Folding and Hydrodynamics of a DNA i-Motif from the c-MYC Promoter Determined by Fluorescent Cytidine Analogs

AbstractThe four-stranded i-motif (iM) conformation of cytosine-rich DNA has importance to a wide variety of biochemical systems that range from their use in nanomaterials to potential roles in oncogene regulation. The iM structure is formed at slightly acidic pH, where hemiprotonation of cytosine results in a stable C-C+ basepair. Here, we performed fundamental studies to examine iM formation from a C-rich strand from the promoter of the human c-MYC gene. We used a number of biophysical techniques to characterize both the hydrodynamic properties and folding kinetics of a folded iM. Our hydrodynamic studies using fluorescence anisotropy decay and analytical ultracentrifugation show that the iM structure has a compact size in solution and displays the rigidity of a double strand. By studying the rates of circular dichroism spectral changes and quenching of fluorescent cytidine analogs, we also established a mechanism for the folding of a random coil oligo into the iM. In the course of determining this folding pathway, we established that the fluorescent dC analogs tC° and PdC can be used to monitor individual residues of an iM structure and to determine the pKa of an iM. We established that the C-C+ hydrogen bonding of certain bases initiates the folding of the iM structure. We also showed that substitutions in the loop regions of iMs give a distinctly different kinetic signature during folding compared with bases that are intercalated. Our data reveal that the iM passes through a distinct intermediate form between the unfolded and folded forms. Taken together, our results lay the foundation for using fluorescent dC analogs to follow structural changes during iM formation. Our technique may also be useful for examining folding and structural changes in more complex iMs

Inference and Evolutionary Analysis of Genome-Scale Regulatory Networks in Large Phylogenies

Changes in transcriptional regulatory networks can significantly contribute to species evolution and adaptation. However, identification of genome-scale regulatory networks is an open challenge, especially in non-model organisms. Here, we introduce multi-species regulatory network learning (MRTLE), a computational approach that uses phylogenetic structure, sequence-specific motifs, and transcriptomic data, to infer the regulatory networks in different species. Using simulated data from known networks and transcriptomic data from six divergent yeasts, we demonstrate that MRTLE predicts networks with greater accuracy than existing methods because it incorporates phylogenetic information. We used MRTLE to infer the structure of the transcriptional networks that control the osmotic stress responses of divergent, non-model yeast species and then validated our predictions experimentally. Interrogating these networks reveals that gene duplication promotes network divergence across evolution. Taken together, our approach facilitates study of regulatory network evolutionary dynamics across multiple poorly studied species. Keywords: regulatory networks; network inference; evolution of gene regulatory networks; evolution of stress response; yeast; probabilistic graphical model; phylogeny; comparative functional genomicsNational Science Foundation (U.S.) (Grant DBI-1350677)National Institutes of Health (U.S.) (Grant R01CA119176-01)National Institutes of Health (U.S.) (Grant DP1OD003958-01

Lung Megakaryocytes are Immune Modulatory Cells that Present Antigen to CD4+ T cells.

Although platelets are the cellular mediators of thrombosis, they are also immune cells. Platelets interact both directly and indirectly with immune cells, impacting their activation and differentiation, as well as all phases of the immune response. Megakaryocytes (Mks) are the cell source of circulating platelets, and until recently Mks were typically only considered bone marrow–resident (BM-resident) cells. However, platelet-producing Mks also reside in the lung, and lung Mks express greater levels of immune molecules compared with BM Mks. We therefore sought to define the immune functions of lung Mks. Using single-cell RNA sequencing of BM and lung myeloid-enriched cells, we found that lung Mks, which we term MkL, had gene expression patterns that are similar to antigen-presenting cells. This was confirmed using imaging and conventional flow cytometry. The immune phenotype of Mks was plastic and driven by the tissue immune environment, as evidenced by BM Mks having an MkL-like phenotype under the influence of pathogen receptor challenge and lung-associated immune molecules, such as IL-33. Our in vitro and in vivo assays demonstrated that MkL internalized and processed both antigenic proteins and bacterial pathogens. Furthermore, MkL induced CD4+ T cell activation in an MHC II–dependent manner both in vitro and in vivo. These data indicated that MkL had key immune regulatory roles dictated in part by the tissue environment.pre-print236 K

The occurrence of tarsal injuries in male mice of C57BL/6N substrains in multiple international mouse facilities.

Dislocation in hindlimb tarsals are being observed at a low, but persistent frequency in group-housed adult male mice from C57BL/6N substrains. Clinical signs included a sudden onset of mild to severe unilateral or bilateral tarsal abduction, swelling, abnormal hindlimb morphology and lameness. Contraction of digits and gait abnormalities were noted in multiple cases. Radiographical and histological examination revealed caudal dislocation of the calcaneus and partial dislocation of the calcaneoquartal (calcaneus-tarsal bone IV) joint. The detection, frequency, and cause of this pathology in five large mouse production and phenotyping centres (MRC Harwell, UK; The Jackson Laboratory, USA; The Centre for Phenogenomics, Canada; German Mouse Clinic, Germany; Baylor College of Medicine, USA) are discussed

Aneuploidy renders cancer cells vulnerable to mitotic checkpoint inhibition

Selective targeting of aneuploid cells is an attractive strategy for cancer treatment(1). Here, we mapped the aneuploidy landscapes of ~1,000 human cancer cell lines, and analyzed genetic and chemical perturbation screens(2–9) to reveal aneuploidy-associated cellular vulnerabilities. We identified and validated an increased sensitivity of aneuploid cancer cells to genetic perturbation of core components of the spindle assembly checkpoint (SAC), which ensures the proper segregation of chromosomes during mitosis(10). Surprisingly, we also found aneuploid cancer cells to be less sensitive to short-term exposures to multiple SAC inhibitors. Indeed, aneuploid cancer cells became increasingly more sensitive to SAC inhibition (SACi) over time. Aneuploid cells exhibited aberrant spindle geometry and dynamics, and kept dividing in the presence of SACi, resulting in accumulating mitotic defects, and in unstable and less fit karyotypes. Therefore, although aneuploid cancer cells could overcome SACi more readily than diploid cells, their long-term proliferation was jeopardized. We identified a specific mitotic kinesin, KIF18A, whose activity was perturbed in aneuploid cancer cells. Aneuploid cancer cells were particularly vulnerable to KIF18A depletion, and KIF18A overexpression restored their response to SACi. Our study reveals a novel, therapeutically-relevant, synthetic lethal interaction between aneuploidy and the SAC

Genetics of randomly bred cats support the cradle of cat domestication being in the Near East

Cat domestication likely initiated as a symbiotic relationship between wildcats (Felis silvestris subspecies) and the peoples of developing agrarian societies in the Fertile Crescent. As humans transitioned from hunter-gatherers to farmers ~12,000 years ago, bold wildcats likely capitalized on increased prey density (i.e., rodents). Humans benefited from the cats’ predation on these vermin. To refine the site(s) of cat domestication, over 1000 random-bred cats of primarily Eurasian descent were genotyped for single-nucleotide variants and short tandem repeats. The overall cat population structure suggested a single worldwide population with significant isolation by the distance of peripheral subpopulations. The cat population heterozygosity decreased as genetic distance from the proposed cat progenitor’s (F.s. lybica) natural habitat increased. Domestic cat origins are focused in the eastern Mediterranean Basin, spreading to nearby islands, and southernly via the Levantine coast into the Nile Valley. Cat population diversity supports the migration patterns of humans and other symbiotic species

Application of In Vivo Induced Antigen Technology (IVIAT) to Bacillus anthracis

In vivo induced antigen technology (IVIAT) is an immuno-screening technique that identifies bacterial antigens expressed during infection and not during standard in vitro culturing conditions. We applied IVIAT to Bacillus anthracis and identified PagA, seven members of a N-acetylmuramoyl-L-alanine amidase autolysin family, three P60 family lipoproteins, two transporters, spore cortex lytic protein SleB, a penicillin binding protein, a putative prophage holin, respiratory nitrate reductase NarG, and three proteins of unknown function. Using quantitative real-time PCR comparing RNA isolated from in vitro cultured B. anthracis to RNA isolated from BALB/c mice infected with virulent Ames strain B. anthracis, we confirmed induced expression in vivo for a subset of B. anthracis genes identified by IVIAT, including L-alanine amidases BA3767, BA4073, and amiA (pXO2-42); the bacteriophage holin gene BA4074; and pagA (pXO1-110). The exogenous addition of two purified putative autolysins identified by IVIAT, N-acetylmuramoyl-L-alanine amidases BA0485 and BA2446, to vegetative B. anthracis cell suspensions induced a species-specific change in bacterial morphology and reduction in viable bacterial cells. Many of the proteins identified in our screen are predicted to affect peptidoglycan re-modeling, and our results support significant cell wall structural remodeling activity during B. anthracis infection. Identification of L-alanine amidases with B. anthracis specificity may suggest new potential therapeutic targets

Life Beyond the Solar System: Remotely Detectable Biosignatures

For the first time in human history, we will soon be able to apply to the scientific method to the question "Are We Alone?" The rapid advance of exoplanet discovery, planetary systems science, and telescope technology will soon allow scientists to search for life beyond our Solar System through direct observation of extrasolar planets. This endeavor will occur alongside searches for habitable environments and signs of life within our Solar System. While these searches are thematically related and will inform each other, they will require separate observational techniques. The search for life on exoplanets holds potential through the great diversity of worlds to be explored beyond our Solar System. However, there are also unique challenges related to the relatively limited data this search will obtain on any individual world

A biomarker-stratified comparison of top-down versus accelerated step-up treatment strategies for patients with newly diagnosed Crohn's disease (PROFILE):a multicentre, open-label randomised controlled trial

Background: Management strategies and clinical outcomes vary substantially in patients newly diagnosed with Crohn's disease. We evaluated the use of a putative prognostic biomarker to guide therapy by assessing outcomes in patients randomised to either top-down (ie, early combined immunosuppression with infliximab and immunomodulator) or accelerated step-up (conventional) treatment strategies. Methods: PROFILE (PRedicting Outcomes For Crohn's disease using a moLecular biomarker) was a multicentre, open-label, biomarker-stratified, randomised controlled trial that enrolled adults with newly diagnosed active Crohn's disease (Harvey-Bradshaw Index ≥7, either elevated C-reactive protein or faecal calprotectin or both, and endoscopic evidence of active inflammation). Potential participants had blood drawn to be tested for a prognostic biomarker derived from T-cell transcriptional signatures (PredictSURE-IBD assay). Following testing, patients were randomly assigned, via a secure online platform, to top-down or accelerated step-up treatment stratified by biomarker subgroup (IBDhi or IBDlo), endoscopic inflammation (mild, moderate, or severe), and extent (colonic or other). Blinding to biomarker status was maintained throughout the trial. The primary endpoint was sustained steroid-free and surgery-free remission to week 48. Remission was defined by a composite of symptoms and inflammatory markers at all visits. Flare required active symptoms (HBI ≥5) plus raised inflammatory markers (CRP &gt;upper limit of normal or faecal calprotectin ≥200 μg/g, or both), while remission was the converse—ie, quiescent symptoms (HBI &lt;5) or resolved inflammatory markers (both CRP ≤ the upper limit of normal and calprotectin &lt;200 μg/g) or both. Analyses were done in the full analysis (intention-to-treat) population. The trial has completed and is registered (ISRCTN11808228). Findings: Between Dec 29, 2017, and Jan 5, 2022, 386 patients (mean age 33·6 years [SD 13·2]; 179 [46%] female, 207 [54%] male) were randomised: 193 to the top-down group and 193 to the accelerated step-up group. Median time from diagnosis to trial enrolment was 12 days (range 0–191). Primary outcome data were available for 379 participants (189 in the top-down group; 190 in the accelerated step-up group). There was no biomarker–treatment interaction effect (absolute difference 1 percentage points, 95% CI –15 to 15; p=0·944). Sustained steroid-free and surgery-free remission was significantly more frequent in the top-down group than in the accelerated step-up group (149 [79%] of 189 patients vs 29 [15%] of 190 patients, absolute difference 64 percentage points, 95% CI 57 to 72; p&lt;0·0001). There were fewer adverse events (including disease flares) and serious adverse events in the top-down group than in the accelerated step-up group (adverse events: 168 vs 315; serious adverse events: 15 vs 42), with fewer complications requiring abdominal surgery (one vs ten) and no difference in serious infections (three vs eight). Interpretation: Top-down treatment with combination infliximab plus immunomodulator achieved substantially better outcomes at 1 year than accelerated step-up treatment. The biomarker did not show clinical utility. Top-down treatment should be considered standard of care for patients with newly diagnosed active Crohn's disease. Funding: Wellcome and PredictImmune Ltd.</p

Congenital Heart Disease–Causing Gata4 Mutation Displays Functional Deficits In Vivo

Defects of atrial and ventricular septation are the most frequent form of congenital heart disease, accounting for almost 50% of all cases. We previously reported that a heterozygous G296S missense mutation of GATA4 caused atrial and ventricular septal defects and pulmonary valve stenosis in humans. GATA4 encodes a cardiac transcription factor, and when deleted in mice it results in cardiac bifida and lethality by embryonic day (E)9.5. In vitro, the mutant GATA4 protein has a reduced DNA binding affinity and transcriptional activity and abolishes a physical interaction with TBX5, a transcription factor critical for normal heart formation. To characterize the mutation in vivo, we generated mice harboring the same mutation, Gata4 G295S. Mice homozygous for the Gata4 G295S mutant allele have normal ventral body patterning and heart looping, but have a thin ventricular myocardium, single ventricular chamber, and lethality by E11.5. While heterozygous Gata4 G295S mutant mice are viable, a subset of these mice have semilunar valve stenosis and small defects of the atrial septum. Gene expression studies of homozygous mutant mice suggest the G295S protein can sufficiently activate downstream targets of Gata4 in the endoderm but not in the developing heart. Cardiomyocyte proliferation deficits and decreased cardiac expression of CCND2, a member of the cyclin family and a direct target of Gata4, were found in embryos both homozygous and heterozygous for the Gata4 G295S allele. To further define functions of the Gata4 G295S mutation in vivo, compound mutant mice were generated in which specific cell lineages harbored both the Gata4 G295S mutant and Gata4 null alleles. Examination of these mice demonstrated that the Gata4 G295S protein has functional deficits in early myocardial development. In summary, the Gata4 G295S mutation functions as a hypomorph in vivo and leads to defects in cardiomyocyte proliferation during embryogenesis, which may contribute to the development of congenital heart defects in humans