Restraint of IFN-γ expression through a distal silencer CNS-28 for tissue homeostasis.

Interferon-γ (IFN-γ) is a key cytokine in response to viral or intracellular bacterial infection in mammals. While a number of enhancers are described to promote IFN-γ responses, to the best of our knowledge, no silencers for the Ifng gene have been identified. By examining H3K4me1 histone modification in naive CD4+ T cells within Ifng locus, we identified a silencer (CNS-28) that restrains Ifng expression. Mechanistically, CNS-28 maintains Ifng silence by diminishing enhancer-promoter interactions within Ifng locus in a GATA3-dependent but T-bet-independent manner. Functionally, CNS-28 restrains Ifng transcription in NK cells, CD4+ cells, and CD8+ T cells during both innate and adaptive immune responses. Moreover, CNS-28 deficiency resulted in repressed type 2 responses due to elevated IFN-γ expression, shifting Th1 and Th2 paradigm. Thus, CNS-28 activity ensures immune cell quiescence by cooperating with other regulatory cis elements within the Ifng gene locus to minimize autoimmunity.

Navigating the multifaceted intricacies of the Na+-Cl- cotransporter, a highly regulated key effector in the control of hydromineral homeostasis.

The Na+-Cl- cotransporter (NCC; SLC12A3) is a highly regulated integral membrane protein that is known to exist as three splice variants in primates. Its primary role in the kidney is to mediate the cosymport of Na+ and Cl- across the apical membrane of the distal convoluted tubule. Through this role and the involvement of other ion transport systems, NCC allows the systemic circulation to reclaim a fraction of the ultrafiltered Na+, K+, Cl-, and Mg+ loads in exchange for Ca2+ and [Formula: see text]. The physiological relevance of the Na+-Cl- cotransport mechanism in humans is illustrated by several abnormalities that result from NCC inactivation through the administration of thiazides or in the setting of hereditary disorders. The purpose of the present review is to discuss the molecular mechanisms and overall roles of Na+-Cl- cotransport as the main topics of interest. On reading the narrative proposed, one will realize that the knowledge gained in regard to these themes will continue to progress unrelentingly no matter how refined it has now become.

Centriole signaling restricts hepatocyte ploidy to maintain liver integrity.

Hepatocyte polyploidization is a tightly controlled process that is initiated at weaning and increases with age. The proliferation of polyploid hepatocytes in vivo is restricted by the PIDDosome-P53 axis, but how this pathway is triggered remains unclear. Given that increased hepatocyte ploidy protects against malignant transformation, the evolutionary driver that sets the upper limit for hepatocyte ploidy remains unknown. Here we show that hepatocytes accumulate centrioles during cycles of polyploidization in vivo. The presence of excess mature centrioles containing ANKRD26 was required to activate the PIDDosome in polyploid cells. As a result, mice lacking centrioles in the liver or ANKRD26 exhibited increased hepatocyte ploidy. Under normal homeostatic conditions, this increase in liver ploidy did not impact organ function. However, in response to chronic liver injury, blocking centriole-mediated ploidy control leads to a massive increase in hepatocyte polyploidization, severe liver damage, and impaired liver function. These results show that hyperpolyploidization sensitizes the liver to injury, posing a trade-off for the cancer-protective effect of increased hepatocyte ploidy. Our results may have important implications for unscheduled polyploidization that frequently occurs in human patients with chronic liver disease.

Regulation of the Renal NaCl Cotransporter and Its Role in Potassium Homeostasis.

Daily dietary potassium (K+) intake may be as large as the extracellular K+ pool. To avoid acute hyperkalemia, rapid removal of K+ from the extracellular space is essential. This is achieved by translocating K+ into cells and increasing urinary K+ excretion. Emerging data now indicate that the renal thiazide-sensitive NaCl cotransporter (NCC) is critically involved in this homeostatic kaliuretic response. This suggests that the early distal convoluted tubule (DCT) is a K+ sensor that can modify sodium (Na+) delivery to downstream segments to promote or limit K+ secretion. K+ sensing is mediated by the basolateral K+ channels Kir4.1/5.1, a capacity that the DCT likely shares with other nephron segments. Thus, next to K+-induced aldosterone secretion, K+ sensing by renal epithelial cells represents a second feedback mechanism to control K+ balance. NCC's role in K+ homeostasis has both physiological and pathophysiological implications. During hypovolemia, NCC activation by the renin-angiotensin system stimulates Na+ reabsorption while preventing K+ secretion. Conversely, NCC inactivation by high dietary K+ intake maximizes kaliuresis and limits Na+ retention, despite high aldosterone levels. NCC activation by a low-K+ diet contributes to salt-sensitive hypertension. K+-induced natriuresis through NCC offers a novel explanation for the antihypertensive effects of a high-K+ diet. A possible role for K+ in chronic kidney disease is also emerging, as epidemiological data reveal associations between higher urinary K+ excretion and improved renal outcomes. This comprehensive review will embed these novel insights on NCC regulation into existing concepts of K+ homeostasis in health and disease.

A role for organ level dynamics in morphogenesis of the C. elegans hermaphrodite distal tip cell.

The morphology of cells in vivo can arise from a variety of mechanisms. In the Caenorhabditis elegans hermaphrodite gonad, the distal tip cell (DTC) elaborates into a complex plexus over a relatively short developmental time period, but the mechanisms underlying this change in cell morphology are not well defined. We correlated the time of DTC elaboration with the L4-to-adult molt, but ruled out a relevant heterochronic pathway as a cue for DTC elaboration. Instead, we found that the timing of gonad elongation and aspects of underlying germline flux influence DTC elaboration. We propose a 'hitch and tow' aspect of organ-level dynamics that contributes to cellular morphogenesis, whereby germline flux drags the flexible DTC cell cortex away from its stationary cell body. More broadly, we speculate that this mechanism may contribute to cell shape changes in other contexts with implications for development and disease.

A mutation in F-actin polymerization factor suppresses the distal arthrogryposis type 5 PIEZO2 pathogenic variant in Caenorhabditis elegans.

The mechanosensitive PIEZO channel family has been linked to over 26 disorders and diseases. Although progress has been made in understanding these channels at the structural and functional levels, the underlying mechanisms of PIEZO-associated diseases remain elusive. In this study, we engineered four PIEZO-based disease models using CRISPR/Cas9 gene editing. We performed an unbiased chemical mutagen-based genetic suppressor screen to identify putative suppressors of a conserved gain-of-function variant pezo-1[R2405P] that in human PIEZO2 causes distal arthrogryposis type 5 (DA5; p. R2718P). Electrophysiological analyses indicate that pezo-1(R2405P) is a gain-of-function allele. Using genomic mapping and whole-genome sequencing approaches, we identified a candidate suppressor allele in the C. elegans gene gex-3. This gene is an ortholog of human NCKAP1 (NCK-associated protein 1), a subunit of the Wiskott-Aldrich syndrome protein (WASP)-verprolin homologous protein (WAVE/SCAR) complex, which regulates F-actin polymerization. Depletion of gex-3 by RNAi, or with the suppressor allele gex-3(av259[L353F]), significantly increased brood size and ovulation rate, as well as alleviating the crushed oocyte phenotype of the pezo-1(R2405P) mutant. Expression of GEX-3 in the soma is required to rescue the brood size defects in pezo-1(R2405P) animals. Actin organization and orientation were disrupted and distorted in the pezo-1 mutants. Mutation of gex-3(L353F) partially alleviated these defects. The identification of gex-3 as a suppressor of the pathogenic variant pezo-1(R2405P) suggests that the PIEZO coordinates with the cytoskeleton regulator to maintain the F-actin network and provides insight into the molecular mechanisms of DA5 and other PIEZO-associated diseases.

Homologous mutations in human ß, embryonic, and perinatal muscle myosins have divergent effects on molecular power generation.

Mutations at a highly conserved homologous residue in three closely related muscle myosins cause three distinct diseases involving muscle defects: R671C in ß-cardiac myosin causes hypertrophic cardiomyopathy, R672C and R672H in embryonic skeletal myosin cause Freeman-Sheldon syndrome, and R674Q in perinatal skeletal myosin causes trismus-pseudocamptodactyly syndrome. It is not known whether their effects at the molecular level are similar to one another or correlate with disease phenotype and severity. To this end, we investigated the effects of the homologous mutations on key factors of molecular power production using recombinantly expressed human ß, embryonic, and perinatal myosin subfragment-1. We found large effects in the developmental myosins but minimal effects in ß myosin, and magnitude of changes correlated partially with clinical severity. The mutations in the developmental myosins dramatically decreased the step size and load-sensitive actin-detachment rate of single molecules measured by optical tweezers, in addition to decreasing overall enzymatic (ATPase) cycle rate. In contrast, the only measured effect of R671C in ß myosin was a larger step size. Our measurements of step size and bound times predicted velocities consistent with those measured in an in vitro motility assay. Finally, molecular dynamics simulations predicted that the arginine to cysteine mutation in embryonic, but not ß, myosin may reduce pre-powerstroke lever arm priming and ADP pocket opening, providing a possible structural mechanism consistent with the experimental observations. This paper presents direct comparisons of homologous mutations in several different myosin isoforms, whose divergent functional effects are a testament to myosin's highly allosteric nature.

Identification of 30 transition fibre proteins in Trypanosoma brucei reveals a complex and dynamic structure.

Transition fibres and distal appendages surround the distal end of mature basal bodies and are essential for ciliogenesis, but only a few of the proteins involved have been identified and functionally characterised. Here, through genome-wide analysis, we have identified 30 transition fibre proteins (TFPs) and mapped their arrangement in the flagellated eukaryote Trypanosoma brucei. We discovered that TFPs are recruited to the mature basal body before and after basal body duplication, with differential expression of five TFPs observed at the assembling new flagellum compared to the existing fixed-length old flagellum. RNAi-mediated depletion of 17 TFPs revealed six TFPs that are necessary for ciliogenesis and a further three TFPs that are necessary for normal flagellum length. We identified nine TFPs that had a detectable orthologue in at least one basal body-forming eukaryotic organism outside of the kinetoplastid parasites. Our work has tripled the number of known transition fibre components, demonstrating that transition fibres are complex and dynamic in their composition throughout the cell cycle, which relates to their essential roles in ciliogenesis and flagellum length regulation.

CHIP inhibits odontoblast differentiation through promoting DLX3 polyubiquitylation and degradation.

Dentin is the major hard tissue of teeth formed by differentiated odontoblasts. How odontoblast differentiation is regulated remains enigmatic. Here, we report that the E3 ubiquitin ligase CHIP is highly expressed in undifferentiated dental mesenchymal cells and downregulated after differentiation of odontoblasts. Ectopic expression of CHIP inhibits odontoblastic differentiation of mouse dental papilla cells, whereas knockdown of endogenous CHIP has opposite effects. Chip (Stub1) knockout mice display increased formation of dentin and enhanced expression of odontoblast differentiation markers. Mechanistically, CHIP interacts with and induces K63 polyubiquitylation of the transcription factor DLX3, leading to its proteasomal degradation. Knockdown of DLX3 reverses the enhanced odontoblastic differentiation caused by knockdown of CHIP. These results suggest that CHIP inhibits odontoblast differentiation by targeting its tooth-specific substrate DLX3. Furthermore, our results indicate that CHIP competes with another E3 ubiquitin ligase, MDM2, that promotes odontoblast differentiation by monoubiquitylating DLX3. Our findings suggest that the two E3 ubiquitin ligases CHIP and MDM2 reciprocally regulate DLX3 activity by catalyzing distinct types of ubiquitylation, and reveal an important mechanism by which differentiation of odontoblasts is delicately regulated by divergent post-translational modifications.

Innate Bacteriostatic Mechanisms Defend the Urinary Tract.

Urinary tract infection (UTI) is the most common type of urogenital disease. UTI affects the urethra, bladder, ureter, and kidney. A total of 13.3% of women, 2.3% of men, and 3.4% of children in the United States will require treatment for UTI. Traditionally, bladder (cystitis) and kidney (pyelonephritis) infections are considered independently. However, both infections induce host defenses that are either shared or coordinated across the urinary tract. Here, we review the chemical and biophysical mechanisms of bacteriostasis, which limit the duration and severity of the illness. Urinary bacteria attempt to overcome each of these defenses, complicating description of the natural history of UTI.

Responses to Pattern-Violating Visual Stimuli Evolve Differently Over Days in Somata and Distal Apical Dendrites.

Scientists have long conjectured that the neocortex learns patterns in sensory data to generate top-down predictions of upcoming stimuli. In line with this conjecture, different responses to pattern-matching vs pattern-violating visual stimuli have been observed in both spiking and somatic calcium imaging data. However, it remains unknown whether these pattern-violation signals are different between the distal apical dendrites, which are heavily targeted by top-down signals, and the somata, where bottom-up information is primarily integrated. Furthermore, it is unknown how responses to pattern-violating stimuli evolve over time as an animal gains more experience with them. Here, we address these unanswered questions by analyzing responses of individual somata and dendritic branches of layer 2/3 and layer 5 pyramidal neurons tracked over multiple days in primary visual cortex of awake, behaving female and male mice. We use sequences of Gabor patches with patterns in their orientations to create pattern-matching and pattern-violating stimuli, and two-photon calcium imaging to record neuronal responses. Many neurons in both layers show large differences between their responses to pattern-matching and pattern-violating stimuli. Interestingly, these responses evolve in opposite directions in the somata and distal apical dendrites, with somata becoming less sensitive to pattern-violating stimuli and distal apical dendrites more sensitive. These differences between the somata and distal apical dendrites may be important for hierarchical computation of sensory predictions and learning, since these two compartments tend to receive bottom-up and top-down information, respectively.

A mechanistic study on the tolerance of PAM distal end mismatch by SpCas9.

The therapeutic application of CRISPR-Cas9 is limited due to its off-target activity. To have a better understanding of this off-target effect, we focused on its mismatch-prone PAM distal end. The off-target activity of SpCas9 depends directly on the nature of mismatches, which in turn results in deviation of the active site of SpCas9 due to structural instability in the RNA-DNA duplex strand. In order to test the hypothesis, we designed an array of mismatched target sites at the PAM distal end and performed in vitro and cell line-based experiments, which showed a strong correlation for Cas9 activity. We found that target sites having multiple mismatches in the 18th to 15th position upstream of the PAM showed no to little activity. For further mechanistic validation, Molecular Dynamics simulations were performed, which revealed that certain mismatches showed elevated root mean square deviation values that can be attributed to conformational instability within the RNA-DNA duplex. Therefore, for successful prediction of the off-target effect of SpCas9, along with complementation-derived energy, the RNA-DNA duplex stability should be taken into account.

Structure and function of distal and subdistal appendages of the mother centriole.

Centrosomes are composed of centrioles surrounded by pericentriolar material. The two centrioles in G1 phase are distinguished by the localization of their appendages in the distal and subdistal regions; the centriole possessing both types of appendage is older and referred to as the mother centriole, whereas the other centriole lacking appendages is the daughter centriole. Both distal and subdistal appendages in vertebrate cells consist of multiple proteins assembled in a hierarchical manner. Distal appendages function mainly in the initial process of ciliogenesis, and subdistal appendages are involved in microtubule anchoring, mitotic spindle regulation and maintenance of ciliary signaling. Mutations in genes encoding components of both appendage types are implicated in ciliopathies and developmental defects. In this Review, we discuss recent advances in knowledge regarding the composition and assembly of centriolar appendages, as well as their roles in development and disease.

Diagnosis of Seronegative and Ultrashort Celiac Disease.

In its conventional form, celiac disease (CeD) is characterized by both positive serology and flat villi in the duodenum, and is well known by gastroenterologists and general practitioners. The aim of this review was to shed light on 2 neglected and not yet well-defined celiac phenotypes, that is, seronegative and ultrashort CeD. Seronegative CeD can be suspected in the presence of flat villi, positive HLA-DQ2 and/or HLA-DQ8, and the absence of CeD antibodies. After ruling out other seronegative enteropathies, the diagnosis can be confirmed by both clinical and histologic improvements after 1 year of a gluten-free diet. Ultrashort CeD is characterized by the finding of flat villi in the duodenal bulb in the absence of mucosal damage in the distal duodenum and with serologic positivity. Data on the prevalence, clinical manifestations, histologic lesions, genetic features, and outcome of seronegative and ultrashort CeD are inconclusive due to the few studies available and the small number of patients diagnosed. Some additional diagnostic tools have been developed recently, such as assessing intestinal transglutaminase 2 deposits, flow cytometry technique, microRNA detection, or proteomic analysis, and they seem to be useful in the identification of complex cases. Further cooperative studies are highly desirable to improve the knowledge of these 2 still-obscure variants of CeD.

Transport activity regulates mitochondrial bioenergetics and biogenesis in renal tubules.

Renal tubules are featured with copious mitochondria and robust transport activity. Mutations in mitochondrial genes cause congenital renal tubulopathies, and changes in transport activity affect mitochondrial morphology, suggesting mitochondrial function and transport activity are tightly coupled. Current methods of using bulk kidney tissues or cultured cells to study mitochondrial bioenergetics are limited. Here, we optimized an extracellular flux analysis (EFA) to study mitochondrial respiration and energy metabolism using microdissected mouse renal tubule segments. EFA detects mitochondrial respiration and glycolysis by measuring oxygen consumption and extracellular acidification rates, respectively. We show that both measurements positively correlate with sample sizes of a few centimeter-length renal tubules. The thick ascending limbs (TALs) and distal convoluted tubules (DCTs) critically utilize glucose/pyruvate as energy substrates, whereas proximal tubules (PTs) are significantly much less so. Acute inhibition of TALs' transport activity by ouabain treatment reduces basal and ATP-linked mitochondrial respiration. Chronic inhibition of transport activity by 2-week furosemide treatment or deletion of with-no-lysine kinase 4 (Wnk4) decreases maximal mitochondrial capacity. In addition, chronic inhibition downregulates mitochondrial DNA mass and mitochondrial length/density in TALs and DCTs. Conversely, gain-of-function Wnk4 mutation increases maximal mitochondrial capacity and mitochondrial length/density without increasing mitochondrial DNA mass. In conclusion, EFA is a sensitive and reliable method to investigate mitochondrial functions in isolated renal tubules. Transport activity tightly regulates mitochondrial bioenergetics and biogenesis to meet the energy demand in renal tubules. The system allows future investigation into whether and how mitochondria contribute to tubular remodeling adapted to changes in transport activity.

Targeting Rap1b signaling cascades with CDNF: Mitigating platelet activation, plasma oxylipins and reperfusion injury in stroke.

Cerebral reperfusion injury in stroke, stemming from interconnected thrombotic and inflammatory signatures, often involves platelet activation, aggregation and its interaction with various immune cells, contributing to microvascular dysfunction. However, the regulatory mechanisms behind this platelet activation and the resulting inflammation are not well understood, complicating the development of effective stroke therapies. Utilizing animal models and platelets from hemorrhagic stroke patients, our research demonstrates that human cerebral dopamine neurotrophic factor (CDNF) acts as an endogenous antagonist, mitigating platelet aggregation and associated neuroinflammation. CDNF moderates mitochondrial membrane potential, reactive oxygen species production, and intracellular calcium in activated platelets by interfering with GTP binding to Rap1b, thereby reducing Rap1b activation and downregulating the Rap1b-MAPK-PLA2 signaling pathway, which decreases release of the pro-inflammatory mediator thromboxane A2. In addition, CDNF reduces the inflammatory response in BV2 microglial cells co-cultured with activated platelets. Consistent with ex vivo findings, subcutaneous administration of CDNF in a rat model of ischemic stroke significantly reduces platelet activation, aggregation, lipid mediator production, infarct volume, and neurological deficits. In summary, our study highlights CDNF as a promising therapeutic target for mitigating platelet-induced inflammation and enhancing recovery in stroke. Harnessing the CDNF pathway may offer a novel therapeutic strategy for stroke intervention.

Dynamic Hybrid Module-Driven NK Cell Stimulation and Release for Tumor Immunotherapy.

Natural killer (NK) cells have become a powerful candidate for adoptive tumor immunotherapy, while their therapeutic efficacy in solid tumors remains unsatisfactory. Here, we developed a hybrid module with an injectable hydrogel and hydroxyapatite (HAp) nanobelts for the controlled delivery of NK cells to enhance the therapy of solid tumors. Surface-functionalized HAp nanobelts modified with agonistic antibodies against NKG2D and 4-1BB and cytokines IL-2 and IL-21 support survival and dynamic activation. Thus, the HAp-modified chitosan (CS) thermos-sensitive hydrogel not only improved the retention of NK cells for more than 20 days in vivo but also increased NK cell function by more than one-fold. The unique architecture of this biomaterial complex protects NK cells from the hostile tumor environment and improves antitumor efficacy. The generation of a transient inflammatory niche for NK cells through a biocompatible hydrogel reservoir may be a conversion pathway to prevent cancer recurrence of resectable tumors.

Disruption of the creb3l1 gene causes defects in caudal fin regeneration and patterning in zebrafish Danio rerio.

BACKGROUND: The gene cAMP-Responsive Element Binding protein 3-like-1 (CREB3L1) has been implicated in bone development in mice, with CREB3L1 knock-out mice exhibiting fragile bones, and in humans, with CREB3L1 mutations linked to osteogenesis imperfecta. However, the mechanism through which Creb3l1 regulates bone development is not fully understood. RESULTS: To probe the role of Creb3l1 in organismal physiology, we used CRISPR-Cas9 genome editing to generate a Danio rerio (zebrafish) model of Creb3l1 deficiency. In contrast to mammalian phenotypes, the Creb3l1 deficient fish do not display abnormalities in osteogenesis, except for a decrease in the bifurcation pattern of caudal fin. Both, skeletal morphology and overall bone density appear normal in the mutant fish. However, the regeneration of caudal fin postamputation is significantly affected, with decreased overall regenerate and mineralized bone area. Moreover, the mutant fish exhibit a severe patterning defect during regeneration, with a significant decrease in bifurcation complexity of the fin rays and distalization of the bifurcation sites. Analysis of genes implicated in bone development showed aberrant patterning of shha and ptch2 in Creb3l1 deficient fish, linking Creb3l1 with Sonic Hedgehog signaling during fin regeneration. CONCLUSIONS: Our results uncover a novel role for Creb3l1 in regulating tissue growth and patterning during regeneration.

Differential requirements for the Eps15 homology domain proteins EHD4 and EHD2 in the regulation of mammalian ciliogenesis.

The endocytic protein EHD1 controls primary ciliogenesis by facilitating fusion of the ciliary vesicle and by removal of CP110 from the mother centriole. EHD3, the closest EHD1 paralog, has a similar regulatory role, but initial evidence suggested that the other two more distal paralogs, EHD2 and EHD4 may be dispensable for ciliogenesis. Herein, we define a novel role for EHD4, but not EHD2, in regulating primary ciliogenesis. To better understand the mechanisms and differential functions of the EHD proteins in ciliogenesis, we first demonstrated a requirement for EHD1 ATP-binding to promote ciliogenesis. We then identified two sequence motifs that are entirely conserved between EH domains of EHD1, EHD3 and EHD4, but display key amino acid differences within the EHD2 EH domain. Substitution of either P446 or E470 in EHD1 with the aligning S451 or W475 residues from EHD2 was sufficient to prevent rescue of ciliogenesis in EHD1-depleted cells upon reintroduction of EHD1. Overall, our data enhance the current understanding of the EHD paralogs in ciliogenesis, demonstrate a need for ATP-binding and identify conserved sequences in the EH domains of EHD1, EHD3 and EHD4 that regulate EHD1 binding to proteins and its ability to rescue ciliogenesis in EHD1-depleted cells.

Development of a Global Metabo-Lipid-Prote-omics Workflow to Compare Healthy Proximal and Distal Colonic Epithelium in Mice.

A multimetabo-lipid-prote-omics workflow was developed to characterize the molecular interplay within proximal (PC) and distal (DC) colonic epithelium of healthy mice. This multiomics data set lays the foundation to better understand the two tissue types and can be used to study, for example, colon-related diseases like colorectal cancer or inflammatory bowel disease. First, the methyl tert-butyl ether extraction method was optimized, so that from a single tissue biopsy >350 reference-matched metabolites, >1850 reference-matched lipids, and >4500 proteins were detected by using targeted and untargeted metabolomics, untargeted lipidomics, and proteomics. Next, each omics-data set was analyzed individually and then merged with the additional omics disciplines to generate a deep understanding of the underlying complex regulatory network within the colon. Our data demonstrates, for example, differences in mucin formation, detected on substrate level as well as on enzyme level, and altered lipid metabolism by the detection of phospholipases hydrolyzing sphingomyelins to ceramides. In conclusion, the combination of the three mass spectrometry-based omics techniques can better entangle the functional and regional differences between PC and DC tissue compared to each single omics technique.