Use of artificial genomes in assessing methods for atypical gene detection

Parametric methods for identifying laterally transferred genes exploit the directional mutational biases unique to each genome. Yet the development of new, more robust methods—as well as the evaluation and proper implementation of existing methods—relies on an arbitrary assessment of performance using real genomes, where the evolutionary histories of genes are not known. We have used the framework of a generalized hidden Markov model to create artificial genomes modeled after genuine genomes. To model a genome, “core” genes—those displaying patterns of mutational biases shared among large numbers of genes—are identified by a novel gene clustering approach based on the Akaike information criterion. Gene models derived from multiple “core” gene clusters are used to generate an artificial genome that models the properties of a genuine genome. Chimeric artificial genomes—representing those having experienced lateral gene transfer—were created by combining genes from multiple artificial genomes, and the performance of the parametric methods for identifying “atypical” genes was assessed directly. We found that a hidden Markov model that included multiple gene models, each trained on sets of genes representing the range of genotypic variability within a genome, could produce artificial genomes that mimicked the properties of genuine genomes. Moreover, different methods for detecting foreign genes performed differently—i.e., they had different sets of strengths and weaknesses—when identifying atypical genes within chimeric artificial genomes.     

Intracellular Ca2+ release underlies the development of phase 2 in mouse ventricular action potentials

The ventricular action potential (AP) is characterized by a fast depolarizing phase followed by a repolarization that displays a second upstroke known as phase 2. This phase is generally not present in mouse ventricular myocytes. Thus we performed colocalized electrophysiological and optical recordings of APs in Langendorff-perfused mouse hearts founding a noticeable phase 2. Ryanodine as well as nifedipine reduced phase 2. Our hypothesis is that a depolarizing current activated by Ca(2+) released from the sarcoplasmic reticulum (SR) rather than the "electrogenicity" of the L-type Ca(2+) current is crucial in the generation of mouse ventricular phase 2. When Na(+) was partially replaced by Li(+) in the extracellular perfusate or the organ was cooled down, phase 2 was reduced. These results suggest that the Na(+)/Ca(2+) exchanger functioning in the forward mode is driving the depolarizing current that defines phase 2. Phase 2 appears to be an intrinsic characteristic of single isolated myocytes and not an emergent property of the tissue. As in whole heart experiments, ventricular myocytes impaled with microelectrodes displayed a large phase 2 that significantly increases when temperature was raised from 22 to 37°C. We conclude that mouse ventricular APs display a phase 2; however, changes in Ca(2+) dynamics and thermodynamic parameters also diminish phase 2, mostly by impairing the Na(+)/Ca(2+) exchanger. In summary, these results provide important insights about the role of Ca(2+) release in AP ventricular repolarization under physiological and pathological conditions.     

Size at first sexual maturity,fecundity, length–weight and length–length relationships of Puntius sophore (Cyprinidae) in Bangladeshi waters

The present study describes the size at first sexual maturity, fecundity, length–weight (LWRs) and length–length relationships (LLRs) of the pool barb, Puntius sophore, using data obtained from different geographical locations in Bangladesh. A total of 905 specimens were caught by traditional fishing gear from March 2010 to February 2011. Additionally, a total of 121 females were collected from a commercial catch of the Padma River during June–July 2011 to estimate size at first maturity and to determine fecundity. Total length (TL), fork length (FL) and standard length (SL) were measured with digital slide calipers. Individual body weights (BW) were determined for all specimens, and gonad weights (GW) from 121 females were weighed to an accuracy of 0.001 g. The female gonadosomatic index (GSI) was calculated as [GSI (%) = (GW/BW) × 100]. Female size at first maturity was estimated using GSI and TL as indicators, and estimated as 5.00 cm TL in the Padma River. Specimens larger than 5.00 cm TL were used to determine fecundity. Mean total fecundity was 5300 ± 2700, ranging from 1580 to 16590. A positive exponential correlation was recorded between total fecundity and total length (r² = 0.421). Relative fecundity ranged from 466 to 4036 (mean 1100 ± 580) in the Padma River. The LWR of pooled data for sexes combined was estimated as BW = 0.0155 TL^2.98 as ancova revealed no significant differences in LWRs between rivers (P > 0.05). All LLRs were highly correlated (r² > 0.983; P < 0.001), and ancova analyses further indicated that LLRs did not differ between rivers (P > 0.05). These results will help in further studies on the population assessment of the species.     

Gonadosomatic index‐based size at first sexual maturity of the catfish Eutropiichthys vacha (Hamilton, 1822) in the Ganges River (NW Bangladesh)

This study describes the size at first sexual maturity, length–weight relationships (LWR) in relation to size at first sexual maturity, and Fulton’s condition factor (K_F) of Eutropiichthys vacha in the Ganges River, northwestern Bangladesh. Sampling was done using traditional fishing gear including cast nets, square lift nets and conical traps during January and April, and July to December 2010. For each individual, total length (TL) was measured to the nearest 0.01 cm, and total weight (BW) was determined to the nearest 0.01 g. The gonadosomatic index (GSI) was calculated by the equation, GSI (%) = (Gonad weight in g/BW) × 100. The size at first sexual maturity of males and females was estimated by the relationship between gonadosomatic index and total length. A total of 583 specimens (289 males; 294 females) ranging from 8.30 to 27.00 cm TL and 3.16 to 159.50 g BW were analyzed. Sizes at first sexual maturity for male and female E. vacha were 13.15 and 14.00 cm TL, respectively. The analysis of covariance (ancova ) revealed significant differences in slope and intercept between early and late phases for males (F = 4.532, P < 0.001) and females (F = 21.984, P < 0.001). The K_F was not significantly correlated with TL for males (r_s = 0.052; P = 0.378), but was highly correlated for females (r_s = ?0.165; P = 0.005). This study establishes a strong base for monitoring changes in length at first sexual maturity attributable to high fishing pressures or other reasons within the Ganges and associated river ecosystems.     

Cloning and expression of cyclophilin from Platanus orientalis pollens in Escherichia coli

Background:

Allergy is a clinical disorder affecting the human population with wide geographical distribution. Platanus orientalis (P. orientalis) trees are planted in many countries and their pollen causes allergic reactions.Cyclophilin has recently been identified as one of the most important allergens of P. orientalis pollen. We aimed to clone and purify this allergen in Escherichia coli for further studies and therapeutic and diagnostic purposes for allergy to P. orientalis.

Methods:

RNA was extracted from P. orientalis. A full-length fragment encoding cyclophilin was prepared by polymerase chain reaction amplification of the first-strand cDNA synthesized from P. orientalis RNA. The cDNA was inserted into the pET32b (+) vector, and the construct transformed into E. coli Top10 and BL21 cells. The expressed protein was purified by the CuSO4 method.

Results:

The cDNA for the cyclophilin of P. orientalis pollen was cloned, and a specific reactivity of recombinant cyclophin was confirmed by immunoblotting using sera from patients allergic to P. orientalis pollen.

Conclusion:

The recombinant cyclophilin has a potential for immunologic assays for evaluation of allergy to P. orientalis pollen.Key Words: Allergy; Recombinant allergen; Cyclophilin, Escherichia coli, Platanus orientalis, Pollen, Cloning     

Multifunctional role of Bcl-2 in malignant transformation and tumorigenesis of Cr(VI)-transformed lung cells

B-cell lymphoma-2 (Bcl-2) is an antiapoptotic protein known to be important in the regulation of apoptosis in various cell types. However, its role in malignant transformation and tumorigenesis of human lung cells is not well understood. We previously reported that chronic exposure of human lung epithelial cells to the carcinogenic hexavalent chromium Cr(VI) caused malignant transformation and Bcl-2 upregulation; however, the role of Bcl-2 in the transformation is unclear. Using a gene silencing approach, we showed that Bcl-2 plays an important role in the malignant properties of Cr(VI)-transformed cells. Downregulation of Bcl-2 inhibited the invasive and proliferative properties of the cells as well as their colony forming and angiogenic activities, which are upregulated in the transformed cells as compared to control cells. Furthermore, animal studies showed the inhibitory effect of Bcl-2 knockdown on the tumorigenesis of Cr(VI)-transformed cells. The role of Bcl-2 in malignant transformation and tumorigenesis was confirmed by gene silencing experiments using human lung carcinoma NCI-H460 cells. These cells exhibited aggressive malignant phenotypes similar to those of Cr(VI)-transformed cells. Knockdown of Bcl-2 in the H460 cells inhibited malignant and tumorigenic properties of the cells, indicating the general role of Bcl-2 in human lung tumorigenesis. Ingenuity Pathways Analysis (IPA) revealed potential effectors of Bcl-2 in tumorigenesis regulation. Additionally, using IPA together with ectopic expression of p53, we show p53 as an upstream regulator of Bcl-2 in Cr(VI)-transformed cells. Together, our results indicate the novel and multifunctional role of Bcl-2 in malignant transformation and tumorigenesis of human lung epithelial cells chronically exposed to Cr(VI).