanding of the interactions between these fluids and the circadian timing system. Ultimately, this review will provide new insight into how alterations in these finely tuned clocks may lead to pathology.

Purification and transcriptomic characterization of proliferative cells of Mesocestoides corti selectively affected by irradiation

Flatworms depend on stem cells for continued tissue growth and renewal during their life cycles, making these cells valuable drug targets. While neoblasts are extensively characterized in the free-living planarian Schmidtea mediterranea, and similar stem cells have been characterized in the trematode Schistosoma mansoni, their identification and characterization in cestodes is just emerging. Since stem cells are generally affected by irradiation, in this work we used this experimental approach to study the stem cells of the model cestode Mesocestoides corti. We found that gamma irradiation produces a dose-dependent decrease in proliferative cells, requiring higher doses than in other flatworms to completely abolish proliferation. The treatment results in the downregulation of candidate marker genes. Transcriptomic studies reveal that several genes downregulated after irradiation are conserved with other flatworms, and are related to cell cycle, DNA replication and repair functions. Furthermore, proliferative cells were isolated by cell sorting and also characterized transcriptomically. We found that the set of genes characteristic of proliferative cells agrees well with those downregulated during irradiation, and have a significant overlap with those expressed in planarian neoblasts or S. mansoni stem cells. Our study highlights that conserved mechanisms of stem cell biology may be functional in flatworms, suggesting that these could be relevant targets to evaluate in the control of parasitic species.

GTP binding protein 2 maintains the quiescence, self-renewal, and chemoresistance of mouse colorectal cancer stem cells via promoting Wnt signaling activation

Colorectal cancer (CRC) is one of the most common cancers and the second most deadly cancer across the globe. Colorectal cancer stem cells (CCSCs) fuel CRC growth, metastasis, relapse, and chemoresistance. A complete understanding of the modulatory mechanisms of CCSC biology is essential for developing efficacious CRC treatment. In the current study, we characterized the expression and function of GTP binding protein 2 (GTPBP2) in a chemical-induced mouse CRC model. We found that GTPBP2 was expressed at a higher level in CD133+CD44+ CCSCs compared with other CRC cells. Using a lentivirus-based Cas9/sgRNA system, GTPBP2 expression was ablated in CRC cells in vitro. GTPBP2 deficiency caused the following effects on CCSCs: 1) Significantly accelerating proliferation and increasing the proportions of cells at G1, S, and G2/M phase; 2) Impairing resistance to 5-Fluorouracil; 3) Weakening self-renewal but not impacting cell migration. In addition, GTPBP2 deficiency remarkably decreased β-catenin expression while increasing β-catenin phosphorylation in CCSCs. These effects of GTPBP2 were present in CCSCs but not in other CRC cell populations. The Wnt agonist SKL2001 completely abolished these changes in GTPBP2-deficient CCSCs. When GTPBP2-deficient CCSCs were implanted in nude mice, they exhibited consistent changes compared with GTPBP2-expressing CCSCs. Collectively, this study indicates that GTPBP2 positively modulates Wnt signaling to reinforce the quiescence, self-renewal, and chemoresistance of mouse CCSCs. Therefore, we disclose a novel mechanism underlying CCSC biology and GTPBP2 could be a therapeutic target in future CRC treatment.

Synthetic biology approaches for enhancing safety and specificity of CAR-T cell therapies for solid cancers

CAR-T cell therapies have been successful in treating numerous hematologic malignancies as the T cell can be engineered to target a specific antigen associated with the disease. However, translating CAR-T cell therapies for solid cancers is proving more challenging due to the lack of truly tumor-associated antigens and the high risk of off-target toxicities. To combat this, numerous synthetic biology mechanisms are being incorporated to create safer and more specific CAR-T cells that can be spatiotemporally controlled with increased precision. Here, we seek to summarize and analyze the advancements for CAR-T cell therapies with respect to clinical implementation, from the perspective of synthetic biology and immunology. This review should serve as a resource for further investigation and growth within the field of personalized cellular therapies.

Deciphering the Role of Post-Translational Modifications and Cellular Location of Hepatitis Delta Virus (HDV) Antigens in HDV-Mediated Liver Damage in Mice.

Hepatitis D virus (HDV) infection represents the most severe form of chronic viral hepatitis. We have shown that the delivery of HDV replication-competent genomes to the hepatocytes using adeno-associated virus (AAV-HDV) as gene delivery vehicles offers a unique platform to investigate the molecular aspects of HDV and associated liver damage. For the purpose of this study, we generated HDV genomes modified by site-directed mutagenesis aimed to (i) prevent some post-translational modifications of HDV antigens (HDAgs) such as large-HDAg (L-HDAg) isoprenylation or short-HDAg (S-HDAg) phosphorylation; (ii) alter the localization of HDAgs within the subcellular compartments; and (iii) inhibit the right conformation of the delta ribozyme. First, the different HDV mutants were tested in vitro using plasmid-transfected Huh-7 cells and then in vivo in C57BL/6 mice using AAV vectors. We found that Ser177 phosphorylation and ribozymal activity are essential for HDV replication and HDAg expression. Mutations of the isoprenylation domain prevented the formation of infectious particles and increased cellular toxicity and liver damage. Furthermore, altering HDAg intracellular localization notably decreased viral replication, though liver damage remained unchanged versus normal HDAg distribution. In addition, a mutation in the nuclear export signal impaired the formation of infectious viral particles. These findings contribute valuable insights into the intricate mechanisms of HDV biology and have implications for therapeutic considerations.

Influenza D virus utilizes both 9-O-acetylated N-acetylneuraminic and 9-O-acetylated N-glycolylneuraminic acids as functional entry receptors.

Influenza D virus (IDV) utilizes bovines as a primary reservoir with periodical spillover to other hosts. We have previously demonstrated that IDV binds both 9-O-acetylated N-acetylneuraminic acid (Neu5,9Ac2) and 9-O-acetylated N-glycolylneuraminic acid (Neu5Gc9Ac). Bovines produce both Neu5,9Ac2 and Neu5Gc9Ac, while humans are genetically unable to synthesize Neu5Gc9Ac. 9-O-Acetylation of sialic acids is catalyzed by CASD1 via a covalent acetyl-enzyme intermediate. To characterize the role of Neu5,9Ac2 and Neu5Gc9Ac in IDV infection and determine which form of 9-O-acetylated sialic acids drives IDV entry, we took advantage of a CASD1 knockout (KO) MDCK cell line and carried out feeding experiments using synthetic 9-O-acetyl sialic acids in combination with the single-round and multi-round IDV infection assays. The data from our studies show that (i) CASD1 KO cells are resistant to IDV infection and lack of IDV binding to the cell surface is responsible for the failure of IDV replication; (ii) feeding CASD1 KO cells with Neu5,9Ac2 or Neu5Gc9Ac resulted in a dose-dependent rescue of IDV infectivity; and (iii) diverse IDVs replicated robustly in CASD1 KO cells fed with either Neu5,9Ac2 or Neu5Gc9Ac at a level similar to that in wild-type cells with a functional CASD1. These data demonstrate that IDV can utilize Neu5,9Ac2- or non-human Neu5Gc9Ac-containing glycan receptor for infection. Our findings provide evidence that IDV has acquired the ability to infect and transmit among agricultural animals that are enriched in Neu5Gc9Ac, in addition to posing a zoonotic risk to humans expressing only Neu5,9Ac2.IMPORTANCEInfluenza D virus (IDV) has emerged as a multiple-species-infecting pathogen with bovines as a primary reservoir. Little is known about the functional receptor that drives IDV entry and promotes its cross-species spillover potential among different hosts. Here, we demonstrated that IDV binds exclusively to 9-O-acetylated N-acetylneuraminic acid (Neu5,9Ac2) and non-human 9-O-acetylated N-glycolylneuraminic acid (Neu5Gc9Ac) and utilizes both for entry and infection. This ability in effective engagement of both 9-O-acetylated sialic acids as functional receptors for infection provides an evolutionary advantage to IDV for expanding its host range. This finding also indicates that IDV has the potential to emerge in humans because Neu5,9Ac2 is ubiquitously expressed in human tissues, including lung. Thus, results of our study highlight a need for continued surveillance of IDV in humans, as well as for further investigation of its biology and cross-species transmission mechanism.

A dual-plasmid CRISPR/Cas9-based method for rapid and efficient genetic disruption in Mycobacterium abscessus.

Mycobacterium abscessus is an opportunistic pathogen of increasing clinical importance due to its poor clinical outcomes and limited treatment options. Drug discovery and development in this highly antibiotic-resistant species will require further understanding of M. abscessus biology, pathogenesis, and antibiotic resistance mechanisms. However, existing methods for facile genetic engineering are relatively inefficient. This study reports on the first application of CRISPR/Cas9 for gene editing in M. abscessus using a dual-plasmid workflow. We establish that our method is easily programmable, efficient, and versatile for genetic disruptions in M. abscessus. This is a critical advancement to facilitating targeted gene function studies in this emerging pathogen.