PCR-based gene synthesis with overlapping unisense-oligomers asymmetric extension supported by a simulator for oligonucleotide extension achieved 1 kbp dsDNA.

We formulated a method to synthesize 1 kbp DNA fragments using 'oligomer unidirectional joining method' via asymmetric extension supported by a simulator for oligonucleotide extension (AESOE). In this study, trials were conducted on 41 sets of different genomic pieces of ten flaviviral genomes, and 31 bacterial 16s rRNA fragments with sizes ranging from 500 bases to 1.0 kbp. Synthetic gene production was found to be successful in all those sets. The synthesis method has three steps: the first step is a seven-linked AESOE, the second step is the linking of the 400-base fragments from the first step, and the third step is the final amplification. Our present approach is highly reproducible and may no longer require optimization of oligomer design.

Prediction of PCR amplification from primer and template sequences using recurrent neural network.

We have developed a novel method to predict the success of PCR amplification for a specific primer set and DNA template based on the relationship between the primer sequence and the template. To perform the prediction using a recurrent neural network, the usual double-stranded formation between the primer and template nucleotide sequences was herein expressed as a five-lettered word. The set of words (pseudo-sentences) was placed to indicate the success or failure of PCR targeted to learn recurrent neural network (RNN). After learning pseudo-sentences, RNN predicted PCR results from pseudo-sentences which were created by primer and template sequences with 70% accuracy. These results suggest that PCR results could be predicted using learned RNN and the trained RNN could be used as a replacement for preliminary PCR experimentation. This is the first report which utilized the application of neural network for primer design and prediction of PCR results.

An improved gene synthesis method with asymmetric directions of oligonucleotides designed using a simulation program.

Artificial gene synthesis based on oligonucleotide augmentation is known as overlap extension PCR which generates a variety of intermediate synthetic products. The orientation and concentration of oligomers can be adjusted to reduce the synthesis of intermediates and optimize the full-length process of DNA synthesis, using a simulation program for serial oligomer extension. The efficiency of the serial oligomer extension process is predicted to be greatest when oligomers are in a 'forward-reverse-reverse-reverse' direction. Oligomers with such designed directions demonstrated generation of the desired product in the shortest time (number of cycles) by repeated annealing and elongation. This method, named Asymmetric Extension supported by a Simulator for Oligonucleotide Extension (AESOE), has shown efficiency and effectiveness with potentials for future improvements and optimal usage in DNA synthesis.

Novel synthetic nucleotides of notifiable dengue (1-4), Japanese encephalitis, yellow fever and Zika flaviviruses.

AIM: To produce synthetic nucleotides of notifiable dengue virus (1-4 types), Japanese encephalitis, yellow fever and Zika flaviviruses. These notifiable flaviviruses, particularly dengue and Zika, are problematic mosquito-borne infections in the Philippines, as well as in those countries with tropical and subtropical climates. METHOD: An algorithmic design formulation of overlap extension - polymerase chain reaction (OE-PCR) was performed to propagate 50-60 oligomer lengths of select notifiable flaviviral RNAs to DNA nucleotides via the two-step process of OE-PCR. RESULT: Algorithmic OE-PCR design formulation efficiently produced 253-256 bp of notifiable flaviviruses. Comparing the newly designed algorithmic OE-PCR with existing executable programs demonstrated it to be efficient and useful in generating accurate sequences of synthetic flaviviral nucleotides. CONCLUSION: The efficiently and accurately produced novel synthetic nucleotides of notifiable dengue virus 1-4, Japanese encephalitis, yellow fever and Zika flaviviruses using OE-PCR is useful in understanding the dynamics of flaviviral species and holds potential for the development of synthetic nucleotide-based immunogens.

Monitoring impacts of air pollution: PIXE analysis and histopathological modalities in evaluating relative risks of elemental contamination.

Environmental toxicants invariably affect all biological organisms resulting to sufferings ranging from subclinical to debilitating clinical conditions. This novel research aimed to determine the toxic burdens of increased environmental elements in some vital organs/tissues of the wild animals (starling, owl, crow and pigeon), exposed to air polluted environment were assessed using particle induced X-ray emission and histopathological approaches. The presence of significantly elevated amounts of elemental toxicants namely: Aluminum (Al), Chlorine (Cl), Iron (Fe), Potassium (K), Magnesium (Mg), Manganese (Mn), Silicon (Si) and Vanadium (V) from the skin, muscle, lungs, liver and kidney of sampled animals were in concurrence with the observed histopathological changes. The skin of sampled starling, owl, pigeon and crow spotlighted highly significant increase (P < 0.001) in Al, Cl, Mg and Si. Muscle samples with myodegenerative lesions and mineral depositions highlighted substantial augmentation (P < 0.001) in the amount of Al, Fe, Mn, Si and V. The lungs of starling, owl, and pigeon were severely intoxicated (P < 0.001) with increased amount of Al, Fe, K, Mn and Si producing pulmonary lesions of congestion, edema, pneumonitis and mineral debris depositions. Liver samples revealed that the sampled animals were laden with Cl, Fe, Mg, Mn and V with histopathological profound degenerative changes and hepatic necrosis. Kidney sections presented severe tubular degenerative and necrotic changes that may be attributed to increased amounts of Cl and Fe. These current findings implied that the environmental/elemental toxicants and the accompanying lesions that were discerned in the organs/tissues of sampled birds may as well be afflicting people living within the polluted area. Further assessment to more conclusively demonstrate correlations of current findings to those of the populace within the area is encouraged.

Detection of spotted fever and typhus group rickettsial infection in wild raccoon dogs (Nyctereutes procyonoides koreensis) in Chonbuk Province, Korea.

Both the spotted fever and the typhus group rickettsioses are potential emerging arthropod-borne infectious disease complexes in Korea. Wild raccoon dogs (Nyctereutes procyonoides koreensis) sheltered at the Korean Society for the Protection of Wild Animals, Chonbuk Branch, Korea, were sampled for the presence of spotted fever and typhus group rickettsial pathogens. Thirty-six serum samples of wild raccoon dogs were examined by an indirect fluorescent antibody test using type-specific YH strain Rickettsia japonica (spotted fever group rickettsia) and Wilmington strain Rickettsia typhi (typhus group rickettsia). The prevalence of sera that were seropositive was 30.5% (11/36) for the spotted fever group and 41.6% (15/36) for the typhus group. Five samples were seropositive for both antigens. This study is first to demonstrate rickettsial antibodies in wild raccoon dogs, and it provides seroepidemiologic evidence of the endemic presence of rickettsial agents in Korea.

Detection of antibodies against spotted fever group Rickettsia (SFGR), typhus group Rickettsia (TGR), and Coxiella burnetii in human febrile patients in the Philippines.

A total of 157 sera from febrile patients in the Philippine General Hospital in Manila, Luzon, and the Northern Samar Provincial Hospital, the Philippines, were used. Serum antibodies against spotted fever group Rickettsia (SFGR) and typhus group Rickettsia (TGR) were detected by indirect immunofluorescence test. Antibody positive rates were 1.3% for SFGR (Rickettsia japonica) and 2.5% for TGR (R. typhus), respectively. Rickettsial antibodies in humans in the Philippines were found for the first time. These results underscore the need for further epidemiological study of clinical rickettsioses in the Philippines.