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.

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.

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.

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.

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.

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.

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.

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.

Isocaloric diets with varying protein levels affected energy metabolism in young adult Sprague-Dawley rats via modifying the gut microbes: A lipid imbalance was brought on by a diet with a particularly high protein content.

Protein is the most important macro-nutrient when it comes to maximizing health, body composition, muscle growth, and recovery of body tissue. In recent years, it has been found that protein also plays an important role in metabolism and gut microbiota. This study was performed to investigate the effects of an isocaloric diet with different crude protein contents on the energy metabolism of Sprague-Dawley (SD) rats. Results revealed that compared with the 20% crude protein (CP; control) diet, the 38% CP diet improved serum parameters that are associated with dyslipidemia and glucose metabolic disorders in SD rats, whereas the 50% CP diet increased liver injury indicators and fatty acid synthesis-related genes and protein expression in the liver. Compared with the control diet, the 14% CP diet increased the abundance of colonic short-chain fatty acid-producing bacteria (Lachnospiraceae_NK4A136_group and Ruminiclostridium_9) and promoted colonic microbial cysteine and methionine metabolism, the 38% CP diet up-regulated colonic microbial lysine biosynthesis and degradation pathways, and the 50% CP diet down-regulated colonic mucosal cholesterol metabolism. Furthermore, the increase of multiple colonic enteropathogenic bacteria in the 50% CP group was associated with higher palmitic acid and stearic acid concentrations in the colonic microbes and lower cholesterol and arachidonic acid concentrations in the colonic mucosa. These findings revealed that the 14% CP and 38% CP diets improved rats' energy metabolism, while the 50% CP diet was accompanied by lipid metabolism imbalances and an increase in the abundance of multiple enteropathogenic bacteria.

METTL3-mediated m6A RNA methylation was involved in aluminum-induced neurotoxicity.

Aluminum (Al) exposure has been linked to the development of a variety of neurodegenerative diseases. However, whether m6A RNA methylation participated in Al-induced neurotoxicity remain to be defined. In this study, mice were administrated with aluminum-lactate at dose of 220 mg/kg. bw by gavage for 3 months. Meanwhile, the primary hippocampal neurons were isolated and treated with 0, 50, 100, 150 µM aluminum-lactate, respectively for 7 days. Al exposure caused neuronal shrinkage, decreased Nissl bodies, and increased apoptosis. In accordance, in vitro studies also showed that Al exposure led to neuronal apoptosis in a dose-dependent manner, together with the decline in m6A RNA methylation levels. Moreover, the mRNA expression of Mettl3, Mettl14, Fto, and Ythdf2 were decreased upon Al exposure. Notably, the protein expression of METTL3 was dramatically down-regulated by 42% and 35% in Al-treated mice and neurons, suggesting METTL3 might exert a crucial role in Al-induced neurotoxicity. We next established a mouse model with hippocampus-specific overexpressing of Mettl3 gene to confirm the regulatory role of RNA methylation and found that METTL3 overexpression relieved the neurological injury induced by Al. The integrated MeRIP-seq and RNA-seq analysis elucidated that 631 genes were differentially expressed at both m6A RNA methylation and mRNA expression. Notably, EGFR tyrosine kinase inhibitor resistance, Rap1 signaling pathway, protein digestion and absorption might be involved in Al-induced neurotoxicity. Moreover, VEGFA, Thbs1, and PDGFB might be the central molecules. Collectively, our findings provide the novel sight into the role of m6A RNA methylation in neurodegenerative disease induced by Al.

Biofortification of iron content by regulating a NAC transcription factor in maize.

Iron (Fe) deficiency remains widespread among people in developing countries. To help solve this problem, breeders have been attempting to develop maize cultivars with high yields and high Fe concentrations in the kernels. We conducted a genome-wide association study and identified a gene, ZmNAC78 (NAM/ATAF/CUC DOMAIN TRANSCRIPTION FACTOR 78), that regulates Fe concentrations in maize kernels. We cultivated maize varieties with both high yield and high Fe concentrations in their kernels by using a molecular marker developed from a 42-base pair insertion or deletion (indel) in the promoter of ZmNAC78. ZmNAC78 expression is enriched in the basal endosperm transfer layer of kernels, and the ZmNAC78 protein directly regulates messenger RNA abundance of Fe transporters. Our results thus provide an approach to develop maize varieties with Fe-enriched kernels.

Quantitative analysis of contrast-enhanced ultrasonography in rat models of hepatic acute graft-versus-host disease.

Background: Hepatic acute graft-versus-host disease (aGVHD) is a major life-threatening complication of allogeneic hematopoietic stem cell transplantation (allo-HSCT). We hypothesized that contrast-enhanced ultrasound (CEUS) could serve as a new imaging biomarker in early diagnosis of hepatic aGVHD by detecting liver microcirculation. Methods: Thirty Wistar rats received allo-HSCT were finally included after excluding 9 rats, and they were randomly divided into 5 groups (1- to 5-week groups, 6 per group). Six rats were used for the control group without any intervention. We observed the clinical scores, serum liver enzyme levels and liver CEUS parameters of rats in each group. Hepatic aGVHD was finally confirmed by histopathologic analysis. The diagnostic performance of CEUS parameters in detecting GVHD was evaluated by comparing the area under the receiver operating curve (AUC) values. Results: After HSCT, the rats developed ruffling of fur, maculopapular rash, weight loss, accompanied by increased clinical scores. Serum liver enzymes were significantly higher than those in the control group from the third week, especially alkaline phosphatase, while CEUS parameters, peak intensity (PI) and mean transit time (MTT), changed in the second week (P<0.001). Compared with non-aGVHD group, the PI was significantly decreased while time to peak and MTT were prolonged in aGVHD group. CEUS parameters were more strongly correlated with pathological grade than serology. PI was an independent predictor for hepatic aGVHD. The AUC of CEUS parameters for diagnosing hepatic aGVHD was 0.933 (95% CI: 0.779-0.992), which was higher than that of clinical scores (AUC =0.748, 95% CI: 0.557-0.888, P=0.032) and serological markers (AUC =0.902, 95% CI: 0.737-0.980, P=0.694). Conclusions: CEUS exhibits promising applications as a quantitative method to detect hepatic aGVHD and early liver damage.

Human endogenous retrovirus K contributes to a stem cell niche in glioblastoma.

Human endogenous retroviruses (HERVs) are ancestral viral relics that constitute nearly 8% of the human genome. Although normally silenced, the most recently integrated provirus HERV-K (HML-2) can be reactivated in certain cancers. Here, we report pathological expression of HML-2 in malignant gliomas in both cerebrospinal fluid and tumor tissue that was associated with a cancer stem cell phenotype and poor outcomes. Using single-cell RNA-Seq, we identified glioblastoma cellular populations with elevated HML-2 transcripts in neural progenitor-like cells (NPC-like) that drive cellular plasticity. Using CRISPR interference, we demonstrate that HML-2 critically maintained glioblastoma stemness and tumorigenesis in both glioblastoma neurospheres and intracranial orthotopic murine models. Additionally, we demonstrate that HML-2 critically regulated embryonic stem cell programs in NPC-derived astroglia and altered their 3D cellular morphology by activating the nuclear transcription factor OCT4, which binds to an HML-2-specific long-terminal repeat (LTR5Hs). Moreover, we discovered that some glioblastoma cells formed immature retroviral virions, and inhibiting HML-2 expression with antiretroviral drugs reduced reverse transcriptase activity in the extracellular compartment, tumor viability, and pluripotency. Our results suggest that HML-2 fundamentally contributes to the glioblastoma stem cell niche. Because persistence of glioblastoma stem cells is considered responsible for treatment resistance and recurrence, HML-2 may serve as a unique therapeutic target.

Oncogene-like addiction to aneuploidy in human cancers.

Most cancers exhibit aneuploidy, but its functional significance in tumor development is controversial. Here, we describe ReDACT (Restoring Disomy in Aneuploid cells using CRISPR Targeting), a set of chromosome engineering tools that allow us to eliminate specific aneuploidies from cancer genomes. Using ReDACT, we created a panel of isogenic cells that have or lack common aneuploidies, and we demonstrate that trisomy of chromosome 1q is required for malignant growth in cancers harboring this alteration. Mechanistically, gaining chromosome 1q increases the expression of MDM4 and suppresses p53 signaling, and we show that TP53 mutations are mutually exclusive with 1q aneuploidy in human cancers. Thus, tumor cells can be dependent on specific aneuploidies, raising the possibility that these "aneuploidy addictions" could be targeted as a therapeutic strategy.

Cell division cycle associated 8 promotes the growth and inhibits the apoptosis of endometrial cancer cells by regulating cell cycle and P53/Rb signaling pathway.

OBJECTIVE: Cell division cycle associated 8 (CDCA8) is over-expressed in a variety of tumors and associated with tumor progression. Nevertheless, the role of CDCA8 in endometrial cancer (EC) is unclear. Therefore, this study aimed to assess the role and mechanism of CDCA8 in EC. METHODS: Immunohistochemical staining was used to evaluate CDCA8 expression in EC, and its relationship with clinicopathology was analyzed. CDCA8 was knocked down or over-expressed to study its effects on cell biological behaviors. Furthermore, the feasible mechanisms of CDCA8 were examined by Western blot. RESULTS: CDCA8 was significantly upregulated in EC tissue (P<0.05) and related to worse tumor grade, Figo stage, tumor (T) stage, and deep myometrial invasion (P<0.05). CDCA8 knockdown inhibited EC cell activities, promoted apoptosis and induced cell cycle arrest (P<0.05), which were reversed by CDCA8 over-expression (P<0.05). Besides, CDCA8 knockdown inhibited the growth of xenograft tumors in nude mice (P<0.05). Furthermore, CDCA8 may affect cell cycle and P53/Rb signaling pathway in EC cells. CONCLUSION: CDCA8 plays a role in the pathogenesis of EC and may be a target for EC treatment.

Neurotoxic properties of the Zika virus envelope protein.

Prenatal Zika virus (ZIKV) infection is a serious global concern as it can lead to brain injury and many serious birth defects, collectively known as congenital Zika syndrome. Brain injury likely results from viral mediated toxicity in neural progenitor cells. Additionally, postnatal ZIKV infections have been linked to neurological complications, yet the mechanisms driving these manifestations are not well understood. Existing data suggest that the ZIKV envelope protein can persist in the central nervous system for extended periods of time, but it is unknown if this protein can independently contribute to neuronal toxicity. Here we find that the ZIKV envelope protein is neurotoxic, leading to overexpression of poly adenosine diphosphate -ribose polymerase 1, which can induce parthanatos. Together, these data suggest that neuronal toxicity resulting from the envelope protein may contribute to the pathogenesis of post-natal ZIKV-related neurologic complications.

An optogenetic-phosphoproteomic study reveals dynamic Akt1 signaling profiles in endothelial cells.

The serine/threonine kinase AKT is a central node in cell signaling. While aberrant AKT activation underlies the development of a variety of human diseases, how different patterns of AKT-dependent phosphorylation dictate downstream signaling and phenotypic outcomes remains largely enigmatic. Herein, we perform a systems-level analysis that integrates methodological advances in optogenetics, mass spectrometry-based phosphoproteomics, and bioinformatics to elucidate how different intensity, duration, and pattern of Akt1 stimulation lead to distinct temporal phosphorylation profiles in vascular endothelial cells. Through the analysis of ~35,000 phosphorylation sites across multiple conditions precisely controlled by light stimulation, we identify a series of signaling circuits activated downstream of Akt1 and interrogate how Akt1 signaling integrates with growth factor signaling in endothelial cells. Furthermore, our results categorize kinase substrates that are preferably activated by oscillating, transient, and sustained Akt1 signals. We validate a list of phosphorylation sites that covaried with Akt1 phosphorylation across experimental conditions as potential Akt1 substrates. Our resulting dataset provides a rich resource for future studies on AKT signaling and dynamics.

Mining of clinical and prognosis related genes in the tumor microenvironment of endometrial cancer: A field synopsis of observational study.

Endometrial cancer (EC) is the sixth most common malignant tumor in women worldwide, and its morbidity and mortality are on the rise. The purpose of this study was to explore potential tumor microenvironment (TME)-related biomarkers associated with the clinical features and prognosis of EC. The Estimating Stromal and Immune Cells in Malignancy Using Expression Data (ESTIMATE) algorithm was used to calculate TME immune and stromal scores of EC samples and to analyze the relationship between immune/stromal scores, clinical features, and prognosis. Heat maps and Venn maps were used to screen for differentially expressed genes (DEGs). The ESTIMATE algorithm revealed immune score was significantly correlated with overall survival and tumor grade in patients with EC. A total of 1448 DEGs were screened, of which 387 were intersecting genes. Gene Ontology (GO) analysis revealed that the biological processes (BP) related to intersecting genes mainly included T cell activation and regulation of lymphocyte activation. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that the intersecting genes were closely related to immune-related signaling pathways. Thirty core genes with more than 7 nodes were identified using protein-protein interaction (PPI) analysis. Six independent prognostic genes of EC were identified using Kaplan-Meier survival analysis and multivariate Cox analysis, namely CD5, BATF, CACNA2D2, LTA, CD52, and NOL4, which are all immune-infiltrating genes that are closely related to clinical features. The current study identified 6 key genes closely related to immune infiltration in the TME of EC that predict clinical outcomes, which may provide new insights into novel prognostic biomarkers and immunotherapy for patients with EC.

METTL3-mediated m6A mRNA modification was involved in cadmium-induced liver injury.

Cadmium is an environmental pollutant that has extensive deleterious effects. However, the mechanisms underlying the hepatotoxicity induced by long-term exposure to cadmium remained undefined. In the present study, we explored the role of m6A methylation in the development of cadmium-induced liver disease. We showed a dynamic change of RNA methylation in liver tissue from mice administrated with cadmium chloride (CdCl2) for 3, 6 and 9 months, respectively. Particularly, the METTL3 expression was declined in a time-dependent manner, associated with the degree of liver injury, indicating the involvement of METTL3 in hepatotoxicity induced by CdCl2. Moreover, we established a mouse model with liver-specific over-expression of Mettl3 and administrated these mice with CdCl2 for 6 months. Notably, METTL3 highly expressed in hepatocytes attenuated CdCl2-induced steatosis and liver fibrosis in mice. In vitro assay also showed METTL3 overexpression ameliorated the CdCl2-induced cytotoxicity and activation of primary hepatic stellate cells. Furthermore, transcriptome analysis identified 268 differentially expressed genes both in mice liver tissue treated with CdCl2 for 3 months and 9 months. Among them, 115 genes were predicted to be regulated by METTL3 determined by m6A2Target database. Further analysis revealed the perturbation of metabolic pathway, glycerophospholipid metabolism, ErbB signaling pathway, Hippo signaling pathway, and choline metabolism in cancer, and circadian rhythm, led to hepatotoxicity induced by CdCl2. Collectively, our findings reveal new insight into the crucial role of epigenetic modifications in hepatic diseases caused by long-term exposure to cadmium.