Bloomchristian6954

Radiation sensitive 52 (RAD52) is an important factor for double-strand break repair (DSBR). However, deficiency in vertebrate/mammalian Rad52 has no apparent phenotype. The underlying mechanism remains elusive. Here, we report that RAD52 deficiency increased cell survival after camptothecin (CPT) treatment. CPT generates single-strand breaks (SSBs) that further convert to double-strand breaks (DSBs) if they are not repaired. RAD52 inhibits SSB repair (SSBR) through strong single-strand DNA (ssDNA) and/or poly(ADP-ribose) (PAR) binding affinity to reduce DNA-damage-promoted X-Ray Repair Cross Complementing 1 (XRCC1)/ligase IIIα (LIG3α) co-localization. The inhibitory effects of RAD52 on SSBR neutralize the role of RAD52 in DSBR, suggesting that RAD52 may maintain a balance between cell survival and genomic integrity. Furthermore, we demonstrate that blocking RAD52 oligomerization that disrupts RAD52's DSBR, while retaining its ssDNA binding capacity that is required for RAD52's inhibitory effects on SSBR, sensitizes cells to different DNA-damaging agents. This discovery provides guidance for developing efficient RAD52 inhibitors in cancer therapy.Animal nervous systems remodel following stress. Although global stress-dependent changes are well documented, contributions of individual neuron remodeling events to animal behavior modification are challenging to study. In response to environmental insults, C. elegans become stress-resistant dauers. Dauer entry induces amphid sensory organ remodeling in which bilateral AMsh glial cells expand and fuse, allowing embedded AWC chemosensory neurons to extend sensory receptive endings. We show that amphid remodeling correlates with accelerated dauer exit upon exposure to favorable conditions and identify a G protein-coupled receptor, REMO-1, driving AMsh glia fusion, AWC neuron remodeling, and dauer exit. REMO-1 is expressed in and localizes to AMsh glia tips, is dispensable for other remodeling events, and promotes stress-induced expression of the remodeling receptor tyrosine kinase VER-1. Our results demonstrate how single-neuron structural changes affect animal behavior, identify key glial roles in stress-induced nervous system plasticity, and demonstrate that remodeling primes animals to respond to favorable conditions.Macrophage-mediated inflammation is critical in the pathogenesis of non-alcoholic steatohepatitis (NASH). Here, we describe that, with high-fat, high-sucrose-diet feeding, mature TIM4pos Kupffer cells (KCs) decrease in number, while monocyte-derived Tim4neg macrophages accumulate. In concert, monocyte-derived infiltrating macrophages enter the liver and consist of a transitional subset that expresses Cx3cr1/Ccr2 and a second subset characterized by expression of Trem2, Cd63, Cd9, and Gpmnb; markers ascribed to lipid-associated macrophages (LAMs). The Cx3cr1/Ccr2-expressing macrophages, referred to as C-LAMs, localize to macrophage aggregates and hepatic crown-like structures (hCLSs) in the steatotic liver. In C-motif chemokine receptor 2 (Ccr2)-deficient mice, C-LAMs fail to appear in the liver, and this prevents hCLS formation, reduces LAM numbers, and increases liver fibrosis. Taken together, our data reveal dynamic changes in liver macrophage subsets during the pathogenesis of NASH and link these shifts to pathologic tissue remodeling.Nucleosomes form heterogeneous groups in vivo, named clutches. Clutches are smaller and less dense in mouse embryonic stem cells (ESCs) compared to neural progenitor cells (NPCs). Using coarse-grained modeling of the pluripotency Pou5f1 gene, we show that the genome-wide clutch differences between ESCs and NPCs can be reproduced at a single gene locus. Larger clutch formation in NPCs is associated with changes in the compaction and internucleosome contact probability of the Pou5f1 fiber. Using single-molecule tracking (SMT), we further show that the core histone protein H2B is dynamic, and its local mobility relates to the structural features of the chromatin fiber. H2B is less stable and explores larger areas in ESCs compared to NPCs. The amount of linker histone H1 critically affects local H2B dynamics. Our results have important implications for how nucleosome organization and H2B dynamics contribute to regulate gene activity and cell identity.Phosphatidylserine (PS) is exposed on the surface of apoptotic cells and is known to promote immunosuppressive signals in the tumor microenvironment (TME). Antibodies that block PS interaction with its receptors have been shown to repolarize the TME into a proinflammatory state. Radiation therapy (RT) is an effective focal treatment of isolated solid tumors but is less effective at controlling metastatic cancers. We found that tumor-directed RT caused an increase in expression of PS on the surface of viable immune infiltrates in mouse B16 melanoma. We hypothesize that PS expression on immune cells may provide negative feedback to immune cells in the TME. Treatment with an antibody that targets PS (mch1N11) enhanced the anti-tumor efficacy of tumor-directed RT and improved overall survival. This combination led to an increase in proinflammatory tumor-associated macrophages. The addition of anti-PD-1 to RT and mch1N11 led to even greater anti-tumor efficacy and overall survival. We found increased PS expression on several immune subsets in the blood of patients with metastatic melanoma after receiving tumor-directed RT. These findings highlight the potential of combining PS targeting with RT and PD-1 pathway blockade to improve outcomes in patients with advanced-stage cancers.Thermoneutral conditions typical for standard human living environments result in brown adipose tissue (BAT) involution, characterized by decreased mitochondrial mass and increased lipid deposition. Low BAT activity is associated with poor metabolic health, and BAT reactivation may confer therapeutic potential. However, the molecular drivers of this BAT adaptive process in response to thermoneutrality remain enigmatic. Using metabolic and lipidomic approaches, we show that endogenous fatty acid synthesis, regulated by carbohydrate-response element-binding protein (ChREBP), is the central regulator of BAT involution. By transcriptional control of lipogenesis-related enzymes, ChREBP determines the abundance and composition of both storage and membrane lipids known to regulate organelle turnover and function. Notably, ChREBP deficiency and pharmacological inhibition of lipogenesis during thermoneutral adaptation preserved mitochondrial mass and thermogenic capacity of BAT independently of mitochondrial biogenesis. In conclusion, we establish lipogenesis as a potential therapeutic target to prevent loss of BAT thermogenic capacity as seen in adult humans.Magnesium (Mg2+) homeostasis depends on active transcellular Mg2+ reuptake from urine in distal convoluted tubules (DCTs) via the Mg2+ channel TRPM6, whose activity has been proposed to be regulated by EGF. Calcium (Ca2+) homeostasis depends on paracellular reabsorption in the thick ascending limbs of Henle (TALs). KCTD1 promotes terminal differentiation of TALs/DCTs, but how its deficiency affects urinary Mg2+ and Ca2+ reabsorption is unknown. Here, this study shows that DCT1-specific KCTD1 inactivation leads to hypomagnesemia despite normal TRPM6 levels because of reduced levels of the sodium chloride co-transporter NCC, whereas Mg2+ homeostasis does not depend on EGF. Moreover, KCTD1 deficiency impairs paracellular urinary Ca2+ and Mg2+ reabsorption in TALs because of reduced NKCC2/claudin-16/-19 and increased claudin-14 expression, leading to hypocalcemia and consequently to secondary hyperparathyroidism and progressive metabolic bone disease. Thus, KCTD1 regulates urinary reabsorption of Mg2+ and Ca2+ by inducing expression of NCC in DCTs and NKCC2/claudin-16/-19 in TALs.Protein kinases lie at the heart of cell-signaling processes and are often mutated in disease. Kinase target recognition at the active site is in part determined by a few amino acids around the phosphoacceptor residue. check details However, relatively little is known about how most preferences are encoded in the kinase sequence or how these preferences evolved. Here, we used alignment-based approaches to predict 30 specificity-determining residues (SDRs) for 16 preferences. These were studied with structural models and were validated by activity assays of mutant kinases. Cancer mutation data revealed that kinase SDRs are mutated more frequently than catalytic residues. We have observed that, throughout evolution, kinase specificity has been strongly conserved across orthologs but can diverge after gene duplication, as illustrated by the G protein-coupled receptor kinase family. The identified SDRs can be used to predict kinase specificity from sequence and aid in the interpretation of evolutionary or disease-related genomic variants.Individuals with malaria exhibit increased morbidity and mortality when infected with Gram-negative (Gr-) bacteria. To explore this experimentally, we performed co-infection of mice with Plasmodium chabaudi and Citrobacter rodentium, an extracellular Gr- bacterial pathogen that infects the large intestine. While single infections are controlled effectively, co-infection results in enhanced virulence that is characterized by prolonged systemic bacterial persistence and high mortality. Mortality in co-infected mice is associated with disrupted iron metabolism, elevated levels of plasma heme, and increased mitochondrial reactive oxygen species (ROS) production by phagocytes. In addition, iron acquisition by the bacterium plays a key role in pathogenesis because co-infection with a mutant C. rodentium strain lacking a critical iron acquisition pathway does not cause mortality. These results indicate that disrupted iron metabolism may drive mortality during co-infection with C. rodentium and P. chabaudi by both altering host immune responses and facilitating bacterial persistence.Vesicular glutamate transporters (VGLUTs) fill synaptic vesicles with glutamate. VGLUTs were originally identified as sodium-dependent transporters of inorganic phosphate (Pi), but the physiological relevance of this activity remains unclear. Heterologous expression of all three VGLUTs greatly augments intracellular Pi levels. Using neuronal models, we show that translocation of VGLUTs to the plasma membrane during exocytosis results in highly increased Pi uptake. VGLUT-mediated Pi influx is counteracted by Pi efflux. Synaptosomes prepared from perinatal VGLUT2-/- mice that are virtually free of VGLUTs show drastically reduced cytosolic Pi levels and fail to import Pi. Glutamate partially competes with sodium (Na+)/Pi (NaPi)-uptake mediated by VGLUTs but does not appear to be transported. A nanobody that blocks glutamate transport by binding to the cytoplasmic domain of VGLUT1 abolishes Pi transport when co-expressed with VGLUT1. We conclude that VGLUTs have a dual function that is essential for both vesicular glutamate loading and Pi restoration in neurons.Motion streaks are smeared representation of fast-moving objects due to temporal integration. Here, we test for motion streak signals in mice with two-photon calcium imaging. For small dots moving at low speeds, neurons in primary visual cortex (V1) encode the component motion, with preferred direction along the axis perpendicular to their preferred orientation. At high speeds, V1 neurons prefer the direction along the axis parallel to their preferred orientation, as expected for encoding motion streaks. Whereas some V1 neurons (∼20%) display a switch of preferred motion axis with increasing speed, others (>40%) respond specifically to high speeds at the parallel axis. Motion streak neurons are also seen in higher visual lateromedial (LM), anterolateral (AL), and rostrolateral (RL) areas, but with higher transition speeds, and many still prefer the perpendicular axis even with fast motion. Our results thus indicate that diverse motion encoding exists in mouse visual cortex, with intriguing differences among visual areas.