Dual miRNA Targeting Restricts Host Range and Attenuates Neurovirulence of Flaviviruses

Mosquito-borne flaviviruses are among the most significant arboviral pathogens worldwide. Vaccinations and mosquito population control programs remain the most reliable means for flavivirus disease prevention, and live attenuated viruses remain one of the most attractive flavivirus vaccine platforms. Some live attenuated viruses are capable of infecting principle mosquito vectors, as demonstrated in the laboratory, which in combination with their intrinsic genetic instability could potentially lead to a vaccine virus reversion back to wild-type in nature, followed by introduction and dissemination of potentially dangerous viral strains into new geographic locations. To mitigate this risk we developed a microRNA-targeting approach that selectively restricts replication of flavivirus in the mosquito host. Introduction of sequences complementary to a mosquito-specific mir-184 and mir-275 miRNAs individually or in combination into the 3’NCR and/or ORF region resulted in selective restriction of dengue type 4 virus (DEN4) replication in mosquito cell lines and adult Aedes mosquitos. Moreover a combined targeting of DEN4 genome with mosquito-specific and vertebrate CNS-specific mir-124 miRNA can silence viral replication in two evolutionally distant biological systems: mosquitoes and mouse brains. Thus, this approach can reinforce the safety of newly developed or existing vaccines for use in humans and could provide an additional level of biosafety for laboratories using viruses with altered pathogenic or transmissibility characteristics.     

Measurement of positional isotope exchange rates in enzyme-catalyzed reactions by fast atom bombardment mass spectrometry: application to argininosuccinate synthetase

Fast atom bombardment mass spectrometry (FAB-MS) has been used to measure positional isotope exchange rates in enzyme-catalyzed reactions. The technique has been applied to the reactions catalyzed by acetyl-CoA synthetase and argininosuccinate synthetase. The FAB technique is also able to quantitatively determine the oxygen-18 or oxygen-17 content of nucleotides on as little as 10 nmol of material with no prior derivatization. Acetyl-CoA synthetase has been shown by FAB-MS to catalyze the positional exchange of an oxygen-18 of ATP from the beta-nonbridge position to the alpha beta-bridge position in the presence of acetate. These results are consistent with acetyl adenylate as a reactive intermediate in this reaction. Argininosuccinate synthetase was shown not to catalyze a positional isotope exchange reaction designed to test for the formation of citrulline adenylate as a reactive intermediate. Argininosuccinate synthetase was also found not to catalyze the transfer of oxygen-18 from [ureido-18O]citrulline to the alpha-phosphorus of ATP in the absence of added aspartate. This experiment was designed to test for the transient formation of carbodiimide as a reactive intermediate. These results suggest that either argininosuccinate synthetase does not catalyze the formation of citrulline adenylate or the enzyme is able to completely suppress the rotation of the phosphoryl groups of PPi.     

Severed intestine in channel catfish

Six cases of severed intestines in farm-raised channel catfish were examined at fish disease diagnostic laboratories in Mississippi and Alabama. This condition has not been reported previously in fish. Affected fish had a 4-7-cm-long intestinal section (hyperemic where it was severed) attached to the stomach. The remainder of the intestine was completely missing in all six cases except for a 1-1.5-cm section of intestine prolapsed from the anus in fish from three of the cases. Ischemia, autodigestion following intussusception, or intestinal epithelium degradation are suggested as possible etiologies for this condition.     

Factors influencing the survival of patients with cancer of the prostate.

During the period 1969 through 1973, 777 new cases of cancer of the prostate in northern Alberta men were registered with the Alberta Cancer Registry. The overall survival rate after 5 years was 41.2%. As expected, the rates were higher for those aged less than 65 years than for those who were older at the time of diagnosis and higher for those without metastases than for those with metastases at that time. Urban residents had a higher survival rate than rural residents (45.3% v. 38.0%), and the survival ratio of the former, 1.31, was significant. Information on occupation, smoking and the interval between appearance of the first symptom and diagnosis was not always available. However, the differentials observed suggest that those in a professional occupation and nonsmokers live longer after the diagnosis of cancer of the prostate but that the interval before diagnosis does not affect the length of survival.