Cell membrane deformation and bioeffects produced by tandem bubble-induced jetting flow
Fang Yuan, Chen Yang, and Pei Zhong
Cavitation plays a pivotal role in ultrasound-generated bioeffects. Here, we report the design of an experimental system based on laser-generated tandem bubbles in a microfluidic chip and surface patterning to investigate the causal relationship between cavitation jetting-induced cell membrane deformation and resultant bioeffects. We have demonstrated that pinpoint membrane poration produced at the cell’s leading edge correlates with area strain integral, which varies significantly with standoff distance to the tandem bubble. By adjusting the standoff distance, distinctly different bioeffects (necrosis, repairable poration, or nonporation) could be produced in individual cells, providing the opportunity to probe mechanotransduction at single cell level with potential applications in disease diagnosis and treatment monitoring based on mechanical characterization of the cell. (See pp. E7039–E7047)
Endohedral gallide cluster superconductors and superconductivity in ReGa5
Weiwei Xie, Huixia Luo, Brendan F. Phelan, Tomasz Klimczuk, Francois Alexandre Cevallos, and Robert Joseph Cava
The prediction of new superconductors remains an elusive goal. It is often chemists who find new superconductors, although it is difficult to translate the physics of superconductivity into chemical requirements for discovering new superconducting compounds. There are many strategies for finding new superconductors, one being to postulate that superconductivity runs in structural families. Here we show that a previously unappreciated structural family, the endohedral gallium cluster phases, is favored for superconductivity, and then use the understanding we develop to find a superconductor. More broadly, our work shows that molecule-based electron counting and stability rules can provide a useful chemistry-based design paradigm for finding new superconductors. Using these ideas to search for new superconductors will be of significant future interest. (See pp. E7048–E7054)
Directed and persistent movement arises from mechanochemistry of the ParA/ParB system
Longhua Hu, Anthony G. Vecchiarelli, Kiyoshi Mizuuchi, Keir C. Neuman, and Jian Liu
Cells typically use processive motor proteins or the growth/shrinkage of cytoskeletal filaments to power directed and persistent movement of cellular structures. What if there are no motor proteins or filaments? Here, we establish a third mechanism of processive motility exemplified by the ParA/ParB system, which faithfully segregates low-copy number plasmids during bacterial cell division. The DNA cargos recruit ParB, which binds to and stimulates the ATPase activity of ParA bound to the nucleoid. ATP hydrolysis dissociates ParA from the nucleoid. The transient tethering arising from the ParA–ParB bonds collectively drives forward movement of the cargo and quenches lateral diffusive motions, producing a strikingly persistent trajectory. This operational principle could be important in early evolution and conserved for many systems. (See pp. E7055–E7064)
Origins of stereoselectivity in evolved ketoreductases
NoeyElizabeth L.Nidhi Tibrewal, Gonzalo Jiménez-Osés, Sílvia Osuna, Jiyong Park, Carly M. Bond, Duilio Cascio, Jack Liang, Xiyun Zhang, Gjalt W. Huisman, Yi Tang, and Kendall N. Houk
Ketoreductases are the most commonly used enzymes in industrial pharmaceutical synthesis. We investigated the nature of enantioselectivity in closely related mutant ketoreductases that reduce almost-symmetrical 3-oxacyclopentanone and 3-thiacyclopentanone, which are difficult to reduce enantioselectively by other means. We present the efficiencies of select variants and their crystallographic structures. Our experimental and theoretical studies reveal how mutations modulate the stereoselectivity of the reduction. Molecular dynamics simulations of the Michaelis–Menten and transition state-bound complexes were used to rationalize the observed stereochemical outcomes. We discovered that the closed conformation of the flexible substrate binding loop is likely the catalytically active one imparting the stereochemical preferences. Our molecular dynamics approach reveals how each enzyme stabilizes the diastereomeric transition structures by altering the active site size. (See pp. E7065–E7072)
Antiinfectives targeting enzymes and the proton motive force
Xinxin Feng, Wei Zhu, Lici A. Schurig-Briccio, Steffen Lindert, Carolyn Shoen, Reese Hitchings, Jikun Li, Yang Wang, Noman Baig, Tianhui Zhou, Boo Kyung Kim, Dean C. Crick, Michael Cynamon, J. Andrew McCammon, Robert B. Gennis, and Eric Oldfield
Uncoupling agents might be expected to be generally cytotoxic, but many US Food and Drug Administration (FDA)-approved drugs do have activity as uncouplers, in addition to targeting enzymes. There is therefore interest in the discovery of antibiotics that have such multitarget activity. Here, we show that some FDA-approved drugs, such as clofazimine, clomiphene, and bedaquiline, with antiinfective activity act as uncouplers. Using molecular dynamics-based in silico screening, we also discovered that the brain cancer drug lead vacquinol is an uncoupler that inhibits an enzyme involved in the formation of tuberculosis (TB) virulence factors, in addition to killing TB bacteria. Our results indicate strong drug–membrane interactions, and that screening for combined enzyme inhibition plus uncoupler activity will lead to new antibiotic leads. (See pp. E7073–E7082)
TMEM110 regulates the maintenance and remodeling of mammalian ER–plasma membrane junctions competent for STIM–ORAI signaling
Ariel Quintana, Vangipurapu Rajanikanth, Suzette Farber-Katz, Aparna Gudlur, Chen Zhang, Ji Jing, Yubin Zhou, Anjana Rao, and Patrick G. Hogan
Close appositions between the endoplasmic reticulum (ER) and the plasma membrane in mammalian cells have essential roles in cellular lipid metabolism and in cytoplasmic calcium signaling. Although recent investigations have yielded considerable insight into the structural basis for lipid transfer at ER–plasma membrane junctions, little is known about the proteins that organize junctions for calcium signaling. Our data show that the ER membrane protein transmembrane protein 110 (TMEM110) supports the maintenance of ER–plasma membrane junctions competent for calcium signaling and acts in concert with other proteins in the dynamic remodeling of the junctions during signaling. (See pp. E7083–E7092)
The ancestral gene repertoire of animal stem cells
AliéAlexandreTetsutaro Hayashi, Itsuro Sugimura, Michaël Manuel, Wakana Sugano, Akira Mano, Nori Satoh, Kiyokazu Agata, and Noriko Funayama
This work reveals the deeply conserved gene repertoire of animal stem cells, from sponges to mammals. This repertoire mostly contains ancient (premetazoan) genes and few novel (metazoan-specific) genes, but the latter point to the importance of genome protection in the origin of stem cells. Additionally, regulators of DNA transcription are only marginally represented among conserved stem-cell genes, whereas genes directly acting upon RNA predominate, including orthologues of RNA-binding proteins that control mammalian embryonic stem cells. Thus posttranscriptional regulation of gene expression has been crucial in animal stem-cell biology across hundreds of millions of years of animal evolution. (See pp. E7093–E7100)
Mandibular and dental characteristics of Late Triassic mammaliaform Haramiyavia and their ramifications for basal mammal evolution
Zhe-Xi Luo, Stephen M. Gatesy, Farish A. Jenkins Jr., William W. Amaral, and Neil H. Shubin
The origins and earliest evolution of mammals can be deciphered by studying Late Triassic fossil relatives of modern mammals. The computed tomography study of Haramiyavia from the Late Triassic has revealed new information about the skull evolution and dental function in the forerunners of mammals. Haramiyavia had a unique way of chewing. Its teeth of multiple cusp-rows were adapted to omnivory or herbivory and are distinctive from the teeth of other early mammal relatives that are presumed to be insectivorous. On the mammal family tree Haramiyavia occupies a position crucial for dating the initial appearance of the major mammalian groups. Our reanalysis affirms that the earliest diversification of mammals occurred in the Jurassic. (See pp. E7101–E7109)
Synthetic CRISPR RNA-Cas9–guided genome editing in human cells
Meghdad Rahdar, Moira A. McMahon, Thazha P. Prakash, Eric E. Swayze, C. Frank Bennett, and Don W. Cleveland
Genome editing with nucleases that recognize specific DNA sequences is a powerful technology for manipulating genomes. This is especially true for the Cas9 nuclease, the site specificity of which is determined by a bound RNA, called a CRISPR RNA (crRNA). Here we develop a chemically modified, 29-nucleotide synthetic CRISPR RNA (scrRNA) and show that it can functionally replace the natural crRNA, producing enhanced cleavage activity at a target DNA site with apparently reduced off-target cleavage. scrRNAs can be synthesized in a commercially feasible manner today and provide a platform for therapeutic applications. (See pp. E7110–E7117)
Human genetic basis of interindividual variability in the course of infection
Jean-Laurent Casanovaa
Pasteur's germ theory of disease initially seemed to have resolved the longstanding antagonism between the proponents of intrinsic and extrinsic disease mechanisms.However, by the turn of the 20th century, it had become clear that each microbe killed only a small minority of infected individuals. Infectious diseases killed half of all children before the age of 15 y, but this enormous burden was caused by the dazzling diversity of pathogens rather than by the potency of individual pathogens. The key problem concerning pediatric infectious diseases thus was identified: their pathogenesis. A human genetic theory of infectious diseases has emerged gradually from clinical and epidemiological studies, building on many elegant studies in plants and animals. (See pp. E7118–E7127.)
Severe infectious diseases of childhood as monogenic inborn errors of immunity
and,Jean-Laurent Casanova
The key problem concerning pediatric infectious diseases, and more generally clinical diseases during primary infection, is their pathogenesis. A plausible and testable human genetic theory of primary infectious diseases has recently emerged, building on elegant studies in plants and animals. Three examples of monogenic resistance to common infections have been discovered. Moreover, a growing range of monogenic single-gene inborn errors of immunity, rarely Mendelian (with complete clinical penetrance) but more commonly non-Mendelian (with incomplete penetrance), have been found to underlie severe infectious diseases striking otherwise healthy children during primary infection. These findings provide a synthetic framework for inherited and infectious diseases and, more generally, for inborn and environmental conditions. (See pp. E7128–E7137)
Amphetamine activates Rho GTPase signaling to mediate dopamine transporter internalization and acute behavioral effects of amphetamine
David S. Wheeler, Suzanne M. Underhill, Donna B. Stolz, Geoffrey H. Murdoch, Edda Thiels, Guillermo Romero, and Susan G. Amara
The dopamine transporter (DAT), a major target for psychostimulant drugs, including cocaine and amphetamines, clears extracellular dopamine and restricts the temporal and spatial extent of neurotransmitter signaling. This study examines the mechanism through which amphetamines trigger internalization of DAT and demonstrates that amphetamine activates the small GTPases, Rho and Rac. Rho activation triggers endocytosis of DAT by a dynamin-dependent, clathrin-independent pathway. Intriguingly, amphetamine must enter the cell to have these effects, and it also increases cAMP, which in turn inactivates Rho and limits carrier internalization. Consistent with these observations, the activation of receptors that couple to protein kinase A in dopamine neurons also antagonizes the behavioral effects of amphetamine in mice, suggesting new pathways to target to disrupt amphetamine action. (See pp. E7138–E7147)
Fasting protects mice from lethal DNA damage by promoting small intestinal epithelial stem cell survival
Kelsey L. Tinkum, Kristina M. Stemler, Lynn S. White, Andrew J. Loza, Sabrina Jeter-Jones, Basia M. Michalski, Catherine Kuzmicki, Robert Pless, Thaddeus S. Stappenbeck, David Piwnica-Worms, and Piwnica-WormsHelen
Cancer patients undergoing chemotherapy experience high rates of dose-limiting morbidity. Recently, short-term fasting prior to chemotherapy was shown to decrease toxicity. Herein we report that fasting protects multiple small intestinal stem cell populations marked by Lgr5, Bmi1, or HopX expression and maintains barrier function to preserve small intestinal architecture from lethal DNA damage. Our findings provide insight into how fasting protects the host from toxicity associated with high-dose chemotherapy. (See pp. E7148–E7154)