Current knowledge of vector-borne zoonotic pathogens in Zambia: A clarion call to scaling-up “One Health” research in the wake of emerging and re-emerging infectious diseases

BackgroundAlthough vector-borne zoonotic diseases are a major public health threat globally, they are usually neglected, especially among resource-constrained countries, including those in sub-Saharan Africa. This scoping review examined the current knowledge and identified research gaps of vector-borne zoonotic pathogens in Zambia.Methods and findingsMajor scientific databases (Web of Science, PubMed, Scopus, Google Scholar, CABI, Scientific Information Database (SID)) were searched for articles describing vector-borne (mosquitoes, ticks, fleas and tsetse flies) zoonotic pathogens in Zambia. Several mosquito-borne arboviruses have been reported including Yellow fever, Ntaya, Mayaro, Dengue, Zika, West Nile, Chikungunya, Sindbis, and Rift Valley fever viruses. Flea-borne zoonotic pathogens reported include Yersinia pestis and Rickettsia felis. Trypanosoma sp. was the only tsetse fly-borne pathogen identified. Further, tick-borne zoonotic pathogens reported included Crimean-Congo Haemorrhagic fever virus, Rickettsia sp., Anaplasma sp., Ehrlichia sp., Borrelia sp., and Coxiella burnetii.ConclusionsThis study revealed the presence of many vector-borne zoonotic pathogens circulating in vectors and animals in Zambia. Though reports of human clinical cases were limited, several serological studies provided considerable evidence of zoonotic transmission of vector-borne pathogens in humans. However, the disease burden in humans attributable to vector-borne zoonotic infections could not be ascertained from the available reports and this precludes the formulation of national policies that could help in the control and mitigation of the impact of these diseases in Zambia. Therefore, there is an urgent need to scale-up “One Health” research in emerging and re-emerging infectious diseases to enable the country to prepare for future epidemics, including pandemics.     

Deuterium isotope effects during carbon-hydrogen bond cleavage by trimethylamine dehydrogenase. Implications for mechanism and vibrationally assisted hydrogen tunneling in wild-type and mutant enzymes

His-172 and Tyr-169 are components of a triad in the active site of trimethylamine dehydrogenase (TMADH) comprising Asp-267, His-172, and Tyr-169. Stopped-flow kinetic studies with trimethylamine as substrate have indicated that mutation of His-172 to Gln reduces the limiting rate constant for flavin reduction approximately 10-fold (Basran, J., Sutcliffe, M. J., Hille, R., and Scrutton, N. S. (1999) Biochem. J. 341, 307-314). A kinetic isotope effect (KIE = k(H)/k(D)) accompanies flavin reduction by H172Q TMADH, the magnitude of which varies significantly with solution pH. With trimethylamine, flavin reduction by H172Q TMADH is controlled by a single macroscopic ionization (pK(a) = 6.8 +/- 0.1). This ionization is perturbed (pK(a) = 7.4 +/- 0.1) in reactions with perdeuterated trimethylamine and is responsible for the apparent variation in the KIE with solution pH. At pH 9.5, where the functional group controlling flavin reduction is fully ionized, the KIE is independent of temperature in the range 277-297 K, consistent with vibrationally assisted hydrogen tunneling during breakage of the substrate C-H bond. Y169F TMADH is approximately 4-fold more compromised than H172Q TMADH for hydrogen transfer, which occurs non-classically. Studies with Y169F TMADH suggest partial thermal excitation of substrate prior to hydrogen tunneling by a vibrationally assisted mechanism. Our studies illustrate the varied effects of compromising mutations on tunneling regimes in enzyme molecules.     

A model in environmental training--The University / Elementary School / Municipality Cooperation

This study targets development of an effective training scheme model that can be implemented at elementary school level with focus on recovery and recycling of wasted papers in Turkey. For this purpose, three schools were chosen from a district within Istanbul. They were separated from one another as full intervention (FI), semi-intervention (SI) and control (C) schools. Different levels of educational activities carried out in the schools, mostly in the FI school, were directed toward being informative as regards recycling and the development of a positive attitude. Afterwards, in order to evaluate the effects of the training, paper wastes were collected in recycle bins placed at appropriate points in schools and weighed on a weekly basis. Quite a significant result was found (p = 0.0001), when the difference was calculated through the Kruskal Wallis Variance Analysis method, regarding the weekly average amount of paper in each of the three schools against per person. Furthermore, when the results were evaluated and compared as to the ones before the 2.5 months summer vacation and the ones after it, the seven measurements taken before (p = 0.001) and the eight taken afterwards (p = 0.0001), were found to have valid differences, once again, as against schools. The results show that the approach we provided to education is an effective method not only for the collection of paper wastes but also for applications in various areas of health education.     

The Caenorhabditis elegans centrosomal protein SPD-2 is required for both pericentriolar material recruitment and centriole duplication

BACKGROUND: The centrosome is composed of a centriole pair and pericentriolar material (PCM). By marking the site of PCM assembly, the centrioles define the number of centrosomes present in the cell. The PCM, in turn, is responsible for the microtubule (MT) nucleation activity of centrosomes. Therefore, in order to assemble a functional bipolar mitotic spindle, a cell needs to control both centriole duplication and PCM recruitment. To date, however, the molecular mechanisms that govern these two processes still remain poorly understood. RESULTS: Here we show that SPD-2 is a novel component of the C. elegans centrosome. SPD-2 localizes to the centriole throughout the cell cycle and accumulates on the PCM during mitosis. We show that SPD-2 requires SPD-5 for its accumulation on the PCM and that in the absence of SPD-2, centrosome assembly fails. We further show that centriole duplication is also defective in spd-2(RNAi) embryos, but not in spd-5(RNAi) embryos, where PCM recruitment is efficiently blocked. CONCLUSIONS: Taken together, our results suggest that SPD-2 may link PCM recruitment and centriole duplication in C. elegans. SPD-2 shares homology with a human centrosome protein, suggesting that this key component of the C. elegans centrosome is evolutionarily conserved.     

Effects of transforming growth factor-alpha (TGF-alpha) in vitro and in vivo on reovirus replication

We have utilized growth factors in in vitro and in vivo systems to examine the role of cellular proliferation in reovirus replication. In vitro, proliferating RIE-1 cells can be infected with whole reovirus virions, but are relatively resistant to infection once confluent (Go arrest). It has been shown that TGF-alpha, which signals through the EGF-receptor (EGF-R), is capable of dramatically increasing the number of RIE-1 cells entering the S-phase in the presence of additional serum factors. Stimulation of the EGF-R without serum results in minimal increases in cells entering the S-phase with a restriction in reovirus replication. Therefore, other factors in serum are essential for fully permissive infection. In vivo, we used metallothionein (MT) promoter/enhancer-TGF-alpha transgenic mice to study the effect of cytokine activation on reovirus type 1 infection. Virus replication decreased following oral infection in these transgenic mice at 1 month of age, concordant with increased mucin production. Titers of reovirus obtained from the livers of 1 year old transgenic mice were approximately 10-fold higher than titers obtained in control mice. Taken together, these data indicate that while growth factor activation ultimately leads to an increase in virus infectivity, other factors may be necessary for reovirus replication.     

Residues glutamate 216 and aspartate 301 are key determinants of substrate specificity and product regioselectivity in cytochrome P450 2D6

Cytochrome P450 2D6 (CYP2D6) metabolizes a wide range of therapeutic drugs. CYP2D6 substrates typically contain a basic nitrogen atom, and the active-site residue Asp-301 has been implicated in substrate recognition through electrostatic interactions. Our recent computational models point to a predominantly structural role for Asp-301 in loop positioning (Kirton, S. B., Kemp, C. A., Tomkinson, N. P., St.-Gallay, S., and Sutcliffe, M. J. (2002) Proteins 49, 216-231) and suggest a second acidic residue, Glu-216, as a key determinant in the binding of basic substrates. We have evaluated the role of Glu-216 in substrate recognition, along with Asp-301, by site-directed mutagenesis. Reversal of the Glu-216 charge to Lys or substitution with neutral residues (Gln, Phe, or Leu) greatly decreased the affinity (K(m) values increased 10-100-fold) for the classical basic nitrogen-containing substrates bufuralol and dextromethorphan. Altered binding was also manifested in significant differences in regiospecificity with respect to dextromethorphan, producing enzymes with no preference for N-demethylation versus O-demethylation (E216K and E216F). Neutralization of Asp-301 to Gln and Asn had similarly profound effects on substrate binding and regioselectivity. Intriguingly, removal of the negative charge from either 216 or 301 produced enzymes (E216A, E216K, and D301Q) with elevated levels (50-75-fold) of catalytic activity toward diclofenac, a carboxylate-containing CYP2C9 substrate that lacks a basic nitrogen atom. Activity was increased still further (>1000-fold) upon neutralization of both residues (E216Q/D301Q). The kinetic parameters for diclofenac (K(m) 108 microm, k(cat) 5 min(-1)) along with nifedipine (K(m) 28 microm, k(cat) 2 min(-1)) and tolbutamide (K(m) 315 microm, k(cat) 1 min(-1)), which are not normally substrates for CYP2D6, were within an order of magnitude of those observed with CYP3A4 or CYP2C9. Neutralizing both Glu-216 and Asp-301 thus effectively alters substrate recognition illustrating the central role of the negative charges provided by both residues in defining the specificity of CYP2D6 toward substrates containing a basic nitrogen.     

Novel isoform of insulin receptor substrate p53/p58 is generated by alternative splicing in the CRIB/SH3-binding region

Insulin receptor substrate p53/p58 (IRSp53) is involved in cytoskeletal dynamics and is a candidate disease sensor in polyglutamine expansion neurodegeneration. It is widely expressed throughout the body, but its levels are dramatically elevated in forebrain regions. IRSp53 functions as a signal transducing adaptor between activated Rho family GTPases and their effectors. There are four known alternatively spliced isoforms of IRSp53 that vary by the identity of the 3'-terminal exon. We report here that there is a fifth alternatively spliced isoform, IRSp53-B, which lacks 40 amino acids abutting the CRIB/SH3 (Cdc42/Rac-interactive binding/Src homology 3)-binding site. We speculate that the novel form has an altered function related to the mechanism of autoinactivation. IRSp53-B has an odd history in the mammalian lineage, which may complicate the use of rodent models to study cytoskeletal reorganization.     

Impact of incorporating the 2C5 crystal structure into comparative models of cytochrome P450 2D6

Cytochrome P450 2D6 (CYP2D6) metabolizes approximately one third of the drugs in current clinical use. To gain insight into its structure and function, we have produced four different sets of comparative models of 2D6: one based on the structures of P450s from four different microorganisms (P450 terp, P450 eryF, P450 cam, and P450 BM3), another on the only mammalian P450 (2C5) structure available, and the other two based on alternative amino acid sequence alignments of 2D6 with all five of these structures. Principal component analysis suggests that inclusion of the 2C5 crystal structure has a profound effect on the modeling process, altering the general topology of the active site, and that the models produced differ significantly from all of the templates. The four models of 2D6 were also used in conjunction with molecular docking to produce complexes with the substrates codeine and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP); this identified Glu 216 [in the F-helix; substrate recognition site (SRS) 2] as a key determinant in the binding of the basic moiety of the substrate. Our studies suggest that both Asp 301 and Glu 216 are required for metabolism of basic substrates. Furthermore, they suggest that Asp 301 (I-helix, SRS-4), a residue thought from mutagenesis studies to bind directly to the basic moiety of substrates, may play a key role in positioning the B'-C loop (SRS-1) and that the loss of activity on mutating Asp 301 may therefore be the result of an indirect effect (movement of the B'-C loop) on replacing this residue.     

Melatonin supplementation can ameliorate the detrimental effects of heat stress on performance and carcass traits of Japanese quail

Melatonin, the major pineal hormone, modulates growth in poultry by influencing hormones involved in growth. We investigated the effects of dietary melatonin supplementation on performance, carcass characteristics, and excretion of nitrogen and some minerals in broiler Japanese quails (Coturnix coturnix japonica) exposed to high-ambient-temperature stress (34°C). One hundred twenty Japanese quails (10 d old) were randomly assigned to 4 treatment groups, 3 replicates of 10 birds each. The birds were kept in either an environment-controlled room at a constant 22°C or were kept at 22°C for 16 h/d and at 34°C for 8 h/d (9:00 am to 5:00 pm). At both temperatures birds were fed either a basal diet or the basal diet supplemented with 40 mg of melatonin per kilogram of diet. The experiment lasted for 32 d. Melatonin improved feed efficiency in both temperatures groups compared with their corresponding controls. Although feed intake was similar in all groups, the improvement in feed efficiency was more noticable in melatonin-fed quails kept at high temperature (p<0.01). Supplemental melatonin significantly increased live weight gain and carcass characteristics under stress conditions (p<0.01) but did not show the same effect at thermoneutral conditions (p>0.05). Heat exposure increased excretion of N, Ca, P, Zn, Fe, and Cr and decreased retention rates for them. Dietary melatonin supplementation returned these values to normal (p<0.01). No interactions between melatonin and temperature were found in the parameters measured. The results of the study show that melatonin supplementation attenuated the retardation in performance as well as the excretion of minerals caused by heat stress in broiler quails. Our data suggest that melatonin might offer protection against heat-stress-related depression in the performance of broiler quails.