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Cancer mortality rates have decreased over the last 48 years attributable to standardized cancer screenings. These screenings were developed without deliberate inclusion of transgender and non-binary populations. While specialists are familiar regarding cancer screening in this distinct population, those in primary care might be more limited. As such, we aimed to develop a screening risk tool that combines the Breast Cancer Risk Assessment Tool (Gail model) with the updated American College of Radiology Appropriateness Criteria-Transgender Breast Cancer Screening, into an online questionnaire designed to accommodate primary care physicians performing routine health screenings to advise appropriate imaging and referral for this population. This new tool can be used for transgender chest/breast risk assessment whereas the Gail model alone was developed without transgender populations in mind, with the aim of early detection and cancer prevention in this historically underserved healthcare population., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© 2024 Bacot-Davis and Moran.)

The spliceosome is a large ribonucleoprotein complex that removes introns from pre-mRNAs. At its functional core lies the essential pre-mRNA processing factor 8 (Prp8) protein. Across diverse eukaryotes, this protein cofactor of RNA catalysis harbors a self-splicing element called an intein. Inteins in Prp8 are extremely pervasive and are found at 7 different sites in various species. Here, we focus on the Prp8 intein from Cryptococcus neoformans (Cne), a human fungal pathogen. We solved the crystal structure of this intein, revealing structural homology among protein splicing sequences in eukaryotes, including the Hedgehog C terminus. Working with the Cne Prp8 intein in a reporter assay, we find that the biologically relevant divalent metals copper and zinc inhibit intein splicing, albeit by 2 different mechanisms. Copper likely stimulates reversible modifications on a catalytically important cysteine, whereas zinc binds at the terminal asparagine and the same critical cysteine. Importantly, we also show that copper treatment inhibits Prp8 protein splicing in Cne. Lastly, an intein-containing Prp8 precursor model is presented, suggesting that metal-induced protein splicing inhibition would disturb function of both Prp8 and the spliceosome. These results indicate that Prp8 protein splicing can be modulated, with potential functional implications for the spliceosome., Competing Interests: The authors have declared that no competing interests exist.

Available data suggest that individual and family well-being are linked to the quality of women's and men's couple relationships, but few tools exist to assess couple relationship functioning in low- and middle-income countries. In response to this gap, Catholic Relief Services has developed a Couple Functionality Assessment Tool (CFAT) to capture valid and reliable data on various domains of relationship quality. This tool is designed to be used by interventions which aim to improve couple and family well-being as a means of measuring the effectiveness of these interventions, particularly related to couple relationship quality. We carried out a validation study of the CFAT among 401 married and cohabiting adults (203 women and 198 men) in rural Chikhwawa District, Malawi. Using psychometric scales, the CFAT addressed six domains of couple relationship quality (intimacy, partner support, sexual satisfaction, gender roles, decision-making, and communication and conflict management), and included questions on intimate partner violence. We used exploratory factor analysis to assess scale performance of each domain and produce a shortened Relationship Quality Index (RQI) composed of items from five relationship quality domains. This article reports the performance of the RQI. Internal reliability and validity of the RQI were found to be good. Regression analyses examined the relationship of the RQI to outcomes important to health and development: intra-household cooperation, positive health behaviors, intimate partner violence, and gender-equitable norms. We found many significant correlations between RQI scores and these couple- and family-level development issues. There is a need to further validate the tool with use in other populations as well as to continue to explore whether the observed linkages between couple functionality and development outcomes are causal relationships.

The aminoglycoside modifying enzyme (AME) ANT(2″)-Ia is a significant target for next generation antibiotic development. Structural studies of a related aminoglycoside-modifying enzyme, ANT(3″)(9), revealed this enzyme contains dynamic, disordered, and well-defined segments that modulate thermodynamically before and after antibiotic binding. Characterizing these structural dynamics is critical for in situ screening, design, and development of contemporary antibiotics that can be implemented in a clinical setting to treat potentially lethal, antibiotic resistant, human infections. Here, the first NMR structural ensembles of ANT(2″)-Ia are presented, and suggest that ATP-aminoglycoside binding repositions the nucleotidyltransferase (NT) and C-terminal domains for catalysis to efficiently occur. Residues involved in ligand recognition were assessed by site-directed mutagenesis. In vitro activity assays indicate a critical role for I129 toward aminoglycoside modification in addition to known catalytic D44, D46, and D48 residues. These observations support previous claims that ANT aminoglycoside sub-class promiscuity is not solely due to binding cleft size, or inherent partial disorder, but can be controlled by ligand modulation on distinct dynamic and thermodynamic properties of ANTs under cellular conditions. Hydrophobic interactions in the substrate binding cleft, as well as solution dynamics in the C-terminal tail of ANT(2″)-Ia, advocate toward design of kanamycin-derived cationic lipid aminoglycoside analogs, some of which have already shown antimicrobial activity in vivo against kanamycin and gentamicin-resistant P. aeruginosa. This data will drive additional in silico, next generation antibiotic development for future human use to combat increasingly prevalent antimicrobial resistance., (© 2017 The Protein Society.)

Cardiovirus Leader (L) proteins induce potent antihost inhibition of active cellular nucleocytoplasmic trafficking by triggering aberrant hyperphosphorylation of nuclear pore proteins (Nup). To achieve this, L binds protein RanGTPase (Ran), a key trafficking regulator, and diverts it into tertiary or quaternary complexes with required kinases. The activity of L is regulated by two phosphorylation events not required for Ran binding. Matched NMR studies on the unphosphorylated, singly, and doubly phosphorylated variants of Mengovirus L (L(M)) show both modifications act together to partially stabilize a short internal α-helix comprising L(M) residues 43-46. This motif implies that ionic and Van der Waals forces contributed by phosphorylation help organize downstream residues 48-67 into a new interface. The full structure of L(M) as bound to Ran (unlabeled) and Ran (216 aa) as bound by L(M) (unlabeled) places L(M) into the BP1 binding site of Ran, wrapped by the conformational flexible COOH tail. The arrangement explains the tight KD for this complex and places the LM zinc finger and phosphorylation interface as surface exposed and available for subsequent reactions. The core structure of Ran, outside the COOH tail, is not altered by L(M) binding and remains accessible for canonical RanGTP partner interactions. Pull-down assays identify at least one putative Ran:L(M) partner as an exportin, Crm1, or CAS. A model of Ran:L(M):Crm1, based on the new structures suggests LM phosphorylation status may mediate Ran's selection of exportin(s) and cargo(s), perverting these native trafficking elements into the lethal antihost Nup phosphorylation pathways.

Unlabelled: The leader (L) and 2A proteins of cardioviruses are the primary antihost agents produced during infection. For encephalomyocarditis virus (EMCV), the prototype of the genus Cardiovirus, these proteins interact independently with key cellular partners to bring about inhibition of active nucleocytoplasmic trafficking and cap-dependent translation, respectively. L and 2A also bind each other and require this cooperation to achieve their effects during infection. Recombinant L and 2A interact with 1:1 stoichiometry at a KD (equilibrium dissociation constant) of 1.5 μM. The mapped contact domains include the amino-proximal third of 2A (first 50 amino acids) and the central hinge region of L. This contact partially overlaps the L segment that makes subsequent contact with Ran GTPase in the nucleus, and Ran can displace 2A from L. The equivalent proteins from Theiler's murine encephalomyelitis virus (TMEV; BeAn) and Saffold virus interact similarly in any subtype combination, with various affinities. The data suggest a mechanism whereby L takes advantage of the nuclear localization signal in the COOH region of 2A to enhance its trafficking to the nucleus. Once there, it exchanges partners in favor of Ran. This required cooperation during infection explains many observed codependent phenotypes of L and 2A mutations., Importance: Cardiovirus pathogenesis phenotypes vary dramatically, from asymptomatic, to mild gastrointestinal (GI) distress, to persistent demyelination and even encephalitic death. Leader and 2A are the primary viral determinants of pathogenesis, so understanding how these proteins cooperate to induce such a wide variety of outcomes for the host is of great important and interest to the field of virology, especially to those who use TMEV as a murine model for multiple sclerosis., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Encephalomyocarditis virus and Theilovirus are species in the Cardiovirus genus of the Picornaviridae family. For all cardioviruses, the viral polyprotein is initiated with a short Leader (L) protein unique to this genus. The nuclear magnetic resonance (NMR) structure of LE from encephalomyocarditis virus (EMCV) has been determined. The protein has an NH2-proximal CHCC zinc finger, a central linker, and a contiguous, highly acidic motif. The theiloviruses encode the same domains, with one or two additional, COOH-proximal domains, characteristic of the human Saffold viruses (SafV) and Theiler's murine encephalomyelitis viruses (TMEV), respectively. The expression of a cardiovirus L, in recombinant form, or during infection/transfection, triggers an extensive, cell-dependent, antihost phosphorylation cascade, targeting nucleoporins (Nups) that form the hydrophobic core of nuclear pore complexes (NPC). The consequent inhibition of active nucleocytoplasmic trafficking is potent and prevents the host from mounting an effective antiviral response. For this inhibition, the L proteins themselves must be phosphorylated. In cells (extracts or recombinant form), LE was shown to be phosphorylated at Thr47 and Tyr41. The first reaction (Thr47), catalyzed by casein kinase 2 (CK2), is an obligatory precedent to the second event (Tyr41), catalyzed by spleen tyrosine kinase (Syk). Site mutations in LE, or kinase-specific inhibitors, prevented LE phosphorylation and subsequent Nup phosphorylation. Parallel experiments with LS (SafV-2) and LT (TMEV BeAn) proteins confirmed the general cardiovirus requirement for L phosphorylation, but CK2 was not the culpable kinase. It is likely that LS and LT are both activated by alternative kinases in different cell types, probably reactive within the Theilo-specific domains. IMPORTANCE An understanding of the diverse methods used by viruses to interfere with cellular processes is important because they can teach us how to control virus infections. This report shows how viruses in the same genus use different cellular enzymes to phosphorylate their proteins. If these processes are interfered with, the viruses are severely disabled.

Evidence suggests a relationship between dietary fat intake, obesity, and colorectal cancer, implying a role for fatty acid metabolism in intestinal tumorigenesis that is incompletely understood. Liver fatty acid-binding protein (L-Fabp), a dominant intestinal fatty acid-binding protein, regulates intestinal fatty acid trafficking and metabolism, and L-Fabp deletion attenuates diet-induced obesity. Here, we examined whether changes in intestinal fatty acid metabolism following L-Fabp deletion modify adenoma development in Apc(Min)(/+) mice. Compound L-Fabp(-/-)Apc(Min)(/+) mice were generated and fed a 10% fat diet balanced equally between saturated, monounsaturated, and polyunsaturated fat. L-Fabp(-/-)Apc(Min)(/+) mice displayed significant reductions in adenoma number and total polyp area compared with Apc(Min)(/+)controls, reflecting a significant shift in distribution toward smaller polyps. Adenomas from L-Fabp(-/-)Apc(Min)(/+) mice exhibited reductions in cellular proliferation, high-grade dysplasia, and nuclear β-catenin translocation. Intestinal fatty acid content was increased in L-Fabp(-/-)Apc(Min)(/+) mice, and lipidomic profiling of intestinal mucosa revealed significant shifts to polyunsaturated fatty acid species with reduced saturated fatty acid species. L-Fabp(-/-)Apc(Min)(/+) mice also showed corresponding changes in mRNA expression of enzymes involved in fatty acid elongation and desaturation. Furthermore, adenomas from L-Fabp(-/-)Apc(Min)(/+) mice displayed significant reductions in mRNA abundance of nuclear hormone receptors involved in cellular proliferation and in enzymes involved in lipogenesis. These findings collectively implicate L-Fabp as an important genetic modifier of intestinal tumorigenesis, and identify fatty acid trafficking and metabolic compartmentalization as an important pathway linking dietary fat intake, obesity, and intestinal tumor formation.

Encephalomyocarditis virus (EMCV), a Cardiovirus, initiates its polyprotein with a short 67 amino acid Leader (L) sequence. The protein acts as a unique pathogenicity factor, with anti-host activities which include the triggering of nuclear pore complex hyperphosphorylation and direct binding inhibition of the active cellular transport protein, Ran GTPase. Chemical modifications and protein mutagenesis now map the Ran binding domain to the L hinge-linker region, and in particular, to amino acids 35-40. Large deletions affecting this region were shown previously to diminish Ran binding. New point mutations, especially K35Q, D37A and W40A, preserve the intact L structure, abolish Ran binding and are deficient for nucleoporin (Nup) hyperphosphorylation. Ran itself morphs through multiple configurations, but reacts most effectively with L when in the GDP format, preferably with an empty nucleotide binding pocket. Therefore, L:Ran binding, mediated by the linker-hinge, is a required step in L-induced nuclear transport inhibition., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Background: Dextran sulfate sodium (DSS) induces experimental colitis and promotes colitis-associated cancer in rodents. Here we document potent inhibition of real-time quantitative polymerase chain reaction (qPCR) using cDNA from DSS-exposed mouse tissues, which complicates gene expression analysis., Methods: We characterize DSS inhibition of qPCR in-vitro and in a wide array of murine tissues following ingestion of DSS. We examine different approaches to RNA purification prior to cDNA synthesis in order to optimize real-time polymerase chain reaction amplification and gene expression analysis., Results: DSS inhibits qPCR amplification of cDNA between 1 and 10 nM. Orally administered DSS interferes with qPCR amplification of cDNA derived from multiple tissues. Poly-A purification of DSS-exposed RNA allows reliable and cost-effective gene expression analysis in DSS-exposed tissue., Conclusions: DSS is a potent inhibitor of real-time qPCR amplification and interferes with tissue-specific gene expression analysis in DSS-exposed mice. Poly-A purification of tissue-derived RNA results in reliable and cost-effective gene expression analysis in DSS-exposed mice., (Copyright © 2011 Crohn's & Colitis Foundation of America, Inc.)

Abnormal sensory processing has been implicated in the pathophysiology of primary dystonia. In the globus pallidus internus (GPi), the primary output structure of the basal ganglia, many neurons respond to sensory (proprioceptive) stimulation. Here we have characterized GPi neuronal responses to passive movement of the contralateral limbs in 22 patients with primary dystonia undergoing microelectrode recording for placement of deep brain stimulator leads. We plotted coordinates of cells responding to limb movement in a common space. We observed distinct representations of leg and arm movement localized to the dorsal and ventral part of the posterior GPi, respectively. Comparing patients with generalized dystonia versus patients with segmental craniocervical dystonia, there was no difference in the volumes or separations of leg and arm related territories. In contrast to parkinsonism, only a small minority of units were responsive to movement across multiple joints. Abnormally increased directional selectivity was found in units responding to dystonic limbs compared with nondystonic limbs. Some affected GPi neurons therefore appear to have altered proprioceptive tuning for movement direction. There is an apparent preservation of GPi somatotopic organization in dystonia in comparison with prior studies of GPi somatotopic organization in non-human primates and humans with Parkinson's disease.

Room-temperature laser action from Cr(2+)-doped Cd(0.85)Mn(0.15)Te has been demonstrated for what is believed to be the first time. We achieved pulsed laser operation centered at ~2.5mu m by pumping into the mid-infrared absorption band of Cr(2+) ions by use of the 1.907- mum output of a H(2) Raman-shifted Nd:YAG laser. The output of the free-running Cr(2+):Cd(0.85)Mn(0.15)Te laser had a width of ~50 nm (FWHM), and the slope efficiency was calculated to be 5.5% under nonoptimum conditions.