Are Internal Fragments Observable in Electron Based Top-Down Mass Spectrometry?

Protein tandem mass spectrometry (MS/MS) often generates sequence-informative fragments from backbone bond cleavages near the termini. This lack of fragmentation in the protein interior is particularly apparent in native top-down MS. Improved sequence coverage, critical for reliable annotation of posttranslational modifications (PTMs) and sequence variants, may be obtained from internal fragments generated by multiple backbone cleavage events. However, internal fragment assignments can be error prone due to isomeric/isobaric fragments from different parts of a protein sequence. Also, internal fragment generation propensity depends on the chosen MS/MS activation strategy. Here, we examine internal fragment formation in electron capture dissociation (ECD) and electron transfer dissociation (ETD) following native and denaturing MS, as well as liquid chromatography (LC)/MS of several proteins. Experiments were undertaken on multiple instruments, including Q-ToF, Orbitrap, and high-field FT-ICR across four laboratories. ECD was performed at both ultrahigh vacuum and at similar pressure to ETD conditions. Two complementary software packages were used for data analysis. When feasible, ETD-higher-energy collision dissociation (ETD-HCD) MS3 was performed to validate/refute potential internal fragment assignments, including differentiating MS3 fragmentation behavior of radical vs. even-electron primary fragments. We show that, under typical operating conditions, internal fragments cannot be confidently assigned in ECD, nor ETD. On the other hand, such fragments, along with some b-type terminal fragments (not typically observed in ECD/ETD spectra) appear at atypical ECD operating conditions, suggesting they originate from a separate ion-electron activation process. Furthermore, atypical fragment ion types, e.g., x ions, are observed at such conditions as well as upon EThcD, presumably due to vibrational activation of radical z-type ions.

Electron Capture vs Transfer Dissociation for Site Determination of Tryptic Peptide Tyrosine Sulfation: Direct Detection of Fibrinogen Sulfation Sites and Identification of Novel Isobaric Interferences.

Tyrosine sulfation, an understudied but crucial post-translational modification, cannot be directly detected in conventional nanoflow liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) due to the extreme sulfate lability. Here, we report the detection of sulfate-retaining fragments from LC-electron capture dissociation (ECD) and nanoLC-electron transfer higher energy collision dissociation (EThcD). Sulfopeptide candidates were identified by Proteome Discoverer and MSFragger analysis of nanoLC-HCD MS/MS data and added to inclusion lists for LC-ECD or nanoLC-EThcD MS/MS. When this approach failed, targeted LC-ECD with fixed m/z isolation windows was performed. For the plasma protein fibrinogen, the known pyroglutamylated sulfopeptide QFPTDYDEGQDDRPK from the beta chain N-terminus was identified despite a complete lack of sulfate-containing fragment ions. The peptide QVGVEHHVEIEYD from the gamma-B chain C-terminus was also identified as sulfated or phosphorylated. This sulfopeptide is not annotated in Uniprot but was previously reported. MSFragger further identified a cysteine-containing peptide from the middle of the gamma chain as sulfated and deamidated. NanoLC-EThcD and LC-ECD MS/MS confirmed the two former sulfopeptides via sulfate-retaining fragment ions, whereas an unexpected fragmentation pattern was observed for the third sulfopeptide candidate. Manual interpretation of the LC-ECD spectrum revealed two additional isobaric identifications: a trisulfide-linked cysteinyl-glycine or a carbamidomethyl-dithiothreiotol covalent adduct. Synthesis of such adducts confirmed the latter identity.

Negative-Ion Electron Capture Dissociation of MALDI-Generated Peptide Anions.

Negative-ion electron capture dissociation (niECD) is an anion MS/MS technique that provides fragmentation analogous to conventional ECD, including high peptide sequence coverage and retention of labile post-translational modifications (PTMs). niECD has been proposed to be the most efficient for salt-bridged zwitterionic precursor ion structures. Several important PTMs, e.g., sulfation and phosphorylation, are acidic and can, therefore, be challenging to characterize in the positive-ion mode. Furthermore, PTM-friendly techniques, such as ECD, require multiple precursor ion-positive charges. By contrast, singly charged ions, refractory to ECD, are most compatible with niECD. Because electrospray ionization (ESI) typically yields multiply charged ions, we sought to explore matrix-assisted laser desorption/ionization (MALDI) in combination with niECD. However, the requirement for zwitterionic gaseous structures may preclude efficient niECD of MALDI-generated anions. Unexpectedly, we found that niECD of anions from MALDI is not only possible but proceeds with similar or higher efficiency compared with ESI-generated anions. Matrix selection did not appear to have a major effect. With MALDI, niECD is demonstrated up to m/z ∼4300. For such larger analytes, multiple electron captures are observed, resulting in triply charged fragments from singly charged precursor ions. Such charge-increased fragments show improved detectability. Furthermore, significantly improved (∼20-fold signal-to-noise increase) niECD spectral quality is achieved with equivalent sample amounts from MALDI vs ESI. Overall, the reported combination with MALDI significantly boosts the analytical utility of niECD.

A pooled-sample draft genome assembly provides insights into host plant-specific transcriptional responses of a Solanaceae-specializing pest, Tupiocoris notatus (Hemiptera: Miridae).

The assembly of genomes from pooled samples of genetically heterogenous samples of conspecifics remains challenging. In this study, we show that high-quality genome assemblies can be produced from samples of multiple wild-caught individuals. We sequenced DNA extracted from a pooled sample of conspecific herbivorous insects (Hemiptera: Miridae: Tupiocoris notatus) acquired from a greenhouse infestation in Tucson, Arizona (in the range of 30-100 individuals; 0.5 mL tissue by volume) using PacBio highly accurate long reads (HiFi). The initial assembly contained multiple haplotigs (>85% BUSCOs duplicated), but duplicate contigs could be easily purged to reveal a highly complete assembly (95.6% BUSCO, 4.4% duplicated) that is highly contiguous by short-read assembly standards (N 50 = 675 kb; Largest contig = 4.3 Mb). We then used our assembly as the basis for a genome-guided differential expression study of host plant-specific transcriptional responses. We found thousands of genes (N = 4982) to be differentially expressed between our new data from individuals feeding on Datura wrightii (Solanaceae) and existing RNA-seq data from Nicotiana attenuata (Solanaceae)-fed individuals. We identified many of these genes as previously documented detoxification genes such as glutathione-S-transferases, cytochrome P450s, and UDP-glucosyltransferases. Together our results show that long-read sequencing of pooled samples can provide a cost-effective genome assembly option for small insects and can provide insights into the genetic mechanisms underlying interactions between plants and herbivorous pests.

Belzutifan-induced regression of retinal capillary hemangioblastoma: A case-series.

Purpose: To report a series of three patients with von Hippel-Lindau (VHL) disease who demonstrated regression of their retinal hemangioblastomas (RH) using belzutifan in conjunction with photocoagulation therapy. Observations: Patient 1, a 23-year-old female, presented with multiple RHs in her right eye (OD) that were lasered. Her left eye (OS) revealed a large inferotemporal RH that measured approximately 2.1 mm2. Systemic belzutifan was administered. Four months after initiation of treatment, the lesion regressed to 1.4 mm2, but belzutifan was not well-tolerated and was discontinued due to side effects. At the date of belzutifan discontinuation, the lesion measured about 1.1 mm2. Focal laser photocoagulation was applied. The lesion regressed to around 0.6 mm2. Two additional laser treatments were applied one month later. On the most recent follow-up, the lesion was completely fibrosed.Patient 2, a 32-year-old male, presented with one RH OD and two RHs OS. Belzutifan was administered for one month before the patient began experiencing side effects of the medication. Consequently, the dose of belzutifan was decreased. After one month with the lowered dose, laser coagulation was applied to OS. In the most recent follow-up, five months after the initial presentation, the lesions remain less vascularized and reduced in size.Patient 3, is a 44-year-old male with a large RH OD. Following seven months of belzutifan daily, there was a significant reduction in the RH size. Conclusions: Belzutifan, a hypoxia-inducible factor inhibitor, is an FDA-approved medication for VHL disease associated with renal cell carcinoma, central nervous system hemangioblastomas, or pancreatic neuroendocrine tumors that do not require immediate surgical resection. Because of the high incidence of VHL-associated RHs, adjuvant laser photocoagulation therapy when belzutifan is suspended or withheld can allow for the regression of large lesions. In this case series, we also propose a reproducible and technically simple method to measure RH lesions size, using Optos fundus imaging.

Females translate male mRNA transferred during mating.

Although RNA is found in the seminal fluid of diverse organisms, it is unknown whether this RNA is functional within females. Here, we develop an experimental proteomic method called VESPA (Variant Enabled SILAC Proteomic Analysis) to test the hypothesis that Drosophila male seminal fluid RNA is translated by females. We find strong evidence for 67 male-derived, female-translated proteins (mdFTPs) in female lower reproductive tracts at six hours postmating, many with predicted functions relevant to reproduction. Gene knockout experiments indicate that genes coding for mdFTPs play diverse roles in postmating interactions, with effects on fertilization efficiency, and the formation and persistence of the insemination reaction mass, a trait hypothesized to be involved in sexual conflict. These findings advance our understanding of reproduction by revealing a novel mechanism of postmating molecular interactions between the sexes that strengthens and extends male influences on reproductive outcomes in previously unrecognized ways. Given the diverse species known to carry RNA in seminal fluid, this discovery has broad significance for understanding molecular mechanisms of cooperation and conflict during reproduction.

Integrated pest management strategies targeting the Florida kissing bug, Triatoma sanguisuga: Preventing this vector of Chagas disease from invading your home.

Triatomines (Hemiptera: Reduviidae: Triatominae), commonly called "kissing bugs", are blood-sucking pests and vectors of the protozoan parasite Trypanosoma cruzi, the causative agent of Chagas disease (CD). Eleven species of kissing bugs occur throughout the southern half of the USA, four of which are well known to invade human dwellings. Certain kissing bugs in the USA are known to transmit T. cruzi to humans and other animals and their bites can also lead to serious allergic reactions, including anaphylaxis. In Florida, the kissing bug Triatoma sanguisuga frequently invades homes, bites residents, and has been found infected with T. cruzi, placing humans and companion animals at risk for CD. This review outlines integrated pest management (IPM) strategies for minimizing human exposure to T. sanguisuga and CD. A comprehensive IPM plan for kissing bugs includes detailed inspections, removal of vertebrate host nesting areas, and kissing bug harborage, home improvements to exclude kissing bugs from entering structures, pest removal, and judicious use of pesticides. This approach can limit or eliminate kissing bug entry into residential structures, thereby preventing kissing bug bites, and CD infections in humans and companion animals.

First Report of Tomato Spotted Wilt Virus infecting Lettuce in Yuma, Arizona.

Tomato spotted wilt virus (TSWV, family Tospoviridae, genus Orthotospovirus) is a thrips-vectored pathogen that infects lettuce (Lactuca sativa) and many vegetable crops (Kuo et al. 2014, Hasegawa et al. 2022). Another thrips-borne pathogen of lettuce, impatiens necrotic spot virus (INSV, Tospoviridae, Orthotospovirus), was first reported in 2021 in Yuma, Arizona (Hasegawa et al. 2022). Symptoms of both viruses in lettuce are similar and include necrotic spotting, leaf chlorosis and plant stunting (Kuo et al. 2014). Beginning February through April of 2022, lettuce displaying symptoms of orthotospovirus infection was collected from romaine lettuce (var. longifolia) fields in three regions of Yuma County. A total of 96 plants were collected (5 from Tacna on 2/21, 5 from Wellton on 2/21, 15 from Wellton on 3/23, 30 from Tacna on 4/4, 20 from Wellton on 4/4, and 21 from Yuma Valley on 4/4). The area of the fields ranged from 10 to 18 acres, and the percent disease incidence ranged from 0.8% (Tacna on 4/4) to 2.75% (Tacna on 2/21). Thrips vector were present in all fields were symptomatic plants were observed. One leaf disk per plant (8 mm in diameter) was sampled with a cork borer and grounded individually with a micro pestle in a 1.7 ml microcentrifuge tube with 150 ul of Tri-reagent (Molecular Research Center). Total RNA was extracted from each sample using the Zymo Direct-zol-96 kit (Zymo Research). Samples were diluted with water to a ratio of 1:10 after RNA extraction. RT-qPCR was performed in 20 ul reactions with 5 ul of input RNA using the PCR Biosystems qPCRBIO Probe 1-Step Go No-ROX for the cDNA/qPCR master mix. RT-qPCR assays were carried out in multiplex reactions using primers specific for TSWV and INSV, in addition to a lettuce internal control gene (LOC111918243), along with negative controls. Primer and probe sequence details are reported in supplemental Table 1. We used a cycle threshold (ct) < 40 to indicate a positive result for both INSV and TSWV (Chen et al. 2013; Boonham et al. 2002). RT-qPCR successfully amplified INSV in 90 out of 96 samples and TSWV in 8 out of 96 samples. These 8 samples tested positive for both TSWV and INSV, showing that INSV and TSWV co-infected lettuce plants. Thus overall, ∼ 95% of symptomatic plants were infected with INSV alone, and ∼ 8% were co-infected with TSWV and INSV. Amplicons of 4 samples testing positive for TSWV were sent for Sanger sequencing (Eurofins Genomics, Louisville, KY). All were identified as TSWV. One amplicon with TSWV was sequenced for INSV and double infection was confirmed. BLAST results from the NCBI nt database show 100% (138 bp) identity to TWSV (MW519211) for the 4 TWSV amplicons and 99.22% (137 bp) identity to INSV (KX790323) for the INSV amplicon. Sanger sequence data are in the GenBank (accession: OQ685940-OQ685944). Based on RT-qPCR results, all TSWV infected plants were also infected with INSV. INSV may have been introduced to Yuma by infected plants or thrips from lettuce transplants produced in California (Hasegawa et al. 2022). TSWV could have been introduced similarly. To our knowledge, this is the first report of TSWV infecting lettuce in Yuma and the first report of INSV and TSWV co-infecting lettuce. TSWV and INSV infections have remained low since their discovery in Yuma, in part due to effective cultural and chemical management by lettuce growers (Palumbo, 2022). However, an increase in disease incidence and severity in the future could have a significant negative impact on production of romaine lettuce in the region.

Trypanosoma cruzi infection in mammals in Florida: New insight into the transmission of T. cruzi in the southeastern United States.

In Latin America, synanthropic mammalian reservoirs maintain Trypanosoma cruzi, a parasitic protozoan, where they facilitate the transmission of the parasite to humans and other reservoir hosts in peridomestic settings. In the United States, raccoons (Procyon lotor) and Virginia opossums (Didelphis virginiana) are known synanthropic T. cruzi reservoir hosts; however, the role these species have in the peridomestic transmission cycle in the US is not well understood. This study aimed to identify the suite of mammalian reservoirs of T. cruzi in Florida. We also compared infection prevalence in raccoon populations sampled from within and outside of the estimated distribution of the common T. cruzi vector in Florida to gain insight into how the arthropod vector distribution impacts the distribution of infected reservoirs in the state. Finally, to investigate the impact of peridomestic landscapes on parasite prevalence, we compared the prevalence of T. cruzi-infected raccoons and opossums across five paired peridomestic and sylvatic sites. We live-trapped and collected peripheral blood samples from 135 raccoons, 112 opossums, 18 nine-banded armadillos (Dasypus novemcinctus), and nine species of rodents in north central Florida. Using quantitative PCR methods, we found that raccoons (42.2%, 95% CI [34.2-50.7%]) and opossums (50.9%, 95% CI [41.8-60.0%]) were infected with T. cruzi and the prevalence across habitats was similar for both raccoons (peridomestic: n = 77, 44.2%, 95% CI [33.6-55.3%], sylvatic: n = 58, 39.7%, 95% CI [28.1-52.5%]) and opossums (peridomestic: n = 66, 48.5%, 95% CI [36.8-60.3%], sylvatic: n = 46, 54.3%, 95% CI [40.2-67.8%]). Raccoons sampled outside the estimated distribution of Triatoma sanguisuga were not infected with T. cruzi (n = 73, 0.0%, 95% CI [0.0-5.0%]). Our study did not indicate that peridomestic habitats in Florida maintained a higher infection prevalence than their sylvatic counterparts; however, we did find a difference in prevalence within vs. outside the estimated vector distribution in Florida.

Identification of the parasite, Trypanosoma cruzi, in multiple tissues of epidemiological significance in the Virginia opossum (Didelphis virginiana): Implications for environmental and vertical transmission routes.

BACKGROUND: Trypanosoma cruzi, a parasitic protozoan, is endemic to the Americas and the causative agent of Chagas disease in humans. In South America, opossums facilitate transmission via infected anal gland secretions in addition to transmission via triatomine vectors. In North America, the Virginia opossum is a reservoir host for the parasite with transmission routes that are not clearly defined. The unique biology of this marsupial provides the opportunity to investigate vertical transmission in this wildlife species in situ. Our objectives were to investigate alternative routes of transmission that may facilitate spillover into other species and to determine if vertical transmission was evident. METHODOLOGY/PRINCIPAL FINDINGS: Virginia opossums were sampled at 10 trapping locations over a 10-month period in a 5-county region of north central Florida. Peripheral blood, fecal swabs, and anal gland secretions were collected from each adult individual, and peripheral blood was collected from joey opossums. Total DNA was extracted from each collected sample type, and T. cruzi infected individuals and the infecting Discrete Typing Unit (DTU) were identified using real time PCR methods. Adult Virginia opossums (n = 112) were infected with T. cruzi (51.8%, 95% CI [42.6-60.8%]) throughout the sampled period and at each location. T. cruzi DNA was found in each of the three biological sample types. Vertical transmission of T. cruzi was inferred in one litter of mother-dependent (n = 20, 5.0%, 95% CI [0.9-23.6%]) joey opossums where 2 joeys from this same litter were rtPCR positive for T. cruzi. CONCLUSIONS/SIGNIFICANCE: We inferred vertical transmission from mother to neonate which may serve to amplify the prevalence of T. cruzi in adult Virginia opossums. T. cruzi DNA was detected in the anal gland secretions of Virginia opossums. Infected anal gland secretions suggest a possible environmental route of transmission for T. cruzi via the deposition of contaminated feces and spraint at wildlife latrines. Only DTU1 was identified in the sampled population which is consistent with human autochthonous cases in the United States.

Differential activity of mGlu7 allosteric modulators provides evidence for mGlu7/8 heterodimers at hippocampal Schaffer collateral-CA1 synapses.

Glutamate acts at eight metabotropic glutamate (mGlu) receptor subtypes expressed in a partially overlapping fashion in distinct brain circuits. Recent evidence indicates that specific mGlu receptor protomers can heterodimerize and that these heterodimers can exhibit different pharmacology when compared to their homodimeric counterparts. Group III mGlu agonist-induced suppression of evoked excitatory potentials and induction of long-term potentiation at Schaffer collateral-CA1 (SC-CA1) synapses in the rodent hippocampus can be blocked by the selective mGlu7 negative allosteric modulator (NAM), ADX71743. Curiously, a different mGlu7 NAM, 6-(4-methoxyphenyl)-5-methyl-3-pyridin-4-ylisoxazonolo[4,5-c]pyridin-4(5H)-one, failed to block these responses in brain slices despite its robust activity at mGlu7 homodimers in vitro. We hypothesized that this might result from heterodimerization of mGlu7 with another mGlu receptor protomer and focused on mGlu8 as a candidate given the reported effects of mGlu8-targeted compounds in the hippocampus. Here, we used complemented donor acceptor-resonance energy transfer to study mGlu7/8 heterodimer activation in vitro and observed that ADX71743 blocked responses of both mGlu7/7 homodimers and mGlu7/8 heterodimers, whereas 6-(4-methoxyphenyl)-5-methyl-3-pyridin-4-ylisoxazonolo[4,5-c]pyridin-4(5H)-one only antagonized responses of mGlu7/7 homodimers. Taken together with our electrophysiology observations, these results suggest that a receptor with pharmacology consistent with an mGlu7/8 heterodimer modulates the activity of SC-CA1 synapses. Building on this hypothesis, we identified two additional structurally related mGlu7 NAMs that also differ in their activity at mGlu7/8 heterodimers, in a manner consistent with their ability to inhibit synaptic transmission and plasticity at SC-CA1. Thus, we propose that mGlu7/8 heterodimers are a key molecular target for modulating the activity of hippocampal SC-CA1 synapses.

Divergent evolutionary trajectories shape the postmating transcriptional profiles of conspecifically and heterospecifically mated cactophilic Drosophila females.

Postmating-prezygotic (PMPZ) reproductive isolation is hypothesized to result from divergent coevolutionary trajectories of sexual selection and/or sexual conflict in isolated populations. However, the genetic basis of PMPZ incompatibilities between species is poorly understood. Here, we use a comparative framework to compare global gene expression in con- and heterospecifically mated Drosophila mojavensis and D. arizonae female reproductive tracts. We find striking divergence between the species in the female postmating transcriptional response to conspecific mating, including differences in differential expression (DE), alternative splicing (AS), and intron retention (IR). As predicted, heterospecific matings produce disrupted transcriptional profiles, but the overall patterns of misregulation are different between the reciprocal crosses. Moreover, we find a positive correlation between postmating transcriptional divergence between species and levels of transcriptional disruption in heterospecific crosses. This result indicates that mating responsive genes that have diverged more in expression also have more disrupted transcriptional profiles in heterospecifically mated females. Overall, our results provide insights into the evolution of PMPZ isolation and lay the foundation for future studies aimed at identifying specific genes involved in PMPZ incompatibilities and the evolutionary forces that have contributed to their divergence in closely related species.

Development and profiling of mGlu7 NAMs with a range of saturable inhibition of agonist responses in vitro.

We describe here a series of metabotropic glutamate receptor 7 (mGlu7) negative allosteric modulators (NAMs) with a saturable range of activity in inhibiting responses to an orthosteric agonist in two distinct in vitro pharmacological assays. The range of inhibition among compounds in this scaffold provides highly structurally related ligands with differential degrees of receptor blockade that can be used to understand inhibitory efficacy profiles in native tissue or in vivo.

Novel genetic basis of resistance to Bt toxin Cry1Ac in Helicoverpa zea.

Crops genetically engineered to produce insecticidal proteins from the bacterium Bacillus thuringiensis have advanced pest management, but their benefits are diminished when pests evolve resistance. Elucidating the genetic basis of pest resistance to Bacillus thuringiensis toxins can improve resistance monitoring, resistance management, and the design of new insecticides. Here, we investigated the genetic basis of resistance to Bacillus thuringiensis toxin Cry1Ac in the lepidopteran Helicoverpa zea, one of the most damaging crop pests in the United States. To facilitate this research, we built the first chromosome-level genome assembly for this species, which has 31 chromosomes containing 375 Mb and 15,482 predicted proteins. Using a genome-wide association study, fine-scale mapping, and RNA-seq, we identified a 250-kb quantitative trait locus on chromosome 13 that was strongly associated with resistance in a strain of Helicoverpa zea that had been selected for resistance in the field and lab. The mutation in this quantitative trait locus contributed to but was not sufficient for resistance, which implies alleles in more than one gene contributed to resistance. This quantitative trait locus contains no genes with a previously reported role in resistance or susceptibility to Bacillus thuringiensis toxins. However, in resistant insects, this quantitative trait locus has a premature stop codon in a kinesin gene, which is a primary candidate as a mutation contributing to resistance. We found no changes in gene sequence or expression consistently associated with resistance for 11 genes previously implicated in lepidopteran resistance to Cry1Ac. Thus, the results reveal a novel and polygenic basis of resistance.

Potent Anti-Inflammatory, Arylpyrazole-Based Glucocorticoid Receptor Agonists That Do Not Impair Insulin Secretion.

Glucocorticoids (GCs) are widely used in medicine for their role in the treatment of autoimmune-mediated conditions, certain cancers, and organ transplantation. The transcriptional activities GCs elicit include transrepression, postulated to be responsible for the anti-inflammatory activity, and transactivation, proposed to underlie the undesirable side effects associated with long-term use. A GC analogue that could elicit only transrepression and beneficial transactivation properties would be of great medicinal value and is highly sought after. In this study, a series of 1-(4-substituted phenyl)pyrazole-based GC analogues were synthesized, biologically screened, and evaluated for SARs leading to the desired activity. Activity observed in compounds bearing an electron deficient arylpyrazole moiety showed promise toward a dissociated steroid, displaying transrepression while having limited transactivation activity. In addition, compounds 11aa and 11ab were found to have anti-inflammatory efficacy comparable to that of dexamethasone at 10 nM, with minimal transactivation activity and no reduction of insulin secretion in cultured rat 832/13 beta cells.

Gene expression and alternative splicing dynamics are perturbed in female head transcriptomes following heterospecific copulation.

BACKGROUND: Despite the growing interest in the female side of copulatory interactions, the roles played by differential expression and alternative splicing mechanisms of pre-RNA on tissues outside of the reproductive tract have remained largely unknown. Here we addressed these questions in the context of con- vs heterospecific matings between Drosophila mojavensis and its sister species, D. arizonae. We analyzed transcriptional responses in female heads using an integrated investigation of genome-wide patterns of gene expression, including differential expression (DE), alternative splicing (AS) and intron retention (IR). RESULTS: Our results indicated that early transcriptional responses were largely congruent between con- and heterospecific matings but are substantially perturbed over time. Conspecific matings induced functional pathways related to amino acid balance previously associated with the brain's physiology and female postmating behavior. Heterospecific matings often failed to activate regulation of some of these genes and induced expression of additional genes when compared with those of conspecifically-mated females. These mechanisms showed functional specializations with DE genes mostly linked to pathways of proteolysis and nutrient homeostasis, while AS genes were more related to photoreception and muscle assembly pathways. IR seems to play a more general role in DE regulation during the female postmating response. CONCLUSIONS: We provide evidence showing that AS genes substantially perturbed by heterospecific matings in female heads evolve at slower evolutionary rates than the genome background. However, DE genes evolve at evolutionary rates similar, or even higher, than those of male reproductive genes, which highlights their potential role in sexual selection and the evolution of reproductive barriers.

Synthesis and SAR of a series of mGlu7 NAMs based on an ethyl-8-methoxy-4-(4-phenylpiperazin-1-yl)quinoline carboxylate core.

A High-Throughput Screening (HTS) campaign identified a fundamentally new mGlu7 NAM chemotype, based on an ethyl-8-methoxy-4-(4-phenylpiperazin-1-yl)quinolone carboxylate core. The initial hit, VU0226390, was a potent mGlu7 NAM (IC50 = 647 nM, 6% L-AP4 min) with selectivity versus the other group III mGlu receptors (>30 µM vs. mGlu4 and mGlu8). A multi-dimensional optimization effort surveyed all regions of this new chemotype, and found very steep SAR, reminiscent of allosteric modulators, and unexpected piperazine mimetics (whereas classical bioisosteres failed). While mGlu7 NAM potency could be improved (IC50s ~ 350 nM), the necessity of the ethyl ester moiety and poor physiochemical and DMPK properties precluded optimization towards in vivo tool compounds or clinical candidates. Still, this hit-to-lead campaign afforded key medicinal chemistry insights and new opportunities.

Discovery of VU6027459: A First-in-Class Selective and CNS Penetrant mGlu7 Positive Allosteric Modulator Tool Compound.

Herein, we report the discovery of the first selective and CNS penetrant mGlu7 PAM (VU6027459) derived from a "molecular switch" within a selective mGlu7 NAM chemotype. VU6027459 displayed CNS penetration in both mice (Kp = 2.74) and rats (Kp= 4.78), it was orally bioavailable in rats (%F = 69.5), and undesired activity at DAT was ablated.

Contributions of cis- and trans-Regulatory Evolution to Transcriptomic Divergence across Populations in the Drosophila mojavensis Larval Brain.

Natural selection on gene expression was originally predicted to result primarily in cis- rather than trans-regulatory evolution, due to the expectation of reduced pleiotropy. Despite this, numerous studies have ascribed recent evolutionary divergence in gene expression predominantly to trans-regulation. Performing RNA-seq on single isofemale lines from genetically distinct populations of the cactophilic fly Drosophila mojavensis and their F1 hybrids, we recapitulated this pattern in both larval brains and whole bodies. However, we demonstrate that improving the measurement of brain expression divergence between populations by using seven additional genotypes considerably reduces the estimate of trans-regulatory contributions to expression evolution. We argue that the finding of trans-regulatory predominance can result from biases due to environmental variation in expression or other sources of noise, and that cis-regulation is likely a greater contributor to transcriptional evolution across D. mojavensis populations. Lastly, we merge these lines of data to identify several previously hypothesized and intriguing novel candidate genes, and suggest that the integration of regulatory and population-level transcriptomic data can provide useful filters for the identification of potentially adaptive genes.