Comprehensive Assessment of the Virulence Factors sub 3, sub 6 and mcpA in the Zoonotic Dermatophyte Trichophyton benhamiae Using FISH and qPCR

Skin infections by keratinophilic fungi are commonly referred to as dermatophytosis and represent a major health burden worldwide. Although patient numbers are on the rise, data on virulence factors, their function and kinetics are scarce. We employed an ex vivo infection model based on guinea pig skin explants (GPSE) for the zoonotic dermatophyte Trichophyton (T.) benhamiae to investigate kinetics of the virulence factors subtilisin (sub) 3, sub 6, metallocarboxypeptidase A (mcpA) and isocitrate lyase (isol) at gene level for ten days. Fluorescence in situ hybridization (FISH) and quantitative polymerase chain reaction (qPCR) were used to detect and quantify the transcripts, respectively. Kingdom-spanning, species-specific and virulence factor-specific probes were successfully applied to isolated fungal elements showing inhomogeneous fluorescence signals along hyphae. Staining results for inoculated GPSE remained inconsistent despite thorough optimization. qPCR revealed a significant increase of sub 3- and mcpA-transcripts toward the end of culture, sub 6 and isol remained at a low level throughout the entire culture period. Sub 3 is tightly connected to the de novo formation of conidia during culture. Since sub 6 is considered an in vivo disease marker. However, the presented findings urgently call for further research on the role of certain virulence factors during infection and disease.

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Baicalin Represses Type Three Secretion System of Pseudomonas aeruginosa through PQS System

Molecules ◽

10.3390/molecules26061497 ◽

2021 ◽

Vol 26 (6) ◽

pp. 1497

Author(s):

Pansong Zhang ◽

Qiao Guo ◽

Zhihua Wei ◽

Qin Yang ◽

Zisheng Guo ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Virulence Factors ◽

Mammalian Cells ◽

Secretion System ◽

Chinese Herbs ◽

Infection Model ◽

Lung Pathology ◽

Promising Alternative ◽

The Impact

Therapeutics that target the virulence of pathogens rather than their viability offer a promising alternative for treating infectious diseases and circumventing antibiotic resistance. In this study, we searched for anti-virulence compounds against Pseudomonas aeruginosa from Chinese herbs and investigated baicalin from Scutellariae radix as such an active anti-virulence compound. The effect of baicalin on a range of important virulence factors in P. aeruginosa was assessed using luxCDABE-based reporters and by phenotypical assays. The molecular mechanism of the virulence inhibition by baicalin was investigated using genetic approaches. The impact of baicalin on P. aeruginosa pathogenicity was evaluated by both in vitro assays and in vivo animal models. The results show that baicalin diminished a plenty of important virulence factors in P. aeruginosa, including the Type III secretion system (T3SS). Baicalin treatment reduced the cellular toxicity of P. aeruginosa on the mammalian cells and attenuated in vivo pathogenicity in a Drosophila melanogaster infection model. In a rat pulmonary infection model, baicalin significantly reduced the severity of lung pathology and accelerated lung bacterial clearance. The PqsR of the Pseudomonas quinolone signal (PQS) system was found to be required for baicalin’s impact on T3SS. These findings indicate that baicalin is a promising therapeutic candidate for treating P. aeruginosa infections.

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Effects of exogenous interleukin-7 on human thymus function

Blood ◽

10.1182/blood.v99.8.2851 ◽

2002 ◽

Vol 99 (8) ◽

pp. 2851-2858 ◽

Cited By ~ 97

Author(s):

Yukari Okamoto ◽

Daniel C. Douek ◽

Richard D. McFarland ◽

Richard A. Koup

Keyword(s):

T Cell ◽

T Cell Receptor ◽

De Novo ◽

Quantitative Polymerase Chain Reaction ◽

Cell Receptor ◽

Cellular Division ◽

Hematopoietic Stem ◽

Human Thymus ◽

Interleukin 7

Abstract Immune reconstitution is a critical component of recovery after treatment of human immunodeficiency virus (HIV) infection, cancer chemotherapy, and hematopoietic stem cell transplantation. The ability to enhance T-cell production would benefit such treatment. We examined the effects of exogenous interleukin-7 (IL-7) on apoptosis, proliferation, and the generation of T-cell receptor rearrangement excision circles (TRECs) in human thymus. Quantitative polymerase chain reaction demonstrated that the highest level of TRECs (14 692 copies/10 000 cells) was present in the CD1a+CD3−CD4+CD8+stage in native thymus, suggesting that TREC generation occurred following the cellular division in this subpopulation. In a thymic organ culture system, exogenous IL-7 increased the TREC frequency in fetal as well as infant thymus, indicating increased T-cell receptor (TCR) rearrangement. Although this increase could be due to the effect of IL-7 to increase thymocyte proliferation and decrease apoptosis of immature CD3− cells, the in vivo experiments using NOD/LtSz-scid mice given transplants of human fetal thymus and liver suggested that IL-7 can also directly enhance TREC generation. Our results provide compelling evidence that IL-7 has a direct effect on increasing TCR-αβ rearrangement and indicate the potential use of IL-7 for enhancing de novo naı̈ve T-cell generation in immunocompromised patients.

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Discovery of novel mechanosensitive genes in vivo using mouse carotid artery endothelium exposed to disturbed flow

Blood ◽

10.1182/blood-2010-04-278192 ◽

2010 ◽

Vol 116 (15) ◽

pp. e66-e73 ◽

Cited By ~ 104

Author(s):

Chih-Wen Ni ◽

Haiwei Qiu ◽

Amir Rezvan ◽

Kihwan Kwon ◽

Douglas Nam ◽

...

Keyword(s):

Carotid Artery ◽

Ex Vivo ◽

Expression Profiles ◽

Quantitative Polymerase Chain Reaction ◽

Gene Expression Profiles ◽

Disturbed Flow ◽

A Genome ◽

Pattern Comparison

Abstract Recently, we showed that disturbed flow caused by a partial ligation of mouse carotid artery rapidly induces atherosclerosis. Here, we identified mechanosensitive genes in vivo through a genome-wide microarray study using mouse endothelial RNAs isolated from the flow-disturbed left and the undisturbed right common carotid artery. We found 62 and 523 genes that changed significantly by 12 hours and 48 hours after ligation, respectively. The results were validated by quantitative polymerase chain reaction for 44 of 46 tested genes. This array study discovered numerous novel mechanosensitive genes, including Lmo4, klk10, and dhh, while confirming well-known ones, such as Klf2, eNOS, and BMP4. Four genes were further validated for protein, including LMO4, which showed higher expression in mouse aortic arch and in human coronary endothelium in an asymmetric pattern. Comparison of in vivo, ex vivo, and in vitro endothelial gene expression profiles indicates that numerous in vivo mechanosensitive genes appear to be lost or dysregulated during culture. Gene ontology analyses show that disturbed flow regulates genes involved in cell proliferation and morphology by 12 hours, followed by inflammatory and immune responses by 48 hours. Determining the functional importance of these novel mechanosensitive genes may provide important insights into understanding vascular biology and atherosclerosis.

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Intravenous Injection of Clinical Grade Human MSCs after Experimental Stroke: Functional Benefit and Microvascular Effect

Cell Transplantation ◽

10.3727/096368916x691132 ◽

2016 ◽

Vol 25 (12) ◽

pp. 2157-2171 ◽

Cited By ~ 12

Author(s):

Anaïck Moisan ◽

Isabelle Favre ◽

Claire Rome ◽

Florence De Fraipont ◽

Emmanuelle Grillon ◽

...

Keyword(s):

Transforming Growth Factor ◽

Ex Vivo ◽

Quantitative Polymerase Chain Reaction ◽

Transforming Growth Factor Β1 ◽

Angiogenic Factors ◽

Experimental Stroke ◽

Vascular Density ◽

Human Mscs ◽

Functional Benefit

Stroke is the leading cause of disability in adults. Many current clinical trials use intravenous (IV) administration of human bone marrow-derived mesenchymal stem cells (BM-MSCs). This autologous graft requires a delay for ex vivo expansion of cells. We followed microvascular effects and mechanisms of action involved after an IV injection of human BM-MSCs (hBM-MSCs) at a subacute phase of stroke. Rats underwent a transient middle cerebral artery occlusion (MCAo) or a surgery without occlusion (sham) at day 0 (D0). At D8, rats received an IV injection of 3 million hBM-MSCs or PBS-glutamine. In a longitudinal behavioral follow-up, we showed delayed somatosensory and cognitive benefits 4 to 7 weeks after hBM-MSC injection. In a separate longitudinal in vivo magnetic resonance imaging (MRI) study, we observed an enhanced vascular density in the ischemic area 2 and 3 weeks after hBM-MSC injection. Histology and quantitative polymerase chain reaction (qPCR) revealed an overexpression of angiogenic factors such as Ang1 and transforming growth factor-β1 (TGF-β1) at D16 in hBM-MSC-treated MCAo rats compared to PBS-treated MCAo rats. Altogether, delayed IV injection of hBM-MSCs provides functional benefits and increases cerebral angiogenesis in the stroke lesion via a release of endogenous angiogenic factors enhancing the stabilization of newborn vessels. Enhanced angiogenesis could therefore be a means of improving functional recovery after stroke.

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Abstract 290: Haemogenic Endocardium Contribute To Definitive Hematopoiesis During Cardiogenesis

Circulation Research ◽

10.1161/res.113.suppl_1.a290 ◽

2013 ◽

Vol 113 (suppl_1) ◽

Author(s):

Haruko Nakano ◽

Xiaoqian Liu ◽

Armin Arshi ◽

Ben van Handel ◽

Rajkumar Sasidharan ◽

...

Keyword(s):

De Novo ◽

Ex Vivo ◽

Embryonic Stem ◽

Circulatory System ◽

Lineage Tracing ◽

Mouse Heart ◽

Heart Tube ◽

Cardiac Progenitors ◽

Definitive Hematopoiesis

The circulatory system is the first functional organ system that develops during mammalian life. Accumulating evidences suggest that cardiac and endocardial cells can arise from a single common progenitor cell during mammalian cardiogenesis. Notably, these early cardiac progenitors express multiple hematopoietic transcription factors, consistent with previous reports. Indeed, a close relationship among cardiac, endocardial and hematopoietic lineages has been suggested in fly, zebrafish, and embryonic stem cell in vitro differentiation models. However, it is unclear when, where and how this hematopoietic gene program is in operation during in vivo mammalian cardiogenesis. Hematopoietic colony assay suggests that mouse heart explants generate myeloids and erythroids in the absence of circulation, suggesting that the heart tube is a de novo site for the definitive hematopoiesis. Lineage tracing revealed that putative cardiac-derived Nkx2-5+/Isl1+ endocardial cells give rise to CD41+ hematopoietic progenitors that contribute to definitive hematopoiesis in vivo and ex vivo during embryogenesis earlier than in the AGM region. Furthermore, Nkx2-5 and Isl1 are both required for the hemogenic activity of the endocardium. Together, identification of Nkx2-5/Isl1-dependent hemogenic endocardial cells (1) adds hematopoietic component in the cardiogenesis lineage tree, (2) changes the long-held dogma that AGM is the only major source of definitive hematopoiesis in the embryo proper, and (3) represents phylogenetically conserved fundamental mechanism of cardio-vasculo-hematopoietic differentiation pathway during the development of circulatory system.

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Angiotensin II overflow from canine skeletal muscle in vivo: importance of plasma angiotensin I

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.1994.266.5.r1664 ◽

1994 ◽

Vol 266 (5) ◽

pp. R1664-R1669 ◽

Cited By ~ 3

Author(s):

J. H. Schwieler ◽

J. Nussberger ◽

T. Kahan ◽

P. Hjemdahl

Keyword(s):

De Novo ◽

Ex Vivo ◽

Plasma Renin ◽

Gracilis Muscle ◽

Ang Ii ◽

Concentration Difference ◽

Beta Adrenoceptor ◽

Arterial Blood ◽

Catheter System

The overflows (i.e., veno-arterial concentration differences multiplied by plasma flow) of angiotensin-(1-10) decapeptide (ANG I) and angiotensin-(1-8) octapeptide (ANG II) from blood-perfused canine gracilis muscle in situ were studied. Special precautions were taken to minimized ex vivo generation and/or degradation of angiotensins in the sampled blood. ANG I was found to be generated in the catheter system supplying the gracilis muscle with arterial blood, but plasma renin activity and ANG II levels were uninfluenced by the catheter system. A positive venoarterial concentration difference over the muscle itself was found for ANG II but not for ANG I under basal conditions. Isoprenaline elicited vasodilatation, reduced ANG I overflow, and tended to increase ANG II overflow, whereas beta-adrenoceptor blockade by propranolol had no effect on these variables. In conclusion, we found no evidence for a local de novo synthesis of ANG II from the gracilis muscle vasculature in vivo. The net overflow of ANG II was most likely caused by local conversion in the tissue of ANG I artifactually generated in the arterial catheter system. beta-Adrenoceptor stimulation enhanced the local conversion of ANG I to ANG II, probably by exposing a greater endothelial surface containing angiotensin-converting enzyme activity.

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Enhancement in Site-Specific Delivery of Carvacrol against Methicillin Resistant Staphylococcus aureus Induced Skin Infections Using Enzyme Responsive Nanoparticles: A Proof of Concept Study

Pharmaceutics ◽

10.3390/pharmaceutics11110606 ◽

2019 ◽

Vol 11 (11) ◽

pp. 606 ◽

Cited By ~ 8

Author(s):

Maria Mir ◽

Naveed Ahmed ◽

Andi Dian Permana ◽

Aoife Maria Rodgers ◽

Ryan F. Donnelly ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Methicillin Resistant Staphylococcus Aureus ◽

Ex Vivo ◽

Infection Model ◽

Skin Infections ◽

Site Specific ◽

Methicillin Resistant ◽

Hydrogel Matrix ◽

Skin Retention

Methicillin resistant Staphylococcus aureus (MRSA) induced skin infections have become a challenging problem due to the escalating antibiotic resistance. Carvacrol (CAR) has been reported to be effective against MRSA. However, due to its characteristics, CAR exhibits low skin retention. In this study, CAR was formulated into site-specific nanoparticle (NPs) delivery system using poly(ε-caprolactone) (PCL), following incorporation into a hydrogel matrix to facilitate dermal delivery. The release study exhibited significantly higher release of CAR from PCL NPs in the presence of bacterial lipase, highlighting its potential for differential delivery. Moreover, encapsulation of CAR in PCL NPs resulted in a two-fold increase in its anti-MRSA activity. Dermatokinetic studies revealed that the NPs loaded hydrogel was able to enhance skin retention of CAR after 24 h (83.29 ± 3.15%), compared to free CAR-loaded hydrogel (0.85 ± 0.14%). Importantly, this novel approach exhibited effective antimicrobial activity in an ex-vivo skin infection model. Hence, these findings have proven the concept that the loading of CAR into a responsive NPs system can lead to sustained antimicrobial effect at the desired site, and may provide a novel effective approach for treatment of MRSA induced skin infections. However, further studies must be conducted to investigate in-vivo efficacy of the developed system in an appropriate infection model.

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Inhibition of Autism-Related Crm1 Disrupts Mitosis and Induces Apoptosis of the Cortical Neural Progenitors

Cerebral Cortex ◽

10.1093/cercor/bhaa011 ◽

2020 ◽

Vol 30 (7) ◽

pp. 3960-3976

Author(s):

Xue Li ◽

Yue Feng ◽

Meifang Yan ◽

Xiaomeng Tu ◽

Bin Xie ◽

...

Keyword(s):

Chromosome Segregation ◽

Spindle Assembly Checkpoint ◽

De Novo ◽

Ex Vivo ◽

Brain Slices ◽

Neural Progenitors ◽

Embryonic Brain ◽

Mitotic Slippage ◽

Developing Mouse Brain

Abstract De novo microdeletion of chromosome 2p15–16.1 presents clinically recognizable phenotypes that include mental retardation, autism, and microcephaly. Chromosomal maintenance 1 (CRM1) is a gene commonly missing in patients with 2p15–16.1 microdeletion and one of two genes found in the smallest deletion case. In this study, we investigate the role and mechanism of Crm1 in the developing mouse brain by inhibiting the protein or knocking down the gene in vivo. Inhibition of Crm1 reduces the proliferation and increases p53-dependent apoptosis of the cortical neural progenitors, thereby impeding the growth of embryonic cerebral cortex. Live imaging of mitosis in ex vivo embryonic brain slices reveals that inhibition of CRM1 arrests the cortical progenitors at metaphase. The arrested cells eventually slip into a pseudo-G1 phase without chromosome segregation. The mitotic slippage cells are marked by persistent expression of the spindle assembly checkpoint (SAC), repressing of which rescues the cells from apoptosis. Our study reveals that activating the SAC and inducing the mitotic slippage may lead to apoptosis of the cortical neural progenitors. The resulting cell death may well contribute to microcephaly associated with microdeletion of chromosome 2p15–16.1 involving CRM1.

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The purine biosynthesis regulator PurR moonlights as a virulence regulator in Staphylococcus aureus

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1904280116 ◽

2019 ◽

Vol 116 (27) ◽

pp. 13563-13572 ◽

Cited By ~ 9

Author(s):

William E. Sause ◽

Divya Balasubramanian ◽

Irnov Irnov ◽

Richard Copin ◽

Mitchell J. Sullivan ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Virulence Factors ◽

Ex Vivo ◽

Purine Biosynthesis ◽

In Vivo Studies ◽

Human Neutrophils ◽

Infection Models ◽

Genes Encoding ◽

Virulence Regulator

The pathogen Staphylococcus aureus colonizes and infects a variety of different sites within the human body. To adapt to these different environments, S. aureus relies on a complex and finely tuned regulatory network. While some of these networks have been well-elucidated, the functions of more than 50% of the transcriptional regulators in S. aureus remain unexplored. Here, we assess the contribution of the LacI family of metabolic regulators to staphylococcal virulence. We found that inactivating the purine biosynthesis regulator purR resulted in a strain that was acutely virulent in bloodstream infection models in mice and in ex vivo models using primary human neutrophils. Remarkably, these enhanced pathogenic traits are independent of purine biosynthesis, as the purR mutant was still highly virulent in the presence of mutations that disrupt PurR’s canonical role. Through the use of transcriptomics coupled with proteomics, we revealed that a number of virulence factors are differentially regulated in the absence of purR. Indeed, we demonstrate that PurR directly binds to the promoters of genes encoding virulence factors and to master regulators of virulence. These results guided us into further ex vivo and in vivo studies, where we discovered that S. aureus toxins drive the death of human phagocytes and mice, whereas the surface adhesin FnbA contributes to the increased bacterial burden observed in the purR mutant. Thus, S. aureus repurposes a metabolic regulator to directly control the expression of virulence factors, and by doing so, tempers its pathogenesis.

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Nanocrystals of Fusidic Acid for Dual Enhancement of Dermal Delivery and Antibacterial Activity: In Vitro, Ex Vivo and In Vivo Evaluation

Pharmaceutics ◽

10.3390/pharmaceutics12030199 ◽

2020 ◽

Vol 12 (3) ◽

pp. 199 ◽

Cited By ~ 2

Author(s):

Iman S. Ahmed ◽

Osama S. Elnahas ◽

Nouran H. Assar ◽

Amany M. Gad ◽

Rania El Hosary

Keyword(s):

Fusidic Acid ◽

Therapeutic Efficacy ◽

Ex Vivo ◽

Skin Permeation ◽

Fold Increase ◽

Spherical Particles ◽

Resistant Bacteria ◽

Infection Model

With the alarming rise in incidence of antibiotic-resistant bacteria and the scarcity of newly developed antibiotics, it is imperative that we design more effective formulations for already marketed antimicrobial agents. Fusidic acid (FA), one of the most widely used antibiotics in the topical treatment of several skin and eye infections, suffers from poor water-solubility, sub-optimal therapeutic efficacy, and a significant rise in FA-resistant Staphylococcus aureus (FRSA). In this work, the physico-chemical characteristics of FA were modified by nanocrystallization and lyophilization to improve its therapeutic efficacy through the dermal route. FA-nanocrystals (NC) were prepared using a modified nanoprecipitation technique and the influence of several formulation/process variables on the prepared FA-NC characteristics were optimized using full factorial statistical design. The optimized FA-NC formulation was evaluated before and after lyophilization by several in-vitro, ex-vivo, and microbiological tests. Furthermore, the lyophilized FA-NC formulation was incorporated into a cream product and its topical antibacterial efficacy was assessed in vivo using a rat excision wound infection model. Surface morphology of optimized FA-NC showed spherical particles with a mean particle size of 115 nm, span value of 1.6 and zeta potential of −11.6 mV. Differential scanning calorimetry and powder X-ray diffractometry confirmed the crystallinity of FA following nanocrystallization and lyophilization. In-vitro results showed a 10-fold increase in the saturation solubility of FA-NC while ex-vivo skin permeation studies showed a 2-fold increase in FA dermal deposition from FA-NC compared to coarse FA. Microbiological studies revealed a 4-fofd decrease in the MIC against S. aureus and S. epidermidis from FA-NC cream compared to commercial Fucidin cream. In-vivo results showed that FA-NC cream improved FA distribution and enhanced bacterial exposure in the infected wound, resulting in increased therapeutic efficacy when compared to coarse FA marketed as Fucidin cream.

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