The structure of the influenza virus neuraminidases, the spatial organization of their active site, the mechanism of carbohydrate chains desialylation by neuraminidase, and its role in the influenza virus function at different stages of the viral infectious cycle are considered in this review. Data on the neuraminidase substrate specificity and different approaches in studying the activity of this enzyme are summarized. In addition, data on neuraminidase inhibitors  are provided, along with considerations on the mechanisms of resistance of modern influenza viruses to those antivirals.  The influenza virus is an enveloped (-)RNA containing a virus with a segmented genome, and its genetic material is coded by eight RNA-segments. All RNA segments are packed in a nucleocapsid protein, and a complex of polymerase proteins is attached to each of the genomic segments. Those RNA-protein complexes are packed in a lipoprotein envelope lined from the inside with a matrix protein, with haemagglutinin, neuraminidase, and M2 proteins exposed on the outer surface of the viral particle.Neuraminidase is an exosialidase EC 3.2.1.18) which cleaves α-ketosidic linkage between the sialic (N-acetylneuraminic) acid and an adjacent sugar residue [.18 whichThe enzyme of the influenza C virus does not belong to the neuraminidase group. It promotes the O-deacetylation of the N-acetyl-9-O-acetylneuraminic acid, i.e. it belongs to the esterase family and will not be considered in this review.The influenza virus NA executes several functions. Firstly, its activity is required at the time of the budding of newly formed viral particles from the surface of the infected cell, to prevent aggregation of viral particles. In addition, NA cleaves neuraminic acid residues from the respiratory tract mucins; by doing so, it facilitates viral movement to the target cell. Those functions will be considered further in more detail.The polypeptide chain of the influenza virus NA comprises 470 amino acid residues. The three-dimensional structure of NA consists of several domains: the cytoplasmic, transmembrane, "head," and also "stem," connecting the head to the transmembrane domain.On the virion surface, NA resembles a homotetramer of a mushroom shape: head of 80*80*40 Å on the thin stem, 15 Å wide and from 60 to 100 Å long . The molThe enzyme active site and calcium binding domain, which stabilizes the enzyme structure at low pH values, are situated in the head of NA ; 8.Homology between the strains inside one subtype attains about 90%, whereas homology between subtypes is 50%, and 30% between А and В types .1 As amino acid sequences of different neuraminidases differ from one another by insertions and deletions, it is common practice to highlight NA subtype according to which the numbering of amino acids is done, usually, as in this case N2 subtype numbering is used.The calcium binding site, which is located inside the molecule  is formed by the oxygen of the main chain residues 297, 345 and 348, as well as by the oxygen of the side chain of Asp324 , 6. AddiThe second neuraminic acid binding site, the so called HBsite, was found in N9 neuraminidases . The a.a367 − 372, which is involved in neuraminic acid binding via serine residues 367, 370 and 372;400 − 403, which interacts with the substrate via the side chain of asparagine 400, the carbonyl oxygen of the main chain of asparagines, and tryptophan 403;430 – 433, which interacts with neuraminic acid via the ε-amino group of lysine 432.All six above-mentioned conservative amino acids were found only in N9 NA. Avian influenza virus NAs of other subtypes usually lack lysine 432, but its absence does not interfere with their haemoadsorption activity. Human influenza virus neuraminidases usually lack the HB-site a.a. sequence. At the same time, two early isolates of human influenza viruses of the Н2N2 subtype (RI+/57 and A/Leningrad/ 134/57) have the HB-site "frame" (serine triplet and tryptophan) , 11, whiThe function of the HB-site has yet to be clarified. It has been suggested that it may play the role of an alternative neuraminic acid binding site, in other words, function as a surrogate of the influenza virus HA; this assumption is based on the existence of viruses with combined HA and NA functions in one protein molecule, such as the ND virus. The HB-site, described earlier, is common for the NAs of viruses which HA interacts with α2-3-sialylated carbohydrate chains (i.e. avian and equine influenza viruses), at the same time, the key amino acid positions of this site are changed in viruses with α2-6-specificity . It is worth mentioning that the H9N2 influenza viruses isolated from poultry in Hong Kong and viruses of H2N2 and H3N2 subtypes, which caused human pandemics, have similar changes in the HB-site sequence. This data allows one to suggest that some species of poultry may act as intermediate hosts in the influenza virus transfer from its natural reservoir (waterfowl population) to humans .2+ binding site [The three-dimensional structure of cytoplasmic, transmembrane and stem domains has not been determined yet  . There iing site .Carbohydrate chains are attached to Asn residues located in the different regions of the NA's head. In particular, glycans attached to asparagines 86 and 234 are headed towards the lipid membrane close to the stem; those attached to Asn146 – are headed from the membrane and are located in close proximity to the active site; finally, glycosylation Asn200 is located on the side surface in the region of the subunits contact. Short oligomannose type chains were found at residues Asn86 and Asn200. Carbohydrate chains of a complex type are attached to Asn146 and Asn234. The glycosylation site at Asn146 is conservative for all NAs, and this carbohydrate chain differs from all other carbohydrates found in all influenza virus glycoproteins: it carries the О-4 sulfated N-acetylgalactosamine . DeletioThere are eight conservative disulfide bonds in the NA structure, and one additional bond in the N2, N8, and N9 subtypes. The invariance of disulfide bonds confirms their importance in the formation of a stable NA structure. It is assumed that, because of its proximity to the symmetry axis of the tetramer, the uncoupled Cys161 of N1 NA takes part in the coupling of subunits. The tetramer assembly mechanism is not universal: for instance, in influenza virus B neuraminidases disulfide bonds are formed by Cys54, whereas Cys78 takes part in polypeptide chains binding (N2 numbering) [The Neu5Ac binding site is located above the first strands of the third and the fourth motifs in a big loop on the NA surface. The active site is located at the N-terminal end of central parallel strands ; The enzyme active site consists of functional amino acid residues Arg118, Asp151, Arg152, Arg224, Glu276, Arg292, Arg371, and Tyr406, and structural amino acid residues Glu119, Arg156, Trp178, Ser179, Asp (or Asn in N7 and N9) 198, Ile222, Glu227, Glu277, Asp293, and Glu425.Functional a.a. are in direct contact with sialic acid, the product of the enzymatic reaction, and they all form polar contacts with it, excluding Arg224, whose aliphatic part forms a nonpolar contact with the glycerol fragment of the Neu5Ac residue  .Recent X-ray studies of neuraminidases from the first phylogenic group have shown that, in comparison with neuraminidases from the second phylogenic group, they have a slightly different structure of the polypeptide chain around the enzyme active centre. In particular, there is a cavity in close proximity to the active site, which is formed by a change in the dimensional orientation of "loop 150." These structural differences allow the launch of the development of influenza virus NA inhibitors, which would specifically interact only with NAs of the first phylogenic group, in particular with the NA of influenza viruses of the H5N1 subtype .The NA's reaction mechanism Formation of the oxocarbonium ion at the С2 atom of Neu5Ac is the key step in the hydrolysis of the oligosaccharide substrate. After the introduction of the Neu5Ac residue into the active centre, Neu5Ac conformation changes from chair to half-chair, i.e. the oxocarbonium ion is formed, due to strong ionic interactions between the carboxylate of the substrate and the guanidine groups of the arginines 118, 292 and 371, eventually leading to glycosidic bond cleavage. The molecule of aglycone leaves the enzyme active site with glycosidic oxygen, protonated by the solvent. Multiple contacts between the intermediate product and the a.a. of the active site (Tyr406 and Asp151 are of minor importance) stabilize the positively charged oxocarbonium ion with preservation of the planar carbon at С2. Neu5Ac2en, in which the C2 atom is in sp2-form, mimics the intermediate reaction product in planar conformation . HydroxyThe presence of invariant residues in the active site, the similarity of the structural organization, and the architecture of complexes with Neu5Ac and with Neu5Ac2en allow to assume that the mechanism of NA functioning for the A and B influenza viruses is identical .The structure of the neuraminidase active site is strictly conservative not only between subtypes, but also between the types of the enzyme, which points to the importance of all its components and the evolutionary stabilized functioning of this system. This observation has allowed to design an NA inhibitor for the influenza virus which mimics the transition state of the hydrolysis reaction, and Neu5Ac2en The success of this drug has initiated a number of studies aimed at designing new NA inhibitors. The main structural elements of the new class of inhibitors (without the oxygen atom in the cycle) are cyclohexane and cyclopentane. One of those structures is the -3-amino-4-acetamido-5-(1-ethylpropoxy)-1-cyclohexene-1-carboxylic acid (oseltamivir or Tamiflu) Besides, a NA inhibitor on the base of a cyclopentane structure has been developed; it has all the functionally important parts of zanamivir  which fit into the NA active centre. BCX-1812 (preamivir) Zanamivir and oseltamivir are already used as drug products, whereas BCX-1812 has entered the last phase of clinical trials.Until recently, it was considered that active uncontrolled use of zanamivir and oseltamivir would not have a significant influence on the development of resistance in influenza virus strains. That is, even if resistant strains emerge, they would not be able to replicate in the absence of the inhibitor . The numHowever, in January 2008 this situation changed dramatically: some H1N1 influenza viruses developed resistance to oseltamivir due to a mutation His274Tyr in NA , and in There is data indicating that NA is relevant at different stages of infection. Firstly, it is considered that it helps the virus approach the target cells by cleavage of sialic acids from respiratory tract mucins . SecondlOne of the most interesting features of the influenza virus is the coexistence of two proteins whose functions are to some extent contradictory, namely: haemagglutinin, which has a receptor-binding function; and neuraminidase, which has a receptor destroying function. Since both of these proteins recognize terminal neuraminic acid residue, this brings up the question of their cooperation or, on the contrary, their competition for receptor/substrate, and of the role of their relations in viral life cycle. Studies of the viruses resistant to NA inhibitors, artificial viral reassortants (which have HA and NA of different origins), and virus particles designed by means of reverse genetics, which lack NA or HA activity, show that the NA and HA of the influenza virus act in concert and their evolution proceeds interdependently -35. AlsoOne of the most popular substrates for NA activity determination is MU-Neu5Ac or MU-NANA The sensitivity of the chemiluminescent method of NA activity determination  is by a The other group of methods is based on cleavage of the neuraminic acid from high molecular weight substrates, such as fetuin, the α1-acid glycoprotein or whole erythrocytes. The amount of free neuraminic acid is usually determined after cleavage ; the mosAn alternative procedure is based on assay of the second product of the hydrolysis, the desialylated glycoprotein, with the help of lectin , which is specific for the unmasked terminal galactose , 42. CarThe substrate specificity of NA is its ability to discriminate between sialic acids  and linkage type with the next residue , as well as the ability to identify internal regions of the oligosaccharide chain. In particular, the following structures have been used for the determination of NA substrate specificity:• free trisaccharides (3' SiaLac or 6'SiaLac)  - 45,• glycoproteins containing only 2-3, or only 2-6-linked neuraminic acid , 46,• glycoproteins or erythrocytes oversialylated with the aid of 2-3- or 2-6- sialyltransferases .Methods based on the use of those substrates achieve only one of the listed goals; in particular, they allow to study specificity at the level of Sia2-3Gal or Sia2-6Gal. More broad specificity can be studied with the use of an analytical procedure which employs a number of synthetic substrates. In , a panelAs already mentioned above, high molecular weight substrates, along with low molecular weight substrates, can be used for studying NA activity and specificity. Low molecular weight substrates allow to study the reaction mechanism and desialylation kinetics without the complications of multivalent interactions (NA is a tetramer) and the possible influence of HA, which interacts with multivalent conjugate 3 – 5 orders of magnitude better than with the monomeric one . High moInvestigation of the evolution of the influenza virus NA substrate specificity for viruses isolated from humans, and its comparison with the substrate specificity of influenza virus NAs isolated from different hosts, such as ducks and pigs, is of great importance. The first undertaking could shed light on the question of the unique character of pandemic strains, while the second could help detect in advance the properties of the enzyme which facilitate the crossing of the interspecies barrier.The specificity of N2 subtype NA of human influenza viruses has gradually changed from 3'SiaLac (H2N2 strains isolated in 1957) to dual specificity, 3'SiaLac/6'SiaLac (strains from 1972 to 1987). Hydrolytic activity towards 6'SiaLac was identified only for viruses isolated in 1967 and further, and starting from 1972 isolates an increase of activity towards this substrate was registered .It has been shown recently that N2 influenza viruses are highly active towards 3'SiaLac, while their activity towards 6'SiaLac varies from extremely low (avian and early human isolates) to high (swine and latter human isolates). It has been shown that NA activity towards 6'SiaLac depends also on the host type and, for human viruses, on the year of isolation .For N1 strains isolated in the 70-80s , 44, it Data on the substrate specificity of N1 and the N2 NAs of several duck, swine and human influenza virus isolates were obtained with the use of BODIPY-labeled synthetic oligosaccharides -51. All With the use of the polyacrylamide conjugates of 3'SiaLac, 6'SiaLac and 6'SiaLacNAc, it has been shown that most of the viruses (H1N1 and H3N2 subtypes) propagated on embryonated chicken eggs and MDCK cells, preferably hydrolyze 3'SiaLac, whereas VER O-isolates of the same viruses preferably hydrolyze 6'SiaLacNAc. In summary, the nature of the host cell line used for virus accumulation influences NA substrate specificity . The reaIt is difficult to compare the results of substrate specificity studies conducted by different authors due to the use of both different influenza virus strains and different substrates in varying concentrations. For example, the Kobasa  group haDespite the limited amount of data published to date, it is already possible to discuss some features. Firstly, the NA substrate specificity of human isolates differs from that of avian isolates. Secondly, the oligosaccharide specificity of the NA of viruses which circulate in different hosts  notably differs, at least for the characteristic "ratio of the hydrolysis velocity of 2-3 oligosaccharides towards 2-6 oligosaccharides." Thirdly, the substrate specificity of influenza virus neuraminidases propagated on different cell lines may be different.Data on NA functioning would be incomplete without taking into account another surface protein of the influenza virus, haemagglutinin. There is only a limited number of publications describing the simultaneous study of HA and NA substrate specificity, and there is virtually no research where the dependence of HA and NA oligosaccharide specificity on one hand and virus infectivity on another hand have been studied. The state-of-the-art analytical procedures for NA introduced in the current review are now up to par with the more advanced analytical methods of HA analysis, which always developed faster; therefore, it is quite easy to predict that one of the main trends in influenza virus studies in the future would be joint studies of HA and NA specificity.


PTEN hamartoma tumour syndrome (PHTS). All the aforementioned syndromes are inherited in an autosomal dominant manner and form a rather heterogenous group both in respect to the number and localization of polyps and the risk of cancer development in the alimentary tract and other organs. Individual syndromes of hamartomatous polyposis frequently manifest similar symptoms, particularly during the early stage of the diseases when in several cases their clinical pictures do not allow for differential diagnosis. The correct diagnosis of the disease using molecular methods allows treatment to be implemented earlier and therefore more effectively since it is followed by a strict monitoring of organs that manifest a predisposition for neoplastic transformation.Hamartomas are tumour-like malformations, consisting of disorganized normal tissues, typical of the site of tumour manifestation. Familial manifestation of hamartomatous polyps can be noted in juvenile polyposis syndrome (JPS), Peutz-Jeghers’ syndrome (PJS), hereditary mixed polyposis syndrome (HMPS) and  PTEN hamartoma tumour syndrome (PHTS). It has also been suggested that the complex includes, i.a., Cowden’s syndrome (CS), Bannayan-Riley-Ruvalcaba syndrome (BRRS), Proteus’es syndrome (PS). All the aforementioned syndromes are inherited in the autosomal dominant manner and are conditioned by mutations in four genes  is a rare disease inherited in an autosomal dominant manner, described by McColl in 1966 900 is a . In mostJuvenile polyposis is diagnosed according the following criteria :– at least 3 polyps detected on colonoscopy– juvenile polyps in the entire digestive tract– in cases of family history of the disease any number of juvenile polyps.Three categories of the disease are distinguished ,10:– juvenile polyposis of infants– juvenile polyposis of the large bowel– general form of juvenile polyposis.The difference between juvenile polyposis of the large bowel and the general form of juvenile polyposis depends on the localization of the polyps. It is estimated that in over 20% patients with JPS inborn errors are detected in various organs. In the alimentary tract Meckel’s diverticula with umbilical fistula andmalrotation of small intestine have been detected. Cases of undescended testes, unilateral renal agenesia and split uterus were diagnosed in the urogenital system. Inborn errors in the chest include a defect in the interatrial septum, arterionevous haemangiomas, stenosis of the pulmonary valve, Fallot’s tetralogy, aortal stenosis, and persisting arterial duct. Macrocephaly, a communicating hydrocephalus and rachischisis were found within the central nervous system. Moreover, osteomas, mesenteric haemangiomas, inherited teleangiectasias, hypertelorism, inborn amniotony, supernumerary toes and acute intermittent porphyria have also been seen.SMAD4 and BMPR1A genes [The manifestation of juvenile polyposis is preconditioned by mutations in 1A genes ,11,12.BMPR1Ac  resides in chromosome 10, in q22-23. The gene consists of 11 exons. It codes for a protein of 532 amino acids, belonging to the family of TGF-β/BMP, representing a type I receptor with properties of serine-threonine kinase [BMPR1A gene includes 3,613 nucleotides [Gene e kinase . The traleotides ,15. It uSMAD4  gene is located in chromosome 18, in the region of q21.1. It consists of 11 exons. The genomic sequence of the gene includes 50 thousand base pairs and its respective mRNA consists of 3,197 nucleotides. It codes for a protein including 552 amino acids. SMAD4 is a suppressor gene and it participates in the passage of a signal along the pathway of transforming growth factor β (TGF β) and its ligands [SMAD4 is included among the “common” SMAD, it contains two conserved domains of MH1 and MH2 (Mad Homology domain). The amine terminus of the SMAD terminus ends with an MH1 hair-pin domain, demonstrating DNA-binding activity. The carboxy terminus of SMAD proteins ends with a strongly conserved domain of MH2. It is responsible for interaction with proteins involved in the translocation of the complex to cell nucleus and interaction with DNA-binding cofactors [nuclear export signal) recognized by CMR1. Interaction of SMAD4 with phosphorylated Co-SMAD masks NES, protecting SMAD4 from recognition by CMR1 and from export to the cell nucleus. Dephosphorylation of receptor SMAD and dissociation of the complex allows SMAD4 to be exported . The import of SMAD proteins to the cell nucleus develops with no involvement of nuclear transport factors. Such an importin-independent transport has also been described in cases of components participating in other transformations, e.g. in the case of β-kateniny along the pathway of Wnt. This is possible due to the direct interaction of SMAD with nucleoporins, mediated by interaction of the hydrophobic corridor in the MH2 domain with the region of FG repetitions in nucleoporins [The  ligands . SMAD4 iofactors . The lineoporins .SMAD4. The complex formed in this way passes to the cell nucleus where it becomes involved in the control of expression including several genes, as either positive or negative regulators of the alterations [SMAD4 with R-SMAD binds to DNA through the MH1 domain, recognizing the palindrome DNA sequence of GTCTAGAC. Such an SMAD-binding sequence (SBE) is frequently noted in genes which undergo expression in the presence of TGF β/BMP ligands. GTCTAGAC SBE is present, on average, in every 1,024 base pairs in the genome or at least one such sequence can be noted in a control region of every moderate size gene [The phosphorylated SMAD bind with Co-SMAD, i.e. erations ,19. Actiize gene . The litize gene . The firSMAD4 gene have been noted in around 20% of patients with familial juvenile polyposis [BMPR1A gene. In these genes over 120 mutations have been detected as leading to the development of polyps linked to juvenile polyposis syndrome. The mutations include first of all, small alterations, point mutations and small deletions. Nevertheless, a high proportion of alterations detected in patients with juvenile polyposis also involve extensive lesions. Large deletions were observed in the region of q22-q23 of chromosome 10. The alterations affected the two neighbouring genes of PTEN and BMPR1A. Mutations in these genes are engaged in the development of distinct syndromes of hamartomatous polyposis.Mutations in the olyposis ,20 whileSMAD4 gene. This mutation is located in a hot spot, a region containing four binucleotide repetitions of AG, where the looping off of a DNA strand fragment probably takes place, which undergoes a deletion.The mutations that have been described so far are of a heterogenous character, with the exception of a single mutation c.1244_1247delAGAC in exon 9 of the SMAD4 gene, who were found with higher frequency to carry large polyps in their stomachs. Germ-line mutations in the SMAD4 gene are responsible for a more aggressive phenotype of juvenile intestinal polyposis, manifesting itself in the form of a vascular malformation within sublayer components when the mutation was located before the codon of 423. It was also noted that polyps with mutation in the SMAD4 gene are detected in both upper and lower parts of the digestive tract while polyps with mutations in BMPR1A gene are restricted to the rectum and the anal canal.Certain correlations were detected between phonotype and genotype in JPS patients carrying a mutation in the Peutz - Jeghers syndrome  is inherited in an autosomal dominant way. The first description of the syndrome was published by L.A.Peutz in 1921, 28 years later H. Jeghers described the clinical presentation of the disease in detail. The first signs in the form of hamartomatous polyps and pigment skin lesions appear in childhood. Incidence of the syndrome ranges from 1/29,000 to 1/120,000 births. The polyps appear during the second or third decade of life in 80-100% of patients. They may be located all along the alimentary tract although the frequency of their manifestation is variable and depends on localization within the digestive tract Table . They arSTK11 (OMIM*602216 Serine/Threonine Protein Kinase 11) gene, located in the short arm of chromosome 19, in the 13.3 region. The gene consists of 10 exons, of which 9 code for a protein. It undergoes comprehensive expression during embryonal development but also in the organs of adults, particularly in the pancreas, the liver and skeletal muscles. The STK11 protein consists of three main domains, the N-terminal non-catalytic domain with two signals for nuclear localization, the highly conserved kinase domain and the regulatory domain at the carboxy end. The kinase domain of this 433 amino acid protein is located between the 49th and 309th amino acid. The STK11 protein contains a few sites which undergo phosphorylation and prenylation and the nuclear localization signal (NLS). Due to the activity of kinases serins are phosphorylated at positions of 31 and 325 and threonine at position 363. STK11 is also capable of undergoing phosphorylation on threonins at positions 185, 189, 336, and on serine at position 402. Autophosphorylation of STK11 at position Thr189 is very important for kinase activity of the protein. On the other hand, the prenylation motive of Cys430-Lys-Gln-Gln433 is positioned at the carboxy terminus of the protein. Loss of STK11 protein function precipitates the development of various defects. This reflects the fact that the STK11 protein is involved in a number of important cellular processes. In Xenopus, a homologue of the STK11 gene, XEEX1, is engaged in the process of early embryonal development. On the other hand, mice lacking the STK11 gene die at around the 8th day of embryogenesis. In the case of STK11+/− mice, manifestation of polyps is observed, which in histopathology are very similar to those noted in PJS. Molecular analysis showed that loss of a single STK11 allele is sufficient for the development of the polyps. Following the 45th week of life in >70% male STK11+/− mice and in 20% female STK11+/− mice histopathologically variable types of liver malignancies are seen. The cancer cells showeda loss of both STK11[STK11 was found to control the TGF β pathway, forming a complex with the SMAD4 protein through LIP1. LIP1 forms a specific bridge between the two proteins [STK11 also interacts with the PTEN protein [STK11 kinase participates in p53-dependent apoptosis.Peutz - Jeghers syndrome is preconditioned by the manifestation of mutations in the oth STK11,27. STK1proteins . STK11 a protein . MoreoveSTK11 have been detected in 70% patients with an inherited form of the disease. In cases of patients with negative anamnesis` detectability of the mutations approximates 50% [STK11 gene over 230 mutations have been described so far, including 70 point mutations. A significant portion of the mutations include small deletions (54) and small insertions (33). Nevertheless, large deletions, including individual exons or even deletions of the entire gene are also frequent in patients with PJS [Germ-line mutations of ates 50% . In the with PJS .Cowden Disease; CD) is a very rare syndrome of hamartomatous polyposis. Its incidence is estimated at 1 per 200,000 deliveries. Its typical trait involves various hamartomatous lesions in tissues originating from three germ layers: the endoderm, ectoderm and mesoderm. Apart from the gastrointestinal region (71% of the patients), the lesions are manifested in the skin, mucous membranes and other organs. An international consortium dealing with Cowden’s syndrome evaluated a diagnostic criteria, which includes dermomucosal lesions, including fibromas of the oral cavity and papillary and hyperkeratotic alterations on the face and extremities [PTEN  gene, responsible for the development of Cowden’s syndrome was mapped in 1997 on chromosome 10, region q23. This is a suppressor gene, coding for a protein consisting of 403 amino acids, representing a phosphatase responsible for the removal of phosphate groups from molecules. Definition of PTEN crystalline structure showed that the N-terminal domain of the phosphatase strictly adheres to the C2 domain at its carboxy terminus. The two domains create a basic catalytic unit, encompassing the almost entire peptide sequence of the protein, with the exception of the small tail in the N-terminal portion of the protein and a longer 50 amino acid-fragment at the carboxy terminus. The domain of phosphatase and C2 creating the catalytic core of the protein are sufficient for its normal function. The remaining parts seem to be involved in the control of activity and/or interaction of PTEN with the other molecules [PTEN can de-phosphorylate both proteins and lipids of cell membrane. It removes the phosphate group from inositol ring position D3, from 3,4,5 phosphatidylinositol triphosphate and 3,4 phosphatidylinositol diphosphate, produced during the transmission of cellular signals through the activity of phosphoinositol 3' kinase (PI3K) [PTEN acts as a specific switch-off for signal transmission along the PI3K pathway, and in this way stops the cell cycle at the G1 phase. The activity of the PTEN gene, involving antagonization of phosphoinositol 3’ kinase action, inhibits the activity of multiple oncoproteins exerting their effect through the PI3K kinase. PTEN-PI3K controls fundamental cellular processes linked to the mechanism of neoplastic transformation. PTEN participates in the control of the cell cycle through Akt kinase. Among substrates of Akt kinase which play a significant role in the cell cycle transcriptional factors such as FKHR (Forkhead transcription factor), AFX, FKHRL1 or GSK3 can be distinguished. PTEN also controls cell divisions. In pten−/− fibroblasts the response to stimulation of apoptosis is lowered due to the augmented transcription of proapoptotic genes, i.e. FAS and Bim. The activity of PTEN protein phosphatase leads to the inhibition of FAK , responsible for cellular adhesion and the capacity of cells to migrate [PTEN protein plays also an important role in the process of angiogenesis and participates in control over the mTOR pathway. In D. melanogaster loss of the dpten gene function results in an increased size of cells and organs while overexpression of dpten in yeasts causes an inverse phenotypic effect. In the case of mice loss of the pten gene in neurons leads to the development of a set of traits resembling those present in Lhermitte-Duclos disease, representing one of the clinical presentations of Cowden’s syndrome [Cowden’s syndrome  . PTEN ac migrate . PTEN prsyndrome .PTEN gene lead to the development of a number of various neoplasms. They are detected in 80% of patients with Cowden’s syndrome. Mutations in the PTEN gene are also detected in other syndromes of hamartomatous polyposity, among others in Bannayan-Riley-Ruvalcaba’s syndrome. The frequency of lesions being detected in the PTEN gene approaches 60%. In the computer base of mutations 208 mutations are described in the PTEN gene, of which the majority involved mutations of altered sense and nonsense mutations (87 mutations), small deletions and insertions (75 mutations) [PTEN gene are noted in the promoter region while deletions of a portion or entire gene are commonly observed in patients with Bannayan-Riley-Ruvalcaba’s syndrome.Somatic mutations in the tations) . In patiThe morbid unit  first appeared in literature in 1971: the case was described of an 11-year-old girl with juvenile polyps and adenomas in the colon and small intestine. However, it was not until 1987 that Sarles suggested the term of mixed polyposis describing the cases of a father and son with numerous various types of polyps in the colon. In the father they included metaplastic polyps and adenomas, in the son juvenile polyps were additionally diagnosed. In the most accurate way clinical traits of mixed polyposity syndrome were presented in a family of a few generations, termed SM96 ,46. AmonCao presented two three-generational families with a course of the disease very similar to that of the SM96 family. In most of the cases polyps were located in the large bowel. In those family members Cao noted no extraintestinal lesions.Individuals with HMPS were found to manifest an augmented predisposition to the development of malignancies in the large bowel.BMPR1A gene. In the remaining family members the mutation could not be detected. Mixed polyposis is the least recognised disease among the hamartomatous polyposity syndromes and it still requires further experimental studies, which would lead to a rapid and simple diagnosis.The gene responsible for the development of mixed polyposity has not yet been identified. Analysis of linkages defined the region strictly bound to the appearance of the morbid signs. This was termed CRAC1 and it is located on chromosome 15, on its long arm. In a proband in one of the families presented by Cao a heterozygous mutation was disclosed, a deletion of 11 nucleotides in exon 2 of the Morbid syndromes linked to hamartomatous polyposities form a rather heterogenous group both in respect to the number and localization of polyps and the risk of cancer development in the alimentary tract and other organs. Even if the diseases are not among the most frequently occurring ones they remain dangerous to patients not only due to their predisposition to manifest themselves as a neoplastic disease but also due to non-neoplastic signs, such as haemorrhages, intussusception and ileus. Every hamartomatous polyposis syndrome manifests its own organ-specific localization of /symptom manifestation and, consequently , each of them requires a different strategy of diagnostic management. Therefore, an accurate qualification of the patients to a specific morbid syndrome represents a basic step towards the appropriate care of patients affected by the predispositions. Recommendations related to diagnostic studies in hamartomatic polyposities are based only on the opinions of experts. To date, no randomised diagnostic studies have been conducted on the efficacy of medical care programmes in the diseases . For PeuSMAD4 so the potential for aberrant vascular development should be taken into account. Attention should be paid to the occurrence of heamorrhages, anaemia, abdominal pains, diarrhea and alterations in the shape or colour of foeces in the patients. Manifestation of such alterations indicates the necessity of performing additional studies, including colonoscopy. In asymptomatic patients endoscopic tests (gastroduodenoscopy and colonoscopy) should be conducted beginning from the 15th year of age. If endoscopy discloses the presence of polyps they should be removed and, in such a situation the test should be repeated and polyps should be removed every year [Beginning at the 24th year of age it is also recommend to subject the patient every 1–2 years to a USG examination of the pancreas. In women the tests include monthly self-examination of breasts from 18 years of age onwards and an according to international guidelines for hereditary breast cancer, annual breast ultrasound and MRI from age 25 and annual mammography from age 35 . The ovaery year . If no pery year ,52. Cowdery year ,53. ExamThe basic method in the care of patients with the above polyposis syndromes involves an endoscopic surveillance with the regular removal of qualifying polyps . In cases of a severe course, with a high number of rapidly growing polyps in the colon, a colectomy with ileo-rectal anastomosis may be recommended . If a grThe authors declare that they have no competing interests.All authors contributed to the literature search and manuscript preparation. All authors read and approved the final manuscript.


ANG II triggered contraction in FPSS and PSS, but Bay K8644 only in PSS. Ischemia reduced and reperfusion intensified the response of the artery to ANG II, but did not change the action of Bay K8644. SNP and 8Br-cGMP reduced the response of the vessels to ANG II and did not change the modulating effect of ischemia, but reduced the intensifying action of reperfusion on contraction caused by the presence of ANG II. SNP lowered the action of Bay K8644 in PSS. In PSS, L-NAME and ODQ intensified the action of ANG II, eliminating the reducing effect of ischemia on the contraction caused by ANG II, but did not influence the intensifying reaction caused by reperfusion. L-NAME and ODQ did not influence the action of Bay K8644. I/R modulated the contraction of arteries triggered by ANG II, but did not influence the response to Bay K8644. The intra- and extracellular pools of calcium ions mediate the action of ANG II, but Bay K8644 stimulated contraction only with participation of calcium ions flowing into the cell. Control of the vascular smooth muscle tone associated with the action of NO and cGMP is subject to modulation under conditions of I/R.Vascular smooth muscle tone changes under the influence of numerous contracting and relaxing factors. The purpose of the present study was to determine the modulating effect of ischemia and reperfusion (I/R) on contraction triggered by angiotensin II (ANG II) and Bay K8644 as well as to investigate the importance of nitric oxide (NO) and cGMP in these reactions. Experiments were performed on isolated and perfused Wistar rat tail arteries. The contraction triggered by ANG II and Bay K8644 with the use of intracellular  and extracellular  pools of calcium ions after I/R and in the presence of sodium nitroprusside (SNP),  The contractibility of blood vessels depends on their normal structure and the availability of calcium ions; it changes under the influence of a number of contracting  and relaxing  factors, which control the activities of various pathways of intracellular and intercellular signaling . The endI/R and oxidative stress are pathological phenomena, which may change the reactivity of the vessels and modulate the effect of substances which control the vascular smooth muscle tone ,11. TissThe purpose of the present study was to establish the modulating effect of I/R on contraction triggered by ANG II (an agonist of the metabotropic AT1 receptor) and Bay K8644  as well as to investigate the role of the signaling pathway associated with NO and cGMP in these reactions.μM/l), a clamp was placed on the proximal segment of the prepared artery for 30 or 60 min and the artery was then removed. Then, a cannula was inserted into the proximal segment of the detached fragment of the rat's caudal artery measuring 2.5–3 cm in length, which was subsequently connected to the perfusion system and the equipment to allow constant measurement and recording of the perfusion pressure. The distal end of the prepared artery was loaded with a weight of 500 mg, and the preparation was placed in a vertical position in a thermostatic dish intended for isolated organs with a volume of 20 ml, oxygenated with saline at a temperature of 37°C. The flow of the perfusion fluid was gradually increased until it reached 1 ml/min. During the next stage, the contraction of the arteries was evaluated after 30, 60 and 120 min of reperfusion.In the experiment, Guiding Principles for the Care and Use of Animals in the Field of Physiological Sciences as well as specific national laws were followed. The Ethical Committee for the Affairs of Experiments on Animals in Bydgoszcz approved the experiments undertaken (No. 1/2008-4). All reagents were purchased from Sigma-Aldrich . Studies were performed on isolated and perfused Wistar rats' tail arteries. Animals, with body weights ranging from 250 to 350 g, were anesthetized with urethane administered intraperitoneally at a dose of 120 mg/kg of body weight. In order to establish the effect of I/R on the reactivity of the vascular smooth muscle triggered by ANG II (30 nM/l) and Bay K8644 (30 μM/l) as a donor of NO, 8Br-cGMP (100 μM/l), an endothelial NO synthase (NOSe) inhibitor  or ODQ , the experiments were conducted using two types of Krebs fluid: i) Fluid without Ca2+-EGTA , with the following composition: NaCl (71.8 mM/l), KCl (4.7 mM/l), NaHCO3 (28.4 mM/l), MgSO4 (2.4 mM/l), KH2PO4 (1.2 mM/l) and glucose (11.1 mM/l) with the addition of EGTA (30 μM/l); ii) Fluid with Ca2+-EGTA  with the the same composition as FPSS with CaCl2 (1.7 mM/l), after emptying the intracellular pool of calcium ions.In order to evaluate the involvement of intracellular and extracellular calcium ion pools in reactions triggered by the studied agonists under control conditions, following I/R and in the presence of sodium nitroprusside .Results are presented as average values and standard deviation. Statistical differences were evaluated using the Student's t-test. P<0.05 was considered to indicate a statistically significant result. Calculations were conducted with Statistica 6.0PL software.ANG II triggered an increase in perfusion pressure in FPSS and PSS, but the values were higher in PSS . After 6μM/l) reduced the control response of the arteries to ANG II with involvement of intra- and extracellular pools of calcium ions and reduced the contraction triggered by Bay K8644 in PSS , and in PSS it was 31 mmHg . The presence of SNP significantly reduced the intensifying effect of reperfusion on the response of the arteries triggered by ANG II. In FPSS, the pressure after 120 min of reperfusion was 79 mmHg , and in PSS it was 93 mmHg . SNP reduced the contraction triggered by Bay K8644 in PSS (P= 0.0088).8Br-cGMP (100 μM/l) changed the response of the arteries to ANG II after I/R in a manner similar to SNP (r to SNP .μM/l) and ODQ  did not significantly influence the perfusion pressure triggered by ANG II with participation of an intracellular pool of Ca2+, but in PSS, an increased reaction was observed  and Bay K8644  was analyzed. In order to establish the significance of calcium ions (from intracellular reserves and the extracellular fluid), the experiments were conducted in fluid without calcium ions  and in standard Krebs fluid, after emptying the cellular reserves of calcium . Then, the effects of signaling pathways associated with NO and cGMP on the aforementioned reactions were evaluated.The presented results reveal that in the case of contraction triggered by ANG II, the two pools of calcium ions mediate the response, but the response in PSS was more intense, and Bay K8644 led to an increase of the perfusion pressure only in the presence of calcium ions entering the cell from outside, since the action of Bay K8644 results from the direct activation of dihydropyridinic calcium channels located in the cellular membrane. Similar effects were also observed in studies which evaluated human mesenteric arteries ,19.2+]i and the smooth muscle sensitivity to calcium ions, these nucleotides cause the vessel to relax (It is known that NO is a basic substance of endothelial origin, which triggers effects via cGMP ,21. Cyclto relax –24. Besito relax –28.Results of assays evaluating the response of the arteries to ANG II following I/R revealed that ischemia reduces and reperfusion intensifies the action of this peptide. The results of previous experiments have indicated that inhibiting the effect of ischemia on the contraction of arteries is associated with the presence of endothelium, NO synthesis and cGMP activation. The experiments which were conducted on rat tail arteries with removed endothelium revealed no reduction in response to ANG II following ischemia. However, intensification of the contraction following reperfusion was observed under these conditions . StudiesA series of experiments conducted on the influence of Bay K8644 revealed no effect of I/R on responses to direct activation of the dihydropyridinic calcium channels. Similarly, experiments using a depolarizing concentration of KCl revealed that ischemia does not influence the response of the arteries to KCl . It shouIn conclusion, I/R modulate the contraction of arteries triggered by ANG II, but do not influence the reactions induced by Bay K8644. Intra- and extracellular pools of calcium ions mediate the action of ANG II, but Bay K8644 stimulates contraction only with the participation of calcium ions entering the cell. Regulation of the vascular smooth muscle tone associated with the action of NO and cGMP is subject to modulation under conditions of I/R.


There are two numerical errors in the penultimate sentence of the abstract. The abstract should read "Among the lifetimes of 100,000 average-risk patients, the number of cancers could be reduced from 4,857 to 1,393 and the number of CRC deaths from 1,782 to 457, while saving CAN$68 per person." The error occurred as the numbers 1,393 and 1,782 were accidentally switched within the sentence. The correct numbers are outlined in Table 5 and properly described in the results and discussion sections.


The cerebellum, also known as small brain, is a special region of the brain and plays critical roles in motor control. The granular layer, which harbors more than 95% of the cerebellar neurons (and more than 50% of the cells in the whole brain), is the input layer of the cerebellum, including two main neuron types: granule cells (GrCs) and Golgi cells (GoCs). From a functional perspective, the cerebral granular layer is responsible for receiving the mossy fiber (MF) inputs originally from sensory systems, cerebral cortex and spinal cord. These MF signals are then transformed and sent to the molecular layer, and eventually the Purkinje cells, to be further integrated to fine tune motor activities. Accordingly, elucidating the spatial/temporal dynamics of the granular layer is fundamental to understand the overall signal processing in the cerebellum. However, there remain many dynamical enigmas at the level of input integration relating to the spatial-temporal integration potentialities.In order to identify them, we built a large-scale 3D network model of the granular layer. The model (in NEURON) has dimensions 1 × 1 × 0.2 mm3, and contains both GrCs and GoCs with realistic cell and synapse counts, and has connectivity tightly constrained by anatomical literature [In this poster, we will present results on the properties of the spread of activity in the granular layer. In addition, we will discuss how the dynamical properties of MF inputs influence the spatial correlation of granule cell firing in the cerebellar cortex. Our results indicate that (1) the spatial correlation of granule cell firing is rather local, and (2) displays very short transient, and (3) exhibits a level of anisotropy. We hope that this work will contribute not only to the basic understanding of the spatial dynamics of the granule layer, but also to the capabilities of the cerebellum to integrate multimodal input.


In order to compare the transmissibility of the 2009 pH1N1 pandemic during successive waves of infections in summer and fall/winter in the Northern Hemisphere, and to assess the temporal changes during the course of the outbreak in relation to the intervention measures implemented, we analyze the epidemiological patterns of the epidemic in Taiwan during July 2009-March 2010.We utilize the multi-phase Richards model to fit the weekly cumulative pH1N1 epidemiological data  as well as the daily number of classes suspended under a unique "325" partial school closing policy in Taiwan, in order to pinpoint the turning points of the summer and fall/winter waves, and to estimate the reproduction numbers R for each wave.Our analysis indicates that the summer wave had slowed down by early September when schools reopened for fall. However, a second fall/winter wave began in late September, approximately 4 weeks after the school reopened, peaking at about 2-3 weeks after the start of the mass immunization campaign in November. R is estimated to be in the range of 1.04-1.27 for the first wave, and between 1.01-1.05 for the second wave.Transmissibility of the summer wave in Taiwan during July-early September, as measured by R, was lower than that of the earlier spring outbreak in North America and Europe, as well as that of the winter outbreak in Southern Hemisphere. Furthermore, transmissibility during fall/winter in Taiwan was noticeably lower than that of the summer, which is attributable to population-level immunity acquired from the earlier summer wave and also to the intervention measures that were implemented prior to and during the fall/winter wave. Although the first known imported case of 2009 pandemic influenza pH1N1) arrived in Taiwan on May 18 from the U.S. via Hong Kong, Serological evidence has indicated that the pH1N1 virus had spread to central Taiwan by April-June N1 arrive implemenAlthough school closing was a widely used method of intervention around the world during the pH1N1 outbreak , its® (MF59® adjuvanted H1N1v from Novartis) were administered, and more than 5 million of the 23 million Taiwanese had been immunized [Moreover, starting November 1, a mass immunization program was initiated in Taiwan sequentially, according to a priority list of 12 target groups , with hemmunized . ChildreA simple mathematical model, the Richards model, is utilized to fit publicly accessible cumulative epidemic data in order to obtain estimates for the turning points (the peaks and volleys of the incidence curve) and the reproduction number R of a particular wave of infections. Examples of applications of the Richards model to infectious diseases include those of SARS ,16, dengThe data was accessed from the Central Epidemic Command Center website of the Taiwan Centers for Disease Control (TCDC). Samples were collected from hospitals and clinics participating in the Taiwan Influenza surveillance system under the Taiwan National Influenza Center (Taiwan NIC), which was established in 2006 to integrate all existing efforts of influenza surveillance and notification with laboratory analysis systems throughout Taiwan in order to enhance the epidemic data collection capacity in Taiwan . The weeWe also accessed the daily record of numbers of classes suspended and number of schools with at least one class suspended during the fall school semester  from the TCDC daily pH1N1 updates  during tC(t) is the cumulative number of cases at time t (in weeks), K is the cumulative case number over a single wave or phase of outbreak, r is the per capita growth rate of the infected population, and a is the exponent of deviation. The explicit solution of the equation is tm is related to the turning point ti of a wave (or the inflection point of the cumulative case curve) by the simple formula tm = ti + lna/(ra), where ln denotes the natural logarithm function.The Richards model  is of thR0 = exp(rT) where T is the generation interval of the disease, or the average time interval from the onset of one infected person to the time when the onset of his or her contacts occurs. It has been shown mathematically [r, the expression R0 = exp(rT) provides an upper bound of the basic reproduction number regardless of the distribution of the generation interval that is being used. In this work, we will use the term effective reproduction number R instead, due to the community-level immunity likely achieved by July and the interventions implemented during the two waves.Moreover, atically  that, gii, and K, based on fitting the explicit solution of the Richards model for C(t) to the epidemic data used in the study, can be obtained easily and efficiently using any standard software with a nonlinear least-squares approximation tool, such as SAS or Matlab. The procedure for locating multiple turning points for multi-wave outbreak, which required the use of the multistage Richards model, is detailed in [The Richards model is a phenomenological model which can be used to describe the phenomenon of a biological growth (of cumulative number in this case) without requiring detailed information on the actual process of disease transmission. The basic premise of the Richards model is that the incidence curve of a single wave of infections contains a single peak of high incidence, resulting in an S-shaped cumulative epidemic curve and a single turning point (or peak incidence) of the outbreak. The turning point, defined as the point in time at which the rate of accumulation changes from increasing to decreasing, or vice versa in the event of a multi-wave outbreak, can be easily pinpointed by locating the inflection point of the cumulative case curve, i.e., the moment at which the trajectory begins to decline, as demonstrated in previous applications  of 2009 to e-week 12 (3/21-3/27) of 2010. The data fit converges to a two-phase Richard model with the first summer wave spanning e-weeks 29-39 (6/28-9/26) of 2009 and the second fall/winter wave spanning e-week 39 of 2009 to e-week 12 of 2010 (9/20/2009-3/27/2010). The estimation results are given in Table The turning points for the two waves are estimated at 8.50 weeks after e-week 29 and 7.96 weeks e-week 39, respectively. Subsequently, the weeks in which the turning points for temporal changes in the weekly confirmed pH1N1 case number took place on e-week 36 (8/30-9/5) for the first wave with a 95% CI range of , and on e-week 47 (11/15-11/21) with a 95% CI range of  for the second wave. We note that the above results were obtained by rounding off the estimates to the next largest integer, e.g., e-week 27+8.50 = 35.50 and hence e-week 36 is the week during which the turning point for the first wave occurred, and similarly for the second wave.0 reflect the uncertainty in the generation time T as well as in the uncertainty in the least-squared estimates for r, and does not reflect the error due to the model itself, which is always difficult to measure.To compute the effective reproduction number R, we use the generation time T = 1.91 days (95% CI: 1.30-2.71) for the 2009 pH1N1 in Mexico estimated by Fraser et al. . We noteWe also fit the weekly confirmed pH1N1 hospitalization data by hospitalization week in Taiwan from e-week 29 (7/12-7/18) of 2009 to e-week 12 3/21-3/27) of 2010 to the Richards model. The results are given in Table /21-3/27 The turning points for the weekly confirmed pH1N1 hospitalizations occurred on e-week 37 (9/6-9/12) for the first wave with a 95% CI range of  and on e-week 46 (11/8-11/14) with a 95% CI range of  for the second wave, which were the same weeks as the case number data turning points. The estimate for R using an estimated generation time T for pH1N1 in Mexico  is againTo further analyze and compare our previous results, we also make use of the daily class suspension data in Taiwan from September 9, 2009 to January 20, 2010, which allows us to ascertain the temporal changes in this intervention measure during the time period. Since this dataset started near the end of the first wave, according to our previous results, only one wave was modeled via the Richards model. The estimation results for model fit using the daily class suspension number data as well as the daily number of schools with at least one class suspended are given in Table i is 50.71  for the daily class suspension number data and 48.81  for the daily number of schools with at least one class suspended. Since the datasets cover only the school days, we account for the holidays to conclude that 50.71 school days after September 9 corresponds to November 19 (95% CI: November 18-20) as the turning point for the daily class suspension data, while ti = 48.81 for the schools with suspension data corresponds to November 17 (95% CI: November 16-18) as the turning point. A graphical illustration of the temporal timelines of the epidemic, as illustrated by the three model fits, is given in Figure The turning point tbasic reproduction number R0.The estimates for effective reproduction number R obtained from the confirmed case and hospitalization data are in good agreement, with R in the range of 1.04-1.27 for the first summer wave during July-September, and 1.01-1.05 for the second wave in fall/winter, using the generation time estimated by  for the A recent modeling study  of the 20  obtained from serological study of a cohort household population in central Taiwan during the same period of time [Transmissibility of the fist pH1N1 wave in Taiwan during the summer in July-September, as measured by R, was lower than that of the earlier spring outbreak in North America ,26,29,30Significantly higher estimate of R (focused on schoolchildren) in the range of 2.0-2.6 was found for the initial pandemic wave in Japan . Using uselected subset of those in the confirmed case time series. Subsequently, the temporal trends of the two time series might not be closely comparable. However, the cumulative curves in Figures The estimates for R using laboratory-confirmed case data by sample receiving weeks are slightly lower than those obtained by using confirmed hospitalization data. Although both the confirmed case and hospitalization datasets identify week 39 as the cutoff week for the two waves, the estimates of turning points for each wave differ by about one week when using the two datasets. Since only the more severe confirmed cases were hospitalized, the individuals in the resulting hospitalization time series is a weekly data can be expected to be most likely not significant. The use of hospitalization data is mainly for the purpose of estimation of reproduction number and comparison with the resulting estimates using the confirmed case data, which is not affected by this delay that might be present in both data.The confirmed case data is generated by sampling week, which could be different from the week of symptom onset and hence pose a potential source of some bias in data. However, samples were typically taken when the physicians diagnosed and reported H1N1 cases. We refer to 2003 SARS outbreak in Taiwan, when it was estimated that the onset-to-diagnosis interval is 1.20 days for previously quarantined persons and 2.89 days for non-quarantined persons . Given tEstimates of R obtained by using other (larger) estimated generation time in literature result in larger values for R, but generally are well within the ranges of the other studies , around the time schools from kindergarten to grade 12 reopened on August 31. However, a second fall/winter wave of cases started to emerge near the end of September around e-week 39 (9/27-10/3), approximately 4 weeks after the schools reopened, which did not reach its peak until mid-November (e-week 46-47 or 11/8-11/21) and lasted until the turn of the year. It is interesting to note that the state-specific fall pandemic waves in Mexico began 2-5 weeks after school reopened , which iThe class suspension data started on September 9 near the end of the first wave when the earliest class suspension occurred, according to our 2-wave fitting in Tables Also of interest is the possible impact of major intervention measures implemented by the Taiwan government during this time period, which including the aforementioned "325 class suspension" policy and the mass immunization program. The daily number of class suspensions started to increase in early September and continued until late November after the implementation of mass immunization campaign Figure . In partUsing routine influenza surveillance data, we modeled the temporal changes of the two waves of pH1N1 epidemic in Taiwan in summer and in fall/winter. The mass H1N1 vaccination program was first initiated sequentially on November 1, where a typical delay of at least two weeks from immunization is needed for protection from the vaccine to take effect in human bodies. Our results suggest that the turning point for the second wave of infections in the fall had occurred around mid-November (e-week 46-47 or 11/8-11/21). Moreover, the class suspension data indicate that the number of class suspensions had peaked by November 20, less than three weeks after the start of mass immunization and most likely before the impact of mass immunizations started to become significant. However, the mass immunization, and perhaps the voluntarily decreased social contacts by the general public in response to the well-publicized mass immunization campaign by the government, could have contributed to the overall mitigation of the disease in the community, as indicated by the early saturation of the winter epidemic by early February. However, this cannot be modeled without detailed vaccination data.The Richards model considers only the cumulative infected population size with saturation in growth as the outbreak progresses, which can be caused by other factors such as implementation of control measures. Although data by reporting date is often and typically scrambled by artificial factors such as health system alertness, public response, and government responsiveness, the Richards model is able to capture the turning points of outbreaks because they are often results of these artificial factors. We note, however, that the skewness in an epidemic curve, as quantified by the exponent of deviation "a" in the Richards model which describes the curvature of a given cumulative case data, also could conceivably arise from various other intrinsic factors such as spatial heterogeneity and individual heterogeneity in contact  are not readily available for the construction of a complete disease transmission model and the reliable estimation of model parameters, as in this study Moreover, the use of cumulative numbers could often, or at least partially, smooth out stochastic variations that typically occur in epidemic data, and hence the Richards model could be a valuable tool in providing clues to the challenging task of public health policy evaluation and planning.The authors declare that they have no competing interests.YHH conceived and organized the study, carried out the analysis, and wrote the first draft. YHH, KFC, TCL, TNW, CYC, and JWC participated in the study and the interpretation of study findings. KFC participated in the writing of the manuscript. MHL participated in the data collection and analysis. All authors have read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/11/332/prepub


FUT3) gene is responsible for the expression of the Lea and Leb blood group antigens. The individuals, who not synthesize these antigens have the phenotype Lewis negative, due to the presence of some single nucleotide polymorphisms (SNPs), such as 59T>G, 508G>A and 1067T>A, whose distribution is different in various ethnic groups. Our aim was to verify the frequencies of these SNPs in an admixed population of Belém-Pará-Brazil.The Lewis , 17.3% (508G>A) and 5.3% (1067T>A).The discrepancies between blood and salivary Lewis phenotypes are related to the relatively high frequencies of 59T>G and the null allele 508G>A. Whereas 38.6% of the individuals were Lewis negative based on blood, only 17.24% also tested negative when their saliva were analyzed.ST values reveal distinct frequencies of the FUT3 SNPs between the present sample and its representative ancestral populations. These observations will help to evaluate the Lewis antigens impact as susceptibility markers, in genetic association studies to certain diseases.We have found a marked consistency between the phenotypes and genotypes of the Lewis blood group system. Furthermore, our obtained F The Lewis antigens were discovered in the erythrocytes, although they are synthesized in the epithelium and then incorporated through the membrane of the erythrocytes from the plasma a and Lewisb are the principal antigens of this system a antigen and a reduction in salivary ABH substances. The Le(a−b+) phenotype, which is found in individuals with the Se gene, results from the association between the Lea specificity and the Led (type H-1) antigen determinant, resulting in the Leb type antigen, a product of the epistatic interaction between the secretor and Lewis loci Le) gene which codify non-functional transferases, and are labeled le. In this case, the individuals, whether secretors or not, do not express the Lea or Leb antigens.The Lewis antigens are biochemically related to the ABH antigens, being produced from the same precursor substances. The Lewisle) contain single nucleotide polymorphism (SNP), many of which inactivate the enzyme 59/508le, which have been detected in Asian (24%) and African (19%) populations, while the 202/314le (17%) and 59/1067le (4%) were found mainly in European populations The silent variant of the Lewis gene (The frequency of the different Lewis phenotypes varies significantly among different ethnic groups. The Le(a-b+) phenotype is the most common overall, but varies from 75% in Europeans to 61% in Africans, and 42% in Asians b in their secretions et al.,Some individuals present changes in the expression of the Lewis antigens  when affected by certain diseases and/or physiological conditions, especially in the erythrocytes, due to their secondary acquisition by these cells. In this case, some individuals that are Le(a−b−) in their blood present LeUnderstanding the complexity of the polymorphisms of the Lewis system is especially important for defining the function of these antigens as markers for the differentiation of the normal condition and pathological alterations observed in human tissue. As the expression of the Lewis antigens in the blood does not always allow the identification of the true phenotype, the present study investigated the frequency of the 59T>G, 508G>A, and 1067T>A SNPs, and their effects on the expression of the phenotypes of these antigens in the population of Belém a city of Pará state in the northern Brazil.et al.,This study analyzed samples of blood and saliva from 150 unrelated and randomly-selected individuals  from the admixed population of Belém in the northeast Brazilian Amazon region. The major groups involving in the admixture process of the Belém population were mainly Portuguese (47%), Africans (23%) and Native Indians (30%). These estimated proportions of ethnic admixture are based on a previous study of Guerreiro In the attempt to control the population heterogeneity and for operational convenience, the sample collection is randomly stratified by the 8 administrative districts that comprise the city of Belém. According to the proportion of the population of each district the individuals have been randomly-selected from the spontaneous demand in the Central Laboratory, which is reference laboratory for clinical analysis in the state of Pará.The study was approved by the Ethics in Research Committee of the Tropical Medicine Nucleus of the Federal University of Pará, and all the study subjects agreed in writing to participate in the study after reading an informed consent form.a, and anti-Leb antibodies (Fresenius Hemocare Brasil Ltda).A 5 ml sample of peripheral blood was obtained from each participant and stored in a tube containing heparin. The phenotypes of the ABO and Lewis systems were determined by the conventional direct hemagglutination technique, using the monoclonal anti-A, anti-B, anti-H, anti-Leet al.,a (1∶500), and anti-Leb(1∶1000) antibodies from Fresenius Hemocare Brazil.The ABH and Lewis secretor status of the subjects was determined by DOT-ELISA assay, based on the technique described by Pflug FUT3 gene was genotyped for the identification of the 59T>G and 508G>A mutations using the sn3-sn4 and sn8-sn9 primers, respectively  by digestion with Mspl. To detect the 508G>A SNP, the sn8-sn9 primer set was used to amplify a fragment of 206 bps and induce a cleavage site for PvuII, which resulted in the production of two fragments, of 104 bps and 102 bps.The ectively . In ordeHindIII in the mutant allele, generating fragments of 85 and 64 bps. The PCRs were based on 1 µL of genomic DNA, 5 pmol of each primer, 1.25 mM of dNTP, 2 mM of MgCl2, 0.5 unit of Taq polymerase in 2.5 µl of buffer . A negative control (amplification with no DNA sample) was included in all runs. The products were analyzed by electrophoresis in 2% agar gel colored with ethidium bromide. Markers of standard DNA with molecular weights of 50 bps and 100 bps from Fermentas Life Science were used in all procedures.To detect the 1067T>A SNP, an initial amplification was conducted using the P1 primers , which pet al. The distribution of frequencies and proportions was processed using the Bioestat 5.0 program of Ayres The analysis of the blood of the 150 subjects returned the following phenotype frequencies for the Lewis system: 57% (85/150) presented the Le(a−b+) phenotype and 4% (7/150) were Le(a+b+), while 39% (58/150) presented phenotype Le(a−b−). In the latter group, 82.76% (48/58) of the individuals expressed the Lewis antigens in the saliva, and were this classified as nongenuine Lewis-negative. The remaining 10 subjects presented no antigens in the saliva, and were classified as genuine Lewis-negative.FUT3 gene. Overall, seven different haplotypes were inferred for the 150 subjects, in which the alleles with the isolated 59Le or 59/508le SNP were the most common. By contrast, alleles with the isolated 508le and 1067le SNPs or the 59/1067le and 59/508/1067le combinations were the rarest. The genotype and haplotype frequencies observed for the Lewis gene (FUT3) are shown in Three very common SNPs –59T>G, 508G>A, and 1067 T>A – were analyzed in the 59Le or 59/508le SNP, in six and four cases, respectively. The distribution of the Lewis genotypes according to the ABO and Lewis blood group phenotypes are presented in The 59T>G polymorphism had an overall frequency of 47.66%, being detected in 121 individuals, of which, 22 were homozygous and 99 heterozygous. The 508G>A SNP was much rarer, with a frequency of 17.33%, being detected in 44 individuals, of which eight were homozygous, included one sample which presented the isolated 508G>A SNP. The 1067T>A SNP was found only in the heterozygous form, at a frequency of 5.3%, with the isolated from being recorded in six individuals, and the combined form, with FUT3 genotype frequencies indicated that the study population was not in Hardy-Weinberg equilibrium . The analysis of linkage disequilibrium between the pairs of combined sites also indicated a highly significant deviation  between the combinations of alleles at sites 59 T>G and 508 G>A.The rates of heterozygosity recorded for the SNPs at positions 59T>G, 508G>A, and 1067T>A were 66%, 24%, and 11%, respectively. Analysis of the FUT3) gene, the different genotypic and phenotypic configurations were grouped in the following categories  in blood and saliva) –10 individuals with the genuine Le(a−b−) blood phenotype, which all presented the a and/or Leb antigens in saliva) – included 48 individuals that had the 59T>G SNP in homozygote (n = 14) or heterozygote (n = 14). In some cases (n = 20), this SNP was combined with the 508G>A and/or 1067T>A null alleles in the heterozygous condition. In this group, 10 individuals secreted only the Lea substance in saliva, so that they were ABH non-secretors, while 38 individuals were ABH and Lewis secretors.Non-genuine Lewis-negative (characterized by Le(a−b−) in blood but with expression of the Le59Le/59Le). Then, the proportion of individuals which are heterozygous for the 59/508le and/or 59/1067le haplotypes is significantly higher for the non-genuine Lewis-negative individuals . On the other hand, the proportion of individuals that are heterozygous for the isolated 59 T>G SNP is significantly higher in the group with the Lewis-positive phenotype .Of the Lewis-positive individuals, 75% (69/92) presented heterozygous genotypes (Le/_), while the other 25% were homozygous for the Le allele (Le/Le). In the case of the non-genuine Lewis-negative individuals, 70.83% (34/48) were heterozygous (Le/−) and 29.17% (14/48) were homozygous for the 59T>G SNP  alleles in the group non-genuine Lewis-negative phenotype is approximately double that found in those with the Lewis-positive phenotype, with the opposite pattern being observed for the mutant On the other hand, in the non-genuine Lewis-negative individuals, non-O blood group phenotypes (in particular A1) were most common, with 54.17% (26/48) of the cases, whereas in the Lewis-positive individuals, non-O phenotypes were present in 38.04% (35/92) of the individuals (p = 0.05 for Fisher’s test). All these differences between positive and non-genuine Lewis-negative individuals can be visualized and conferred in ST values  presented the Lewis antigen in their saliva, contradicting the negative result from the blood samples.In the present study, 92 of the 150 study subjects had Lewis-positive phenotypes, which were identified by the presence of at least one FUT3 gene resulted in the identification of three principal SNPs, at positions 59T>G, 508G>A, and 1067T>A, which are known to account for 90% of the Lewis-negative phenotypes in Europeans, confirming the presence of the corresponding genotype combination, as predicted by existing knowledge of the effects of the isolated SNPs or haplotypes. However, the haplotypes corresponding to the Lewis genotypes cannot be determined reliably in all cases.The molecular analyses of the 59Le SNP, and the other with SNPs at positions 59T>G and 508G>A (59/508le). This distribution of alleles is effectively ambiguous, because heterozygous individuals that were either 59Le/Le or 59/508Le/le were recorded in both positive and negative Lewis blood groups. In addition, when null alleles, that is, 59/508le, 59/1067le, 1067le, 508le, and 59/508/1067le, were homozygous, the individuals presented the genuine Lewis-negative phenotype, which determines the absence of the expression of Lewis antigens in both the blood and the saliva, confirming the inactivating effect of these SNPs located in the catalytic domain Based on the available data In the present study, the 59T>G SNP, either homo- or heterozygous and isolated or in combination with other null mutations, was recorded in all individuals with Lewis antigens in the saliva, but with negative results for the blood samples. Given this, the pattern of blood-saliva discrepancies in the characterization of the Lewis phenotypes is consistent with that observed in previous studies, in which the non-genuine Lewis-negative trait was related to the presence of this type of mutation in the trans-membranous region 59Le allele does not act on the glycolipids, due to its differentiated activity in relation to the acceptor substrates, that is, glycolipids versus glycoproteins a and Leb factors in the plasma and, thus, an Le(a−b−) blood phenotype. Experimental molecular studies have shown that the isolated 59T>G SNP presents a reduction of approximately 50% in the levels of the FUT3 enzyme, which is probably a result of the reduction in its potential for retention in the Golgi complex, although it is normally present in conjunction with the 508G>A and 1067T>A SNPs In addition, studies in enzymatic kinetics have shown that the enzyme produced by the a antigens in the intestinal mucosa, in particular, that the quantity of Lea in the samples of Le/Le individuals was approximately twice that of the Le/le heterozygotes, indicating a regulatory effect of the Lewis gene a antigens in the saliva varying considerably, but randomly between the heterozygotes and homozygotes, invariably being reduced.One other aspect that has been analyzed in previous studies is the correlation between the amount of Lewis enzyme and the level of expression of the Leet al.,It seems likely that a set of genetic and epigenetic factors interact to limit the activity of the FUT3 enzyme, which may then synthesize quantities of antigens that are undetectable in serological tests, resulting in the apparent expression of the Lewis-negative phenotype. In addition, this data also provides the different proportions between the inactive alleles, which compose the heterozygote frequencies of Lewis-positive and non-genuine Lewis-negative individuals, illustrating the known genetic dosage effect, which has been demonstrated in a previous study by Nishihara b epitopes in comparison with group O individuals, which can interfere with the power of recognition of some Lewis antibodies, resulting in positive individuals which are characterized as being negative One interesting result of the present study was the predominance of non-O blood groups in non-genuine Lewis-negative individuals. This may be explained by the fact that the biosynthetic routes of the ABH and Lewis antigens are related, and cause competition between the A/B glycosyltransferases and the Lewis fucosyltransferases for the common type 1 H precursor. This leads to a reduction in the number of LeFUT3 genotype and/or blood group A1 are the most likely to express the non-genuine Lewis-negative phenotype, indicating that the negatively modulating effect of the 59T>G SNP may be accentuated under certain circumstances, either pathological  or physiological conditions, such as pregnancy, which may cause a reduction in the circulatory concentration of active Lewis glycolipids, resulting in the incompatible expression of Lewis antigens in the blood Given this, individuals that have the heterozygous FUT3 gene. In the present study, by contrast, the isolated form of the 59Le allele was present at a relatively high frequency, which indicates the possibility that other combinations of 59T>G may exist, involving mutations that were not investigated in this population. An even more likely explanation may be the ethnic composition of the sample and the dynamics of the population, considering the possible contribution of its Amerindian component to the frequency of the 59Le allele, although this group has not been studied in detail. The differences in the frequencies of the FUT3 SNPs observed between the present sample and its representative ancestral populations (According to the distribution of frequencies recorded in previous studies ulations  may be tST values reflect the distinct frequencies of the FUT3 SNPs between Belém and its ancestral populations Overall, the results of the present study indicated a marked consistency between the phenotypes and genotypes of the Lewis blood group system. The molecular analyses conducted in the present study helped to elucidate the serological discrepancies in phenotypes through the identification of the different mutant alleles and their respective frequencies, especially those related to the individuals classified as non-genuine Lewis-negative. In addition, our obtained F