Experim mental Proc cedures

2. Experim mental Proc cedures

inte erface is able e to accomm modate large er substituted d

2.1 Protein P Preparation

side e-chain residu ues.

Overprod duction and p purification o of wild-type and variant Ps s3 αHSDprote eins were performed as described pr reviously [20 ]. Protein pur rity was analy yzed with SDS-P PAGE on a a 12% poly yacrylamide gel. Protein con ncentrations w were estimat ted by assum ming

A 280 of 0.54 (wild-type a and T254F), 0

0.82 (R251W W and

R251W/T25 54F), and 0.6 1 (R251Y and d R251Y/T25 54F) for 1 mg·mL L -1 of protein. .

Fig. . 1 Crystal structure of f Pseudomona as sp. B-0831 1

2.2 Gel Filtr ration Chrom matography

3 -h hydroxysteroid d dehydrogen nase (Ps3 αHS SD) obtained d with h the program PyMol. The s ubstituted site es, Arg251 and d

For comp parison of m molecular mas sses, variant and

Thr r254, are indica ated.

A Test f for Stabilizati ion of an Olig gomeric Prote ein by Introdu uction of Aro omatic Residu ues into the I nterface 355 5

Table 1 Elu ution time in g gel filtration c chromatograph hy of Tab le 2 T m values of Pseudomonas sp. B-0831 1 Pseudomonas s sp. B-0831 3 -hydroxystero  oid dehydroge enase

3 -h hydroxysteroid d dehydrogen nase (Ps3 αHS SD) and its s (Ps3 αHSD) an nd its variants .

vari iants.

ΔT m m (ºC) WT HSD

Concentr ration (mg·mL -1 1 ) Time (mi n)

T m (ºC)

58.6 - R251Y

0.76 17.99 WT T HSD

44.9 -13. 7 R251W

1.10 17.97 R25 51Y

44.6 -14. 0 T254F

1.00 18.00 R25 51W

58.4 -0. 2 R251Y/T254

1.00 18.01 T25 54F

42.9 -15. 7 R251W/T254 4F 0.70

F 1.00

17.97 R25 51Y/T254F

18.29 R25 51W/T254F

42.4 -16. 2

Next, the e authors exa amined the h heat denatura ation vari iants with do ouble mutatio ons at positi ons 251 and d properties o of wild-type a and variant P Ps3 αHSDprot teins

254 4 displayed ~ ~16 ºC lower r T m than w ild-type, and d using DSC C to det termine the e changes in

thus s considerab bly less sta ability. Base ed on these e conformatio onal stability induced by t the mutations s, as

find dings, the a authors prop pose that th he aromatic c shown in Fi ig. 2. Heat d denaturation was irrevers ible,

resi idues introdu uced do not i interact stron ngly but have e allowing est timation of th he T m value ( (Table 2). The eT m slig ght unfavorab ble interactio ons with each h other. The e

values rang ged from -0

resu ults suggest t that “hot spo ot” residues s hould not be e signifying lo ower stability y of all the va ariants, comp ared

0.2 ºC to -1

16.2 ºC, cle early

repl laced.

to the wild-t type protein. S Substitutions of small to la arge side-chai ins in protein n Arg251 (R R251Y and R R251W) muta ants exhibite d an

core es often lead d to destabiliz zation due to o unfavorable e approximate e 14 ºC decre ease in T m , su uggesting that t the

inte eractions or c conformationa al strain, sinc ce the protein n residue is cr ritical for the ermostability of Ps3 αHSD

core e is tightly p acked [22-26 6]. In oligom er interfaces, , contrast, the eT m value wa as not signifi icantly decre ased

D. In

larg ger side-cha ains may b be accommo odated more e in the T254 4F mutant ( (-0.2 ºC), in ndicating tha at at

favo orably, owing g to the highe er flexibility o of this region n. position 254, an a 2 aromatic re sidue can be How wever, the present res sults demon nstrated that t

accommoda ted within th he interface w with no nega ative intr roduction of l large aromatic c residues de stabilizes the e effects. Th hese data suggest co ontext-depen ndent

prot tein, leading g to the c conclusion that protein n contribution n to interfac ce formation n and hot spot

inte erfaces are n naturally wel ll designed w with optimal l

residues for oligomerizat tion [21]. Bo oth the Ps3 αH HSD

inte eractions and no conforma ational constra aints.

Fig. 2 Therm mal denaturati ion curves of P Ps3 Αhsd and it ts variants.

A Test for Stabilization of an Oligomeric Protein by Introduction of Aromatic Residues into the Interface

4. Conclusion

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Apr. 2013, Vol. 7, No. 4, pp. 358-362 Journal of Life Sciences, ISSN 1934-7391, USA

Automated Classification of Segmented Cancerous Cells in Multispectral Images

Alaa Hilal, Jamal Charara, Ali Al Houseini, Walid Hassan and Mohamad Nassreddine Signal and Image Processing Lab, Faculty of Sciences I, Lebanese University, Beirut, Lebanon

Received: December 13, 2012 / Accepted: January 24, 2013 / Published: April 30, 2013.

Abstract: Automatic reading procedures in colon cells biopsies allow a faster and precise reading of microscopic biopsies. These procedures implement automatic image segmentation in order to classify cell types as cancerous or noncancerous. The authors have developed a new approach aiming to detect colon cancer cells derived from the “Snake” method but using a progressive division of the dimensions of the image to achieve rapid segmentation. The aim of the present paper was to classify different cancerous cell types based on nine morphological parameters and on probabilistic neural network. Three types of cells were used to assess the efficiency of our classifications models, including BH (Benign Hyperplasia), IN (Intraepithelial Neoplasia) that is a precursor state for cancer, and Ca (Carcinoma) that corresponds to abnormal tissue proliferation (cancer). Results showed that among the nine parameters used to classify cells, only three morphologic parameters (area, Xor convex and solidity) were found to be effective in distinguishing the three types of cells. In addition, classification of unknown cells was possible using this method.

Key words: Multispectral image, classification, morphologic parameters, probabilistic neural network.