TOM-1/tomosyn acts with the UNC-6/netrin receptor UNC-5 to inhibit growth cone protrusion in Caenorhabditis elegans

In the polarity/protrusion model of growth cone repulsion from UNC-6/netrin, UNC-6 first polarizes the growth cone of the VD motor neuron axon via the UNC-5 receptor, and then regulates protrusion asymmetrically across the growth cone based on this polarity. UNC-6 stimulates protrusion dorsally through the UNC-40/DCC receptor, and inhibits protrusion ventrally through UNC-5, resulting in net dorsal growth. Previous studies showed that UNC-5 inhibits growth cone protrusion via the flavin monooxygenases and potential destabilization of F-actin, and via UNC-33/CRMP and restriction of microtubule plus-end entry into the growth cone. We show that UNC-5 inhibits protrusion through a third mechanism involving TOM-1/tomosyn. A short isoform of TOM-1 inhibited protrusion downstream of UNC-5, and a long isoform had a pro-protrusive role. TOM-1/tomosyn inhibits formation of the SNARE complex. We show that UNC-64/syntaxin is required for growth cone protrusion, consistent with a role of TOM-1 in inhibiting vesicle fusion. Our results are consistent with a model whereby UNC-5 utilizes TOM-1 to inhibit vesicle fusion, resulting in inhibited growth cone protrusion, possibly by preventing the growth cone plasma membrane addition required for protrusion

A short isoform of the UNC-6/Netrin receptor UNC-5 is required for growth cone polarity and robust growth cone protrusion in Caenorhabditis elegans

Introduction: UNC-6/Netrin is a conserved bi-functional guidance cue which regulates dorsal-ventral axon guidance in C. elegans. In the Polarity/Protrusion model of UNC-6/Netrin mediated dorsal growth away from UNC-6/Netrin, The UNC-5 receptor first polarizes the VD growth cone such that filopodial protrusions are biased dorsally. Based on this polarity, the UNC-40/DCC receptor stimulates growth cone lamellipodial and filopodial protrusion dorsally. The UNC-5 receptor maintains dorsal polarity of protrusion, and inhibits growth cone protrusion ventrally, resulting in net dorsal growth cone advance.Methods: Growth cone imaging in mutants, combined with Cas9 genome editing and genetic analysis, were used to analyze the role of a novel short isoform on unc-5 in growth cone polarity and protrusion.Results: Work presented here demonstrates a novel role of a previously undescribed, conserved short isoform of UNC-5 (UNC-5B). UNC-5B lacks the cytoplasmic domains of UNC-5 long, including the DEATH domain, the UPA/DB domain, and most of the ZU5 domain. Mutations that specifically affect only the unc-5 long isoforms were hypomorphic, suggesting a role of unc-5B short. A mutation specifically affecting unc-5B caused loss of dorsal polarity of protrusion and reduced growth cone filopodial protrusion, the opposite of unc-5 long mutations. Transgenic expression of unc-5B partially rescued unc-5 axon guidance defects, and resulted in large growth cones. Tyrosine 482 (Y482) in the cytoplasmic juxtamembrane region has been shown to be important for UNC-5 function, and is present in both UNC-5 long and UNC-5B short. Results reported here show that Y482 is required for the function of UNC-5 long and for some functions of UNC-5B short. Finally, genetic interactions with unc-40 and unc-6 suggest that UNC-5B short acts in parallel to UNC-6/Netrin to ensure robust growth cone lamellipodial protrusion.Discussion: These results demonstrate a previously-undescribed role for the UNC-5B short isoform, which is required for dorsal polarity of growth cone filopodial protrusion and to stimulate growth cone protrusion, in contrast to the previously-described role of UNC-5 long in inhibiting growth cone protrusion

The SYNTHESIS, SCREENING OF NOVEL 1-SUBSTITUTED-3-(4-OXO-2-PHENYLQUINAZOLIN-3(4H)-YL) UREA AND THIOUREA ANALOGUES AS POTENT ANTIBACTERIALS

Objective: The proposed study is an attempt to determine antibacterial activity of synthesized novel 1-substituted-3-(4-oxo-2-phenylquinazolin-3(4H)-yl) urea and thiourea analogues as potent antibacterials against S. aureus and E. coli bacteria. Methods: The present study reports new series of 1-substituted-3-(4-oxo-2-phenylquinazolin-3(4H)-yl) urea and thiourea derivatives as potent antibacterial agents. Reagents used in the present study were of synthetic grade and solvents were used after distillation. Novel quinazolinone analogues were synthesized by considering substitution pattern, characterization of the synthesized analogues was performed using various techniques like Thin layer chromatography, Melting point, Infrared spectroscopy, Proton NMR spectrometry and Mass spectrometry. TLC of the synthesized analogues was carried out by using (toluene: methanol in the ratio 2:1), melting point was found by open capillary method, IR spectrum was recorded on JASCO V-530, 1H NMR was recorded on Bruker Avance Spectrometer and Mass spectra were obtained from G6460A, triple quadrupole/MS/MS system. In vitro antibacterial activity was performed against S. aureus and E. coli. Results: Six derivatives of quinazolinone analogues were synthesized. The structures of 1-substituted-3-(4-oxo-2-phenylquinazolin-3(4H)-yl) urea and thiourea derivatives were confirmed by physical and spectral analysis. Synthesized molecules showed Rf of 0.45-0.80 in toluene: methanol mobile phase, melting point was carried out by open capillary method and were in range of 90-210 ° C, IR spectrum was recorded in range of 14000-400 cm-1and showed characteristic peaks of NH and of C-O-NH, 1H NMR of the compounds was distinct to confirm structures with delta values in the range of 7.53-11.960, Mass spectra proved parent peaks of synthesized compounds confirming molecular weight. The compounds were assayed for antibacterial activity against S. aureus and E. coli using ciprofloxacin as standard. The synthesized analogues have shown good yield and comparable antibacterial. Conclusion: The present study delivers a convenient and efficient protocol for the quinazolinone analogues synthesis

Establishment of 3D Co-Culture Models from Different Stages of Human Tongue Tumorigenesis: Utility in Understanding Neoplastic Progression

<div><p>To study multistep tumorigenesis process, there is a need of <i>in-vitro</i> 3D model simulating <i>in-vivo</i> tissue. Present study aimed to reconstitute <i>in-vitro</i> tissue models comprising various stages of neoplastic progression of tongue tumorigenesis and to evaluate the utility of these models to investigate the role of stromal fibroblasts in maintenance of desmosomal anchoring junctions using transmission electron microscopy. We reconstituted <i>in-vitro</i> models representing normal, dysplastic, and malignant tissues by seeding primary keratinocytes on either fibroblast embedded in collagen matrix or plain collagen matrix in growth factor-free medium. The findings of histomorphometry, immunohistochemistry, and electron microscopy analyses of the three types of 3D cultures showed that the stratified growth, cell proliferation, and differentiation were comparable between co-cultures and their respective native tissues; however, they largely differed in cultures grown without fibroblasts. The immunostaining intensity of proteins, viz., desmoplakin, desmoglein, and plakoglobin, was reduced as the disease stage increased in all co-cultures as observed in respective native tissues. Desmosome-like structures were identified using immunogold labeling in these cultures. Moreover, electron microscopic observations revealed that the desmosome number and their length were significantly reduced and intercellular spaces were increased in cultures grown without fibroblasts when compared with their co-culture counterparts. Our results showed that the major steps of tongue tumorigenesis can be reproduced <i>in-vitro</i>. Stromal fibroblasts play a role in regulation of epithelial thickness, cell proliferation, differentiation, and maintenance of desmosomalanchoring junctions in <i>in-vitro</i> grown tissues. The reconstituted co-culture models could help to answer various biological questions especially related to tongue tumorigenesis.</p></div

Cell proliferation and differentiation staining pattern in <i>in-vitro</i> grown tissues.

<p>Immunohistochemical staining for cell proliferation and differentiation specific proteins in Fib+ and Fib- OCs reconstructed from normal, dysplastic and malignant tongue tissues along with their respective native tissues: PCNA nuclear staining was seen in the basal proliferating cells of normal- native tissue, Fib+ and Fib- OCs (a, b, c) while cytoplasmic involucrin staining was seen in the upper differentiated cells of normal- native tissue, Fib+ and Fib- OCs (d, e, f). PCNA nuclear staining was seen in the basal as well as supra basal cells of dysplastic-native tissue, Fib+ and Fib- OCs (g, h, i) while cytoplasmic involucrin staining was seen in the native dysplastic tissue but not in Fib+ and Fib- OCs (j, k, l). PCNA staining was observed throughout the epithelium of malignant-native tissue, Fib+ and Fib- OCs (m, n, o), while involucrin staining was seen in the differentiated cells of malignant-native tissue but not in the Fib+ and Fib- OCs (p, q, r). The experiments were performed at least three times.</p

List of antibodies used in immunohistochemistry /immunofluorescence/immunogold staining protocols.

<p>List of antibodies used in immunohistochemistry /immunofluorescence/immunogold staining protocols.</p

Isolation and characterization of primary keratinocytes and fibroblasts from tongue tissue.

<p>Representative phase contrast microscopic images showing the growth of primary keratinocytes/fibroblasts from human normal tongue tissues at different time points–day 2 (a, f), day 6 (b, g) and day 9 (c, h). Immunofluorescence stained images showed the positive expression (green) for cytokeratin (d) and negative expression for vimentin (e) in normal keratinocytes while, fibroblasts showed negative expression for keratin (j) and positive expression (red) for vimentin (i). Nuclei are counterstained with DAPI (blue). Bars-50 μm.</p

Validation of endogenous lineage phenotype.

<p>Immunohistochemistry of normal OC showing keratin (b) and vimentin (f) staining in the epithelium and stromal fibroblasts respectively and staining of integrin β4 (d) in the basement membrane as seen in native tissues (a, c, e). The experiments were performed in triplicates.</p

Ultrastructural analysis of altered desmosomal assembly.

<p>Electron micrographs depicting desmosomes (D) and intercellular spaces (ICS) in native tissues (a-c) along with Fib+ OCs (d-f) and Fib- OCs(g-i). Native tissue and Fib+ OCs show similar ultrastructure having widened intercellular spaces (ICS) and reduced number of desmosomes as disease stage progressed, whereas Fib- OCs showed complete collapse of ultrastructure with more widened ICS and reduced desmosomes even at normal stage. Normal desmosome structure was seen in normal-native tissues as well as Fib+ OCs (a’, d’) but in normal-Fib- OCs desmosomes were found to be in aggregated forms (g’). Desmosome structures were confirmed by immunogold labeling in normal native tissue (a”) normal Fib+ OC (d”) and normal Fib- OC (g”). Bars 1 μm.</p