scholarly journals AutoNeuriteJ: An ImageJ plugin for measurement and classification of neuritic extensions

2020 ◽  
Author(s):  
Benoit Boulan ◽  
Anne Beghin ◽  
Charlotte Ravanello ◽  
Jean-Christophe Deloulme ◽  
Sylvie Gory-Fauré ◽  
...  
Keyword(s):  
Neuronal Cultures ◽  
Neuronal Growth ◽  
Wild Type ◽  
Phenotypic Difference ◽  
Phenotypic Differences ◽  
Large Numbers ◽  
Neuritic Growth ◽  
Imagej Plugin

AbstractMorphometry characterization is an important procedure in describing neuronal cultures and identifying phenotypic differences. This task usually requires labor-intensive measurements and the classification of numerous neurites from large numbers of neurons in culture. To automate these measurements, we wrote AutoNeuriteJ, an imageJ/Fiji plugin that measures and classifies neurites from a very large number of neurons. We showed that AutoNeuriteJ is able to detect variations of neuritic growth induced by several compounds known to affect the neuronal growth. In these experiments measurement of more than 5000 mouse neurons per conditions was obtained within a few hours. Moreover, by analyzing mouse neurons deficient for the microtubule associated protein 6 (MAP6) and wild type neurons we illustrate that AutoNeuriteJ is capable to detect subtle phenotypic difference in axonal length. Overall the use of AutoNeuriteJ will provide rapid, unbiased and accurate measurement of neuron morphologies.

Phenotypic differences in PFIC2 and BRIC2 correlate with protein stability of mutant Bsep and impaired taurocholate secretion in MDCK II cells

2008 ◽  
Vol 294 (1) ◽  
pp. G58-G67 ◽  
Author(s):  
Tatehiro Kagawa ◽  
Norihito Watanabe ◽  
Kaori Mochizuki ◽  
Asano Numari ◽  
Yoshie Ikeno ◽  
...  
Keyword(s):  
Protein Expression ◽  
Apical Membrane ◽  
Bile Salt Export Pump ◽  
Transport Activity ◽  
Wild Type ◽  
Phenotypic Difference ◽  
Mutant Proteins ◽  
Phenotypic Differences ◽  
Benign Recurrent Intrahepatic Cholestasis ◽  
Taurocholate Transport

Progressive familial cholestasis (PFIC) 2 and benign recurrent intrahepatic cholestasis (BRIC) 2 are caused by mutations in the bile salt export pump (BSEP, ABCB11) gene; however, their prognosis differs. PFIC2 progresses to cirrhosis and requires liver transplantation, whereas BRIC2 is clinically benign. To identify the molecular mechanism(s) responsible for the phenotypic differences, eight PFIC2 and two BRIC2 mutations were introduced in rat Bsep, which was transfected in MDCK II cells. Taurocholate transport activity, protein expression, and subcellular distribution of these mutant proteins were studied in a polarized MDCK II monolayer. The taurocholate transport activity was approximately half of the wild-type (WT) in BRIC2 mutants (A570T and R1050C), was substantially less in two PFIC2 mutants (D482G and E297G), and was almost abolished in six other PFIC2 mutants (K461E, G982R, R1153C, R1268Q, 3767–3768insC, and R1057X). Bsep protein expression levels correlated closely with transport activity, except for R1057X. The half-life of the D482G mutant was shorter than that of the WT (1.35 h vs. 3.49 h in the mature form). BRIC2 mutants and three PFIC mutants (D482G, E297G, and R1057X) were predominantly distributed in the apical membrane. The other PFIC2 mutants remained intracellular. The R1057X mutant protein was stably expressed and trafficked to the apical membrane, suggesting that the COOH-terminal tail is required for transport activity but not for correct targeting. In conclusion, taurocholate transport function was impaired in proportion to rapid degradation of Bsep protein in the mutants, which were aligned in the following order: A570T and R1050C > D482G > E297G > K461E, G982R, R1153C, R1268Q, 3767–3768insC, and R1057X. These results may explain the phenotypic difference between BRIC2 and PFIC2.

scholarly journals Activation of Kinin B1R Upregulates ADAM17 and Results in ACE2 Shedding in Neurons

2020 ◽  
Vol 22 (1) ◽  
pp. 145
Author(s):  
Rohan Umesh Parekh ◽  
Srinivas Sriramula
Keyword(s):  
Protein Expression ◽  
Renin Angiotensin System ◽  
Neuronal Cultures ◽  
Wild Type ◽  
High Concentration ◽  
Neurogenic Hypertension ◽  
B1 Receptor ◽  
Gene And Protein Expression ◽  
Kinin B1 Receptor ◽  
Specific Antagonist

Angiotensin converting enzyme 2 (ACE2) is a critical component of the compensatory axis of the renin angiotensin system. Alterations in ACE2 gene and protein expression, and activity mediated by A Disintegrin And Metalloprotease 17 (ADAM17), a member of the “A Disintegrin And Metalloprotease” (ADAM) family are implicated in several cardiovascular and neurodegenerative diseases. We previously reported that activation of kinin B1 receptor (B1R) in the brain increases neuroinflammation, oxidative stress and sympathoexcitation, leading to the development of neurogenic hypertension. We also showed evidence for ADAM17-mediated ACE2 shedding in neurons. However, whether kinin B1 receptor (B1R) activation has any role in altering ADAM17 activity and its effect on ACE2 shedding in neurons is not known. In this study, we tested the hypothesis that activation of B1R upregulates ADAM17 and results in ACE2 shedding in neurons. To test this hypothesis, we stimulated wild-type and B1R gene-deleted mouse neonatal primary hypothalamic neuronal cultures with a B1R-specific agonist and measured the activities of ADAM17 and ACE2 in neurons. B1R stimulation significantly increased ADAM17 activity and decreased ACE2 activity in wild-type neurons, while pretreatment with a B1R-specific antagonist, R715, reversed these changes. Stimulation with specific B1R agonist Lys-Des-Arg9-Bradykinin (LDABK) did not show any effect on ADAM17 or ACE2 activities in neurons with B1R gene deletion. These data suggest that B1R activation results in ADAM17-mediated ACE2 shedding in primary hypothalamic neurons. In addition, stimulation with high concentration of glutamate significantly increased B1R gene and protein expression, along with increased ADAM17 and decreased ACE2 activities in wild-type neurons. Pretreatment with B1R-specific antagonist R715 reversed these glutamate-induced effects suggesting that indeed B1R is involved in glutamate-mediated upregulation of ADAM17 activity and ACE2 shedding.

scholarly journals The mystery of Anser neglectus Sushkin, 1897. Victim of the Tunguska disaster? A Hungarian story

2019 ◽  
Vol 27 (2) ◽  
pp. 20-58
Author(s):  
Jacques Van Impe
Keyword(s):  
Distinct Species ◽  
Genetic Mutations ◽  
Zoological Garden ◽  
Large Numbers ◽  
Bean Goose ◽  
Field Identification ◽  
Breeding Grounds ◽  
The Republic ◽  
The Town

Abstract The well-known Russian ornithologist Prof. Peter Sushkin described it as a distinct species from Bashkortostan (Bashkiria) in 1897, a highly acclaimed discovery. However, its breeding grounds never been discovered. Since then, there has been a long-standing debate over the taxonomic position of Anser neglectus. Taxonomists have argued that Anser neglectus belongs to the group of A. fabalis Lath. because of its close resemblance with A. f. fabalis. At the beginning of the 20th century, large numbers of the Sushkin’s goose were observed in three winter quarters: on two lakes in the Republic of Bachkortostan, in the surroundings of the town of Tashkent in the Republic Uzbekistan, and in the puszta Hortobágy in eastern Hungary. It is a pity that taxonomists did not thoroughly compare the Russian and Hungarian ornithological papers concerning the former presence of Anser neglectus in these areas, because these rich sources refer to characteristics that would cast serious doubt on the classification of Anser neglectus as a subspecies, an individual variation or mutation of A. f. fabalis. Sushkin’s goose, though a typical Taiga Bean Goose, distinguished itself from other taxa of the Bean Goose by its plumage, its field identification, by its specific “Gé-gé” call, the size of its bill, and by its preference for warm and dry winter haunts. A. neglectus should therefore be considered a separate, fully distinct species, sensu Stegmann (1935) and Stegmann in Schenk (1931/34), if we follow the established criteria in bird systematics of Tobias et al. (2010). Between 1908 and 1911, an estimation of up to 150.000 individuals of A. neglectus wintered in the Hortobágy puszta. Approximate counts for both other winter quarters are not available. The last living birds were seen in the zoological garden of Budapest in 1934. Since then, A. f. fabalis and A. s. rossicus “Type neglectus” (i.e. A. f. fabalis and A. s. rossicus with a color of the bill and the legs, similar to the former A. neglectus) have been observed sporadically on the breeding grounds and in the winter quarters of both taxa. However, the true A. neglectus seems to be extinct. Its sudden disappearance may be related to the Tunguska event, the catastrophe in 1908 that may have caused genetic mutations. This hypothesis is considered to be the most likely, among other available hypotheses about its extinction.

A Dictystelium mutant with defective aggregate size determination

Development ◽  
1996 ◽  
Vol 122 (9) ◽  
pp. 2569-2578 ◽  
Author(s):  
D.A. Brock ◽  
G. Buczynski ◽  
T.P. Spann ◽  
S.A. Wood ◽  
J. Cardelli ◽  
...  
Keyword(s):  
Mrna Level ◽  
Phosphodiesterase Inhibitor ◽  
Aggregate Size ◽  
Conditioned Media ◽  
Wild Type ◽  
Significant Similarity ◽  
Western Blots ◽  
Cytosolic Protein ◽  
Large Numbers ◽  
Cell Fractions

Starved Dictyostelium cells aggregate into groups of roughly 10(5) cells. We have identified a gene which, when repressed by antisense transformation or homologous recombination, causes starved cells to form large numbers of small aggregates. We call the gene smlA for small aggregates. A roughly 1.0 kb smlA mRNA is expressed in vegetative and early developing cells, and the mRNA level then decreases at about 10 hours of development. The sequence of the cDNA and the derived amino acid sequence of the SmlA protein show no significant similarity to any known sequence. There are no obvious motifs in the protein or large regions of hydrophobicity or charge. Immunofluorescence and staining of Western blots of cell fractions indicates that SmlA is a 35x10(3) Mr cytosolic protein present in all vegetative and developing cells and is absent from smlA cells. The absence of SmlA does not affect the growth rate, cell cycle, motility, differentiation, or developmental speed of cells. Synergy experiments indicate that mixing 5% smlA cells with wild-type cells will cause the wild-type cells to form smaller fruiting bodies and aggregates. Although there is no detectable SmlA protein secreted from cells, starvation medium conditioned by smlA cells will cause wild-type cells to form large numbers of small aggregates. The component in the smlA-conditioned media that affects aggregate size is a molecule with a molecular mass greater than 100x10(3) Mr that is not conditioned media factor, phosphodiesterase or the phosphodiesterase inhibitor. The data thus suggest that the cytosolic protein SmlA regulates the secretion or processing of a secreted factor that regulates aggregate size.

scholarly journals A Novel Secreted Cysteine-Rich Anionic (Sca) Protein from the Citrus Postharvest Pathogen Penicillium digitatum Enhances Virulence and Modulates the Activity of the Antifungal Protein B (AfpB)

2020 ◽  
Vol 6 (4) ◽  
pp. 203
Author(s):  
Sandra Garrigues ◽  
Jose F. Marcos ◽  
Paloma Manzanares ◽  
Mónica Gandía
Keyword(s):  
Citrus Fruit ◽  
Penicillium Digitatum ◽  
Antifungal Protein ◽  
Penicillium Expansum ◽  
Wild Type ◽  
Phenotypic Differences ◽  
Antifungal Proteins ◽  
Ascomycete Fungi ◽  
In Vitro Antifungal Activity

Antifungal proteins (AFPs) from ascomycete fungi could help the development of antimycotics. However, little is known about their biological role or functional interactions with other fungal biomolecules. We previously reported that AfpB from the postharvest pathogen Penicillium digitatum cannot be detected in the parental fungus yet is abundantly produced biotechnologically. While aiming to detect AfpB, we identified a conserved and novel small Secreted Cysteine-rich Anionic (Sca) protein, encoded by the gene PDIG_23520 from P. digitatum CECT 20796. The sca gene is expressed during culture and early during citrus fruit infection. Both null mutant (Δsca) and Sca overproducer (Scaop) strains show no phenotypic differences from the wild type. Sca is not antimicrobial but potentiates P. digitatum growth when added in high amounts and enhances the in vitro antifungal activity of AfpB. The Scaop strain shows increased incidence of infection in citrus fruit, similar to the addition of purified Sca to the wild-type inoculum. Sca compensates and overcomes the protective effect of AfpB and the antifungal protein PeAfpA from the apple pathogen Penicillium expansum in fruit inoculations. Our study shows that Sca is a novel protein that enhances the growth and virulence of its parental fungus and modulates the activity of AFPs.

Clinical importance of molecular markers of adult diffuse glioma

2019 ◽  
Vol 19 (5) ◽  
pp. 412-416 ◽  
Author(s):  
Emanuela Molinari ◽  
Olimpia E Curran ◽  
Robin Grant
Keyword(s):  
Molecular Markers ◽  
Molecular Genetic ◽  
Clinical Importance ◽  
Response To Treatment ◽  
Low Grade ◽  
Diffuse Glioma ◽  
Wild Type ◽  
Genetic Classification ◽  
Cns Tumours

In 2016, the WHO incorporated molecular markers, in addition to histology, into the diagnostic classification of central nervous system (CNS) tumours. This improves diagnostic accuracy and prognostication: oligo-astrocytoma no longer exists as a clinical entity; isocitrate dehydrogenase (IDH) mutant and 1p/19q co-deleted oligodendroglioma is a smaller category with better prognosis; IDH wild-type ‘low-grade’ glioma has a much poorer prognosis; and glioblastoma is divided into IDH mutant (with an better prognosis than pre-2016 glioblastoma) and IDH wild type (with a poorer prognosis). Previous advice based on phenotype alone will change with respect to median survival, best management plan and response to treatment. There are implications for routine neuropathology reporting and future trial design. Cases that are difficult to classify may need more advanced molecular genetic classification through DNA methylation-based classification of CNS tumours (Heidelberg Classifier). We discuss the practical implications.

scholarly journals Repair of the UL21 Locus in Pseudorabies Virus Bartha Enhances the Kinetics of Retrograde, Transneuronal Infection In Vitro and In Vivo

2008 ◽  
Vol 83 (3) ◽  
pp. 1173-1183 ◽  
Author(s):  
D. Curanović ◽  
M. G. Lyman ◽  
C. Bou-Abboud ◽  
J. P. Card ◽  
L. W. Enquist
Keyword(s):  
Pseudorabies Virus ◽  
Particle Production ◽  
Neural Circuit ◽  
Neuronal Cultures ◽  
Wild Type ◽  
Infectious Particle ◽  
Viral Spread ◽  
Kinetics Of

ABSTRACT The attenuated pseudorabies virus (PRV) strain Bartha contains several characterized mutations that affect its virulence and ability to spread through neural circuits. This strain contains a small genomic deletion that abrogates anterograde spread and is widely used as a retrograde-restricted neural circuit tracer. Previous studies showed that the retrograde-directed spread of PRV Bartha is slower than that of wild-type PRV. We used compartmented neuronal cultures to characterize the retrograde defect and identify the genetic basis of the phenotype. PRV Bartha is not impaired in retrograde axonal transport, but transneuronal spread among neurons is diminished. Repair of the UL21 locus with wild-type sequence restored efficient transneuronal spread both in vitro and in vivo. It is likely that mutations in the Bartha UL21 gene confer defects that affect infectious particle production, causing a delay in spread to presynaptic neurons and amplification of infection. These events manifest as slower kinetics of retrograde viral spread in a neural circuit.

scholarly journals AutoNeuriteJ: An ImageJ plugin for measurement and classification of neuritic extensions

PLoS ONE ◽  
2020 ◽  
Vol 15 (7) ◽  
pp. e0234529
Author(s):  
Benoit Boulan ◽  
Anne Beghin ◽  
Charlotte Ravanello ◽  
Jean-Christophe Deloulme ◽  
Sylvie Gory-Fauré ◽  
...  
Keyword(s):  
Imagej Plugin

scholarly journals Allorestricted cytotoxic T cells. Large numbers of allo-H-2Kb-restricted antihapten and antiviral cytotoxic T cell populations clonally develop in vitro from murine splenic precursor T cells.

1985 ◽  
Vol 162 (2) ◽  
pp. 592-606 ◽  
Author(s):  
J Reimann ◽  
D Kabelitz ◽  
K Heeg ◽  
H Wagner
Keyword(s):  
T Cells ◽  
High Probability ◽  
Cytotoxic T Lymphocyte ◽  
Cytotoxic T Cells ◽  
Large Fraction ◽  
Cytotoxic T Cell ◽  
Wild Type ◽  
Large Numbers ◽  
Simplex Virus

Cytotoxic T lymphocyte (CTL) responses of splenic T cells from C57BL/6 B6) mice and mutant H-2Kbm1 (bm1) mice to haptenic (trinitrophenyl [TNP] ) and herpes simplex virus (HSV) determinants in the context of an allogenic (wild-type or mutant) H-2Kb molecule were analyzed in a modified limiting dilution system. In the B6-anti-bm1TNP mixed leukocyte reaction (MLR), estimated frequencies for precursors of CTL clones that lysed bm1TNP targets ranged from 1/120 to 1/400; in the bm1-anti-B6TNP MLR, estimated frequencies of precursors of CTL clones that lysed B6TNP targets ranged from 1/500 to 1/1,300. Estimated frequencies for precursors of CTL clones that lysed the respective unmodified and TNP-modified allogeneic targets were two- to three-fold lower. Lytic specificity patterns determined by split-well analysis showed that at least 20-30% of the generated CTL populations (selected for a high probability of clonality) in both MLR displayed allorestricted lysis of TNP-modified concanavalin A blast targets. In the B6-anti-bm1HSV MLR, estimated frequencies for precursors of CTL clones that lysed bm1HSV targets ranged from 1/70 to 1/300; in the bm1-anti-B6HSV MLR, estimated frequencies for precursors of CTL clones that lysed B6HSV targets ranged from 1/300 to 1/1,200. Again, estimated frequencies for precursors of CTL clones that lysed the respective noninfected and virus-infected allogeneic targets were two- to fourfold lower. Of the CTL populations selected for a high probability of clonality at least 30-60% displayed allorestricted lysis of virus-infected lipopolysaccharide blast targets in both MLR. It is concluded that a large fraction of clonally developing CTL populations stimulated with TNP-modified or HSV-infected allo-H-2Kb-bearing cells displayed an allorestricted pattern of recognition. It was further evident that the estimated frequencies of splenic precursors that generated allorestricted CTL clones was two- to threefold higher than the estimated frequencies of precursors that gave rise to the respective alloreactive CTL populations.

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