High-density hapten labeling and HRP conjugation of oligonucleotides for use as in situ hybridization probes to detect mRNA targets in cells and tissues.

Oligonucleotides that carry a detectable label can be used to probe for mRNA targets in in situ hybridization experiments. Oligonucleotide probes (OPs) have several advantages over cDNA probes and riboprobes. These include the easy synthesis of large quantities of probe, superior penetration of probe into cells and tissues, and the ability to design gene- or allele-specific probes. One significant disadvantage of OPs is poor sensitivity, in part due to the constraints of adding and subsequently detecting multiple labels per oligonucleotide. In this study, we compared OPs labeled with multiple detectable haptens (such as biotin, digoxigenin, or fluorescein) to those directly conjugated with horseradish peroxidase (HRP). We used branching phosphoramidites to add from two to 64 haptens per OP and show that in cells, 16-32 haptens per OP give the best detection sensitivity for mRNA targets. OPs were also made by directly conjugating the same oligonucleotide sequences to HRP. In general, the HRP-conjugated OPs were more sensitive than the multihapten versions of the same sequence. Both probe designs work well both on cells and on formaldehyde-fixed, paraffin-embedded tissues. We also show that a cocktail of OPs further increases sensitivity and that OPs can be designed to detect specific members of a gene family. This work demonstrates that multihapten-labeled and HRP-conjugated OPs are sensitive and specific and can make superior in situ hybridization probes for both research and diagnostic applications.     

Survival of Escherichia coli O157:H7 on cattle hides

The objective of this study was to determine the time period that Escherichia coli O157:H7 survives on the hides of cattle. Extensive research has been conducted and is ongoing to identify and develop novel preharvest intervention strategies to reduce the presence of E. coli O157:H7 on live cattle and subsequent transfer to processed carcasses. If a reduction of E. coli O157:H7 levels in feces can be achieved through preharvest intervention, it is not known how long it would take for such reductions to be seen on the hide. In the study presented herein, three trials were conducted to follow E. coli O157:H7 hide prevalence over time. For each trial, 36 animals were housed in individual stanchions to minimize or prevent hide contamination events. Through prevalence determination and isolate genotyping with pulsed-field gel electrophoresis, survival of E. coli O157:H7 on the hides of live cattle was determined to be short lived, with an approximate duration of 9 days or less. The results of this study suggest that any preharvest interventions that are to be administered at the end of the finishing period will achieve maximum effect in reducing E. coli O157:H7 levels on cattle hides if given 9 days before the cattle are presented for processing. However, it should be noted that interventions reducing pathogen shedding would also contribute to decreasing hide contamination through lowering the contamination load of the processing plant lairage environment, regardless of the time of application.     

Impact of rapid urbanization on the rates of infection by Vibrio cholerae O1 and enterotoxigenic Escherichia coli in Dhaka, Bangladesh

Background

In Bangladesh, increases in cholera epidemics are being documented with a greater incidence and severity. The aim of this prospective study was to identify the prevalence and importance of V. cholerae O1 and enterotoxigenic Escherichia coli (ETEC) as causal agents of severe diarrhea in a high diarrhea prone urban area in Dhaka city.

Methodology

Systematic surveillance was carried out on all diarrheal patients admitted from Mirpur between March 2008 to February 2010 at the ICDDR, B hospital. Stool or rectal swabs were collected from every third diarrheal patient for microbiological evaluation.

Principal Findings

Of diarrheal patients attending the hospital from Mirpur, 41% suffered from severe dehydration with 39% requiring intravenous rehydration therapy. More diarrheal patients were above five years of age (64%) than those below five years of age (36%). About 60% of the patients above five years of age had severe dehydration compared with only 9% of patients under five years of age. The most prevalent pathogen isolated was Vibrio cholerae O1 (23%) followed by ETEC (11%). About 8% of cholera infection was seen in infants with the youngest children being one month of age while in the case of ETEC the rate was 11%. Of the isolated ETEC strains, the enterotoxin type were almost equally distributed; ST accounted for 31% of strains; LT/ST for 38% and LT for 31%.

Conclusion

V. cholerae O1 is the major bacterial pathogen and a cause of severe cholera disease in 23% of patients from Mirpur. This represents a socioeconomic group that best reflects the major areas of high cholera burden in the country. Vaccines that can target such high risk groups in the country and the region will hopefully be able to reduce the disease morbidity and the transmission of pathogens that impact the life and health of people.     

Distribution of Copper, Iron, and Zinc in Biological Samples (Scalp Hair, Serum, Blood, and Urine) of Pakistani Viral Hepatitis (A–E) Patients and Controls

The aim of the present study was to compare the level of copper (Cu), iron (Fe) and zinc (Zn) in biological samples (serum, blood, urine, and scalp hair) of patients suffering from different viral hepatitis (A, B, C, D, and E; n?=?521) of both gender age ranged 31–45 years. For comparative study, 255 age-matched control subjects, of both genders residing in the same city were selected as referents. The elements in the biological samples were analyzed by flame atomic absorption spectrophotometry, prior to microwave-assisted acid digestion. The validity and accuracy of the methodology was checked by using certified reference materials (CRMs) and with those values obtained by conventional wet acid digestion method on same CRMs. The results of this study showed that the mean values of Cu and Fe were higher in blood, sera, and scalp hair samples of hepatitis patients, while Zn level was found to be lower than age-matched control subjects. The urinary levels of these elements were found to be higher in the hepatitis patients than in the age-matched healthy controls (p?<?0.05). These results are consistent with literature-reported data, confirming that the deficiency of zinc and hepatic iron and copper overload can directly cause lipid peroxidation and eventually hepatic damage.     

Continuous Distributed Representation of Biological Sequences for Deep Proteomics and Genomics

We introduce a new representation and feature extraction method for biological sequences. Named bio-vectors (BioVec) to refer to biological sequences in general with protein-vectors (ProtVec) for proteins (amino-acid sequences) and gene-vectors (GeneVec) for gene sequences, this representation can be widely used in applications of deep learning in proteomics and genomics. In the present paper, we focus on protein-vectors that can be utilized in a wide array of bioinformatics investigations such as family classification, protein visualization, structure prediction, disordered protein identification, and protein-protein interaction prediction. In this method, we adopt artificial neural network approaches and represent a protein sequence with a single dense n-dimensional vector. To evaluate this method, we apply it in classification of 324,018 protein sequences obtained from Swiss-Prot belonging to 7,027 protein families, where an average family classification accuracy of 93%±0.06% is obtained, outperforming existing family classification methods. In addition, we use ProtVec representation to predict disordered proteins from structured proteins. Two databases of disordered sequences are used: the DisProt database as well as a database featuring the disordered regions of nucleoporins rich with phenylalanine-glycine repeats (FG-Nups). Using support vector machine classifiers, FG-Nup sequences are distinguished from structured protein sequences found in Protein Data Bank (PDB) with a 99.8% accuracy, and unstructured DisProt sequences are differentiated from structured DisProt sequences with 100.0% accuracy. These results indicate that by only providing sequence data for various proteins into this model, accurate information about protein structure can be determined. Importantly, this model needs to be trained only once and can then be applied to extract a comprehensive set of information regarding proteins of interest. Moreover, this representation can be considered as pre-training for various applications of deep learning in bioinformatics. The related data is available at Life Language Processing Website: http://llp.berkeley.edu and Harvard Dataverse: http://dx.doi.org/10.7910/DVN/JMFHTN.     

Synergistic effect of PEG-400 and cyclodextrin to enhance solubility of progesterone

Conclusion  PEG-400, polysorbate 80, and 2 CDs (Trappsol HPB and Captisol) were used in an attempt to improve the aqueous solubility of a model hydrophobic drug, progesterone. The aqueous solubility of progesterone improved significantly from 0.007 mg/mL by the addition of PEG-400, CDs, and polysorbate 80. In systems containing various amounts of PEG-400 and 3% Trappsol HPB in water (% wt/wt), the theoretical solubility was calculated by adding the solubilities in the individual systems. The observed solubility values were up to 96% higher than the theoretical values. The effect of synergism was significant in 5% to 50% PEG-400/water systems containing Trappsol HPB. Systems containing Captisol did not show such synergistic effects. In general, the addition of polysorbate 80 to the PEG-400/water systems containing CDs affected synergism negatively.     

Phenotypic characteristics and population genetics of Enterococcus faecalis cultured from patients in Tehran during 2000-2001

Conventional bacteriology techniques were used to identify enterococci isolates cultured from patients at different hospitals in Tehran during 2000-2001. The identification was confirmed using species-specific PCR targeting the D-alanyl-D-alanine ligase gene. A total of 59 isolates of Enterococcus faecalis were identified. The rates of resistance to different antibiotics were in the following order: penicillin 84%, ciprofloxacin 42%, high-level gentamicin 30%, nitrofurantoin 14%, imipenem 4%, and chloramphenicol 2%. Resistance to ampicillin was found to be rare among the Iranian isolates of E. faecalis. Multi-locus enzyme electrophoresis was then used to analyze the strains. Forty-five electrophoretic types were obtained when 10 enzyme loci were screened. Although the collection of bacterial isolates was limited in time and location, considerable heterogeneity was found. Analysis of strains for linkage disequilibrium demonstrated that the studied population is not clonal, since the index of association was not significantly different from zero (Ia = 0.0296). Enterococcus faecalis isolates recovered from patients in Tehran were genetically diverse and seemed to possess a high potential for genetic recombinations, though none were resistant to vancomycin.     

N-terminally fusion of Her2/neu to HSP70 decreases efficiency of Her2/neu DNA vaccine

DNA vaccines consisted of tumor-associated antigen (TAA) are well suited for immunotherapy against tumor. The construct can contain TAA fused to an appropriate molecule (biologic adjuvant) to improve the efficacy of anti-tumor immune response. Heat shock protein 70 (HSP70) has been shown to be an excellent candidate, capable of cross-priming TAA by antigen presenting cells leading to a robust T-cell response. However, the relationship between strong T-cell responses and tumor rejection is not always mutually exclusive, for which TAA loss or activation of suppressive mechanisms may occur. HSP70 fused to downstream of Her2/neu as DNA vaccine has been shown to be efficient against Her2-expressing tumors. In this study, we examined if N-terminally fusion of Her2/neu to HSP70 could also improve efficiency of Her2/neu DNA vaccine. Therefore, mice with an established Her2/neu expressing tumor were immunized with DNA vaccine consisting of extracellular and trans-membrane domain (EC+TM) of rat Her2/neu alone or N-terminally fused to HSP70 and immune response was evaluated. Administration of rat Her2/neu led to partial control of tumor progression. Surprisingly, fusion of HSP70 to N-terminal of rat Her2/neu led to tumor progression. Our result proposes that fusion direction of biologic adjuvant is an important consideration when Her2/neu is used.     

Experimental modelling of thermal inactivation of urease

Thermal inactivation of jack bean urease (EC 3.5.1.5) was investigated in a 0.1 M phosphate buffer with pH 7. An injection flow calorimetry method was adapted for the measurement of the enzyme activity. The inactivation curves were measured in the temperature range of 55 to 87.5 degrees C. The curves exhibited a biphasic pattern in the whole temperature range and they were well fitted with a biexponential model. A simultaneous fit of all inactivation data was based on kinetic models that were derived from different inactivation mechanisms and comprised the material balances of several enzyme forms and the enthalpy balance characterizing the initial heating period of enzyme solution. The multitemperature evaluation revealed that an adequate model had to incorporate at least three reaction steps. It was concluded that the key reaction steps at urease thermal inactivation were the reversible dissociation/denaturation of native form into an inactive denatured form, and irreversible association reactions of both the denatured and native forms.