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Jove V, Venkataraman K, Gabel TM, Duvall LB
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Feeding and Quantifying Animal-Derived Blood and Artificial Meals in Aedes aegypti Mosquitoes

Females of certain mosquito species can spread diseases while biting vertebrate hosts to obtain protein-rich blood meals required for egg development. In the laboratory, researchers can deliver animal-derived and artificial blood meals to mosquitoes via membrane feeders, which allow for manipulation of meal composition. Here, we present methods for feeding blood and artificial blood meals to Aedes aegypti mosquitoes and quantifying the volume consumed by individual females. Targeted feeding and quantification of artificial/blood meals have broad uses, including testing the effects of meal components on mosquito behavior and physiology, delivering pharmacological compounds without injection, and infecting mosquitoes with specific pathogens. Adding fluorescein dye to the meal prior to feeding allows for subsequent meal size quantification. The meal volume consumed by mosquitoes can be measured either by weight, if the females are to be used later for behavioral experiments, or by homogenizing individual females in 96-well plates and measuring fluorescence levels using a plate reader as an endpoint assay. Meal size quantification can be used to determine whether changing the meal components alters the meal volume ingested or if meal consumption differs between mosquito strains. Precise meal size quantification is also critical for downstream assays, such as those measuring effects on host attraction or fecundity. The methods presented here can be further adapted to track meal digestion over the course of days or to include multiple distinguishable markers added to different meals (like nectar and blood) to quantify the consumption of each meal by a single mosquito. These methods allow researchers to singlehandedly perform high-throughput measurements to compare the meal volume consumed by hundreds of individual mosquitoes. These tools will therefore be broadly useful to the community of mosquito researchers for answering diverse biological questions.
Dou YH, Barbosa I, Jiang H, Iasillo C, Molloy KR, Schulze WM, Cusack S, Schmid M, Le Hir H, LaCava J, Jensen TH
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NCBP3 positively impacts mRNA biogenesis

NUCLEIC ACIDS RESEARCH 2020 OCT 9; 48(18):10413-10427
The nuclear Cap-Binding Complex (CBC), consisting of Nuclear Cap-Binding Protein 1 (NCBP1) and 2 (NCBP2), associates with the nascent 5' cap of RNA polymerase II transcripts and impacts RNA fate decisions. Recently, the C17orf85 protein, also called NCBP3, was suggested to form an alternative CBC by replacing NCBP2. However, applying protein-protein interaction screening of NCBP1, 2 and 3, we find that the interaction profile of NCBP3 is distinct. Whereas NCBP1 and 2 identify known CBC interactors, NCBP3 primarily interacts with components of the Exon Junction Complex (EJC) and the TRanscription and EXport (TREX) complex. NCBP3-EJC association in vitro and in vivo requires EJC core integrity and the in vivo RNA binding profiles of EJC and NCBP3 overlap. We further show that NCBP3 competes with the RNA degradation factor ZC3H18 for binding CBC-bound transcripts, and that NCBP3 positively impacts the nuclear export of polyadenylated RNAs and the expression of large multi-exonic transcripts. Collectively, our results place NCBP3 with the EJC and TREX complexes in supporting mRNA expression.
Arsenault SV, King JT, Kay S, Lacy KD, Ross KG, Hunt BG
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Simple inheritance, complex regulation: Supergene-mediated fire ant queen polymorphism

MOLECULAR ECOLOGY 2020 OCT; 29(19):3622-3636
The fire antSolenopsis invictaexists in two alternate social forms: monogyne nests contain a single reproductive queen and polygyne nests contain multiple reproductive queens. This colony-level social polymorphism corresponds with individual differences in queen physiology, queen dispersal patterns and worker discrimination behaviours, all evidently regulated by an inversion-based supergene that spans more than 13 Mb of a "social chromosome," contains over 400 protein-coding genes and rarely undergoes recombination. The specific mechanisms by which this supergene influences expression of the many distinctive features that characterize the alternate forms remain almost wholly unknown. To advance our understanding of these mechanisms, we explore the effects of social chromosome genotype and natal colony social form on gene expression in queens sampled as they embarked on nuptial flights, using RNA-sequencing of brains and ovaries. We observe a large effect of natal social form, that is, of the social/developmental environment, on gene expression profiles, with similarly substantial effects of genotype, including: (a) supergene-associated gene upregulation, (b) allele-specific expression and (c) pronounced extra-supergenetrans-regulatory effects. These findings, along with observed spatial variation in differential and allele-specific expression within the supergene region, highlight the complex gene regulatory landscape that emerged following divergence of the inversion-mediatedSbhaplotype from its homologue, which presumably largely retained the ancestral gene order. The distinctive supergene-associated gene expression trajectories we document at the onset of a queen's reproductive life expand the known record of relevant molecular correlates of a complex social polymorphism and point to putative genetic factors underpinning the alternate social syndromes.
Jin SC, Lewis SA, Bakhtiari S, Zeng X, Sierant MC, Shetty S, Nordlie SM, Elie A, Corbett MA, Norton BY, van Eyk CL, Haider S, Guida BS, Magee H, Liu JM, Pastore S, Vincent JB, Brunstrom-Hernandez J, Papavasileiou A, Fahey MC, Berry JG, Harper K, Zhou CC, Zhang JH, Li BY, Heim J, Webber DL, Frank MSB, Xia L, Xu YR, Zhu DN, Zhang BH, Sheth AH, Knight JR, Castaldi C, Tikhonova IR, Lopez-Giraldez F, Keren B, Whalen S, Buratti J, Doummar D, Cho MG, Retterer K, Millan F, Wang YG, Waugh JL, Rodan L, Cohen JS, Fatemi A, LinE, Phillips JP, Feyma T, MacLennan SC, Vaughan S, Crompton KE, Reid SM, Reddihough DS, Shang Q, Gao C, Novak I, Badawi N, Wilson YA, McIntyre SJ, Mane SM, Wang XY, Amor DJ, Zarnescu DC, Lu QS, Xing QH, Zhu CL, Bilguvar K, Padilla-Lopez S, Lifton RP, Gecz J, MacLennan AH, Kruer MC
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Mutations disrupting neuritogenesis genes confer risk for cerebral palsy

NATURE GENETICS 2020 OCT; 52(10):1046-1056
Whole-exome sequencing of 250 parent-offspring trios identifies an enrichment of rare damaging de novo mutations in individuals with cerebral palsy and implicates genetically mediated dysregulation of early neuronal connectivity in the etiology of this disorder. In addition to commonly associated environmental factors, genomic factors may cause cerebral palsy. We performed whole-exome sequencing of 250 parent-offspring trios, and observed enrichment of damaging de novo mutations in cerebral palsy cases. Eight genes had multiple damaging de novo mutations; of these, two (TUBA1AandCTNNB1) met genome-wide significance. We identified two novel monogenic etiologies,FBXO31andRHOB, and showed that theRHOBmutation enhances active-state Rho effector binding while theFBXO31mutation diminishes cyclin D levels. Candidate cerebral palsy risk genes overlapped with neurodevelopmental disorder genes. Network analyses identified enrichment of Rho GTPase, extracellular matrix, focal adhesion and cytoskeleton pathways. Cerebral palsy risk genes in enriched pathways were shown to regulate neuromotor function in aDrosophilareverse genetics screen. We estimate that 14% of cases could be attributed to an excess of damaging de novo or recessive variants. These findings provide evidence for genetically mediated dysregulation of early neuronal connectivity in cerebral palsy.
Rosenbaum E, Seier K, Bandlamudi C, Dickson M, Gounder M, Keohan ML, Chi P, Kelly C, Movva S, Nacev B, Simeone N, Donoghue M, Slotkin EK, Qin LX, Antonescu CR, Tap WD, D'Angelo SP
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HLA Genotyping in Synovial Sarcoma: Identifying HLA-A*02 and Its Association with Clinical Outcome

CLINICAL CANCER RESEARCH 2020 OCT 15; 26(20):5448-5455
Purpose: To determine if a targeted exome panel utilizing matched normal DNA can accurately detect germline and somatic HLA genes in patients with synovial sarcoma (SS) and whether select HLA-A*02 genotypes are prognostic or predictive of outcome in metastatic SS. Experimental Design: Patients with metastatic SS consented to HLA typing by a Clinical Laboratory Improvement Amendments (CLIA)-certified test to determine eligibility for a clinical trial of NY-ESO-1-specific engineered T cells restricted to carriers of HLAA* 02:01,-A*02:05, or-A*02:06 (HLA-A*02 eligible). HLA genotype was determined from Memorial Sloan Kettering Integrated Molecular Profiling of Actionable Cancer Targets (MSK-IMPACT), where feasible, and somatic loss of heterozygosity (LOH) in HLA alleles was identified. Overall survival (OS) was estimated and stratified by HLA-A*02 eligibility. Results: A total of 23 patients had HLA genotyping by a CLIA-certified lab and MSK-IMPACT. Ninety percent (108/ 110) of the sequenced alleles were concordant between IMPACT and the outside lab. LOH of HLA genes was detected in three tumors, one had loss of HLA-A*02:01. In total, 66 patients were screened for T-cell therapy and 20 (30%) were HLA-A *02 eligible on outside testing. Univariate analysis of OS from the time of metastasis found HLA-A *02 eligibility was marginally associated with shorter OS [HR = 1.95; 95% confidence interval (CI), 0.995-3.813; P = 0.052]. On multivariate analysis, older age and larger tumor size, but not HLA-A*02 eligibility, were significantly associated with decreased OS. HLA-A *02 eligibility did not impact OS after chemotherapy or pazopanib in the metastatic setting. Conclusions: Targeted gene panels like MSK-IMPACT may accurately report HLA type and identify loss of somatic HLA alleles. In a multivariable model, HLA-A*02 eligibility was not significantly associated with OS in patients with metastatic SS.
Frew JW, Jiang CS, Singh N, Grand D, Navrazhina K, Vaughan R, Krueger JG
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Malignancy and infection risk during adalimumab therapy in hidradenitis suppurativa

Background The association of adalimumab therapy with malignancy and infection is established in other inflammatory diseases; however, rates of hidradenitis suppurativa (HS) are based on case reports or retrospective healthcare data and the effect of adalimumab therapy on these rates is unknown. Previously reported rates in the PIONEER OLE Phase 3 study reported on rates only in a subpopulation of 88 participants rather than the entire cohort. Aim To quantify rates of malignancy and serious infection in all patients with HS treated with adalimumab 40 mg weekly. Methods Reanalysis was undertaken of individual patient data from the PIONEER 1, PIONEER 2 and PIONEER open-label extension Phase 3 trial data encompassing 591 unique patients with HS administered adalimumab 40 mg weekly without concurrent antibiotic exposure. Incidence rates of serious infection and malignancy were calculated. Results Incidence rates of serious infection and malignancy were 2.14 and 0.46 per 100 patient-years, respectively. Rates of infection and malignancy were comparable to those in other inflammatory conditions examined. Conclusion Incidence of serious infection in patients with HS on adalimumab is comparable to those with psoriasis and inflammatory arthropathies, but the incidence of malignancy is increased. This may reflect disease-specific malignancy risk rather than an effect of adalimumab.
Flamholz AI, Dugan E, Blikstad C, Gleizer S, Ben-Nissan R, Amram S, Antonovsky N, Ravishankar S, Noor E, Bar-Even A, Milo R, Savage DF
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Functional reconstitution of a bacterial CO2 concentrating mechanism in Escherichia coli

ELIFE 2020 OCT 21; 9(?):? Article e59882
Many photosynthetic organisms employ a CO2 concentrating mechanism (CCM) to increase the rate of CO2 fixation via the Calvin cycle. CCMs catalyze approximate to 50% of global photosynthesis, yet it remains unclear which genes and proteins are required to produce this complex adaptation. We describe the construction of a functional CCM in a non-native host, achieved by expressing genes from an autotrophic bacterium in an Escherichia coli strain engineered to depend on rubisco carboxylation for growth. Expression of 20 CCM genes enabled E. coli to grow by fixing CO2 from ambient air into biomass, with growth in ambient air depending on the components of the CCM. Bacterial CCMs are therefore genetically compact and readily transplanted, rationalizing their presence in diverse bacteria. Reconstitution enabled genetic experiments refining our understanding of the CCM, thereby laying the groundwork for deeper study and engineering of the cell biology supporting CO2 assimilation in diverse organisms.
Bavley CC, Fetcho RN, Burgdorf CE, Walsh AP, Fischer DK, Hall BS, Sayles NM, Contoreggi NH, Hackett JE, Antigua SA, Babij R, Garc?a NDV, Kash TL, Milner TA, Liston C, Rajadhyaksha AM
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Cocaine- and stress-primed reinstatement of drug-associated memories elicit differential behavioral and frontostriatal circuit activity patterns via recruitment of L-type Ca(2+)channels

MOLECULAR PSYCHIATRY 2020 OCT; 25(10):2373-2391
Cocaine-associated memories are critical drivers of relapse in cocaine-dependent individuals that can be evoked by exposure to cocaine or stress. Whether these environmental stimuli recruit similar molecular and circuit-level mechanisms to promote relapse remains largely unknown. Here, using cocaine- and stress-primed reinstatement of cocaine conditioned place preference to model drug-associated memories, we find that cocaine drives reinstatement by increasing the duration that mice spend in the previously cocaine-paired context whereas stress increases the number of entries into this context. Importantly, both forms of reinstatement require Ca(v)1.2 L-type Ca(2+)channels (LTCCs) in cells of the prelimbic cortex that project to the nucleus accumbens core (PrL -> NAcC). Utilizing fiber photometry to measure circuit activity in vivo in conjunction with the LTCC blocker, isradipine, we find that LTCCs drive differential recruitment of the PrL -> NAcC pathway during cocaine- and stress-primed reinstatement. While cocaine selectively activates PrL -> NAcC cells prior to entry into the cocaine-paired chamber, a measure that is predictive of duration in that chamber, stress increases persistent activity of this projection, which correlates with entries into the cocaine-paired chamber. Using projection-specific chemogenetic manipulations, we show that PrL -> NAcC activity is required for both cocaine- and stress-primed reinstatement, and that activation of this projection in Ca(v)1.2-deficient mice restores reinstatement. These data indicate that LTCCs are a common mediator of cocaine- and stress-primed reinstatement. However, they engage different patterns of behavior and PrL -> NAcC projection activity depending on the environmental stimuli. These findings establish a framework to further study how different environmental experiences can drive relapse, and supports further exploration of isradipine, an FDA-approved LTCC blocker, as a potential therapeutic for the prevention of relapse in cocaine-dependent individuals.
Orange DE, Darnell RB
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PRIME Cells Predicting Rheumatoid Arthritis Flares

NEW ENGLAND JOURNAL OF MEDICINE 2020 OCT 15; 383(16):1594-1596
Chen J, Boyaci H, Campbell EA
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Diverse and unified mechanisms of transcription initiation in bacteria

In this Review, Chen, Boyaci and Campbell examine universal pathways and diverse regulatory mechanisms in transcription initiation in evolutionarily divergent bacteria, and they discuss the mechanisms whereby antimicrobials inhibit transcription initiation and the insights those mechanisms provide into the transcription cycle. Transcription of DNA is a fundamental process in all cellular organisms. The enzyme responsible for transcription, RNA polymerase, is conserved in general architecture and catalytic function across the three domains of life. Diverse mechanisms are used among and within the different branches to regulate transcription initiation. Mechanistic studies of transcription initiation in bacteria are especially amenable because the promoter recognition and melting steps are much less complicated than in eukaryotes or archaea. Also, bacteria have critical roles in human health as pathogens and commensals, and the bacterial RNA polymerase is a proven target for antibiotics. Recent biophysical studies of RNA polymerases and their inhibition, as well as transcription initiation and transcription factors, have detailed the mechanisms of transcription initiation in phylogenetically diverse bacteria, inspiring this Review to examine unifying and diverse themes in this process.