A Complex Interplay Between Nutrition and Alcohol Use Disorder: Implications for Breaking the Vicious Cycle

Approximately 16.5% of the United States population met the diagnostic criteria for substance use disorder (SUD) in 2021, including 29.5 million individuals with alcohol use disorder (AUD). Individuals with AUD are at increased risk for malnutrition, and impairments in nutritional status in chronic alcohol users can be detrimental to physical and emotional well-being. Furthermore, these nutritional deficiencies could contribute to the never-ending cycle of alcoholism and related pathologies, thereby jeopardizing the prospects of recovery and treatment outcomes. Improving nutritional status in AUD patients may not only compensate for general malnutrition but could also reduce adverse symptoms during recovery, thereby promoting abstinence and successful treatment of AUD. In this review, we briefly summarize alterations in the nutritional status of people with addictive disorders, in addition to the underlying neurobiological mechanisms and clinical implications regarding the role of nutritional intervention in recovery from alcohol use disorder.

cGAS-STING Pathway Activation during Trypanosoma cruzi Infection Leads to Tissue-Dependent Parasite Control.

Host cell invasion by Trypanosoma cruzi is a markedly silent process, with limited host transcriptional changes indicative of innate immune recognition, except for a modest type I IFN (IFN-I) response. In this study, we show that T. cruzi-induced IFN-ß production was nearly abolished in primary murine cGAS-/- or stimulator of IFN genes (STING)-deficient (STINGGt) macrophages and fibroblasts. T. cruzi infection did not impact the ability of IFN-regulatory factor reporter macrophages to respond to classical cGAS-STING agonists, indicating that the limited IFN-ß induction is not due to active parasite suppression. cGAS-/-, STINGGt, and IFN-α/ß receptor-/- (IFNAR-/-) macrophages infected with T. cruzi yielded significantly higher numbers of amastigotes compared with wild-type macrophages; however, the impact of the STING pathway during infection in vivo is more complex. Despite an initial increase in parasite growth, STINGGt and IFNAR-/- mice ultimately had lower parasite burden in footpads as compared with wild-type mice, demonstrating a role for IFN-I expression in potentiating parasite growth at the infection site. STING pathway activation had little impact on parasite levels in the skeletal muscle; however, in the heart, cGAS-/- and STINGGt mice, but not IFNAR-/- mice, accumulated higher acute parasite loads, suggesting a protective role of STING sensing of T. cruzi in this organ that was independent of IFN-I. Together, these results demonstrate that host cGAS-STING senses T. cruzi infection, enhancing parasite growth at the site of entry, and contributes to acute-phase parasite restriction in the heart, a major site of tissue damage in chronic T. cruzi infection.

DIAGNOSIS AND TREATMENT OF A NATURAL INFECTION WITH TRYPANOSOMA CRUZI (CHAGAS DISEASE) IN A SYMPTOMATIC DE BRAZZA'S MONKEY (CERCOPITHECUS NEGLECTUS) IN ALABAMA.

Trypanosoma cruzi, the causative agent of Chagas disease, is a zoonotic, vector-borne, protozoan hemoflagellate with a wide host range. An 11-yr-old, captive-bred male De Brazza's monkey (Cercopithecus neglecus) presented with weight loss despite normal appetite. Examination revealed hypoglycemia, nonregenerative anemia, and many trypanosomes on a blood smear. A whole blood sample was PCR-positive for T. cruzi discrete typing unit TcIV and the monkey seroconverted using two different methods. The monkey was treated with the standard human dose of benznidazole twice daily for 60 d; however, blood obtained over the next 1.5 yr posttreatment remained PCR-positive for T. cruzi. A second course of benznidazole at a higher dose but lower frequency for 26 wk was required for the monkey to convert to sustained PCR-negative status. The monkey recovered with no apparent lasting effects.

Frequency Variation and Dose Modification of Benznidazole Administration for the Treatment of Trypanosoma cruzi Infection in Mice, Dogs, and Nonhuman Primates.

Trypanosoma cruzi naturally infects a broad range of mammalian species and frequently results in the pathology that has been most extensively characterized in human Chagas disease. Currently employed treatment regimens fail to achieve parasitological cure of T. cruzi infection in the majority of cases. In this study, we have extended our previous investigations of more effective, higher dose, intermittent administration protocols using the FDA-approved drug benznidazole (BNZ), in experimentally infected mice and in naturally infected dogs and nonhuman primates (NHP). Collectively, these studies demonstrate that twice-weekly administration of BNZ for more than 4 months at doses that are ~2.5-fold that of previously used daily dosing protocols, provided the best chance to obtain parasitological cure. Dosing less frequently or for shorter time periods was less dependable in all species. Prior treatment using an ineffective dosing regimen in NHPs did not prevent the attainment of parasitological cure with an intensified BNZ dosing protocol. Furthermore, parasites isolated after a failed BNZ treatment showed nearly identical susceptibility to BNZ as those obtained prior to treatment, confirming the low risk of induction of drug resistance with BNZ and the ability to adjust the treatment protocol when an initial regimen fails. These results provide guidance for the use of BNZ as an effective treatment for T. cruzi infection and encourage its wider use, minimally in high value dogs and at-risk NHP, but also potentially in humans, until better options are available.

Frequency variation and dose modification of benznidazole administration for the treatment of Trypanosoma cruzi infection in mice, dogs and non-human primates.

Trypanosoma cruzi naturally infects a broad range of mammalian species and frequently results in the pathology that has been most extensively characterized in human Chagas disease. Currently employed treatment regimens fail to achieve parasitological cure of T. cruzi infection in the majority of cases. In this study, we have extended our previous investigations of more effective, higher dose, intermittent administration protocols using the FDA-approved drug benznidazole (BNZ), in experimentally infected mice and in naturally infected dogs and non-human primates (NHP). Collectively these studies demonstrate that twice-weekly administration of BNZ for more than 4 months at doses that are ∻2.5-fold that of previously used daily dosing protocols, provided the best chance to obtain parasitological cure. Dosing less frequently or for shorter time periods was less dependable in all species. Prior treatment using an ineffective dosing regimen in NHPs did not prevent the attainment of parasitological cure with an intensified BNZ dosing protocol. Furthermore, parasites isolated after a failed BNZ treatment showed nearly identical susceptibility to BNZ as those obtained prior to treatment, confirming the low risk of induction of drug resistance with BNZ and the ability to adjust the treatment protocol when an initial regimen fails. These results provide guidance for the use of BNZ as an effective treatment for T. cruzi infection and encourage its wider use, minimally in high value dogs and at-risk NHP, but also potentially in humans, until better options are available.

Epitopes in the Glycosylphosphatidylinositol Attachment Signal Peptide of Trypanosoma cruzi Mucin Proteins Generate Robust but Delayed and Nonprotective CD8+ T Cell Responses.

Infection with the protozoan parasite Trypanosoma cruzi elicits substantial CD8+ T cell responses that disproportionately target epitopes encoded in the large trans-sialidase (TS) gene family. Within the C57BL/6 infection model, a significant proportion (30-40%) of the T. cruzi-specific CD8+ T cell response targets two immunodominant TS epitopes, TSKb18 and TSKb20. However, both TS-specific CD8+ T cell responses are dispensable for immune control, and TS-based vaccines have no demonstrable impact on parasite persistence, a determinant of disease. Besides TS, the specificity and protective capacity of CD8+ T cells that mediate immune control of T. cruzi infection are unknown. With the goal of identifying alternative CD8+ T cell targets, we designed and screened a representative set of genome-wide, in silico-predicted epitopes. Our screen identified a previously uncharacterized, to our knowledge, T cell epitope MUCKb25, found within mucin family proteins, the third most expanded large gene family in T. cruzi. The MUCKb25-specific response was characterized by delayed kinetics, relative to TS-specific responses, and extensive cross-reactivity with a large number of endogenous epitope variants. Similar to TS-specific responses, the MUCKb25 response was dispensable for control of the infection, and vaccination to generate MUCK-specific CD8+ T cells failed to confer protection. The lack of protection by MUCK vaccination was partly attributed to the fact that MUCKb25-specific T cells exhibit limited recognition of T. cruzi-infected host cells. Overall, these results indicate that the CD8+ T cell compartment in many T. cruzi-infected mice is occupied by cells with minimal apparent effector potential.

Prophylactic low-dose, bi-weekly benznidazole treatment fails to prevent Trypanosoma cruzi infection in dogs under intense transmission pressure.

Trypanosoma cruzi naturally infects a wide variety of wild and domesticated mammals, in addition to humans. Depending on the infection dose and other factors, the acute infection can be life-threatening, and in all cases, the risk of chagasic heart disease is high in persistently infected hosts. Domestic, working, and semi-feral dogs in the Americas are at significant risk of T. cruzi infection and in certain settings in the southern United States, the risk of new infections can exceed 30% per year, even with the use of vector control protocols. In this study, we explored whether intermittent low-dose treatment with the trypanocidal compound benznidazole (BNZ) during the transmission season, could alter the number of new infections in dogs in an area of known, intense transmission pressure. Preliminary studies in mice suggested that twice-weekly administration of BNZ could prevent or truncate infections when parasites were delivered at the mid-point between BNZ doses. Pre-transmission season screening of 126 dogs identified 53 dogs (42.1%) as T. cruzi infection positive, based upon blood PCR and Luminex-based serology. Serial monitoring of the 67 uninfected dogs during the high transmission season (May to October) revealed 15 (22.4%) new infections, 6 in the untreated control group and 9 in the group receiving BNZ prophylaxis, indicating no impact of this prophylaxis regimen on the incidence of new infections. Although these studies suggest that rigorously timed and more potent dosing regimen may be needed to achieve an immediate benefit of prophylaxis, additional studies would be needed to determine if drug prophylaxis reduced disease severity despite this failure to prevent new infections.

Discovery of an orally active benzoxaborole prodrug effective in the treatment of Chagas disease in non-human primates.

Trypanosoma cruzi, the agent of Chagas disease, probably infects tens of millions of people, primarily in Latin America, causing morbidity and mortality. The options for treatment and prevention of Chagas disease are limited and underutilized. Here we describe the discovery of a series of benzoxaborole compounds with nanomolar activity against extra- and intracellular stages of T. cruzi. Leveraging both ongoing drug discovery efforts in related kinetoplastids, and the exceptional models for rapid drug screening and optimization in T. cruzi, we have identified the prodrug AN15368 that is activated by parasite carboxypeptidases to yield a compound that targets the messenger RNA processing pathway in T. cruzi. AN15368 was found to be active in vitro and in vivo against a range of genetically distinct T. cruzi lineages and was uniformly curative in non-human primates (NHPs) with long-term naturally acquired infections. Treatment in NHPs also revealed no detectable acute toxicity or long-term health or reproductive impact. Thus, AN15368 is an extensively validated and apparently safe, clinically ready candidate with promising potential for prevention and treatment of Chagas disease.

A modified drug regimen clears active and dormant trypanosomes in mouse models of Chagas disease.

A major contributor to treatment failure in Chagas disease, caused by infection with the protozoan parasite Trypanosoma cruzi, is that current treatment regimens do not address the drug insensitivity of transiently dormant T. cruzi amastigotes. Here, we demonstrated that use of a currently available drug in a modified treatment regimen of higher individual doses, given less frequently over an extended treatment period, could consistently extinguish T. cruzi infection in three mouse models of Chagas disease. Once per week administration of benznidazole at a dose 2.5 to 5 times the standard daily dose rapidly eliminated actively replicating parasites and ultimately eradicated the residual, transiently dormant parasite population in mice. This outcome was initially confirmed in "difficult to cure" mouse infection models using immunological, parasitological, and molecular biological approaches and ultimately corroborated by whole organ analysis of optically clarified tissues using light sheet fluorescence microscopy (LSFM). This tool was effective for monitoring pathogen load in intact organs, including detection of individual dormant parasites, and for assessing treatment outcomes. LSFM-based analysis also suggested that dormant amastigotes of T. cruzi may not be fully resistant to trypanocidal compounds such as benznidazole. Collectively, these studies provide important information on the phenomenon of dormancy in T. cruzi infection in mice, demonstrate methods to therapeutically override dormancy using a currently available drug, and provide methods to monitor alternative therapeutic approaches for this, and possibly other, low-density infectious agents.

A phylogenetic examination of host use evolution in the quinaria and testacea groups of Drosophila.

Adaptive radiations provide an opportunity to examine complex evolutionary processes such as ecological specialization and speciation. While a well-resolved phylogenetic hypothesis is critical to completing such studies, the rapid rates of evolution in these groups can impede phylogenetic studies. Here we study the quinaria and testacea species groups of the immigrans-tripunctata radiation of Drosophila, which represent a recent adaptive radiation and are a developing model system for ecological genetics. We were especially interested in understanding host use evolution in these species. In order to infer a phylogenetic hypothesis for this group we sampled loci from both the nuclear genome and the mitochondrial DNA to develop a dataset of 43 protein-coding loci for these two groups along with their close relatives in the immigrans-tripunctata radiation. We used this dataset to examine their evolutionary relationships along with the evolution of feeding behavior. Our analysis recovers strong support for the monophyly of the testacea but not the quinaria group. Results from our ancestral state reconstruction analysis suggests that the ancestor of the testacea and quinaria groups exhibited mushroom-feeding. Within the quinaria group, we infer that transition to vegetative feeding occurred twice, and that this transition did not coincide with a genome-wide change in the rate of protein evolution.

Fine-scale geographic patterns of gene flow and reproductive character displacement in Drosophila subquinaria and Drosophila recens.

When two species are incompletely isolated, strengthening premating isolation barriers in response to the production of low fitness hybrids may complete the speciation process. Here, we use the sister species Drosophila subquinaria and Drosophila recens to study the conditions under which this reinforcement of species boundaries occurs in natural populations. We first extend the region of known sympatry between these species, and then we conduct a fine-scale geographic survey of mate discrimination coupled with estimates of gene flow within and admixture between species. Within D. subquinaria, reinforcement is extremely effective: we find variation in mate discrimination both against D. recens males and against conspecific allopatric males on the scale of a few kilometres and in the face of gene flow both from conspecific populations and introgression from D. recens. In D. recens, we do not find evidence for increased mate discrimination in sympatry, even where D. recens is rare, consistent with substantial gene flow throughout the species' range. Finally, we find that introgression between species is asymmetric, with more from D. recens into D. subquinaria than vice versa. Within each species, admixture is highest in the geographic region where it is rare relative to the other species, suggesting that when hybrids are produced they are of low fitness. In sum, reinforcement within D. subquinaria is effective at maintaining species boundaries, but even when reinforcing selection is strong it may not always result in a pattern of strong reproductive character displacement due to variation in the frequency of hybridization and gene flow from neighbouring populations.

Reproductive character displacement of epicuticular compounds and their contribution to mate choice in Drosophila subquinaria and Drosophila recens.

Interactions between species can alter selection on sexual displays used in mate choice within species. Here we study the epicuticular pheromones of two Drosophila species that overlap partially in geographic range and are incompletely reproductively isolated. Drosophila subquinaria shows a pattern of reproductive character displacement against Drosophila recens, and partial behavioral isolation between conspecific sympatric versus allopatric populations, whereas D. recens shows no such variation in mate choice. First, using manipulative perfuming experiments, we show that females use pheromones as signals for mate discrimination both between species and among populations of D. subquinaria. Second, we show that patterns of variation in epicuticular compounds, both across populations and between species, are consistent with those previously shown for mating probabilities: pheromone compositions differ between populations of D. subquinaria that are allopatric versus sympatric with D. recens, but are similar across populations of D. recens regardless of overlap with D. subquinaria. We also identify differences in pheromone composition among allopatric regions of D. subquinaria. In sum, our results suggest that epicuticular compounds are key signals used by females during mate recognition, and that these traits have diverged among D. subquinaria populations in response to reinforcing selection generated by the presence of D. recens.

Epicuticular compounds of Drosophila subquinaria and D. recens: identification, quantification, and their role in female mate choice.

The epicuticle of various Drosophila species consists of long-chain cuticular hydrocarbons (CHCs) and their derivatives that play a role in waterproofing and a dynamic means of chemical communication. Here, via gas chromatography and mass spectrometry, we identified and quantified the epicuticular composition of D. recens and D. subquinaria, two closely related species that show a pattern of reproductive character displacement in nature. Twenty-four compounds were identified with the most abundant, 11-cis-Vaccenyl acetate, present only in males of each species. Also exclusive to males were five tri-acylglycerides. The 18 remaining compounds were CHCs, all shared between the sexes and species. These CHCs were composed of odd carbon numbers (C29, C31, C33, and C35), with an increase in structural isomers in the C33 and C35 groups. Saturated hydrocarbons comprise only methyl-branched alkanes and were found only in the C29 and C31 groups. Alkenes were the least prevalent, with alkadienes dominating the chromatographic landscape in the longer chain lengths. Sexual dimorphism was extensive with 6/8 of the logcontrast CHCs differing significantly in relative concentration between males and females in D. recens and D. subquinaria, respectively. Males of the two species also differed significantly in relative concentration of six CHCs, while females differed in none. Female-choice mating trials revealed directional sexual selection on male CHCs in a population of each species, consistent with female mate preferences for these traits. The sexual selection vectors differed significantly in multivariate trait space, suggesting that different pheromone blends determine male attractiveness in each species.

Cytoskeleton assembly in Toxoplasma gondii cell division.

Cell division across members of the protozoan parasite phylum Apicomplexa displays a surprising diversity between different species as well as between different life stages of the same parasite. In most cases, infection of a host cell by a single parasite results in the formation of a polyploid cell from which individual daughters bud in a process dependent on a final round of mitosis. Unlike other apicomplexans, Toxoplasma gondii divides by a binary process consisting of internal budding that results in only two daughter cells per round of division. Since T. gondii is experimentally accessible and displays the simplest division mode, it has manifested itself as a model for apicomplexan daughter formation. Here, we review newly emerging insights in the prominent role that assembly of the cortical cytoskeletal scaffold plays in the process of daughter parasite formation.

Cactin is essential for G1 progression in Toxoplasma gondii.

Toxoplasma gondii is an obligate intracellular protozoan parasite whose rapid lytic replication cycles define its pathogenicity. We identified a temperature-sensitive growth mutant, FV-P6, which irreversibly arrests before the middle of the G1 stage of the tachyzoite cell cycle. This arrest is caused by a point mutation in a gene conserved across eukaryotes, Cactin, whose product localizes to the nucleus. To elucidate the role of TgCactin we performed genome-wide expression profiling. Besides the expected G1 expression profile, many genes associated with the extracellular state as well as with the bradyzoite cyst stage were identified. Consistent with these profiles were the expression of AP2 transcription factors typically associated with extracellular and bradyzoite stage parasites. This suggests a role for TgCactin in control of gene expression. As TgCactin does not contain any functionally defined domains we reasoned TgCactin exerts its function through interactions with other proteins. In support of this model we demonstrated that TgCactin is present in a protein complex and can oligomerize. Taken together, these results suggest that TgCactin acts as a pivotal protein potentially regulating gene expression at several transition points in parasite development.

A DOC2 protein identified by mutational profiling is essential for apicomplexan parasite exocytosis.

Exocytosis is essential to the lytic cycle of apicomplexan parasites and required for the pathogenesis of toxoplasmosis and malaria. DOC2 proteins recruit the membrane fusion machinery required for exocytosis in a Ca(2+)-dependent fashion. Here, the phenotype of a Toxoplasma gondii conditional mutant impaired in host cell invasion and egress was pinpointed to a defect in secretion of the micronemes, an apicomplexan-specific organelle that contains adhesion proteins. Whole-genome sequencing identified the etiological point mutation in TgDOC2.1. A conditional allele of the orthologous gene engineered into Plasmodium falciparum was also defective in microneme secretion. However, the major effect was on invasion, suggesting that microneme secretion is dispensable for Plasmodium egress.

A family of intermediate filament-like proteins is sequentially assembled into the cytoskeleton of Toxoplasma gondii.

The intracellular protozoan parasite Toxoplasma gondii divides by a unique process of internal budding that involves the assembly of two daughter cells within the mother. The cytoskeleton of Toxoplasma, which is composed of microtubules associated with an inner membrane complex (IMC), has an important role in this process. The IMC, which is directly under the plasma membrane, contains a set of flattened membranous sacs lined on the cytoplasmic side by a network of filamentous proteins. This network contains a family of intermediate filament-like proteins or IMC proteins. In order to elucidate the division process, we have characterized a 14-member subfamily of Toxoplasma IMC proteins that share a repeat motif found in proteins associated with the cortical alveoli in all alveolates. By creating fluorescent protein fusion reporters for the family members we determined the spatiotemporal patterns of all 14 IMC proteins through tachyzoite development. This revealed several distinct distribution patterns and some provide the basis for novel structural models such as the assembly of certain family members into the basal complex. Furthermore we identified IMC15 as an early marker of budding and, lastly, the dynamic patterns observed throughout cytokinesis provide a timeline for daughter parasite development and division.

Molecular evolution of a Y chromosome to autosome gene duplication in Drosophila.

In contrast to the rest of the genome, the Y chromosome is restricted to males and lacks recombination. As a result, Y chromosomes are unable to respond efficiently to selection, and newly formed Y chromosomes degenerate until few genes remain. The rapid loss of genes from newly formed Y chromosomes has been well studied, but gene loss from highly degenerate Y chromosomes has only recently received attention. Here, we identify and characterize a Y to autosome duplication of the male fertility gene kl-5 that occurred during the evolution of the testacea group species of Drosophila. The duplication was likely DNA based, as other Y-linked genes remain on the Y chromosome, the locations of introns are conserved, and expression analyses suggest that regulatory elements remain linked. Genetic mapping reveals that the autosomal copy of kl-5 resides on the dot chromosome, a tiny autosome with strongly suppressed recombination. Molecular evolutionary analyses show that autosomal copies of kl-5 have reduced polymorphism and little recombination. Importantly, the rate of protein evolution of kl-5 has increased significantly in lineages where it is on the dot versus Y linked. Further analyses suggest this pattern is a consequence of relaxed purifying selection, rather than adaptive evolution. Thus, although the initial fixation of the kl-5 duplication may have been advantageous, slightly deleterious mutations have accumulated in the dot-linked copies of kl-5 faster than in the Y-linked copies. Because the dot chromosome contains seven times more genes than the Y and is exposed to selection in both males and females, these results suggest that the dot suffers the deleterious effects of genetic linkage to more selective targets compared with the Y chromosome. Thus, a highly degenerate Y chromosome may not be the worst environment in the genome, as is generally thought, but may in fact be protected from the accumulation of deleterious mutations relative to other nonrecombining regions that contain more genes.