Identification of an IL-1 receptor mutation driving autoinflammation directs IL-1-targeted drug design.

The interleukin 1 (IL-1) pathway signals through IL-1 receptor type 1 (IL-1R1) and emerges as a central mediator for systemic inflammation. Aberrant IL-1 signaling leads to a range of autoinflammatory diseases. Here, we identified a de novo missense variant in IL-1R1 (p.Lys131Glu) in a patient with chronic recurrent multifocal osteomyelitis (CRMO). Patient PBMCs showed strong inflammatory signatures, particularly in monocytes and neutrophils. The p.Lys131Glu substitution affected a critical positively charged amino acid, which disrupted the binding of the antagonist ligand, IL-1Ra, but not IL-1α or IL-1ß. This resulted in unopposed IL-1 signaling. Mice with a homologous mutation exhibited similar hyperinflammation and greater susceptibility to collagen antibody-induced arthritis, accompanied with pathological osteoclastogenesis. Leveraging the biology of the mutation, we designed an IL-1 therapeutic, which traps IL-1ß and IL-1α, but not IL-1Ra. Collectively, this work provides molecular insights and a potential drug for improved potency and specificity in treating IL-1-driven diseases.

Cyclin A participates in the TSO1-MYB3R1 regulatory module to maintain shoot meristem size and fertility in Arabidopsis.

The stem cell pools at the shoot apex and root tip give rise to all the above- and below-ground tissues of a plant. Previous studies in Arabidopsis identified a TSO1-MYB3R1 transcriptional module that controls the number and size of the stem cell pools at the shoot apex and root tip. As TSO1 and MYB3R1 are homologous to components of an animal cell cycle regulatory complex, DREAM, Arabidopsis mutants of TSO1 and MYB3R1 provide valuable tools for investigations into the link between cell cycle regulation and stem cell maintenance in plants. In this study, an Arabidopsis cyclin A gene, CYCA3;4, was identified as a member of the TSO1-MYB3R1 regulatory module and cyca3;4 mutations suppressed the tso1-1 mutant phenotype specifically in the shoot. The work reveals how the TSO1-MYB3R1 module is integrated with the cell cycle machinery to control cell division at the shoot meristem.

Unveiling the role of PIK3R1 in cancer: A comprehensive review of regulatory signaling and therapeutic implications.

Phosphoinositide 3-kinase (PI3K) is responsible for phosphorylating phosphoinositides to generate secondary signaling molecules crucial for regulating various cellular processes, including cell growth, survival, and metabolism. The PI3K is a heterodimeric enzyme complex comprising of a catalytic subunit (p110α, p110ß, or p110δ) and a regulatory subunit (p85). The binding of the regulatory subunit, p85, with the catalytic subunit, p110, forms an integral component of the PI3K enzyme. PIK3R1 (phosphoinositide-3-kinase regulatory subunit 1) belongs to class IA of the PI3K family. PIK3R1 exhibits structural complexity due to alternative splicing, giving rise to distinct isoforms, prominently p85α and p55α. While the primary p85α isoform comprises multiple domains, including Src homology 3 (SH3) domains, a Breakpoint Cluster Region Homology (BH) domain, and Src homology 2 (SH2) domains (iSH2 and nSH2), the shorter isoform, p55α, lacks certain domains present in p85α. In this review, we will highlight the intricate regulatory mechanisms governing PI3K signaling along with the impact of PIK3R1 alterations on cellular processes. We will further delve into the clinical significance of PIK3R1 mutations in various cancer types and their implications for prognosis and treatment outcomes. Additionally, we will discuss the evolving landscape of targeted therapies aimed at modulating PI3K-associated pathways. Overall, this review will provide insights into the dynamic interplay of PIK3R1 in cancer, fostering advancements in precision medicine and the development of targeted interventions.

Ectodomain shedding of PLA2R1 is mediated by the metalloproteases ADAM10 and ADAM17.

Phospholipase A2 receptor 1 (PLA2R1) is a 180-kDa transmembrane protein that plays a role in inflammation and cancer and is the major autoantigen in membranous nephropathy, a rare but severe autoimmune kidney disease. A soluble form of PLA2R1 has been detected in mouse and human serum. It is likely produced by proteolytic shedding of membrane-bound PLA2R1 but the mechanism is unknown. Here, we show that human PLA2R1 is cleaved by A Disintegrin And Metalloprotease 10 (ADAM10) and ADAM17 in HEK293 cells, mouse embryonic fibroblasts, and human podocytes. By combining site-directed mutagenesis and sequencing, we determined the exact cleavage site within the extracellular juxtamembrane stalk of human PLA2R1. Orthologs and paralogs of PLA2R1 are also shed. By using pharmacological inhibitors and genetic approaches with RNA interference and knock-out cellular models, we identified a major role of ADAM10 in the constitutive shedding of PLA2R1 and a dual role of ADAM10 and ADAM17 in the stimulated shedding. We did not observe evidence for cleavage by ß- or γ-secretase, suggesting that PLA2R1 may not be a substrate for regulated intramembrane proteolysis. PLA2R1 shedding occurs constitutively and can be triggered by the calcium ionophore ionomycin, the protein kinase C activator PMA, cytokines, and lipopolysaccharides, in vitro and in vivo. Altogether, our results show that PLA2R1 is a novel substrate for ADAM10 and ADAM17, producing a soluble form that is increased in inflammatory conditions and likely exerts various functions in physiological and pathophysiological conditions including inflammation, cancer, and membranous nephropathy.

A novel three-layer module BoMYB1R1-BoMYB4b/BoMIEL1-BoDFR1 regulates anthocyanin accumulation in kale.

Anthocyanin is an important pigment responsible for plant coloration and beneficial to human health. Kale (Brassica oleracea var. acephala), a primary cool-season flowers and vegetables, is an ideal material to study anthocyanin biosynthesis and regulation mechanisms due to its anthocyanin-rich leaves. However, the underlying molecular mechanism of anthocyanin accumulation in kale remains poorly understood. Previously, we demonstrated that BoDFR1 is a key gene controlling anthocyanin biosynthesis in kale. Here, we discovered a 369-bp InDel variation in the BoDFR1 promoter between the two kale inbred lines with different pink coloration, which resulted in reduced transcriptional activity of the BoDFR1 gene in the light-pink line. With the 369-bp insertion as a bait, an R2R3-MYB repressor BoMYB4b was identified using the yeast one-hybrid screening. Knockdown of the BoMYB4b gene led to increased BoDFR1 expression and anthocyanin accumulation. An E3 ubiquitin ligase, BoMIEL1, was found to mediate the degradation of BoMYB4b, thereby promoting anthocyanin biosynthesis. Furthermore, the expression level of BoMYB4b was significantly reduced by light signals, which was attributed to the direct repression of the light-signaling factor BoMYB1R1 on the BoMYB4b promoter. Our study revealed that a novel regulatory module comprising BoMYB1R1, BoMIEL1, BoMYB4b, and BoDFR1 finely regulates anthocyanin accumulation in kale. The findings aim to establish a scientific foundation for genetic improvement of leaf color traits in kale, meanwhile, providing a reference for plant coloration studies.

Loss of Calpain 3 dysregulates store-operated calcium entry and its exercise response in mice.

Limb-Girdle Muscular Dystrophy R1/2A (LGMD R1/2A) is caused by mutations in the CAPN3 gene encoding Calpain 3, a skeletal-muscle specific, Ca2+-dependent protease. Localization of Calpain 3 within the triad suggests it contributes to Ca2+ homeostasis. Through live-cell Ca2+ measurements, muscle mechanics, immunofluorescence, and electron microscopy (EM) in Capn3 deficient (C3KO) and wild-type (WT) mice, we determined whether loss of Calpain 3 altered Store-Operated Calcium Entry (SOCE) activity. Direct Ca2+ influx measurements revealed loss of Capn3 elicits elevated resting SOCE and increased resting cytosolic Ca2+, supported by high incidence of calcium entry units (CEUs) observed by EM. C3KO and WT mice were subjected to a single bout of treadmill running to elicit SOCE. Within 1HR post-treadmill running, C3KO mice exhibited diminished force production in extensor digitorum longus muscles and a greater decay of Ca2+ transients in flexor digitorum brevis muscle fibers during repetitive stimulation. Striking evidence for impaired exercise-induced SOCE activation in C3KO mice included poor colocalization of key SOCE proteins, stromal-interacting molecule 1 (STIM1) and ORAI1, combined with disappearance of CEUs in C3KO muscles. These results demonstrate that Calpain 3 is a key regulator of SOCE in skeletal muscle and identify SOCE dysregulation as a contributing factor to LGMD R1/2A pathology.

Notoginsenoside R1 targets PPAR-γ to inhibit hepatic stellate cell activation and ameliorates liver fibrosis.

BACKGROUND: Hepatic fibrosis, a common pathological process that occurs in end-stage liver diseases, is a serious public health problem and lacks effective therapy. Notoginsenoside R1 (NR1) is a small molecule derived from the traditional Chinese medicine Sanqi, exhibiting great potential in treating diverse metabolie disorders. Here we aimed to enquired the role of NR1 in liver fibrosis and its underlying mechanism in hepatoprotective effects. METHODS: We investigated the anti-fibrosis effect of NR1 using CCl4-induced mouse mode of liver fibrosis as well as TGF-ß1-activated JS-1, LX-2 cells and primary hepatic stellate cell. Cell samples treated by NR1 were collected for transcriptomic profiling analysis. PPAR-γ mediated TGF-ß1/Smads signaling was examined using PPAR-γ selective inhibitors and agonists intervention, immunofluorescence staining and western blot analysis. Additionally, we designed and studied the binding of NR1 to PPAR-γ using molecular docking. RESULTS: NR1 obviously attenuated liver histological damage, reduced serum ALT, AST levels, and decreased liver fibrogenesis markers in mouse mode. Mechanistically, NR1 elevated PPAR-γ and decreased TGF-ß1, p-Smad2/3 expression. The TGF-ß1/Smads signaling pathway and fibrotic phenotype were altered in JS-1 cells after using PPAR-γ selective inhibitors and agonists respectively, confirming PPAR-γ played a pivotal protection role inNR1 treating liver fibrosis. Further molecular docking indicated NR1 had a strong binding tendency to PPAR-γ with minimum free energy. CONCLUSIONS: NR1 attenuates hepatic stellate cell activation and hepatic fibrosis by elevating PPAR-γ to inhibit TGF-ß1/Smads signalling. NR1 may be a potential candidate compound for reliving liver fibrosis.

CENPA knockdown restrains cell progression and tumor growth in breast cancer by reducing PLA2R1 promoter methylation and modulating PLA2R1/HHEX axis.

BACKGROUND: Breast cancer is a lethal malignancy affecting females worldwide. It has been reported that upregulated centromere protein A (CENPA) expression might indicate unfortunate prognosis and can function as a prognostic biomarker in breast cancer. This study aimed to investigate the accurate roles and downstream mechanisms of CENPA in breast cancer progression. METHODS: CENPA protein levels in breast cancer tissues and cell lines were analyzed by Western blot and immunohistochemistry assays. We used gain/loss-of-function experiments to determine the potential effects of CENPA and phospholipase A2 receptor (PLA2R1) on breast cancer cell proliferation, migration, and apoptosis. Co-IP assay was employed to validate the possible interaction between CENPA and DNA methyltransferase 1 (DNMT1), as well as PLA2R1 and hematopoietically expressed homeobox (HHEX). PLA2R1 promoter methylation was determined using methylation-specific PCR assay. The biological capabilities of CENPA/PLA2R1/HHEX axis in breast cancer cells was determined by rescue experiments. In addition, CENPA-silenced MCF-7 cells were injected into mice, followed by measurement of tumor growth. RESULTS: CENPA level was prominently elevated in breast cancer tissues and cell lines. Interestingly, CENPA knockdown and PLA2R1 overexpression both restrained breast cancer cell proliferation and migration, and enhanced apoptosis. On the contrary, CENPA overexpression displayed the opposite results. Moreover, CENPA reduced PLA2R1 expression through promoting DNMT1-mediated PLA2R1 promoter methylation. PLA2R1 overexpression could effectively abrogate CENPA overexpression-mediated augment of breast cancer cell progression. Furthermore, PLA2R1 interacted with HHEX and promoted HHEX expression. PLA2R1 knockdown increased the rate of breast cancer cell proliferation and migration but restrained apoptosis, which was abrogated by HHEX overexpression. In addition, CENPA silencing suppressed tumor growth in vivo. CONCLUSION: CENPA knockdown restrained breast cancer cell proliferation and migration and attenuated tumor growth in vivo through reducing PLA2R1 promoter methylation and increasing PLA2R1 and HHEX expression. We may provide a promising prognostic biomarker and novel therapeutic target for breast cancer.

LPA suppresses T cell function by altering the cytoskeleton and disrupting immune synapse formation.

Cancer and chronic infections often increase levels of the bioactive lipid, lysophosphatidic acid (LPA), that we have demonstrated acts as an inhibitory ligand upon binding LPAR5 on CD8 T cells, suppressing cytotoxic activity and tumor control. This study, using human and mouse primary T lymphocytes, reveals how LPA disrupts antigen-specific CD8 T cell:target cell immune synapse (IS) formation and T cell function via competing for cytoskeletal regulation. Specifically, we find upon antigen-specific T cell:target cell formation, IP3R1 localizes to the IS by a process dependent on mDia1 and actin and microtubule polymerization. LPA not only inhibited IP3R1 from reaching the IS but also altered T cell receptor (TCR)­induced localization of RhoA and mDia1 impairing F-actin accumulation and altering the tubulin code. Consequently, LPA impeded calcium store release and IS-directed cytokine secretion. Thus, targeting LPA signaling in chronic inflammatory conditions may rescue T cell function and promote antiviral and antitumor immunity.

The protein phosphatase PP6 promotes RIPK1-dependent PANoptosis.

BACKGROUND: The innate immune system serves as the first line of host defense. Transforming growth factor-ß-activated kinase 1 (TAK1) is a key regulator of innate immunity, cell survival, and cellular homeostasis. Because of its importance in immunity, several pathogens have evolved to carry TAK1 inhibitors. In response, hosts have evolved to sense TAK1 inhibition and induce robust lytic cell death, PANoptosis, mediated by the RIPK1-PANoptosome. PANoptosis is a unique innate immune inflammatory lytic cell death pathway initiated by an innate immune sensor and driven by caspases and RIPKs. While PANoptosis can be beneficial to clear pathogens, excess activation is linked to pathology. Therefore, understanding the molecular mechanisms regulating TAK1 inhibitor (TAK1i)-induced PANoptosis is central to our understanding of RIPK1 in health and disease. RESULTS: In this study, by analyzing results from a cell death-based CRISPR screen, we identified protein phosphatase 6 (PP6) holoenzyme components as regulators of TAK1i-induced PANoptosis. Loss of the PP6 enzymatic component, PPP6C, significantly reduced TAK1i-induced PANoptosis. Additionally, the PP6 regulatory subunits PPP6R1, PPP6R2, and PPP6R3 had redundant roles in regulating TAK1i-induced PANoptosis, and their combined depletion was required to block TAK1i-induced cell death. Mechanistically, PPP6C and its regulatory subunits promoted the pro-death S166 auto-phosphorylation of RIPK1 and led to a reduction in the pro-survival S321 phosphorylation. CONCLUSIONS: Overall, our findings demonstrate a key requirement for the phosphatase PP6 complex in the activation of TAK1i-induced, RIPK1-dependent PANoptosis, suggesting this complex could be therapeutically targeted in inflammatory conditions.

Effectiveness of a novel rat model of off-target PLA2R1 knockout to renal impairment.

Phospholipase A2 receptor 1 (PLA2R1) plays a crucial role in various diseases, including membranous nephropathy. However, the precise implications of PLA2R1 deficiency remain poorly understood. In this study, we created PLA2R1 knockout rats to explore potential consequences resulting from the loss of the PLA2R1 gene. Unexpectedly, our PLA2R1 knockout rats exhibited symptoms resembling those of chronic kidney disease after an 8-week observation period. Notably, several rats developed persistent proteinuria, a hallmark of renal dysfunction. Immunohistochemical and immunofluorescence analyses revealed insignificant glomerular fibrosis, reduced podocyte count, and augmented glomerular expression of complement C3 (C3) compared to immunoglobin A (IgA) and immunoglobin G(IgG) in the rat model. These findings suggest that the loss of PLA2R1 may contribute to the pathogenesis of membranous nephropathy and related conditions. Our knockout rat model provides a valuable tool for investigating the underlying pathology of PLA2R1-associated diseases, and may facilitate the development of targeted therapies for membranous nephropathy and other related disorders.

Interim analysis: Open-label extension study of leniolisib for patients with APDS.

BACKGROUND: Activated phosphoinositide 3-kinase delta (PI3Kδ) syndrome (APDS; or p110δ-activating mutations causing senescent T cells, lymphadenopathy, and immunodeficiency) is an inborn error of immunity caused by PI3Kδ hyperactivity. Resultant immune deficiency and dysregulation lead to recurrent sinopulmonary infections, herpes viremia, autoimmunity, and lymphoproliferation. OBJECTIVE: Leniolisib, a selective PI3Kδ inhibitor, demonstrated favorable impact on immune cell subsets and lymphoproliferation over placebo in patients with APDS over 12 weeks. Here, we report results from an interim analysis of an ongoing open-label, single-arm extension study. METHODS: Patients with APDS aged 12 years or older who completed NCT02435173 or had previous exposure to PI3Kδ inhibitors were eligible. The primary end point was safety, assessed via investigator-reported adverse events (AEs) and clinical/laboratory evaluations. Secondary and exploratory end points included health-related quality of life, inflammatory markers, frequency of infections, and lymphoproliferation. RESULTS: Between September 2016 and August 2021, 37 patients (median age, 20 years; 42.3% female) were enrolled. Of these 37 patients, 26, 9, and 2 patients had previously received leniolisib, placebo, or other PI3Kδ inhibitors, respectively. At the data cutoff date (December 13, 2021), median leniolisib exposure was 102 weeks. Overall, 32 patients (87%) experienced an AE. Most AEs were grades 1 to 3; none were grade 4. One patient with severe baseline comorbidities experienced a grade 5 AE, determined as unrelated to leniolisib treatment. While on leniolisib, patients had reduced annualized infection rates (P = .004), and reductions in immunoglobulin replacement therapy occurred in 10 of 27 patients. Other observations include reduced lymphadenopathy and splenomegaly, improved cytopenias, and normalized lymphocyte subsets. CONCLUSIONS: Leniolisib was well tolerated and maintained durable outcomes with up to 5 years of exposure in 37 patients with APDS. CLINICALTRIALS: gov identifier: NCT02859727.

Target Site of Prepulse Inhibition of the Trigeminal Blink Reflex in Humans.

Despite the clinical significance of prepulse inhibition (PPI), the mechanisms are not well understood. Herein, we present our investigation of PPI in the R1 component of electrically induced blink reflexes. The effect of a prepulse was explored with varying prepulse test intervals (PTIs) of 20-600 ms in 4 females and 12 males. Prepulse-test combinations included the following: stimulation of the supraorbital nerve (SON)-SON [Experiment (Exp) 1], sound-sound (Exp 2), the axon of the facial nerve-SON (Exp 3), sound-SON (Exp 4), and SON-SON with a long trial-trial interval (Exp 5). Results showed that (1) leading weak SON stimulation reduced SON-induced ipsilateral R1 with a maximum effect at a PTI of 140 ms, (2) the sound-sound paradigm resulted in a U-shaped inhibition time course of the auditory startle reflex (ASR) peaking at 140 ms PTI, (3) facial nerve stimulation showed only a weak effect on R1, (4) a weak sound prepulse facilitated R1 but strongly inhibited SON-induced late blink reflexes (LateRs) with a similar U-shaped curve, and (5) LateR in Exp 5 was almost completely absent at PTIs >80 ms. These results indicate that the principal sensory nucleus is responsible for R1 PPI. Inhibition of ASR or LateR occurs at a point in the startle reflex circuit where auditory and somatosensory signals converge. Although the two inhibitions are different in location, their similar time courses suggest similar neural mechanisms. As R1 has a simple circuit and is stable, R1 PPI helps to clarify PPI mechanisms.SIGNIFICANCE STATEMENT Prepulse inhibition (PPI) is a phenomenon in which the startle response induced by a startle stimulus is suppressed by a preceding nonstartle stimulus. This study demonstrated that the R1 component of the trigeminal blink reflex shows clear PPI despite R1 generation within a circuit consisting of the trigeminal and facial nuclei, without startle reflex circuit involvement. Thus, PPI is not specific to the startle reflex. In addition, PPI of R1, the auditory startle reflex, and the trigeminal late blink reflex showed similar time courses in response to the prepulse test interval, suggesting similar mechanisms regardless of inhibition site. R1 PPI, in conjunction with other paradigms with different prepulse-test combinations, would increase understanding of the underlying mechanisms.

Deep learning identified genetic variants for COVID-19-related mortality among 28,097 affected cases in UK Biobank.

Analysis of host genetic components provides insights into the susceptibility and response to viral infection such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19). To reveal genetic determinants of susceptibility to COVID-19 related mortality, we train a deep learning model to identify groups of genetic variants and their interactions that contribute to the COVID-19 related mortality risk using the UK Biobank data (28,097 affected cases and 1656 deaths). We refer to such groups of variants as super variants. We identify 15 super variants with various levels of significance as susceptibility loci for COVID-19 mortality. Specifically, we identify a super variant (odds ratio [OR] = 1.594, p = 5.47 × 10-9 ) on Chromosome 7 that consists of the minor allele of rs76398985, rs6943608, rs2052130, 7:150989011_CT_C, rs118033050, and rs12540488. We also discover a super variant (OR = 1.353, p = 2.87 × 10-8 ) on Chromosome 5 that contains rs12517344, rs72733036, rs190052994, rs34723029, rs72734818, 5:9305797_GTA_G, and rs180899355.

Whole-brain mapping of increased manganese levels in welders and its association with exposure and motor function.

Although manganese (Mn) is a trace metal essential for humans, chronic exposure to Mn can cause accumulation of this metal ion in the brain leading to an increased risk of neurological and neurobehavioral health effects. This is a concern for welders exposed to Mn through welding fumes. While brain Mn accumulation in occupational settings has mostly been reported in the basal ganglia, several imaging studies also revealed elevated Mn in other brain areas. Since Mn functions as a magnetic resonance imaging (MRI) T1 contrast agent, we developed a whole-brain MRI approach to map in vivo Mn deposition differences in the brains of non-exposed factory controls and exposed welders. This is a cross-sectional analysis of 23 non-exposed factory controls and 36 exposed full-time welders from the same truck manufacturer. We collected high-resolution 3D MRIs of brain anatomy and R1 relaxation maps to identify regional differences using voxel-based quantification (VBQ) and statistical parametric mapping. Furthermore, we investigated the associations between excess Mn deposition and neuropsychological and motor test performance. Our results indicate that: (1) Using whole-brain MRI relaxometry methods we can generate excess Mn deposition maps in vivo, (2) excess Mn accumulation due to occupational exposure occurs beyond the basal ganglia in cortical areas associated with motor and cognitive functions, (3) Mn likely diffuses along white matter tracts in the brain, and (4) Mn deposition in specific brain regions is associated with exposure (cerebellum and frontal cortex) and motor metrics (cerebellum and hippocampus).

Vitamin D promotes the folate transport and metabolism in zebrafish (Danio rerio).

Vitamin D (VD) is a fat-soluble sterol that possesses a wide range of physiological functions. The present study aimed to evaluate the effects of VD on folate metabolism in zebrafish and further investigated the underlying mechanism. Wild-type (WT) zebrafish were fed with a diet containing 0 IU/kg VD3 or 800 IU/kg VD3 for 3 wk. Meanwhile, cyp2r1 mutant zebrafish with impaired VD metabolism was used as another model of VD deficiency. Our results showed that VD deficiency in zebrafish suppressed the gene expression of folate transporters, including reduced folate carrier (RFC) and proton-coupled folate transporter (PCFT) in the intestine. Moreover, VD influenced the gene expression of several enzymes related to cellular folate metabolism in the intestine and liver of zebrafish. Importantly, VD-deficient zebrafish contained a remarkably lower level of folate content in the liver. Notably, VD was incapable of altering folate metabolism in zebrafish when gut microbiota was depleted by antibiotic treatment. Further studies proved that gut commensals from VD-deficient fish displayed a lower capacity to produce folate than those from WT fish. Our study revealed the potential correlation between VD and folate metabolism in zebrafish, and gut microbiota played a key role in VD-regulated folate metabolism in zebrafish.NEW & NOTEWORTHY Our study has identified that VD influences intestinal uptake and transport of folate in zebrafish while also altering hepatic folate metabolism and storage. Interestingly, the regulatory effects of VD on folate transport and metabolism diminished after the gut flora was interrupted by antibiotic treatment, suggesting that the regulatory effects of VD on folate metabolism in zebrafish are most likely dependent on the intestinal flora.

N6-methyladenosine-modified circPLPP4 sustains cisplatin resistance in ovarian cancer cells via PIK3R1 upregulation.

BACKGROUND: Cisplatin (CDDP) is the first-line chemotherapeutic strategy to treat patients with ovarian cancer (OC). The development of CDDP resistance remains an unsurmountable obstacle in OC treatment and frequently induces tumor recurrence. Circular RNAs (circRNAs) are noncoding RNAs with important functions in cancer progression. Whether circRNAs function in CDDP resistance of OC is unclear. METHODS: Platinum-resistant circRNAs were screened via circRNA deep sequencing and examined using in situ hybridization (ISH) in OC. The role of circPLPP4 in CDDP resistance was assessed by clone formation and Annexin V assays in vitro, and by OC patient-derived xenografts and intraperitoneal tumor models in vivo. The mechanism underlying circPLPP4-mediated activation of miR-136/PIK3R1 signaling was examined by luciferase reporter assay, RNA pull-down, RIP, MeRIP and ISH. RESULTS: circPLPP4 was remarkably upregulated in platinum resistant OC. circPLPP4 overexpression significantly enhanced, whereas circPLPP4 silencing reduced, OC cell chemoresistance. Mechanistically, circPLPP4 acts as a microRNA sponge to sequester miR-136, thus competitively upregulating PIK3R1 expression and conferring CDDP resistance. The increased circPLPP4 level in CDDP-resistant cells was caused by increased RNA stability, mediated by increased N6-methyladenosine (m6A) modification of circPLPP4. In vivo delivery of an antisense oligonucleotide targeting circPLPP4 significantly enhanced CDDP efficacy in a tumor model. CONCLUSIONS: Our study reveals a plausible mechanism by which the m6A -induced circPLPP4/ miR-136/ PIK3R1 axis mediated CDDP resistance in OC, suggesting that circPLPP4 may serve as a promising therapeutic target against CDDP resistant OC. A circPLPP4-targeted drug in combination with CDDP might represent a rational regimen in OC.

Pitfalls in measurements of R1 relaxation rates of protein backbone 15N nuclei.

The dynamics of the backbone and side-chains of protein are routinely studied by interpreting experimentally determined 15N spin relaxation rates. R1(15N), the longitudinal relaxation rate, reports on fast motions and encodes, together with the transverse relaxation R2, structural information about the shape of the molecule and the orientation of the amide bond vectors in the internal diffusion frame. Determining error-free 15N longitudinal relaxation rates remains a challenge for small, disordered, and medium-sized proteins. Here, we show that mono-exponential fitting is sufficient, with no statistical preference for bi-exponential fitting up to 800 MHz. A detailed comparison of the TROSY and HSQC techniques at medium and high fields showed no statistically significant differences. The least error-prone DD/CSA interference removal technique is the selective inversion of amide signals while avoiding water resonance. The exchange of amide with solvent deuterons appears to affect the rate R1 of solvent-exposed amides in all fields tested and in each DD/CSA interference removal technique in a statistically significant manner. In summary, the most accurate R1(15N) rates in proteins are achieved by selective amide inversion, without the addition of D2O. Importantly, at high magnetic fields stronger than 800 MHz, when non-mono-exponential decay is involved, it is advisable to consider elimination of the shortest delays (typically up to 0.32 s) or bi-exponential fitting.

GHS-R1a deficiency protects against lipopolysaccharide-induced spatial memory impairment in mice.

Neuroinflammation has been implicated in cognitive deficits of neurological and neurodegenerative diseases. There is abundant evidence that the application of ghrelin, an orexigenic hormone regulating appetite and energy balance, abrogates neuroinflammation and rescues associated memory impairment. However, the underlying mechanism is uncertain. In this study, we find that both intraperitoneal (i.p.) and intracerebroventricular (i.c.v.) administration of lipopolysaccharide (LPS) impairs spatial memory in mice. LPS treatment causes neuroinflammation and microglial activation in the hippocampus. Ghsr1a deletion suppresses LPS-induced microglial activation and neuroinflammation, and rescued LPS-induced memory impairment. Our findings thus suggest that GHS-R1a signaling may promote microglial immunoactivation and contribute to LPS-induced neuroinflammation. GHS-R1a may be a new therapeutic target for cognitive dysfunction associated with inflammatory conditions.

Comparative genomic analysis of PIK3R1-mutated and wild-type breast cancers.

PURPOSE: The PIK3R1 gene encodes the regulatory subunit-p85a-of the PI3K signaling complex. Prior studies have found that pathogenic somatic alterations in PIK3R1 are enriched in human breast cancers but the genomic landscape of breast cancer patients harboring PIK3R1 mutations has not been extensively characterized. METHODS: We retrospectively analyzed 6,009 patient records that underwent next-generation sequencing (NGS) using the Tempus xT solid tumor assay. All patients had breast cancer with known HER2 (+/-) and hormone receptor (HR; +/-) status and were classified according to the presence of PIK3R1 mutations including short variants and copy number alterations. RESULTS: The frequency of PIK3R1 mutations varied according to subtype: 6% in triple negative (TNBC, 89/1,475), 2% in HER2-/HR+ (80/3,893) and 2.3% in HER2+ (15/641) (p < 0.001). Co-mutations in PTEN, TP53 and NF1 were significantly enriched, co-mutations in PIK3CA were significantly less prevalent, and tumor mutational burden was significantly higher in PIK3R1-mutated HER2- samples relative to PIK3R1 wild-type. At the transcriptional-level, PIK3R1 RNA expression in HER2- disease was significantly higher in PIK3R1-mutated (excluding copy number loss) samples, regardless of subtype. CONCLUSION: This is the largest investigation of the PIK3R1 mutational landscape in breast cancer patients (n = 6,009). PIK3R1 mutations were more common in triple-negative breast cancer (~ 6%) than in HER2 + or HER2-/HR + disease (approximately 2%). While alterations in the PI3K/AKT pathway are often actionable in HER2-/HR + breast cancer, our study suggests that PIK3R1 could be an important target in TNBC as well.