Nobiletin promotes lipolysis of white adipose tissue in a circadian clock-dependent manner.

Nobiletin has been reported to protect against obesity-related metabolic disorders by enhancing the circadian rhythm; however its effects on lipid metabolism in adipose tissue are unclear. In this study, mice were fed with high-fat diet (HFD) for four weeks firstly and gavaged with 50 or 200 mg/kg bodyweight/day nobiletin at Zeitgeber time (ZT) 4 for another four weeks while still receiving HFD. At the end of the 8-week experimental period, the mice were sacrificed at ZT4 or ZT8 on the same day. Mature 3T3-L1 adipocytes were treated with nobiletin in the presence or absence of siBmal1, siRora, siRorc, SR8278 or SR9009. Nobiletin reduced the weight of white adipose tissue (WAT) and the size of adipocytes in WAT. At ZT4, nobiletin decreased the TG, TC and LDL-c levels and increased serum FFA level and glucose tolerance. Nobiletin triggered the lipolysis of mesenteric and epididymal WAT at both ZT4 and ZT16. Nobiletin increased the level of RORγ at ZT16, that of BMAL1 and PPARγ at ZT4, and that of ATGL at both ZT4 and ZT16. Nobiletin increased lipolysis and ATGL levels in 3T3-L1 adipocytes in Bmal1- or Rora/c- dependent manner. Dual luciferase assay indicated that nobiletin enhanced the transcriptional activation of RORα/γ on Atgl promoter and decreased the repression of RORα/γ on PPARγ-binding PPRE. Promoter deletion analysis indicated that nobiletin inhibited the suppression of PPARγ-mediated Atgl transcription by RORα/γ. Taken together, nobiletin elevated lipolysis in WAT by increasing ATGL levels through activating the transcriptional activity of RORα/γ and decreasing the repression of RORα/γ on PPARγ-binding PPRE.

Aluminum causes irreversible damage to the development of hippocampal neurons by regulating m6A RNA methylation.

The underlying mechanism of the aluminum (Al) on neurotoxicity remains unclear. We explored whether the impairment of hippocampal neurons induced by developmental Al exposure was associated with the m6A RNA modification in mice. In this study, the pregnant female mice were administered 4 mg/mL aluminum-lactate from gestational day (GD) 6 to postnatal day (PND) 21. On PND 21, 10 offsprings per group were euthanized by exsanguination from the abdominal aorta after deep anesthetization. The other offsprings which treated with aluminum-lactate on maternal generation were divided into two groups and given 0 (PND60a) and 4 mg/mL (PND60b) aluminum-lactate in their drinking water until PND 60. Significant neuronal injuries of hippocampus as well as a reduction in the m6A RNA modification and the expression of methylase were observed at PND 21 and PND 60a mice. The results indicated that Al-induced developmental neurotoxicity could persist into adulthood despite no sustained Al accumulation. m6A RNA modification had a crucial role in developmental neurotoxicity induced by Al. In addition, Al exposure during the embryonic to adult stages can cause more severe nerve damage and decline of m6A RNA modification. Collectively, these results suggest that the mechanism underlying Al-induced neurotoxicity appears to involve m6A RNA modification.

Nobiletin Protects Against Alcoholic Liver Disease in Mice via the BMAL1-AKT-Lipogenesis Pathway.

SCOPE: Alcoholic liver disease (ALD) is a global public health concern. Nobiletin, a polymethoxyflavone abundant in citrus fruits, enhances circadian rhythms and ameliorates diet-induced hepatic steatosis, but its influences on ALD are unknown. This study investigates the role of brain and muscle Arnt-like protein-1 (Bmal1), a key regulator of the circadian clock, in nobiletin-alleviated ALD. METHODS AND RESULTS: This study uses chronic ethanol feeding plus an ethanol binge to establish ALD models in Bmal1flox/flox and Bmal1 liver-specific knockout (Bmal1LKO) mice. Nobiletin mitigates ethanol-induced liver injury (alanine aminotransferase [ALT]), glucose intolerance, hepatic apoptosis, and lipid deposition (triglyceride [TG], total cholesterol [TC]) in Bmal1flox/flox mice. Nobiletin fails to modulated liver injury (ALT, aspartate aminotransferase [AST]), apoptosis, and TG accumulation in Bmal1LKO mice. The expression of lipogenic genes (acetyl-CoA carboxylase alpha [Acaca], fatty acid synthase [Fasn]) and fatty acid oxidative genes (carnitine pamitoyltransferase [Cpt1a], cytochrome P450, family 4, subfamily a, polypeptide 10 [Cyp4a10], and cytochrome P450, family4, subfamily a, polypeptide 14 [Cyp4a14]) is inhibited, and the expression of proapoptotic genes (Bcl2 inteacting mediator of cell death [Bim]) is enhanced by ethanol in Bmal1flox/flox mice. Nobiletin antagonizes the expression of these genes in Bmal1flox/flox mice and not in Bmal1LKO mice. Nobiletin activates protein kinase B (PKB, also known as AKT) phosphorylation, increases the levels of the carbohydrate response element binding protein (ChREBP), ACC1, and FASN, and reduces the level of sterol-regulatory element binding protein 1 (SREBP1) and phosphorylation of ACC1 in a Bmal1-dependent manner. CONCLUSION: Nobiletin alleviates ALD by increasing the expression of genes involved in fatty acid oxidation by increasing AKT phosphorylation and lipogenesis in a Bmal1-dependent manner.

Nutrition strategies to control post-weaning diarrhea of piglets: From the perspective of feeds.

Post-weaning diarrhea (PWD) is a globally significant threat to the swine industry. Historically, antibiotics as well as high doses of zinc oxide and copper sulfate have been commonly used to control PWD. However, the development of bacterial resistance and environmental pollution have created an interest in alternative strategies. In recent years, the research surrounding these alternative strategies and the mechanisms of piglet diarrhea has been continually updated. Mechanically, diarrhea in piglets is a result of an imbalance in intestinal fluid and electrolyte absorption and secretion. In general, enterotoxigenic Escherichia coli (ETEC) and diarrheal viruses are known to cause an imbalance in the absorption and secretion of intestinal fluids and electrolytes in piglets, resulting in diarrhea when Cl- secretion-driven fluid secretion surpasses absorptive capacity. From a perspective of feedstuffs, factors that contribute to imbalances in fluid absorption and secretion in the intestines of weaned piglets include high levels of crude protein (CP), stimulation by certain antigenic proteins, high acid-binding capacity (ABC), and contamination with deoxynivalenol (DON) in the diet. In response, efforts to reduce CP levels in diets, select feedstuffs with lower ABC values, and process feedstuffs using physical, chemical, and biological approaches are important strategies for alleviating PWD in piglets. Additionally, the diet supplementation with additives such as vitamins and natural products can also play a role in reducing the diarrhea incidence in weaned piglets. Here, we examine the mechanisms of absorption and secretion of intestinal fluids and electrolytes in piglets, summarize nutritional strategies to control PWD in piglets from the perspective of feeds, and provide new insights towards future research directions.

Network-based elucidation of colon cancer drug resistance mechanisms by phosphoproteomic time-series analysis.

Aberrant signaling pathway activity is a hallmark of tumorigenesis and progression, which has guided targeted inhibitor design for over 30 years. Yet, adaptive resistance mechanisms, induced by rapid, context-specific signaling network rewiring, continue to challenge therapeutic efficacy. Leveraging progress in proteomic technologies and network-based methodologies, we introduce Virtual Enrichment-based Signaling Protein-activity Analysis (VESPA)-an algorithm designed to elucidate mechanisms of cell response and adaptation to drug perturbations-and use it to analyze 7-point phosphoproteomic time series from colorectal cancer cells treated with clinically-relevant inhibitors and control media. Interrogating tumor-specific enzyme/substrate interactions accurately infers kinase and phosphatase activity, based on their substrate phosphorylation state, effectively accounting for signal crosstalk and sparse phosphoproteome coverage. The analysis elucidates time-dependent signaling pathway response to each drug perturbation and, more importantly, cell adaptive response and rewiring, experimentally confirmed by CRISPR knock-out assays, suggesting broad applicability to cancer and other diseases.

TDP-43 proteinopathy in ALS is triggered by loss of ASRGL1 and associated with HML-2 expression.

TAR DNA-binding protein 43 (TDP-43) proteinopathy in brain cells is the hallmark of amyotrophic lateral sclerosis (ALS) but its cause remains elusive. Asparaginase-like-1 protein (ASRGL1) cleaves isoaspartates, which alter protein folding and susceptibility to proteolysis. ASRGL1 gene harbors a copy of the human endogenous retrovirus HML-2, whose overexpression contributes to ALS pathogenesis. Here we show that ASRGL1 expression was diminished in ALS brain samples by RNA sequencing, immunohistochemistry, and western blotting. TDP-43 and ASRGL1 colocalized in neurons but, in the absence of ASRGL1, TDP-43 aggregated in the cytoplasm. TDP-43 was found to be prone to isoaspartate formation and a substrate for ASRGL1. ASRGL1 silencing triggered accumulation of misfolded, fragmented, phosphorylated and mislocalized TDP-43 in cultured neurons and motor cortex of female mice. Overexpression of ASRGL1 restored neuronal viability. Overexpression of HML-2 led to ASRGL1 silencing. Loss of ASRGL1 leading to TDP-43 aggregation may be a critical mechanism in ALS pathophysiology.

Intermolecular interactions probed by rotational dynamics in gas-phase clusters.

The rotational dynamics of a molecule is sensitive to neighboring atoms or molecules, which can be used to probe the intermolecular interactions in the gas phase. Here, we real-time track the laser-driven rotational dynamics of a single N2 molecule affected by neighboring Ar atoms using coincident Coulomb explosion imaging. We find that the alignment trace of N-N axis decays fast and only persists for a few picoseconds when an Ar atom is nearby. We show that the decay rate depends on the rotational geometry of whether the Ar atom stays in or out of the rotational plane of the N2 molecule. Additionally, the vibration of the van der Waals bond is found to be excited through coupling with the rotational N-N axis. The observations are well reproduced by solving the time-dependent Schrödinger equation after taking the interaction potential between the N2 and Ar into consideration. Our results demonstrate that environmental effects on a molecular level can be probed by directly visualizing the rotational dynamics.

Reciprocal antagonism of PIN1-APC/CCDH1 governs mitotic protein stability and cell cycle entry.

Induced oncoproteins degradation provides an attractive anti-cancer modality. Activation of anaphase-promoting complex (APC/CCDH1) prevents cell-cycle entry by targeting crucial mitotic proteins for degradation. Phosphorylation of its co-activator CDH1 modulates the E3 ligase activity, but little is known about its regulation after phosphorylation and how to effectively harness APC/CCDH1 activity to treat cancer. Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (PIN1)-catalyzed phosphorylation-dependent cis-trans prolyl isomerization drives tumor malignancy. However, the mechanisms controlling its protein turnover remain elusive. Through proteomic screens and structural characterizations, we identify a reciprocal antagonism of PIN1-APC/CCDH1 mediated by domain-oriented phosphorylation-dependent dual interactions as a fundamental mechanism governing mitotic protein stability and cell-cycle entry. Remarkably, combined PIN1 and cyclin-dependent protein kinases (CDKs) inhibition creates a positive feedback loop of PIN1 inhibition and APC/CCDH1 activation to irreversibly degrade PIN1 and other crucial mitotic proteins, which force permanent cell-cycle exit and trigger anti-tumor immunity, translating into synergistic efficacy against triple-negative breast cancer.

Chk2 sustains PLK1 activity in mitosis to ensure proper chromosome segregation.

Polo-like kinase 1 (PLK1) protects against genome instability by ensuring timely and accurate mitotic cell division. PLK1 activity is tightly regulated throughout the cell cycle. Although the pathways that initially activate PLK1 in G2 are well-characterized, the factors that directly regulate PLK1 in mitosis remain poorly understood. Here, we identify that human PLK1 activity is sustained by the DNA damage response kinase Checkpoint kinase 2 (Chk2) in mitosis. Chk2 directly phosphorylates PLK1 T210, a residue on its T-loop whose phosphorylation is essential for full PLK1 kinase activity. Loss of Chk2-dependent PLK1 activity causes increased mitotic errors, including chromosome misalignment, chromosome missegregation, and cytokinetic defects. Moreover, Chk2 deficiency increases sensitivity to PLK1 inhibitors, suggesting that Chk2 status may be an informative biomarker for PLK1 inhibitor efficacy. This work demonstrates that Chk2 sustains mitotic PLK1 activity and protects genome stability through discrete functions in interphase DNA damage repair and mitotic chromosome segregation.

Synergy of Subgroup J Avian Leukosis Virus and Chicken Infectious Anemia Virus Enhances the Pathogenicity in Chickens.

Subgroup J avian leukemia virus (ALV-J) and chicken infectious anemia virus (CIAV) are widely acknowledged as significant immunosuppressive pathogens that commonly co-infect chickens, causing substantial economic losses in the poultry industry. However, whether co-infection of ALV-J and CIAV have synergistic pathogenicity remains uncertain. To explore their synergistic pathogenesis, we established a co-infection model of ALV-J and CIAV in HD11 cells and specific-pathogen-free (SPF) chickens. We discovered that ALV-J and CIAV can synergistically promote the secretion of IL-6, IL-10, IFN-α, and IFN-γ and apoptosis in HD11 cells. In vivo, compared to the ALV-J and CIAV mono-infected group, the mortality increased significantly by 27% (20 to 47%) and 14% (33 to 47%) in the co-infected group, respectively. We also discovered that ALV-J and CIAV synergistically inhibited weight gain and exhibited more severe organ damage in co-infected chickens. Furthermore, we found that CIAV can promote the replication of ALV-J in HD11 cells and significantly enhance ALV-J viral load in blood and tissues of co-infected chickens, but ALV-J cannot promote the replication of CIAV. Moreover, by measuring the immune organ indexes and proportions of blood CD3+CD4+ and CD3+CD8+ lymphocytes, more serious instances of immunosuppression were observed in ALV-J and CIAV co-infected chickens than in mono-infected chickens. Taken together, our findings demonstrate that ALV-J and CIAV synergistically enhance pathogenicity and immunosuppression.

A New Indole Derivative, LWX-473, Overcomes Glucocorticoid Resistance in Jurkat Cells by Activating Mediators of Apoptosis.

BACKGROUND: Glucocorticoids (GCs) are commonly used as the primary chemotherapy for lymphoid malignancies, including acute lymphoblastic leukemia (ALL). However, the development of GC resistance limits their prolonged use. METHODS: In this study, we investigated the potential of a newly synthesized indole derivative called LWX-473, in combination with the classic GC Dexamethasone (DEX), to enhance the responsiveness of Jurkat cells to GC treatment. RESULTS: Our findings demonstrate that LWX-473 alone or in combination with DEX significantly improves GC-induced cell apoptosis and arrests the cell cycle in the G1 phase. Notably, the combination of LWX-473 and DEX exhibits superior efficacy in killing Jurkat cells compared to LWX-473 alone. Importantly, this compound demonstrates reduced toxicity towards normal cells. CONCLUSIONS: Our study reveals that LWX-473 has the ability to restore the sensitivity of Jurkat cells to DEX by modulating the mitochondrial membrane potential, activating the expression of DEX-liganded glucocorticoid receptor (GR), and inhibiting key molecules in the JAK/STAT signaling pathway. These findings suggest that LWX-473 could be a potential therapeutic agent for overcoming GC resistance in lymphoid malignancies.

Wnt5 controls splenic myelopoiesis and neutrophil functional ambivalency during DSS-induced colitis.

Neutrophils are important innate immune cells with plasticity, heterogenicity, and functional ambivalency. While bone marrow is often regarded as the primary source of neutrophil production, the roles of extramedullary production in regulating neutrophil plasticity and heterogenicity in autoimmune diseases remain poorly understood. Here, we report that the lack of wingless-type MMTV integration site family member 5 (WNT5) unleashes anti-inflammatory protection against colitis in mice, accompanied by reduced colonic CD8+ T cell activation and enhanced splenic extramedullary myelopoiesis. In addition, colitis upregulates WNT5 expression in splenic stromal cells. The ablation of WNT5 leads to increased splenic production of hematopoietic niche factors, as well as elevated numbers of splenic neutrophils with heightened CD8+ T cell suppressive capability, in part due to elevated CD101 expression and attenuated pro-inflammatory activities. Thus, our study reveals a mechanism by which neutrophil plasticity and heterogenicity are regulated in colitis through WNT5 and highlights the role of splenic neutrophil production in shaping inflammatory outcomes.

EGFR targeting PhosTACs as a dual inhibitory approach reveals differential downstream signaling.

We recently developed a heterobifunctional approach [phosphorylation targeting chimeras (PhosTACs)] to achieve the targeted protein dephosphorylation (TPDephos). Here, we envisioned combining the inhibitory effects of receptor tyrosine kinase inhibitors (RTKIs) and the active dephosphorylation by phosphatases to achieve dual inhibition of kinases. We report an example of tyrosine phosphatase-based TPDephos and the effective epidermal growth factor receptor (EGFR) tyrosine dephosphorylation. We also used phosphoproteomic approaches to study the signaling transductions affected by PhosTAC-related molecules at the proteome-wide level. This work demonstrated the differential signaling pathways inhibited by PhosTAC compared with the TKI, gefitinib. Moreover, a covalent PhosTAC selective for mutated EGFR was developed and showed its inhibitory potential for dysregulated EGFR. Last, EGFR PhosTACs, consistent with EGFR dephosphorylation profiles, induced apoptosis and inhibited cancer cell viability during prolonged PhosTAC treatment. PhosTACs showcased their potential of modulating RTKs activity, expanding the scope of bifunctional molecule utility.

A biomimetic approach to lycogarubin C, lynamicin D and related analogues.

An efficient and biomimetic synthetic approach to 3,4-diindolylpyrrole-2,5-dicarboxylate derivatives, including lycogarubin C, lynamicin D and related analogues, was discovered. The crucial transformation included the one-pot formation of two C-N bonds and one C-C bond to construct characteristic pyrrole rings.

High-SNR mid-infrared dual-comb spectroscopy using active phase control cooperating with CWs-dependent phase correction.

Mid-infrared (MIR) dual-comb spectroscopy (DCS) is a highly effective method for molecular metrology of rovibrational transition spectra in a quick accurate manner. However, due to limited comb frequency instability, manipulating coherence between two frequency combs to accomplish high-quality spectral analysis in the MIR region is a huge challenge. Here, we developed a comb-teeth resolved MIR DCS based on active phase control cooperating with a CWs-dependent (CWD) interferogram timing correction. Firstly, four meticulously engineered actuators were individually integrated into two near-infrared (NIR) seed combs to facilitate active coherence maintenance. Subsequently, two PPLN waveguides were adopted to achieve parallel difference frequency generations (DFG), directly achieving a coherent MIR dual-comb spectrometer. To improve coherence and signal-to-noise ratio (SNR), a CWD resampled interferogram timing correction was used to optimize the merit of DCS from 7.5 × 105 to 2.5 × 106. Meanwhile, we carried out the measurement of MIR DCS on the methane hot-band absorption spectra (v3 band), which exhibited a good agreement with HITRAN by a standard deviation on recording residual of 0.76%. These experimental results confirm that this MIR DCS with CWD interferogram timing correction has significant potential to characterize the rovibrational transitions of MIR molecules.

Periodic self-triggered strategy for highly non-linear hybrid stochastic delayed networks based on impulsive control.

This article investigates the stabilization issue of highly non-linear hybrid stochastic delayed networks (HSDNs) via periodic self-triggered control under impulse (PS-TCI). Firstly, the existence of a unique global solution for highly non-linear HSDNs under PS-TCI is studied. Then, a stabilization criterion for highly non-linear HSDNs is established, by combining a graph-theoretic approach with a novel Lyapunov-based analysis, based on a 'genuine' Lyapunov function defined by introducing an auxiliary timer. Therein, the less conservative polynomial growth condition and local Lipschitz condition for the drift and diffusion coefficients are used than the linear growth condition and global Lipschitz condition. Meanwhile, the design idea of PS-TCI is based on the evolution of an upper bound of the mathematical expectation for Lyapunov function (not directly Lyapunov function or system state), which implies that the triggered instant of PS-TCI is not a random variable. Finally the theoretical results are employed to study the stability of a class of FitzHugh-Nagumo circuits networks and the central pattern generators networks of a hexapod robot, and correlative numerical simulations are provided for demonstration.

A proteome-wide quantitative platform for nanoscale spatially resolved extraction of membrane proteins into native nanodiscs.

The intricate molecular environment of the native membrane profoundly influences every aspect of membrane protein (MP) biology. Despite this, the most prevalent method of studying MPs uses detergent-like molecules that disrupt and remove this vital local membrane context. This severely impedes our ability to quantitatively decipher the local molecular context and comprehend its regulatory role in the structure, function, and biogenesis of MPs. Using a library of membrane-active polymers we have developed a platform for the high-throughput analysis of the membrane proteome. The platform enables near-complete spatially resolved extraction of target MPs directly from their endogenous membranes into native nanodiscs that maintain the local membrane context. We accompany this advancement with an open-access quantitative database that provides the most efficient extraction conditions of 2065 unique mammalian MPs. Our method enables rapid and near-complete extraction and purification of target MPs directly from their endogenous organellar membranes at physiological expression levels while maintaining the nanoscale local membrane environment. Going beyond the plasma membrane proteome, our platform enables extraction from any target organellar membrane including the endoplasmic reticulum, mitochondria, lysosome, Golgi, and even transient organelles such as the autophagosome. To further validate this platform we took several independent MPs and demonstrated how our resource can enable rapid extraction and purification of target MPs from different organellar membranes with high efficiency and purity. Further, taking two synaptic vesicle MPs, we show how the database can be extended to capture multiprotein complexes between overexpressed MPs. We expect these publicly available resources to empower researchers across disciplines to capture membrane 'nano-scoops' containing a target MP efficiently and interface with structural, functional, and other bioanalytical approaches. We demonstrate an example of this by combining our extraction platform with single-molecule TIRF imaging to demonstrate how it can enable rapid determination of homo-oligomeric states of target MPs in native cell membranes.

A light-sensitive protein-based wearable pH biometer.

Bacteriorhodopsin is a biological material with excellent photosensitivity properties. It can directly convert optical signals into electrical signals and is widely used in various biosensors. Here, we present a bR-based wearable pH biometer that can be used to monitor wound infection. The mechanism of the pH-sensitive effect of the bR electrode is explained, which generates a transient photovoltage under light irradiation and a negative photovoltage when the lamp is turned off. Since the photoelectric signal of bR is affected by different pH values, the photovoltage is changed by adjusting the pH value. The ratio (Vn/Vp) of negative photovoltage (Vn) to positive photovoltage (Vp) has a good linear relationship (R2 = 0.9911) in the pH range of 4.0-10.0. In vitro experiments using rats as a model confirmed that this wearable pH biometer can monitor pH changes that occur in wound infection.

The CUL5 E3 ligase complex negatively regulates central signaling pathways in CD8+ T cells.

CD8+ T cells play an important role in anti-tumor immunity. Better understanding of their regulation could advance cancer immunotherapies. Here we identify, via stepwise CRISPR-based screening, that CUL5 is a negative regulator of the core signaling pathways of CD8+ T cells. Knocking out CUL5 in mouse CD8+ T cells significantly improves their tumor growth inhibiting ability, with significant proteomic alterations that broadly enhance TCR and cytokine signaling and their effector functions. Chemical inhibition of neddylation required by CUL5 activation, also enhances CD8 effector activities with CUL5 validated as a major target. Mechanistically, CUL5, which is upregulated by TCR stimulation, interacts with the SOCS-box-containing protein PCMTD2 and inhibits TCR and IL2 signaling. Additionally, CTLA4 is markedly upregulated by CUL5 knockout, and its inactivation further enhances the anti-tumor effect of CUL5 KO. These results together reveal a negative regulatory mechanism for CD8+ T cells and have strong translational implications in cancer immunotherapy.