Piperidinols that show anti-tubercular activity as inhibitors of arylamine N-acetyltransferase: an essential enzyme for mycobacterial survival inside macrophages

Latent M. tuberculosis infection presents one of the major obstacles in the global eradication of tuberculosis (TB). Cholesterol plays a critical role in the persistence of M. tuberculosis within the macrophage during latent infection. Catabolism of cholesterol contributes to the pool of propionyl-CoA, a precursor that is incorporated into cell-wall lipids. Arylamine N-acetyltransferase (NAT) is encoded within a gene cluster that is involved in the cholesterol sterol-ring degradation and is essential for intracellular survival. The ability of the NAT from M. tuberculosis (TBNAT) to utilise propionyl-CoA links it to the cholesterol-catabolism pathway. Deleting the nat gene or inhibiting the NAT enzyme prevents intracellular survival and results in depletion of cell-wall lipids. TBNAT has been investigated as a potential target for TB therapies. From a previous high-throughput screen, 3-benzoyl-4-phenyl-1-methylpiperidinol was identified as a selective inhibitor of prokaryotic NAT that exhibited antimycobacterial activity. The compound resulted in time-dependent irreversible inhibition of the NAT activity when tested against NAT from M. marinum (MMNAT). To further evaluate the antimycobacterial activity and the NAT inhibition of this compound, four piperidinol analogues were tested. All five compounds exert potent antimycobacterial activity against M. tuberculosis with MIC values of 2.3-16.9 µM. Treatment of the MMNAT enzyme with this set of inhibitors resulted in an irreversible time-dependent inhibition of NAT activity. Here we investigate the mechanism of NAT inhibition by studying protein-ligand interactions using mass spectrometry in combination with enzyme analysis and structure determination. We propose a covalent mechanism of NAT inhibition that involves the formation of a reactive intermediate and selective cysteine residue modification. These piperidinols present a unique class of antimycobacterial compounds that have a novel mode of action different from known anti-tubercular drugs

Automatsko brojanje putnika u javnom željezničkom prijevozu uporabom waveleta

Previously, we introduced a passengers’ counting algorithm in public rail transport. The main disadvantage of that algorithm is it lacks automatic event detection. In this article, we implement two automatic wavelet-based passengers counting algorithms. The new algorithms employ the spatial-domain Laplacian-of-Gaussian-based wavelet, and the frequency-domain applied Non-Linear Difference of Gaussians-based wavelet bandpass video scene filters to extract illumination invariant scene features and to combine them efficiently into the background reference frame. Manual segmentation of the scene into rectangles and tiles for detecting an object as seated is no longer needed as we now apply a boundary box tracker on the segmented moving objects’ blobs. A scene map is combined with the wavelet-based methods and the boundary box for multi-camera object registration. We have developed a novel holistic geometrical approach for exploiting the scene map and the recorded video sequences from both cameras installed in each train coach to separate the detected objects and locate their positions on the scene map. We test all the algorithms with several video sequences recorded from the both cameras installed in each train coach. We compare the previously developed non-automatic passengers’ counting algorithm with the two new automatic wavelet-based passengers’ counting algorithms, and an additional spatial-domain automatic non-wavelet based Simple Mixture of Gaussian Models algorithm.U prethodnim radovima uveli smo algoritam za brojanje putnika u javnom željezničkom prijevozu. Glavna manjkavost dosadašnjeg algoritma odsustvo je sustava za automatsko otkrivanje događaja. U ovom radu implementirali smo dva algoritma za automatsko brojanje putnika temeljena na waveletima. Novi algoritmi koriste LoG (Laplacian-of-Gaussian-based) wavelete u prostornoj domeni i pojasne filtre temeljene na waveletima nastalim na nelinearnim razlikama Gaussovih funkcija u frekvencijskoj domeni, pomoću kojih se izdvajaju značajke neosjetljive na razlike u osvjetljenju iz pojedine scene. Te značajke kombiniraju se u referentnu sliku koja prikazuje pozadinu scene. Ručna segmentacija scene u pravokutnike korištena u prethodnom algoritmu više nije potrebna jer se sada koristi automatsko praćenje rubova na segmentiranim objektima. Mapa scene kombinirana je s wavelet metodama i okvirom granica slike u svrhu registracije objekata pomoću više kamera. Razvili smo i novi cjeloviti geometrijski pristup koji koristi mapu scene i snimljeni videozapis iz dvije kamere postavljene u svakom vagonu vlaka pomoću kojeg možemo odvojiti detektirane objekte i locirati njihove položaje na mapi scene. Algoritmi su ispitani na nekoliko videosekvenci snimljenih s dvije kamere u vagonima. Usporedili smo ranije razvijene neautomatske algoritme za brojanje putnika s dva nova algoritma i s jednostavnim MoG algoritmom u prostornoj domeni

Syndecan-1 Enhances Proliferation, Migration and Metastasis of HT-1080 Cells in Cooperation with Syndecan-2

Syndecans are transmembrane heparan sulphate proteoglycans. Their role in the development of the malignant phenotype is ambiguous and depends upon the particular type of cancer. Nevertheless, syndecans are promising targets in cancer therapy, and it is important to elucidate the mechanisms controlling their various cellular effects. According to earlier studies, both syndecan-1 and syndecan-2 promote malignancy of HT-1080 human fibrosarcoma cells, by increasing the proliferation rate and the metastatic potential and migratory ability, respectively. To better understand their tumour promoter role in this cell line, syndecan expression levels were modulated in HT-1080 cells and the growth rate, chemotaxis and invasion capacity were studied. For in vivo testing, syndecan-1 overexpressing cells were also inoculated into mice. Overexpression of full length or truncated syndecan-1 lacking the entire ectodomain but containing the four juxtamembrane amino acids promoted proliferation and chemotaxis. These effects were accompanied by a marked increase in syndecan-2 protein expression. The pro-migratory and pro-proliferative effects of truncated syndecan-1 were not observable when syndecan-2 was silenced. Antisense silencing of syndecan-2, but not that of syndecan-1, inhibited cell migration. In vivo, both full length and truncated syndecan-1 increased tumour growth and metastatic rate. Based on our in vitro results, we conclude that the tumour promoter role of syndecan-1 observed in HT-1080 cells is independent of its ectodomain; however, in vivo the presence of the ectodomain further increases tumour proliferation. The enhanced migratory ability induced by syndecan-1 overexpression is mediated by syndecan-2. Overexpression of syndecan-1 also leads to activation of IGF1R and increased expression of Ets-1. These changes were not evident when syndecan-2 was overexpressed. These findings suggest the involvement of IGF1R and Ets-1 in the induction of syndecan-2 synthesis and stimulation of proliferation by syndecan-1. This is the first report demonstrating that syndecan-1 enhances malignancy of a mesenchymal tumour cell line, via induction of syndecan-2 expression