Geographical Distribution of Aedes aegypti aegypti and Aedes aegypti formosus (Diptera: Culicidae) in Kenya and Environmental Factors Related to Their Relative Abundance.

The mosquito Aedes aegypti (L.) is the primary vector of various infectious viruses and is typified by a polymorphic color and abundance of white scales on the body. It has been conventionally separated into two subspecies, Ae. aeg. formosus (Walker) (Aaf) and Ae. aeg. aegypti (L.) (Aaa), with Aaf considered a 'sylvan' form and Aaa a 'domestic' form. Because the two subspecies show different susceptibilities to dengue viruses it is important to understand their distribution. In this study, we collected larvae from artificial and natural habitats in southern Kenya and reared them to adults to morphologically identify subspecies. We describe the geographical distribution and relative abundance of Aaa and Aaf in Kenya, and estimate the environmental factors associated with their distributions by GIS using climate and environment data. A total of 5,243 Ae. aegypti adults were collected from 249 sites, with Aaa accounting for 22% of the specimens. The relative abundance of Aaa was higher in coastal areas versus sites in western Kenya. Aaa abundance was also higher in urbanized than forested areas, which is consistent with known ecology. In contrast and inconsistent with previous studies, both Aaa and Aaf were sympatric in artificial and natural habitats. The high relative abundance of Aaa in coastal areas might derive from old populated cities, climate, and/or introduction from abroad.

New and Common Haplotypes Shape Genetic Diversity in Asian Tiger Mosquito Populations from Costa Rica and Panamá.

The Asian tiger mosquito, Aedes albopictus (Skuse) (Diptera: Culicidae), is a vector of several human pathogens. Ae. albopictus is also an invasive species that, over recent years, has expanded its range out of its native Asia. Ae. albopictus was suspected to be present in Central America since the 1990s, and its presence was confirmed by most Central American nations by 2010. Recently, this species has been regularly found, yet in low numbers, in limited areas of Panamá and Costa Rica (CR). Here, we report that short sequences (∼558 bp) of the mitochondrial cytochrome oxidase subunit 1 (COI) and NADH dehydrogenase subunit 5 genes of Ae. albopictus, had no haplotype diversity. Instead, there was a common haplotype for each gene in both CR and Panamá. In contrast, a long COI sequence (∼1,390 bp) revealed that haplotype diversity (±SD) was relatively high in CR (0.72±0.04) when compared with Panamá (0.33±0.13), below the global estimate for reported samples (0.89±0.01). The long COI sequence allowed us to identify seven (five new) haplotypes in CR and two (one new) in Panamá. A haplotype network for the long COI gene sequence showed that samples from CR and Panamá belong to a single large group. The long COI gene sequences suggest that haplotypes in Panamá and CR, although similar to each other, had a significant geographic differentiation (Kst=1.33; P<0.001). Thus, most of our results suggest a recent range expansion in CR and Panamá.

Synthesis of DNA fragments containing 2'-deoxy-4'-selenonucleoside units using DNA polymerases: comparison of dNTPs with O, S and Se at the 4'-position in replication.

4'-SelenoDNA fragments were synthesized for the first time using 4'-selenothymidine triphosphate (SeTTP) by taking advantage of its bioequivalence against DNA polymerases. DNA fragments each with a homologous element (O, S or Se) at the 4'-position of the thymidine units were effectively amplified using KOD Dash DNA polymerase.

Climate change is catchy--but when will it really hurt?

Concern and general awareness about the impacts of climate change in all sectors of the social-ecological-economic system is growing as a result of improved climate science products and information, as well as increased media coverage of the apparent manifestations of the phenomenon in our society. However, scales of climate variability and change, in space and time, are often confused and so attribution of impacts on various sectors, including the health sector, can be misunderstood and misrepresented. In this review, we assess the mechanistic links between climate and infectious diseases in particular, and consider how this relationship varies, and may vary according to different time scales, especially for aetiologically climate-linked diseases. While climate varies in the medium (inter-annual) time frame, this variability itself may be oscillating and/or trending on cyclical and long-term (climate change) scales because of regional and global scale climate phenomena such as the El-Nino southern oscillation coupled with global-warming drivers of climate change. As several studies have shown, quantifying and modelling these linkages and associations at appropriate time and space scales is both necessary and increasingly feasible with improved climate science products and better epidemiological data. The application of this approach is considered for South Africa, and the need for a more concerted effort in this regard is supported.

Habitat characteristics of Anopheles gambiae s.s. larvae in a Kenyan highland.

Anopheline larval habitats associated with a swamp, were examined in a highland area (1910 m elevation) of western Kenya. A significant association was found between occurrence of Anopheles gambiae Giles s.s. (Diptera: Culicidae) larvae and two factors, habitat size and vegetation type. Over 80% of An. gambiae s.s. larvae were found in small isolated pools, characterized by short plants, occurring in both swamp margins and roadside ditches. However, Anopheles gambiae s.s. was not found in habitats marked by papyrus and floating plants. The larval habitat of An. gambiae s.s. was characterized by warmer daytime temperatures of water, which were significantly affected by habitat size and plant size. The density of indoor resting An. gambiae s.s. was 0.22 per house and negatively associated with distance from the swamp. These results indicate that the practice of swamp cultivation, in populated areas of the African highlands, increases availability and enhances habitat conditions for the malaria vector.

Synthesis and biological activities of cyclic ADP-carbocyclic-ribose and its analogs.

An efficient synthesis of cyclic ADP-carbocyclic-ribose (2), as a stable mimic for cyclic ADP-ribose, was achieved. Treatment of N1-carbocyclic-ribosyladenosine bisphosphate derivative 10 with AgNO3 in the presence of molecular sieves 3A in pyridine gave the desired cyclic product in 93% yield, which was deprotected to give the target cyclic ADP-carbocyclic-ribose (2).

Anopheline mosquito survival strategies during the dry period in western Kenya.

The dry season survival mechanism of Anopheles gambiae Giles is one of the most vexing deficiencies in our understanding of the biology of the major malaria vectors. In this study, we examined the dynamics of anopheline adult mosquitoes, their larval habitats, and egg survival potential during the dry season in the basin region of Lake Victoria, western Kenya. Through field surveys, we demonstrated two survival strategies of An. gambiae sensu stricto during the dry season: continuous reproduction throughout the year and embryo dormancy in moist soil for at least several days. We further demonstrated that An. gambiae shows a strong preference for moist soil as an oviposition substrate rather than dry soil substrate under the insectary conditions. The observation that anopheline eggs remain a dormant stage to resist desiccation clearly contrasts the conventional wisdom that anopheline eggs hatch shortly after they are laid. Our results from western Kenya are consistent with the suggestion that anopheline mosquitoes do not necessarily suffer a severe population bottleneck during the dry season and thus maintain a large effective population size.

Effect of 9-(6,7-dideoxy-beta-D-allo-hept-5-ynofuranosyl)adenine on noradrenaline release from vascular sympathetic nerves.

1. The effects of 9-(6,7-dideoxy-beta-D-allo-hept-5-ynofuranosyl)adenine (HAK2701), a selective and potent ligand for P3 receptor-like protein, on the release of endogenous noradrenaline (NA) from electrically stimulated rat mesenteric artery and rabbit ear artery were compared with those of a number of purinoceptor agonists. 2. In the rat mesenteric artery, the P1 receptor agonists 2-chloroadenosine (2CA) and 5'-N-ethylcarboxamidoadenosine (NECA) and the P2 purinoceptor agonists beta,gamma-methylene ATP (betagammamATP) and 2-methylthio ATP (2mSATP) significantly inhibited the release of NA in a xanthine-sensitive manner. HAK2701 did not significantly inhibit the release of NA, the relative order of potency being betagammamATP > NECA > 2CA > 2mSATP >> HAK2701. 3. In the rabbit ear artery, both P1 and P2 receptor agonists significantly facilitated the release of NA in a xanthine-sensitive manner. HAK2701 also significantly facilitated the release of NA, the relative order of potency being HAK2701 > betagammamATP > 2CA > 2mSATP > NECA. 4. These findings suggest that HAK2701 may be a potent and selective agonist for facilitatory prejunctional purinoceptors, but not for inhibitory purinoceptors, on adrenergic nerve terminals.

Nucleosides and nucleotides. 200. Reinvestigation of 5'-N-ethylcarboxamidoadenosine derivatives: structure-activity relationships for P(3) purinoceptor-like proteins.

The non-P(1) and non-P(2) muscle relaxant effect of ATP in rabbit thoracic aorta has recently been attributed to a putative P(3) purinoceptor, which is activated by either adenosine or ATP. Since the physiological roles of this putative P(3) purinoceptor and of a new [(3)H]-5'-N-ethylcarboxamidoadenosine (NECA)-binding protein from rat brain membranes called P(3) purinoceptor-like protein (P(3)LP), due to its ligand specificity, have not been fully elucidated, we needed a specific ligand to obtain further information about the receptor. We examined the structure-activity relationship (SAR) of various 5'-N-substituted-carboxamidoadenosine derivatives toward P(3)LP and discovered a hydrophobic binding region near the 5'-N-substituted-carboxamide group. From the linear alkyl N-substituted derivatives, the N-n-pentyl derivative 10 was found to be the most potent ligand with a K(i) value of 12 nM. In the series of the N-cycloalkyl derivatives, the N-cyclohexyl derivative 27 was the strongest ligand with a K(i) value of 18 nM. On the other hand, the N-substituents having branched alkyl side chains and bulky cycloalkyl groups did not show any potent affinities for P(3)LP. Therefore, the hydrophobic pocket accommodates approximately a 10-carbon-atom-long linear alkyl side chain, while a considerably stronger hydrophobic binding region of about a 5-carbon-atom-long depth exists near the nitrogen atom of the amide group. This pocket also allows substitution with bulky hydrophobic groups since the 5'-N-cycloalkyl derivatives have high affinities. We also examined the receptor selectivity for the selected nucleosides 10 and 27 with 1 [9-(6,7-dideoxy-beta-D-allo-hept-5-ynofuranosyl)adenine, HAK2701] and NECA versus P(1) purinoceptor subtypes, such as adenosine A(1), A(2A), A(2B), and A(3) receptors, and found that 27 is the most selective ligand for P(3)LP.

Synthesis of sugar-modified 4'-thiocytidine derivatives.

An improved and large-scale synthesis of 1,4-anhydro-2,3,5-tri-O-benzyl-4-thio-D-ribitol (4) has been achieved via dibromination of 2,3,5-tri-O-benzyl-D-ribitol (1). Using the Pummerer reaction, 4'-thiocytidine derivative (10) was successfully prepared in multigram scale. After deprotection of the 2,4-dimethoxybenzoyl group of 10, the resulting 11 was converted into 2'-deoxy-4'-thiocytidine (16) via the radical deoxgenation. The sugar-modified 4'-thiocytidine derivatives, 17 and 18, were also prepared.

An efficient synthesis of cyclic IDP- and cyclic 8-bromo-IDP-carbocyclic-riboses using a modified Hata condensation method to form an intramolecular pyrophosphate linkage as a key step. An entry to a general method for the chemical synthesis of cyclic ADP-ribose analogues

An efficient synthesis of cyclic IDP-carbocyclic-ribose (3) and its 8-bromo derivative 6, as stable mimics of cyclic ADP-ribose, was achieved, and a condensation reaction with phenylthiophosphate-type substrate 15 or 16 to form an intramolecular pyrophosphate linkage was a key step. N-1-Carbocyclic-ribosylinosine derivative 28 and the corresponding 8-bromo congener 24 were prepared via condensation between N-1-(2,4-dinitrophenyl)inosine derivative 17 and a known optically active carbocyclic amine 18. Compounds 24 and 28 were then converted to the corresponding 5"-phosphoryl-5'-phenylthiophosphate derivatives 15 and 16, respectively, which were substrates for the condensation reaction to form an intramolecular pyrophosphate linkage. Treatment of 8-bromo substrate 15 with I2 or AgNO3 in the presence of molecular sieves 3A (MS 3A) in pyridine at room temperature gave the desired cyclic product 12 quantitatively, while the yield was quite low without MS. The similar reaction of 8-unsubstituted substrate 16 gave the corresponding cyclized product 32 in 81% yield. Acidic treatment of these cyclic pyrophosphates 12 and 32 readily gave the targets 6 and 3, respectively. This result suggests that the construction of N-1-substituted hypoxanthine nucleoside structures from N-1-(2,4-dinitrophenyl)inosine derivatives and the intramolecular condensation by activation of the phenylthiophosphate group with I2 or AgNO3/MS 3A combine to provide a very efficient route for the synthesis of analogues of cyclic ADP-ribose such as 3 and 6. Thus, this may be an entry to a general method for synthesizing biologically important cyclic nucleotides of this type.

Use of cotton rats to evaluate the efficacy of antivirals in treatment of measles virus infections.

No practical animal models for the testing of chemotherapeutic or biologic agents identified in cell culture assays as being active against measles virus (MV) are currently available. Cotton rats may serve this purpose. To evaluate this possibility, 5-ethynyl-1-beta-D-ribofuranosylimidazole-4-carboxamide (EICAR) and poly(acrylamidomethyl propanesulfonate) (PAMPS), two compounds that have been reported to inhibit MV in vitro, and ribavirin, an established antiviral drug with MV-inhibitory activity, were evaluated for their antiviral activities against MV and respiratory syncytial virus (RSV) in tissue culture and in hispid cotton rats. A single administration of PAMPS markedly inhibited pulmonary RSV or MV replication (>3 log(10) reduction in pulmonary titer compared to that for controls), but only if this compound was administered intranasally at about the time of virus inoculation. Both EICAR and ribavirin exhibited therapeutic activity against RSV and MV in cotton rats when they were administered parenterally. However, both of these compounds were less effective against MV. On the basis of the pulmonary virus titers on day 4 after virus inoculation, the minimal efficacious dose of EICAR against MV (120 mg/kg of body weight/day when delivered intraperitoneally twice daily) appeared to be three times lower against this virus than that of ribavirin delivered at a similar dose (i.e., 360 mg/kg/day). These findings correlated with those obtained in vitro. The data obtained suggest that cotton rats may indeed be useful for the initial evaluation of the activities of antiviral agents against MV.

Synthesis and structure-activity relationships of selective ligands for P3 purinoceptor-like protein (P3LP).

We examined the structure-activity relationship of various 5'-N-substituted-carboxamidoadenosine derivatives toward P3 purinoceptor-like protein (P3LP), which has affinity for both adenine nucleosides and nucleotides. We discovered a hydrophobic binding region near the 5'-N-substituted-carboxamide group. From the linear alkyl N-substituted derivatives, 1-(adenin-9-yl)-1-deoxy-N-n-pentyl-beta-D-ribofuranuronamide (6) was found to be the most potent ligand. In the series of the N-cycloalkyl derivatives, 1-(adenin-9-yl)-1-deoxy-N-cyclohexyl-beta-D-ribofuranuronamide (8) was the strongest ligand. We also examined the receptor selectivity for the selected nucleosides 6 and 8 with 1 (HAK2701) and N-ethylcarboxamidoadenosine (NECA) versus P1 purinoceptor subtypes, such as adenosine A1, A2A, A2B, and A3 receptors and found 8 is the most selective ligand for P3LP.

Transcriptional control of Borna disease virus (BDV) in persistently BDV-infected cells.

Regulation of viral RNA levels in infected cells is considered important in the investigation of viral transcription and replication. Amounts of Borna disease virus (BDV) RNAs were increased in confluent persistently BDV-infected MDCK cells (MDCK/BDV) cells, while maintained at low levels in growing cells. The amount of 1.9-kb RNA without cap formation and polyadenylation at the 5' and 3' ends respectively were remarkably increased (200% per day) in confluent MDCK/BDV cells. Both the full-length genomic and anti-genomic RNAs were increased accompained by 1.9-kb RNA, suggesting the transcription of the 1.9-kb RNA was important for replication of BDV. Ribavirin has an inhibitory effect on replication and transcription of BDV at concentrations from 1 to 10 microgram/ml [Mizutani T et al., Arch Virol (1998)143: 2039-2 044]. BDV transcripts were decreased with ribavirin treatment and increased after its removal which indicated that ribavirin has a reversible inhibitory effect on BDV transcription. Furthermore, BDV transcription was also decreased by two agents, RMNPA and EICAR, which selectively inhibit enzyme activity related to cap formation at the 5' end of mRNA. On the contrary, when the growing MDCK/BDV cells were treated with actinomycin D, transcripts of BDV RNA were increased for 24 h. These agents and culture conditions in this study were found to be useful tools for up-and down-regulation of BDV transcription in persistently BDV-infected cells.

Mechanism-based design of inosine 5-monophosphate dehydrogenase inhibitors: synthesis and biological activities of 5-ethynyl-1-beta-D-ribofuranosylimidazole-4-carboxamide (EICAR) and its derivatives.

Inosine 5 -monophosphate dehydrogenase (IMPDH) catalyzes the nicotinamide adenine dinucleotide (NAD)-dependent oxidation of inosine 5 -monophosphate (IMP) to xanthosine 5 -monophosphate (XMP), and is one of the key rate-determining enzymes of de novo guanine nucleotide biosynthesis in mammalian systems. Based on the proposed catalytic mechanism of IMPDH, we designed and synthesized 5-ethynyl-1-beta-D-ribofuranosylimidazole-4-carboxamide (5b, EICAR) from 5-amino-1-beta-D-ribofuranosylimida-zole-4-carboxamide (AICAR) via palladium chemistry. EICAR is a potent cytostatic agent that inhibits various tumor cells in culture including human solid tumor cells in vitro and in vivo. EICAR also showed broad-spectrum antiviral activities, about 10- to 100-fold greater than those of ribavirin. An examination of the structure-activity relationships revealed that an alkynyl group, especially an ethynyl group at the 5-position, is important for its activity due to the inhibition of IMPDH. The mode of action of EICAR is also discussed.

Tremorgenic indole alkaloids. Studies directed toward the assembly of the A, F, and I rings of penitrem D: observation of an unexpected stereochemical outcome.

[formula: see text] In this Letter we demonstrate the viability of a highly stereoselective tandem Mannich cyclization-grammine fragmentation/addition cascade, critical for assembly of the A and F rings of penitrem D. We also explored simultaneous execution of this tactic with concurrent construction of ring I. Reinvestigation of a model system provided an explanation for the unanticipated stereochemical outcome at C(28).

Synthetic study on carbocyclic analogs of cyclic ADP-ribose, a novel second messenger: an efficient synthesis of cyclic IDP-carbocyclic-ribose.

An efficient synthesis of cyclic IDP-carbocyclic-ribose, as a stable mimic for cyclic ADP-ribose, was achieved. 8-Bromo-N1-carbocyclic-ribosylinosine derivative 10, prepared from N1-(2,4-dinitrophenyl)inosine derivative 5 and an optically active carbocyclic amine 6, was converted to 8-bromo-N1-carbocyclic-ribosylinosine bisphosphate derivative 15. Treatment of 15 with I2 in the presence of molecular sieves in pyridine gave the desired cyclic product 16 quantitatively, which was deprotected and reductively debrominated to give the target cyclic IDP-carbocyclic-ribose (3).

Spatial distribution and habitat characterization of anopheline mosquito larvae in Western Kenya.

Studies were conducted to characterize larval habitats of anopheline mosquitoes and to analyze spatial heterogeneity of mosquito species in the Suba District of western Kenya. A total of 128 aquatic habitats containing mosquito larvae were sampled, and 2,209 anopheline and 10,538 culicine larvae were collected. The habitats were characterized based on size, pH, distance to the nearest house and to the shore of Lake Victoria, coverage of canopy, surface debris, algae and emergent plants, turbidity, substrate, and habitat types. Microscopic identification of third- and fourth-instar anopheline larvae did not yield any Anopheles funestus or other anophelines. A total of 829 An. gambiae s.l. larvae from all habitats were analyzed further by rDNA-polymerase chain reaction to identify individual species within the An. gambiae species complex. Overall, An. arabiensis was the predominant species (63.4%), and An. gambiae was less common (31.4%). The species composition of An. gambiae s.l. varied significantly among the sampling sites throughout Suba District. The larval habitats in the southern area of the district had a higher proportion of An. gambiae than in the northern area. Multiple logistic analysis did not detect any significant association between the occurrence of anopheline larvae and habitat variables, and principal component analysis did not identify key environmental factors associated with the abundance of An. gambiae. However, significant spatial heterogeneity in the relative abundance of An. gambiae within the Suba district was detected. When the effect of larval habitat locality was considered in the analysis, we found that the distance to the nearest house and substrate type were significantly associated with the relative abundance of An. gambiae. Future studies integrating detailed water chemistry analysis, remote sensing technology, and the ecology of predators may be required to further elucidate the mechanisms underlying the observed spatial variation of anopheline larval distribution.

Synthesis and incorporation of tricyclic-imidazo[5',4':4,5]pyrido[2,3-d]pyrimidine nucleosides into oligonucleotides and their thermal stability.

We designed and synthesized four novel tricyclicnucleosides 1-4, which have imidazo[5',4':4,5]pyrido[2,3-d]pyrimidine structures, to construct theramallystable, non-natural, DNA duplexes. Both 1:2 and 3:4 possess four complementary hydrogen-bond pairs and are expected to form more stable base-pairs than the naturally occurring Watson-Crick base-pairs when incorporated into oligodeoxynucleotides (ODNs). NMR studies of these base-pairs at the nucleoside level in CDCl3 identified the expected base-pair formation with four hydrogen bonds. We also synthesized several ODNs containing these nucleosides and measured their thermal stability. We found that if several of the novel base-pairs were incorporated on complementary strands, the resulting DNA duplexes had very high thermal stability.