• Microbiology and Biotechnology Letters
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• v.49 no.3
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• pp.329-336
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• 2021

Cellulophga sp. J9-3, is a gram-negative, aerobic marine bacterium belonging to the family Flavobacteriaceae. In addition to cellulose degradability, the J9-3 strain is also capable of hydrolyzing agar in the solid and liquid medium, and the production of agarase in the presence of agarose can be remarkably induced by the bacterium. From the cell culture broth of Cellulophga sp. J9-3, ammonium sulfate precipitation and three kinds of column chromatography were successively performed to purify a specific agarase protein, the AgaJ93. Purified AgaJ93 showed the strongest hydrolyzing activity towards agarose (approximately 22%), and even displayed activity towards starch. AgaJ93 hydrolyzed agarose into neoagarotetraose and neoagarohexaose via various oligosaccharide intermediates, indicating that AgaJ93 is an endo-type β-agarase. AgaJ93 showed maximum activity at a pH of 7.0 and temperature of 35 ℃. Its activity increased by more than six times in the presence of Co²⁺ ions. The N-terminal sequence of AgaJ93 showed 82% homology with the heat-resistant endo-type β-agarase Aga2 of Cellulophaga sp. W5C. However, the biochemical properties of the two enzymes were different. Therefore, AgaJ93 is expected to be a novel agarose, different from the previously reported β-agarases.

• Journal of Microbiology and Biotechnology
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• v.29 no.2
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• pp.235-243
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• 2019

A special eosinophilic agarase exo-type ${\beta}$-agarase gene, AgaDL6, was cloned from a marine agar-degrading bacterium, Flammeovirga sp. HQM9. The gene comprised 1,383-bp nucleotides encoding a putative agarase AgaDL6 of 461 amino acids with a calculated molecular mass of 52.8 kDa. Sequence analysis revealed a ${\beta}$-agarase domain that belongs to the glycoside hydrolase family (GH) 16 and a carbohydrate-binding module (CBM_4_9) unique to agarases. AgaDL6 was heterologously expressed in Escherichia coli BL21 (DE3). Enzyme activity analysis of the purified protein showed that the optimal temperature and pH of AgaDL6 were $50^{\circ}C$ and 3.0, respectively. AgaDL6 showed thermal stability by retaining more than 98% of activity after incubation for 2 h at $50^{\circ}C$, a feature quite different from other agarases. AgaDL6 also exhibited outstanding acid stability, retaining 100% of activity after incubation for 24 h at pH 2.0 to 5.0, a property distinct from other agarases. This is the first agarase characterized to have such high acid stability. In addition, we observed no obvious stimulation or inhibition of AgaDL6 in the presence of various metal ions and denaturants. AgaDL6 is an exo-type ${\beta}$-1,4 agarase that cleaved agarose into neoagarotetraose and neoagarohexaose as the final products. These characteristics make AgaDL6 a potentially valuable enzyme in the cosmetic, food, and pharmaceutical industries.

• Food Science and Biotechnology
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• v.17 no.5
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• pp.1115-1118
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• 2008

${\alpha}$-Galactosidase gene (aga) from Leuconostoc mesenteroides SY1 was expressed in a heterologous host, Lactobacillus brevis 2.14 using an Escherichia coli-Leuconostoc shuttle vector, pSJE. pSJEaga (pSJE carrying aga) was introduced into Lactobacillus brevis 2.14 by electroporation and transformation efficiency was $1.1{\times}10^3$ per ${\mu}g$ DNA. L. brevis transformants (TFs) showed higher ${\alpha}$-galactosidase (${\alpha}$-Gal) activities than cells containing pSJE. Transcription levels of aga in L. brevis 2.14 grown on different carbon sources (1%, w/v) were examined by slot blot analysis. Aga transcript levels and ${\alpha}$-Gal activities were higher in cells grown on melibiose, raffinose, and galactose than cells on glucose, sucrose, and fructose. Western blot result showed that L. brevis 2.14 harboring pSJEaga produced much more ${\alpha}$-Gal when grown on melibiose than on glucose.

• Asian Pacific Journal of Cancer Prevention
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• v.16 no.17
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• pp.7541-7546
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• 2015

Aim: To report the histologic findings on Whipple resection specimens and thus determine the extent and spread of carcinomas of ampullary region and head of pancreas in our population. Setting: Section of Histopathology, Department of Pathology, Aga Khan University Hospital (AKUH), Karachi, Pakistan. Materials and Methods: A case series of 311 consecutive Whipple resection specimens received between January 1,2003 and December 31, 2014. Specimens processed for histologic sections and representative sections submitted and histologically examined as per established and standard protocols. All relevant tumor parameters including histologic type, histologic grade, pathologic T and N stage and tumor size were assessed. Epidemiologic data were also recorded. All findings were analysed using SPSS 19.0 software. Results: Ampullary (periampullary) carcinomas were much more common than carcinomas of the head of the pancreas, especially in males, with an average age of 53 years. Mean tumor size was 2.5 cms, over 54% were well differentiated. A large majority were pT2 or pT3 and N0. Carcinomas of pancreatic head were also more common in males, mean age was 55 years, mean tumor size was 3.5cms, and over 65% were moderately differentiated. The majority were T2 or T3 and pN1. Prognostically, significant statistical correlation was seen with tumor grade and pathologic T and N stage (p values statistically significant). However, tumor size was not statistically significant. Conclusions: Ampullary carcinomas are more common compared to pancreatic carcinomas. Majority of ampullary carcinomas were well differentiated while majority of pancreatic carcinomas were moderately differentiated. Large majority of both types of cases were pT2 or T3. Histologic tumor grade and pathologic T and N stage are significantly related to prognosis in Pakistani patients with ampullary and pancreatic cancers.

• Clinical and Experimental Pediatrics
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• v.62 no.3
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• pp.102-107
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• 2019

Purpose: This study compared the iron statuses of small for gestational age (SGA) and appropriate for gestational age (AGA) infants at birth. Methods: The clinical data of 904 newborn infants admitted to the neonatal intensive care unit were reviewed. Blood samples were drawn from the infants within 24 hours after birth. Serum ferritin level was used as a marker of total iron status. Results: In this study, 115 SGA (GA, $36.5{\pm}2.9weeks$; birth weight [BW], $1,975{\pm}594.5g$) and 717 AGA (GA, $35.1{\pm}3.5weeks$; BW, $2,420.3{\pm}768.7g$) infants were included. The SGA infants had higher hematocrit levels ($50.6%{\pm}5.8%$ vs. $47.7%{\pm}5.7%$, P<0.05) than the AGA infants. No difference in serum ferritin level (ng/mL) was found between the groups (mean [95% confidence interval]: SGA vs. AGA infants, 139.0 [70.0-237.0] vs. 141.0 [82.5-228.5]). After adjusting for gestational age, the SGA infants had lower ferritin levels (147.1 ng/mL [116.3-178.0 ng/mL] vs. 189.4 ng/mL [178.0-200.8 ng/mL], P<0.05). Total body iron stores were also lower in the SGA infants than in the AGA infants (185.6 [153.4-211.7] vs 202.2 [168.7-241.9], P<0.05). Conclusion: The SGA infants had lower ferritin and total body iron stores than the AGA infants. The SGA infants affected by maternal hypertension who were born at late preterm had an additional risk of inadequate iron store. Iron deficiency should be monitored in these infants during follow-up.

• Journal of Microbiology and Biotechnology
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• v.28 no.2
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• pp.284-292
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• 2018

A novel ${\beta}$-agarase, AgaJ5, was identified from an agar-degrading marine bacterium, Gayadomonas joobiniege G7. It belongs to the glycoside hydrolase family 86 and is composed of 805 amino acids with a 30-amino-acid signal peptide. Zymogram analysis showed that purified AgaJ5 has agarase activity. The optimum temperature and pH for AgaJ5 activity were determined to be $30^{\circ}C$ and 4.5, respectively. AgaJ5 was an acidic ${\beta}$-agarase that had strong activity at a narrow pH range of 4.5-5.5, and was a cold-adapted enzyme, retaining 40% of enzymatic activity at $10^{\circ}C$. AgaJ5 required monovalent ions such as $Na^+$ and $K^+$ for its maximum activity, but its activity was severely inhibited by several metal ions. The $K_m$ and $V_{max}$ of AgaJ5 for agarose were 8.9 mg/ml and 188.6 U/mg, respectively. Notably, thin-layer chromatography, mass spectrometry, and agarose-liquefication analyses revealed that AgaJ5 was an endo-type ${\beta}$-agarase producing neoagarohexaose as the final main product of agarose hydrolysis. Therefore, these results suggest that AgaJ5 from G. joobiniege G7 is a novel endo-type neoagarohexaose-producing ${\beta}$-agarase having specific biochemical features that may be useful for industrial applications.

• Asian Pacific Journal of Cancer Prevention
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• v.13 no.6
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• pp.2473-2476
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• 2012

Background: In papillary and follicular thyroid cancers (PTC, FTC), nodal and distant metastasis are generally considered important determinants of recurrence and survival, respectively. However, there is no consensus about the threshold primary tumour size (PTS) for these determinants. The aim of this study was to assess size relationships for developing nodal, pulmonary, bone and overall distant metastases. Methods: This prospective study covered 139 (93 females and 46 males) consecutive biopsy proven patients with PTC (114/139, mean age $41.0{\pm}15.7$ years, M: F, 35%:65%) and FTC (25/139, mean age $39.2{\pm}14.3$ years, M: F: 24%:76%). Results: Average primary tumor size was $23.4{\pm}11.1$ mm and $26.5{\pm}13.1$ mm for PTC and FTC respectively (p value=0.223). Nodal metastasis was found more common in PTC than FTC (49% vs 28%, p value <0.05), whereas overall distant metastasis was approximately the same (13% and 24%, p value=0.277); however, bone metastasis was significantly higher in FTC than PTC (24% vs 5%, p value <0.05). Cumulative risk for nodal and distant metastases for FTC and PTC starts at PTS <20 mm and may indicate an unusual aggressive tumor behavior in the studied population. Highest cumulative risk for nodal and pulmonary metastases in PTC and for bone metastasis in FTC was found to be ${\geq}50$ mm PTS. Conclusion: We conclude that a PTS of <20 mm may indicate an unusual aggressive tumor behavior with highest cumulative risk for nodal and pulmonary metastases in PTC and for bone metastasis in FTC with a cutoff of ${\geq}50$ mm.

• Journal of Microbiology and Biotechnology
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• v.22 no.11
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• pp.1532-1539
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• 2012

The ${\alpha}$-galactosidase-coding gene agaAJB13 was cloned from Sphingomonas sp. JB13 showing 16S rDNA (1,343 bp) identities of ${\leq}97.2%$ with other identified Sphingomonas strains. agaAJB13 (2,217 bp; 64.9% GC content) encodes a 738-residue polypeptide (AgaAJB13) with a calculated mass of 82.3 kDa. AgaAJB13 showed the highest identity of 61.4% with the putative glycosyl hydrolase family 36 ${\alpha}$-galactosidase from Granulicella mallensis MP5ACTX8 (EFI56085). AgaAJB13 also showed <37% identities with reported protease-resistant or Sphingomonas ${\alpha}$-galactosidases. A sequence analysis revealed different catalytic motifs between reported Sphingomonas ${\alpha}$-galactosidases (KXD and RXXXD) and AgaAJB13 (KWD and SDXXDXXXR). Recombinant AgaAJB13 (rAgaAJB13) was expressed in Escherichia coli BL21 (DE3). The purified rAgaAJB13 was characterized using p-nitrophenyl-${\alpha}$-D-galactopyranoside as the substrate and showed an apparent optimum at pH 5.0 and $60^{\circ}C$ and strong resistance to trypsin and proteinase K digestion. Compared with reported proteaseresistant ${\alpha}$-galactosidases showing thermolability at $50^{\circ}C$ or $60^{\circ}C$ and specific activities of <71 U/mg with or without protease treatments, rAgaAJB13 exhibited a better thermal stability (half-life of >60 min at $60^{\circ}C$) and higher specific activities (225.0-256.5 U/mg). These sequence and enzymatic properties suggest AgaAJB13 is the first identified and characterized Sphingomonas ${\alpha}$-galactosidase, and shows novel protease resistance with a potential value for basic research and industrial applications.

• Clinical and Experimental Pediatrics
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• v.54 no.6
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• pp.246-252
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• 2011

Purpose: The outcomes of small for gestational age (SGA) infants especially in extremely low birth weight infants (ELBWIs) are controversial. This study evaluated the mortality and morbidity of ELBWIs, focusing on whether or not they were also SGA. Methods: The medical records of 415 ELBWIs (birth weight<1,000 g), who were inborn and admitted to the Samsung Medical Center neonatal intensive care unit from January 2000 to December 2008, were reviewed retrospectively. Mortality and morbidities were compared by body size groups: very SGA (VSGA), birth weight ${\leq}$3rd percentile; SGA, 3rd to 10th percentile; and appropriate for gestational age (AGA) infants, >10th percentile for gestational age. For gestational subgroup analysis, groups were divided into infants with gestational age ${\leq}24^{+6}$ weeks (subgroup I), $25^{+0}$ to $26^{+6}$ weeks (subgroup II), and ${\geq}27^{+0}$ weeks (subgroup III) Results: Gestational age was $29^{+2}{\pm}2^{+6}$ weeks in the VSGA infants (n=49), $27^{+5}{\pm}2^{+2}$weeks in the SGA infants (n=45), and $25^{+4}{\pm}1^{+4}$ weeks in AGA infants (n=321). Birth weight was $692{\pm}186.6$ g, $768{\pm}132.9$ g, and $780{\pm}142.5$ g in the VSGA, SGA, and AGA groups, respectively. Cesarean section rate and maternal pregnancy-induced hypertension were more common in the VSGA and SGA than in AGA pregnancies. However, chorioamnionitis was more common in the AGA group. The mortalities of the lowest gestational group (subgroup I), and also of the lower gestational group (subgroup I+II) were significantly higher in the VSGA group than the SGA or AGA groups (P=0.020 and P=0.012, respectively). VSGA and SGA infants showed lower incidence in respiratory distress syndrome, ductal ligation, bronchopulmonary dysplasia, intraventricular hemorrhage than AGA group did. However, by multiple logistic regression analysis of each gestational subgroup, the differences were not significant. Conclusion: Of ELBWIs, extremely SGA in the lower gestational subgroups, had an impact on mortality, which may provide information useful for prenatal counseling.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.21
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• pp.9495-9498
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• 2014

Background: The difference in prognosis of adult and childhood acute lymphoblastic leukemia (ALL) can be attributed largely to variation in cytogenetic abnormalities with age groups. Cytogenetic analysis in acute leukemia is now routinely used to assist patient management, particularly in terms of diagnosis, disease monitoring, prognosis and risk stratification. Knowing about cytogenetic profile at the time of diagnosis is important in order to take critical decisions in management of the patients. Aim and Objectives: To determine the frequency of cytogenetic abnormalities in Pakistani adult patients with ALL in order to have insights regarding behavior of the disease. Materials and Methods: A retrospective analysis of all the cases of ALL (${\geq}15$years old) diagnosed at Aga Khan University from January 2006 to June 2014 was performed. Phenotype (B/T lineage) was confirmed in all cases by flow cytometry. Cytogenetic analysis was made for all cases using the trypsin-Giemsa banding technique. Karyotypes were interpreted using the International System for Human Cytogenetic Nomenclature (ISCN) criteria. Results: A total of 166 patients were diagnosed as ALL during the study period, of which 151 samples successfully yielded metaphase chromosomes. The male to female ratio was 3.4:1. The majority (n=120, 72.3%) had a B-cell phenotype. A normal karyotype was present in 51% (n=77) of the cases whereas 49% (n=74) had an abnormal karyotype. Of the abnormal cases, 10% showed Philadelphia chromosome; t(9;22)(q34;q11.2). Other poor prognostic cytogenetic subgroups were t(4;11)(q21;q23), hypodiploidy (35-45 chromosomes) and complex karyotype. Hyperdiploidy (47-57 chromosomes) occurred in 6.6%; all of whom were younger than 30 years. Conclusions: This study showed a relatively low prevalence of Philadelphia chromosome in Pakistani adults with ALL with an increase in frequency with age (p=0.003). The cumulative prevalence of Philadelphianegative poor cytogenetic aberrations in different age groups was not significant (p=0.6).