Isolation of a Transformation-Defective Deletion Mutant of Moloney Murine Sarcoma Virus

A transformation-defective (td) deletion mutant of Moloney murine sarcoma virus (td Mo-MSV) and a transforming component termed Mo-MSV 3 were cloned from a stock of clone 3 Mo-MSV. To define the defect of the transforming function, the RNA of td Mo-MSV was compared with those of Mo-MSV 3 and of another transforming variant termed Mo-MSV 124 and with helper Moloney murine leukemia virus (Mo-MuLV). The RNA monomers of td Mo-MSV and Mo-MSV 3 comigrated on polyacrylamide gels and were estimated to be 4.8 kilobases (kb) in length. In agreement with previous analyses, the RNA of Mo-MSV 124 measured 5.5 kb and that of Mo-MuLV measured 8.5 kb. The interrelationships among the viral RNAs were studied by fingerprinting and mapping of RNase T₁-resistant oligonucleotides (T₁-oligonucleotides) and by identification of T₁-oligonucleotides present in hybrids formed by a given viral RNA with cDNA's made from another virus. The nontransforming td Mo-MSV RNA lacked most of the Mo-MSV-specific sequence, i.e., the four 3′-proximal T₁-oligonucleotides of the six T₁-oligonucleotides that are shared by the Mo-MSV-specific sequences of Mo-MSV 3 and Mo-MSV 124. The remaining two Mo-MSV-specific oligonucleotides identified td Mo-MSV as a deletion mutant of MSV rather than a deletion mutant of Mo-MuLV. td Mo-MSV and Mo-MSV 124 exhibited similar deletions of gag, pol, and env sequences which were less extensive than those of Mo-MSV 3. Hence, td Mo-MSV is not simply a deletion mutant of Mo-MSV 3. In addition to their MSV-specific sequences, all three MSV variants, including td Mo-MSV, shared the terminal sequences probably encoding the proviral long terminal repeat, which differed from their counterpart in Mo-MuLV. This may indirectly contribute to the oncogenic potential of MSV. A comparison of td Mo-MSV sequences with either Mo-MSV 124 or Mo-MSV 3 indicated directly, in a fashion similar to the deletion analyses which defined the src gene of avian sarcoma viruses, that Mo-MuLV-unrelated sequences of Mo-MSV are necessary for transformation. A definition of transformation-specific sequences of Mo-MSV by deletion analysis confirmed and extended previous analyses which have identified Mo-MuLV-unrelated sequences in Mo-MSV RNA and other studies which have described transformation of mouse 3T3 fibroblasts upon transfection with DNAs containing the Mo-MSV-specific sequence.     

Synthesis of 3′,5′-Dithymidylyl-α-hydroxyphosphonate Dimer Building Blocks for Oligonucleotide Synthesis—A New Pro-oliguncleotide

Abstract

The synthesis of the dimer building blocks 1 and 2 and their introduction into (T)₁₅-oligonucleotides is described. The stability against 3′-exonuclease digestion (SVP) as well as the hybridization properties (T_m values) were examined.     

A linear function for the melting behavior of lipids

The melting behavior of a variety of saturated long chain compounds is shown to be related to hydrocarbon chain length by the equation TN = C₀ + T_∞N where T is the absolute melting temperature, and N is the number of long chain carbon atoms. The constants C₀ and T_∞ are determined graphically or analytically from TN vs. N data. The linear relationship, derived from fundamental thermodynamic principles, is empirically satisfied. For each homologous series considered, coefficients of the equation provide a rational means for correlation and comparison with other polymorphs and indicate the relative importance of chain length, chain parity (even or odd), and headgroup polarity to melting behavior.     

Physical Factors Influencing Pleasant Touch during Passive Fingertip Stimulation

Objective

Tactile explorations with the fingertips provide information regarding the physical properties of surfaces and their relative pleasantness. Previously, we performed an investigation in the active touch domain and linked several surface properties (i.e. frictional force fluctuations and net friction) with their pleasantness levels. The aim of the present study was to investigate physical factors being important for pleasantness perception during passive fingertip stimulation. Specifically we were interested to see whether factors, such as surfaces'' topographies or their frictional characteristics could influence pleasantness. Furthermore, we ascertained how the stimulus pleasantness level was impacted by (i) the normal force of stimulus application (F_N) and (ii) the stimulus temperature (T_S).

Methods and Results

The right index fingertips of 22 blindfolded participants were stimulated using 27 different stimuli, which varied in average roughness (Ra) and T_S. A 4-axis robot moved the stimuli horizontally under participants'' fingertips with three levels of F_N. The robot was equipped with force sensors, which recorded the F_N and friction force (F_T) during stimulation. Participants rated each stimulus according to a three-level pleasantness scale, as very pleasant (scored 0), pleasant (scored 1), or unpleasant (scored 2). These ordinal pleasantness ratings were logarithmically transformed into linear and unidimensional pleasantness measures with the Rasch model. Statistical analyses were conducted to investigate a possible link between the stimulus properties (i.e. Ra, F_N, F_T, and T_S) and their respective pleasantness levels. Only the mean Ra and F_T values were negatively correlated with pleasantness. No significant correlation was detected between F_N or T_S and pleasantness.

Conclusion

Pleasantness perception, resulting from passive fingertip stimulation, seems to be influenced by the surfaces'' average roughness levels and average F_T occurring during fingertip stimulation.     

Deviations from Hardy-Weinberg Proportions: Sampling Variances and Use in Estimation of Inbreeding Coefficients

An analysis is made of the distribution of deviations from Hardy-Weinberg proportions with k alleles and of estimates of inbreeding coefficients (f) obtained from these deviations.—If f is small, the best estimate of f in large samples is shown to be 2Σ _i(T_ii/N_i)/(k - 1), where T_ii is an unbiased measure of the excess of the ith homozygote and N_i the number of the ith allele in the sample [frequency = N_i/(2N)]. No extra information is obtained from the T_ij, where these are departures of numbers of heterozygotes from expectation. Alternatively, the best estimator can be computed from the T_ij, ignoring the T_ii. Also (1) the variance of the estimate of f equals 1/(N(k - 1)) when all individuals in the sample are unrelated, and the test for f = 0 with 1 d.f. is given by the ratio of the estimate to its standard error; (2) the variance is reduced if some alleles are rare; and (3) if the sample consists of full-sib families of size n, the variance is increased by a proportion (n - 1)/4 but is not increased by a half-sib relationship.—If f is not small, the structure of the population is of critical importance. (1) If the inbreeding is due to a proportion of inbred matings in an otherwise random-breeding population, f as determined from homozygote excess is the same for all genes and expressions are given for its sampling variance. (2) If the homozygote excess is due to population admixture, f is not the same for all genes. The above estimator is probably close to the best for all f values.     

Characterization of 10S RNA: a new stable rna molecule from Escherichia coli.

Summary When cells of Escherichia coli are labeled with ³²P_i for long periods of time and the cell content is subjected to electrophoresis in polyacrylamide gels, an RNA band appears which is about 10S in size. This band seems to contain three conformers. After treatment with formamide only a single band appears in this region of the gel, which contains 550 nucleotides as determined from its mobility. The complexity of the fingerprint of this material, after digestion with T₁-RNase, is in agreement with the size as determined by the mobility, this confirming that indeed it is a single molecule. Composition of the T₁-oligonucleotides was determined by digesting the T₁-generated oligonucleotides with pancreatic RNase and T₂-RNase. The quantitative and qualitative analysis of these digestions suggests that 10S RNA contains 609 nucleotides. The molecule contains, besides the four regular bases, one copy per molecule of the modified base pseudouridine.10S RNA cannot be processed by cell extracts to tRNA-sized molecules and does not bind significantly to ribosomes, hence it is unlikely to be a tRNA precursor or an mRNA.     

Ctp1 and the MRN-Complex Are Required for Endonucleolytic Rec12 Removal with Release of a Single Class of Oligonucleotides in Fission Yeast

DNA double-strand breaks (DSBs) are formed during meiosis by the action of the topoisomerase-like Spo11/Rec12 protein, which remains covalently bound to the 5′ ends of the broken DNA. Spo11/Rec12 removal is required for resection and initiation of strand invasion for DSB repair. It was previously shown that budding yeast Spo11, the homolog of fission yeast Rec12, is removed from DNA by endonucleolytic cleavage. The release of two Spo11 bound oligonucleotide classes, heterogeneous in length, led to the conjecture of asymmetric cleavage. In fission yeast, we found only one class of oligonucleotides bound to Rec12 ranging in length from 17 to 27 nucleotides. Ctp1, Rad50, and the nuclease activity of Rad32, the fission yeast homolog of Mre11, are required for endonucleolytic Rec12 removal. Further, we detected no Rec12 removal in a rad50S mutant. However, strains with additional loss of components localizing to the linear elements, Hop1 or Mek1, showed some Rec12 removal, a restoration depending on Ctp1 and Rad32 nuclease activity. But, deletion of hop1 or mek1 did not suppress the phenotypes of ctp1Δ and the nuclease dead mutant (rad32-D65N). We discuss what consequences for subsequent repair a single class of Rec12-oligonucleotides may have during meiotic recombination in fission yeast in comparison to two classes of Spo11-oligonucleotides in budding yeast. Furthermore, we hypothesize on the participation of Hop1 and Mek1 in Rec12 removal.     

P relaxation responses associated with n(2)/o(2) diffusion in soybean nodule cortical cells and excised cortical tissue

N₂-fixing Bradyrhizobium japonicum nodules and cortical tissue derived from these nodules were examined in vivo by ³¹P nuclear magnetic resonance (NMR) spectroscopy. Perfusion of the viable nodules and excised cortical tissue with O₂ followed by N₂ or Ar caused a loss of orthophosphate (Pi) resonance magnetization associated with the major portion of acidic Pi (δ 0.9 ppm, pH 5.5) residing in the cortical cells. Resumption of O₂ perfusion restored approximately 80% of the intensity of this peak. Detailed examination of the nuclear relaxation processes, spin-lattice relaxation time (T₁), and spin-spin relaxation time (T₂), under perfusion with N₂ or Ar as opposed to O₂, indicated that loss of signal was due to T₁ saturation of the acidic Pi signal under the rapid-pulsed NMR recycling conditions. In excised cortical tissue, Pi T₁, values derived from biexponential relaxation processes under perfusing O₂ were 59% 3.72 ± 0.93 s and 41% 0.2 ± 0.08 s, whereas under N₂ these values were 85% 7.07 ± 1.36 s and 15% 0.39 ± 0.07 s. The T₁ relaxation behavior of whole nodule vacuolar Pi showed the same trend, but the overall values were somewhat shorter. T₂ values for cortical tissue were also biexponential but were essentially the same under O₂ (38% 0.066 ± 0.01 s and 63% 0.41 ± 0.08 s) and N₂ (39% 0.07 ± 0.01 s and 61% 0.37 ± 0.01 s) perfusion. Soybean (Glycine max) root tissue as well as Pi solutions exhibited single exponential T₁ decay values that were not altered by changes in the perfusing gas. These data indicate that oxygen induces a change in the physical environment of phosphate in the cortical cell tissue. Although under certain conditions oxygen has been observed to act as a paramagnetic relaxation agent, model T₁ experiments demonstrate that O₂ does not significantly influence Pi relaxation in this manner. Alternatively, we suggest that an increase in solution viscosity brought on by the production of an occlusion glycoprotein (under O₂ perfusion) is responsible for the observed relaxation changes.     

Thermal preferences of hatchling saltwater crocodiles (Crocodylus porosus) in response to time of day,social aggregation and feeding

Three month old hatchling Crocodylus porosus with data loggers in their stomachs were placed in thermal gradients, in isolation (N=16) and in groups of 4 (N=8 groups; 32 individuals). Mean T_b and variation in T_b (SD) was not different whether individual crocodiles in isolation were fasted or fed, or if individuals were housed in isolation (I) or in groups (G). However, individuals in isolation (N=16) maintained slightly lower T_bs than those in groups (N=32) during the early morning (06:00–11:00 h). The overall mean T_b recorded for fasted individuals in the isolated and group treatments (N=48) was 30.9±2.3 °C SD, with 50% of T_bs (T_set) between 29.4 °C and 32.6 °C, and a voluntary maximum and minimum of 37.6 °C and 23.2 °C respectively. During the day (11:00–17:00 h), individuals in isolation and in groups selected the warmer parts of the gradient on land, where they moved little. Outside of this quiescent period (QP), activity levels were much higher and they used the water more. There was a strong diurnal cycle for fasted individuals in isolation and in groups, with T_b during the QP (31.9±2.09 °C; N=48) significantly higher than during the non-quiescent period (NQP: 30.6±2.31 °C). Thermal variation (SD) in T_b was relatively stable throughout the day, with the highest variation at around dusk and early evening (18:00–20:00 h), which coincided with a period of highest activity. The diurnal activity cycle appears innate, and may reflect the need to engage in feeding activity at the water's edge in the early evening, despite ambient temperatures being cooler, with reduced activity and basking during the day. If so, preferred T_b may be more accurately defined as the mean T_b during the QP rather than the NQP. Implications for the thermal environment best suited for captive C. porosus hatchlings are discussed.     

Incorporation of cytokinin N-benzyladenine into tobacco callus transfer ribonucleic Acid and ribosomal ribonucleic Acid preparations

The incorporation of the cytokinin N⁶-benzyladenine into tobacco (Nicotiana tabacum) callus tRNA and rRNA preparations isolated from tissue grown on medium containing either N⁶-benzyladenine-8-¹⁴C or N⁶-benzyladenine-8-¹⁴C: benzene-³H(G) has been examined. N⁶-benzyladenine was incorporated into both the tRNA and rRNA preparations as the intact base. Over 90% of the radioactive N⁶-benzyladenosine recovered from the RNA preparations was associated with the rRNA. Purification of the crude rRNA by either MAK chromatography or Sephadex G-200 gel filtration had no effect on the N⁶-benzyladenosine content of the RNA preparation. The distribution of N⁶-benzyladenosine moieties in tobacco callus tRNA fractionated by BD-cellulose chromatography did not correspond to the distribution of ribosylzeatin activity. N⁶-benzyladenosine was released from the rRNA preparation by treatment with venom phosphodiesterase and phosphatase, ribonuclease T₂ and phosphatase, or ribonuclease T₂ and a 3′-nucleotidase. N⁶-benzyladenosine was not released from the RNA preparation by treatment with either ribonuclease T₂ or phosphatase alone or by successive treatment with ribonuclease T₂ and a 5′-nucleotidase. Brief treatment of the rRNA preparation with ribonuclease T₁ and pancreatic ribonuclease converted the N⁶-benzyladenosine moieties into an ethyl alcohol soluble form. On the basis of these and earlier results, the N⁶-benzyladenosine recovered from the tobacco callus RNA preparations appears to be present as a constituent of RNA and not as a nonpolynucleotide contaminant.     

Niabella thaonhiensis sp. nov., Isolated From the Forest Soil of Kyonggi University in Korea

Strain NHI-24^T was isolated from forest soil by a polycarbonate membrane transwell plate. It is a Gram-negative, rod-shaped, non-motile, non-spore-forming bacterium. Phylogenetic analysis based on 16S rRNA gene sequence data indicated that strain NHI-24^T is closely related to members of the genus Niabella: N. drilacis E90^T (97.7 %), N. tibetensis 15-4^T (96.7 %), N. aurantiaca R2A15-11^T (96.5 %), N. hirudinis E96^T (95.8 %), N. soli JS13-8^T (94.7 %), N. ginsengisoli NBRC106414^T (94.4 %), and N. yanshanensis CCBAU 05354^T (94.2 %). Growth temperatures range widely, from 15 to 37 °C, with 30 °C as the optimum. Salt tolerance ranges from 0 to 2 %. The strain grows at pH 6.5–11.0, with an optimal range of pH 7.0–9.5. Cells produce flexirubin-type pigments, and the predominant menaquinone is MK-7. The major fatty acids of strain NHI-24^T are iso-C_15:0 (36.72 %), iso-C_15:1 G (20.8 %), and summed feature 3 (C_16:1 ω7c/C_16:1 ω6c; 15.2 %). DNA–DNA hybridization values between strain NHI-24^T and members of the genus Niabella range from 37 to 53 %. Based on these results, it is proposed that strain NHI-24^T represents a novel species of the genus Niabella with the name Niabella thaonhiensis (= KACC 17215^T = KEMB 9005-018^T = JCM 18864^T).     

A proposed mechanism for the thermal denaturation of a recombinant Bacillus halmapalus α-amylase—the effect of calcium ions

The thermal stability of a recombinant α-amylase from Bacillus halmapalus α-amylase (BHA) has been investigated using circular dichroism spectroscopy (CD) and differential scanning calorimetry (DSC). This α-amylase is homologous to other Bacillus α-amylases where crystallographic studies have identified the existence of three calcium binding sites in the structure. Denaturation of BHA is irreversible with a T_m of approximately 89 °C and DSC thermograms can be described using a one-step irreversible model. A 5 °C increase in T_m in the presence of 10-fold excess CaCl₂ was observed. However, a concomitant increase in the tendency to aggregate was also observed. The presence of 30–40-fold excess calcium chelator (ethylenediaminetetraacetic acid (EDTA) or ethylene glycol-bis[β-aminoethyl ether] N,N,N′,N′-tetraacetic acid (EGTA)) results in a large destabilization of BHA, corresponding to about 40 °C lower T_m as determined by both CD and DSC. Ten-fold excess EGTA reveals complex DSC thermograms corresponding to both reversible and irreversible transitions, which probably originate from different populations of BHA/calcium complexes. Combined interpretation of these observations and structural information on homologous α-amylases forms the basis for a suggested mechanism underlying the inactivation mechanism of BHA. The mechanism includes irreversible thermal denaturation of different BHA/calcium complexes and the calcium binding equilibria. Furthermore, the model accounts for a temperature-induced reversible structural change associated with calcium binding.     

Observation of the Oxygen Diffusion Barrier in Soybean (Glycine max) Nodules with Magnetic Resonance Microscopy

The effects of selected gas perfusion treatments on the spinlattice relaxation times (T₁) of the soybean (Glycine max) nodule cortex and inner nodule tissue were studied with ¹H high resolution magnetic resonance microscopy. Three gas treatments were used: (a) perfusion with O₂ followed by N₂; (b) O₂ followed by O₂; and (c) air followed by N₂. Soybean plants with intact attached nodules were placed into the bore of a superconducting magnet and a selected root with nodules was perfused with the gas of interest. Magnetic resonance images were acquired with repetition times from 50 to 3200 ms. The method of partial saturation was used to calculate T₁ times on selected regions of the image. Calculated images based on T₁ showed longer T₁ values in the cortex than in the inner nodule during all of the gas perfusions. When nodules were perfused with O₂-O₂, there was no significant change in the T₁ of the nodule between the two gas treatments. When the nodule was perfused with O₂-N₂ or air-N₂, however, the T₁ of both the cortex and inner nodule increased. In these experiments, the increase in T₁ of the cortex was 2- to 3-fold greater than the increase observed in the inner nodule. A similar change in T₁ was found in detached live nodules, but there was no change in T₁ with selective gas perfusion of detached dead nodules. These observations suggest that cortical cells respond differently to selected gas perfusion than the inner nodule, with the boundary of T₁ change sharply delineated at the interface of the inner nodule and the inner cortex.     

Polymorphisms in Plasmodium falciparum chloroquine resistance transporter (Pfcrt) and multidrug-resistant gene 1 (Pfmdr-1) in Nigerian children 10 years post-adoption of artemisinin-based combination treatments

The emergence and spread of Plasmodium falciparum parasites resistant to artemisinin derivatives and their partners in southeastern Asia threatens malaria control and elimination efforts, and heightens the need for an alternative therapy. We have explored the distribution of P. falciparum chloroquine resistance transporter (Pfcrt) and multidrug-resistant gene 1 (Pfmdr-1) haplotypes 10 years following adoption of artemisinin-based combination therapies in a bid to investigate the possible re-emergence of Chloroquine-sensitive parasites in Nigeria, and investigated the effect of these P. falciparum haplotypes on treatment outcomes of patients treated with artemisinin-based combination therapies. A total of 271 children aged <5 years with uncomplicated falciparum malaria were included in this study. Polymorphisms on codons 72–76 of the Pfcrt gene and codon 86 and 184 of Pfmdr-1 were determined using the high resolution melting assay. Of 240 (88.6%) samples successfully genotyped with HRM for Pfcrt, wildtype C₇₂M₇₄N₇₅K₇₆ (42.9%) and mutant C₇₂I₇₄E₇₅T₇₆ (53.8%) were observed. Also, wildtype N₈₆Y₁₈₄ (62.9%) and mutant N₈₆F₁₈₄ (21.1%), Y₈₆Y₁₈₄ (6.4%), and Y₈₆F₁₈₄ (0.4%) haplotypes of Pfmdr-1 were observed. Measures of responsiveness to ACTs were similar in children infected with P. falciparum crt haplotypes (C₇₂I₇₄E₇₅T₇₆ and C₇₂M₇₄N₇₅K₇₆) and major mdr-1 haplotypes (N₈₆Y₁₈₄, N₈₆F₁₈₄ and Y₈₆Y₁₈₄). Despite a 10 year gap since the malaria treatment policy changed to ACTs, over 50% of the P. falciparum parasites investigated in this study harboured the Chloroquine-resistant C₇₂I₇₄E₇₅T₇₆ haplotype, however this did not compromise the efficacy of artemisinin-based combination therapies. Should complete artemisinin resistance emerge from or spread to Nigeria, chloroquine might not be a good alternative therapy.     

<Emphasis Type="Italic">Novosphingobium profundi</Emphasis> sp. nov. isolated from a deep-sea seamount

A marine bacterial strain, F72^T, was isolated from a solitary scleractinian coral, collected in Yap seamounts in the Pacific Ocean. Strain F72^T is a Gram-negative, light-yellow-pigmented, motile, rod-shaped bacterium. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain F72^T is related to the genus Novosphingobium and has high 16S rRNA gene sequence similarities with the type strains of Novosphingobium pentaromativorans US6-1^T (97.7 %), Novosphingobium panipatense SM16^T (97.6 %), Novosphingobium mathurense SM117^T (97.2 %) and Novosphingobium barchaimii LL02^T (97.1 %). Ubiquinone Q-10 was detected as the dominant quinone. The predominant cellular fatty acids were C_18:1ω7c and C_17:1ω6c. The genomic DNA G+C content of strain F72^T was 63.4 mol %. The polar lipids profile contained phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylmethylethanolamine, phosphatidylcholine, sphingoglycolipid and one uncharacterized lipid. Strain F72^T shared DNA relatedness of 25 % with N. pentaromativorans JCM 12182^T, 31 % with N. panipatense DSM 22890^T, 21 % with N. mathurense DSM 23374^T and 26 % with N. barchaimii DSM 25411^T. Combined data from phenotypic, phylogenetic and DNA–DNA relatedness studies demonstrated that the strain F72^T is a representative of a novel species of the genus Novosphingobium, for which we propose the name Novosphingobium profundi sp. nov. (type strain F72^T = KACC 18566^T = CGMCC 1.15390^T).     

Utilizing Chemical Genomics to Identify Cytochrome b as a Novel Drug Target for Chagas Disease

Unbiased phenotypic screens enable identification of small molecules that inhibit pathogen growth by unanticipated mechanisms. These small molecules can be used as starting points for drug discovery programs that target such mechanisms. A major challenge of the approach is the identification of the cellular targets. Here we report GNF7686, a small molecule inhibitor of Trypanosoma cruzi, the causative agent of Chagas disease, and identification of cytochrome b as its target. Following discovery of GNF7686 in a parasite growth inhibition high throughput screen, we were able to evolve a GNF7686-resistant culture of T. cruzi epimastigotes. Clones from this culture bore a mutation coding for a substitution of leucine by phenylalanine at amino acid position 197 in cytochrome b. Cytochrome b is a component of complex III (cytochrome bc₁) in the mitochondrial electron transport chain and catalyzes the transfer of electrons from ubiquinol to cytochrome c by a mechanism that utilizes two distinct catalytic sites, Q_N and Q_P. The L197F mutation is located in the Q_N site and confers resistance to GNF7686 in both parasite cell growth and biochemical cytochrome b assays. Additionally, the mutant cytochrome b confers resistance to antimycin A, another Q_N site inhibitor, but not to strobilurin or myxothiazol, which target the Q_P site. GNF7686 represents a promising starting point for Chagas disease drug discovery as it potently inhibits growth of intracellular T. cruzi amastigotes with a half maximal effective concentration (EC₅₀) of 0.15 µM, and is highly specific for T. cruzi cytochrome b. No effect on the mammalian respiratory chain or mammalian cell proliferation was observed with up to 25 µM of GNF7686. Our approach, which combines T. cruzi chemical genetics with biochemical target validation, can be broadly applied to the discovery of additional novel drug targets and drug leads for Chagas disease.