Expression and function of ABC-transporter protein ABCB1 correlates with inhibitory capacity of Ruxolitinib in vitro and in vivo

ABSTRACTTheSinorhizobium melilotiBacA ABC transporter protein plays an important role in its nodulating symbiosis with the legume alfalfa (Medicago sativa). TheMycobacterium tuberculosisBacA homolog was found to be important for the maintenance of chronic murine infections, yet itsin vivofunction is unknown. In the legume plant as well as in the mammalian host, bacteria encounter host antimicrobial peptides (AMPs). We found that theM. tuberculosisBacA protein was able to partially complement the symbiotic defect of anS. melilotiBacA-deficient mutant on alfalfa plants and to protect this mutantin vitrofrom the antimicrobial activity of a synthetic legume peptide, NCR247, and a recombinant human β-defensin 2 (HBD2). This finding was also confirmed using anM. tuberculosisinsertion mutant. Furthermore,M. tuberculosisBacA-mediated protection of the legume symbiontS. melilotiagainst legume defensins as well as HBD2 is dependent on its attached ATPase domain. In addition, we show thatM. tuberculosisBacA mediates peptide uptake of the truncated bovine AMP, Bac71-16. This process required a functional ATPase domain. We therefore suggest thatM. tuberculosisBacA is important for the transport of peptides across the cytoplasmic membrane and is part of a complete ABC transporter. Hence, BacA-mediated protection against host AMPs might be important for the maintenance of latent infections.

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2072-P: Deletion of the Mitofusins 1 and 2 (Mfn1 and Mfn2) in the Pancreatic Beta Cell Disrupts Mitochondrial Structure and Function In Vitro and Strongly Impairs Glucose-Stimulated Insulin Secretion In Vivo

Diabetes ◽

10.2337/db20-2072-p ◽

2020 ◽

Vol 69 (Supplement 1) ◽

pp. 2072-P

Author(s):

ELENI GEORGIADOU ◽

PAULINE L. CHABOSSEAU ◽

ALEJANDRA TOMAS ◽

ISABELLE LECLERC ◽

GUY A. RUTTER

Keyword(s):

Insulin Secretion ◽

Beta Cell ◽

Pancreatic Beta Cell ◽

Structure And Function ◽

Insulin Secretion In Vivo ◽

Mitochondrial Structure ◽

Glucose Stimulated Insulin Secretion ◽

And Function

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Grafts Derived from an α-Synuclein Triplication Patient Mediate Functional Recovery but Develop Disease-Associated Pathology in the 6-OHDA Model of Parkinson’s Disease

Journal of Parkinson s Disease ◽

10.3233/jpd-202366 ◽

2020 ◽

pp. 1-14

Author(s):

Shelby Shrigley ◽

Fredrik Nilsson ◽

Bengt Mattsson ◽

Alessandro Fiorenzano ◽

Janitha Mudannayake ◽

...

Keyword(s):

Parkinson’S Disease ◽

Parkinson's Disease ◽

Cell Lines ◽

Functional Recovery ◽

Embryonic Stem ◽

Hesc Line ◽

Alternative Source ◽

And Function

Background: Human induced pluripotent stem cells (hiPSCs) have been proposed as an alternative source for cell replacement therapy for Parkinson’s disease (PD) and they provide the option of using the patient’s own cells. A few studies have investigated transplantation of patient-derived dopaminergic (DA) neurons in preclinical models; however, little is known about the long-term integrity and function of grafts derived from patients with PD. Objective: To assess the viability and function of DA neuron grafts derived from a patient hiPSC line with an α-synuclein gene triplication (AST18), using a clinical grade human embryonic stem cell (hESC) line (RC17) as a reference control. Methods: Cells were differentiated into ventral mesencephalic (VM)-patterned DA progenitors using an established GMP protocol. The progenitors were then either terminally differentiated to mature DA neurons in vitro or transplanted into 6-hydroxydopamine (6-OHDA) lesioned rats and their survival, maturation, function, and propensity to develop α-synuclein related pathology, were assessed in vivo. Results: Both cell lines generated functional neurons with DA properties in vitro. AST18-derived VM progenitor cells survived transplantation and matured into neuron-rich grafts similar to the RC17 cells. After 24 weeks, both cell lines produced DA-rich grafts that mediated full functional recovery; however, pathological changes were only observed in grafts derived from the α-synuclein triplication patient line. Conclusion: This data shows proof-of-principle for survival and functional recovery with familial PD patient-derived cells in the 6-OHDA model of PD. However, signs of slowly developing pathology warrants further investigation before use of autologous grafts in patients.

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Heterodimer Formation of the Homodimeric ABC Transporter OpuA

International Journal of Molecular Sciences ◽

10.3390/ijms22115912 ◽

2021 ◽

Vol 22 (11) ◽

pp. 5912

Author(s):

Patricia Alvarez-Sieiro ◽

Hendrik R. Sikkema ◽

Bert Poolman

Keyword(s):

Abc Transporter ◽

Conformational Dynamics ◽

Host Organism ◽

Affinity Tag ◽

Practical Applications ◽

Fundamental Research ◽

Protein Multimerization ◽

Biochemical Analyses

Many proteins have a multimeric structure and are composed of two or more identical subunits. While this can be advantageous for the host organism, it can be a challenge when targeting specific residues in biochemical analyses. In vitro splitting and re-dimerization to circumvent this problem is a tedious process that requires stable proteins. We present an in vivo approach to transform homodimeric proteins into apparent heterodimers, which then can be purified using two-step affinity-tag purification. This opens the door to both practical applications such as smFRET to probe the conformational dynamics of homooligomeric proteins and fundamental research into the mechanism of protein multimerization, which is largely unexplored for membrane proteins. We show that expression conditions are key for the formation of heterodimers and that the order of the differential purification and reconstitution of the protein into nanodiscs is important for a functional ABC-transporter complex.

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Extracellular vesicles as mediators and markers of acute organ injury: current concepts

European Journal of Trauma and Emergency Surgery ◽

10.1007/s00068-021-01607-1 ◽

2021 ◽

Author(s):

Birte Weber ◽

Niklas Franz ◽

Ingo Marzi ◽

Dirk Henrich ◽

Liudmila Leppik

Keyword(s):

Extracellular Vesicles ◽

Inflammatory Diseases ◽

Organ Injury ◽

Isolation And Characterization ◽

Communication Processes ◽

Incidence And Mortality ◽

New Biomarkers ◽

And Function

AbstractDue to the continued high incidence and mortality rate worldwide, there is a need to develop new strategies for the quick, precise, and valuable recognition of presenting injury pattern in traumatized and poly-traumatized patients. Extracellular vesicles (EVs) have been shown to facilitate intercellular communication processes between cells in close proximity as well as distant cells in healthy and disease organisms. miRNAs and proteins transferred by EVs play biological roles in maintaining normal organ structure and function under physiological conditions. In pathological conditions, EVs change the miRNAs and protein cargo composition, mediating or suppressing the injury consequences. Therefore, incorporating EVs with their unique protein and miRNAs signature into the list of promising new biomarkers is a logical next step. In this review, we discuss the general characteristics and technical aspects of EVs isolation and characterization. We discuss results of recent in vitro, in vivo, and patients study describing the role of EVs in different inflammatory diseases and traumatic organ injuries. miRNAs and protein signature of EVs found in patients with acute organ injury are also debated.

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Allelopathic Effects of Three Herb Species on Phytophthora cinnamomi, a Pathogen Causing Severe Oak Decline in Mediterranean Wood Pastures

Forests ◽

10.3390/f12030285 ◽

2021 ◽

Vol 12 (3) ◽

pp. 285

Author(s):

Manuela Rodríguez-Romero ◽

Belén Godoy-Cancho ◽

Isabel M. Calha ◽

José António Passarinho ◽

Ana Cristina Moreira

Keyword(s):

Phytophthora Cinnamomi ◽

Raphanus Raphanistrum ◽

Inhibitory Capacity ◽

Pathogen Suppression ◽

Pathogen Populations ◽

Defensive Chemical ◽

Chemical Response ◽

Herb Species

The ability of three herbaceous plants (Diplotaxis tenuifolia (L.) DC., Eruca vesicaria L. and Raphanus raphanistrum L.) from Iberian wood pastures to reduce Phytophthora cinnamomi Rands pathogen populations through allelopathic relationships is studied. The inhibitory capacity of their aqueous root extracts (AREs) on mycelial growth and production of P. cinnamomi reproductive structures is analysed in vitro. In addition, Quercus seedlings were grown in infested by P. cinnamomi-soils and with the presence or absence of allelopathic and susceptible herb species to the pathogen to assess the defensive chemical response of Quercus seedlings through their leaf phenolic compounds. Results show a strong inhibitory capacity of AREs on P. cinnamomi activity in vitro and a protective effect of these herb species on Quercus plants against P. cinnamomi in vivo. D. tenuifolia would be especially suited for biological control in the pathogen suppression.

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Modelling the Human Placental Interface In Vitro—A Review

Micromachines ◽

10.3390/mi12080884 ◽

2021 ◽

Vol 12 (8) ◽

pp. 884

Author(s):

Marta Cherubini ◽

Scott Erickson ◽

Kristina Haase

Keyword(s):

Drug Testing ◽

Cell Types ◽

In Vitro Models ◽

Placental Development ◽

Scientific Challenge ◽

Induced Pluripotent ◽

And Function ◽

And Control

Acting as the primary link between mother and fetus, the placenta is involved in regulating nutrient, oxygen, and waste exchange; thus, healthy placental development is crucial for a successful pregnancy. In line with the increasing demands of the fetus, the placenta evolves throughout pregnancy, making it a particularly difficult organ to study. Research into placental development and dysfunction poses a unique scientific challenge due to ethical constraints and the differences in morphology and function that exist between species. Recently, there have been increased efforts towards generating in vitro models of the human placenta. Advancements in the differentiation of human induced pluripotent stem cells (hiPSCs), microfluidics, and bioprinting have each contributed to the development of new models, which can be designed to closely match physiological in vivo conditions. By including relevant placental cell types and control over the microenvironment, these new in vitro models promise to reveal clues to the pathogenesis of placental dysfunction and facilitate drug testing across the maternal–fetal interface. In this minireview, we aim to highlight current in vitro placental models and their applications in the study of disease and discuss future avenues for these in vitro models.

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Therapeutic Application of Brain-Specific Angiogenesis Inhibitor 1 for Cancer Therapy

Cancers ◽

10.3390/cancers13143562 ◽

2021 ◽

Vol 13 (14) ◽

pp. 3562

Author(s):

Mitra Nair ◽

Chelsea Bolyard ◽

Tae Jin Lee ◽

Balveen Kaur ◽

Ji Young Yoo

Keyword(s):

Angiogenesis Inhibitor ◽

Suppressor Gene ◽

Chemotherapeutic Agents ◽

Tumor Models ◽

In Vivo Models ◽

Herpes Simplex Viruses ◽

Multiple Tumor ◽

And Function

Brain-specific angiogenesis inhibitor 1 (BAI1/ADGRB1) is an adhesion G protein-coupled receptor that has been found to play key roles in phagocytosis, inflammation, synaptogenesis, the inhibition of angiogenesis, and myoblast fusion. As the name suggests, it is primarily expressed in the brain, with a high expression in the normal adult and developing brain. Additionally, its expression is reduced in brain cancers, such as glioblastoma (GBM) and peripheral cancers, suggesting that BAI1 is a tumor suppressor gene. Several investigators have demonstrated that the restoration of BAI1 expression in cancer cells results in reduced tumor growth and angiogenesis. Its expression has also been shown to be inversely correlated with tumor progression, neovascularization, and peri-tumoral brain edema. One method of restoring BAI1 expression is by using oncolytic virus (OV) therapy, a strategy which has been tested in various tumor models. Oncolytic herpes simplex viruses engineered to express the secreted fragment of BAI1, called Vasculostatin (Vstat120), have shown potent anti-tumor and anti-angiogenic effects in multiple tumor models. Combining Vstat120-expressing oHSVs with other chemotherapeutic agents has also shown to increase the overall anti-tumor efficacy in both in vitro and in vivo models. In the current review, we describe the structure and function of BAI1 and summarize its application in the context of cancer treatment.

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Alcohol-and-HIV-Induced Lysosomal Dysfunction Regulates Extracellular Vesicles Secretion in Vitro and in Liver-Humanized Mice

Biology ◽

10.3390/biology10010029 ◽

2021 ◽

Vol 10 (1) ◽

pp. 29

Author(s):

Raghubendra Singh Dagur ◽

Moses New-Aaron ◽

Murali Ganesan ◽

Weimin Wang ◽

Svetlana Romanova ◽

...

Keyword(s):

Oxidative Stress ◽

Extracellular Vesicles ◽

Treated Group ◽

Human Hepatocytes ◽

In Vivo Studies ◽

People Living With Hiv ◽

Humanized Mouse Model ◽

And Function

Background: Alcohol abuse is common in people living with HIV-1 and dramaticallyenhances the severity of HIV-induced liver damage by inducing oxidative stress and lysosomaldysfunction in the liver cells. We hypothesize that the increased release of extracellular vesicles(EVs) in hepatocytes and liver humanized mouse model is linked to lysosome dysfunction. Methods:The study was performed on primary human hepatocytes and human hepatoma RLWXP-GFP (Huh7.5 cells stably transfected with CYP2E1 and XPack-GFP) cells and validated on ethanol-fed liverhumanizedfumarylacetoacetate hydrolase (Fah)-/-, Rag2-/-, common cytokine receptor gamma chainknockout (FRG-KO) mice. Cells and mice were infected with HIV-1ADA virus. Results: We observedan increase in the secretion of EVs associated with a decrease in lysosomal activity and expressionof lysosomal-associated membrane protein 1. Next-generation RNA sequencing of primary humanhepatocytes revealed 63 differentially expressed genes, with 13 downregulated and 50 upregulatedgenes in the alcohol–HIV-treated group. Upstream regulator analysis of differentially expressedgenes through Ingenuity Pathway Analysis identified transcriptional regulators affecting downstreamgenes associated with increased oxidative stress, lysosomal associated disease, and function andEVs biogenesis. Our in vitro findings were corroborated by in vivo studies on human hepatocytetransplantedhumanized mice, indicating that intensive EVs’ generation by human hepatocytes andtheir secretion to serum was associated with increased oxidative stress and reduction in lysosomalactivities triggered by HIV infection and ethanol diet. Conclusion: HIV-and-ethanol-metabolisminducedEVs release is tightly controlled by lysosome status in hepatocytes and participates in thedevelopment of double-insult-induced liver injury.

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Phosphorylation-dependent interaction of the synaptic vesicle proteins cysteine string protein and synaptotagmin I

Biochemical Journal ◽

10.1042/bj20020123 ◽

2002 ◽

Vol 364 (2) ◽

pp. 343-347 ◽

Cited By ~ 51

Author(s):

Gareth J.O. EVANS ◽

Alan MORGAN

Keyword(s):

Rat Brain ◽

Direct Interaction ◽

Secretory Vesicle ◽

Brain Membrane ◽

Synaptotagmin I ◽

Phosphorylated Form ◽

Order Of Magnitude ◽

And Function

The secretory vesicle cysteine string proteins (CSPs) are members of the DnaJ family of chaperones, and function at late stages of Ca2+-regulated exocytosis by an unknown mechanism. To determine novel binding partners of CSPs, we employed a pull-down strategy from purified rat brain membrane or cytosolic proteins using recombinant hexahistidine-tagged (His6-)CSP. Western blotting of the CSP-binding proteins identified synaptotagmin I to be a putative binding partner. Furthermore, pull-down assays using cAMP-dependent protein kinase (PKA)-phosphorylated CSP recovered significantly less synaptotagmin. Complexes containing CSP and synaptotagmin were immunoprecipitated from rat brain membranes, further suggesting that these proteins interact in vivo. Binding assays in vitro using recombinant proteins confirmed a direct interaction between the two proteins and demonstrated that the PKA-phosphorylated form of CSP binds synaptotagmin with approximately an order of magnitude lower affinity than the non-phosphorylated form. Genetic studies have implicated each of these proteins in the Ca2+-dependency of exocytosis and, since CSP does not bind Ca2+, this novel interaction might explain the Ca2+-dependent actions of CSP.

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