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This letter to the editor discusses a case of chronic lymphocytic leukemia (CLL) with MDM2 amplification as an alternative pathway to TP53 dysfunction. The patient had a complex karyotype and no detectable TP53 mutation or deletion, but was found to have a high level MDM2 amplification that was predicted to mediate TP53 dysregulation. The letter emphasizes the importance of understanding alternative mechanisms of TP53 dysregulation in CLL for better patient outcomes. The article titled "Molecular map of chronic lymphocytic leukemia and its impact on outcome" provides a comprehensive understanding of CLL and its genetic characteristics, while the article "Cytogenetic complexity in chronic lymphocytic leukemia: definitions, associations, and clinical impact" focuses on the cytogenetic complexity of CLL and its associations with clinical outcomes. Lastly, the article "Characterizing ATM aberrations in Chronic Lymphocytic Leukemia (CLL): Prognostic Implications and Sensitivity to PARP Inhibition" examines the prognostic implications and sensitivity to PARP inhibition of ATM aberrations in CLL. These articles offer valuable insights for researchers and healthcare professionals studying CLL and its treatment options. [Extracted from the article]

Huntington's disease and juvenile-onset schizophrenia have long been regarded as distinct disorders. However, both manifest cell-intrinsic abnormalities in glial differentiation, with resultant astrocytic dysfunction and hypomyelination. To assess whether a common mechanism might underlie the similar glial pathology of these otherwise disparate conditions, we used comparative correlation network approaches to analyse RNA-sequencing data from human glial progenitor cells (hGPCs) produced from disease-derived pluripotent stem cells. We identified gene sets preserved between Huntington's disease and schizophrenia hGPCs yet distinct from normal controls that included 174 highly connected genes in the shared disease-associated network, focusing on genes involved in synaptic signalling. These synaptic genes were largely suppressed in both schizophrenia and Huntington's disease hGPCs, and gene regulatory network analysis identified a core set of upstream regulators of this network, of which OLIG2 and TCF7L2 were prominent. Among their downstream targets, ADGRL3 , a modulator of glutamatergic synapses, was notably suppressed in both schizophrenia and Huntington's disease hGPCs. Chromatin immunoprecipitation sequencing confirmed that OLIG2 and TCF7L2 each bound to the regulatory region of ADGRL3 , whose expression was then rescued by lentiviral overexpression of these transcription factors. These data suggest that the disease-associated suppression of OLIG2 and TCF7L2-dependent transcription of glutamate signalling regulators may impair glial receptivity to neuronal glutamate. The consequent loss of activity-dependent mobilization of hGPCs may yield deficient oligodendrocyte production, and hence the hypomyelination noted in these disorders, as well as the disrupted astrocytic differentiation and attendant synaptic dysfunction associated with each. Together, these data highlight the importance of convergent glial molecular pathology in both the pathogenesis and phenotypic similarities of two otherwise unrelated disorders, Huntington's disease and schizophrenia. [ABSTRACT FROM AUTHOR]

Human papillomavirus (HPV) infections are common among reproductive-age women. The use of primary HPV testing has proven more effective in identifying high-grade precancerous lesions, leading to better clinical outcomes in women. HPV integration into the host genome is a multifactorial process. Various host, viral, and environmental cofactors, such as smoking, cervicovaginal microbiota, estrogen levels, and coinfection with HIV, influence this process. The disruption of the E2 gene is identified as a hallmark of HPV integration and cancer development, leading to the overexpression of oncogenic proteins (E6/E7) and the formation of superenhancers, thereby contributing to cellular transformation and cervical neoplasia. HPV integration results in host genomic instability, chromosomal rearrangements, loss of function in tumor suppressor genes, and epigenome dysregulation. Countless efforts have been made to eradicate cervical cancer worldwide, including improving disease screening and human papillomavirus (HPV) vaccination programs. Nevertheless, cervical cancer still claims the lives of more than 300 000 women every year. Persistent infections with high-risk HPV genotypes 16 and 18 are the main cause of cancer and may result in HPV integration into the host genome. The central dogma is that HPV integration is an important step in oncogenesis, but in fact, it impedes the virus from replicating and spreading. HPV causing cervical cancer can therefore be perceived as a failed evolutionary viral trait. Here we outline the occurrence and mechanisms of HPV integration and how this process results in oncogenic transformation. [ABSTRACT FROM AUTHOR]

Background: Adenoid cystic carcinoma is a rare subtype of triple‐negative breast carcinoma. These low‐grade tumours, which are treated by simple mastectomy and have an excellent prognosis compared to other triple‐negative breast carcinomas. Solid‐variant adenoid cystic carcinomas have basaloid features and are difficult to distinguish morphologically from other triple‐negative breast cancers. Breast adenoid cystic carcinoma exhibits MYB protein overexpression, which can be detected by immunohistochemistry (IHC). Aim: We compared the IHC expression of MYB in solid‐variant adenoid cystic carcinoma with that in other triple‐negative breast cancers. Methods: We conducted IHC staining of 210 samples of triple‐negative breast cancers, including solid‐variant adenoid cystic carcinoma (n = 17), metaplastic breast carcinoma (n = 44), basaloid triple‐negative breast cancer (n = 21), and other triple‐negative invasive ductal carcinoma (n = 128). We classified nuclear staining of MYB as diffuse/strong (3+), focal moderate (2+), focal weak (1+), or none (0). Results: All 17 solid/basaloid adenoid cystic carcinoma cases exhibited 3+ MYB expression. Of the 21 solid/basaloid triple‐negative breast cancers, one (5%) had 2+ expression, seven (33%) 1+ expression, and 13 (62%) 0 expression. Of the 44 metaplastic carcinoma cases, 39 cases (89%) had no (0) staining, and the other five cases had focal weak (1+) or moderate (2+) staining. Among the 128 triple‐negative invasive ductal carcinoma cases, 92 cases (72%) had no (0) staining, 36 cases (28%) exhibited focal weak (1+) or moderate (2+) staining. Conclusions: Our study revealed diffuse/strong MYB staining (3+) only in solid/basaloid adenoid cystic carcinomas. Thus, we recommend routine MYB IHC staining in triple‐negative breast carcinoma with solid/basaloid morphology to improve diagnostic accuracy. [ABSTRACT FROM AUTHOR]

Aims: Human epidermal growth factor receptor 2 (HER2) expression is an important biomarker in breast cancer (BC). Most BC cases categorised as HER2‐negative (HER2−) express low levels of HER2 [immunohistochemistry (IHC) 1+ or IHC 2+/in‐situ hybridisation not amplified (ISH−)] and represent a clinically relevant therapeutic category that is amenable to targeted therapy using a recently approved HER2‐directed antibody–drug conjugate. A group of practising pathologists, with expertise in breast pathology and BC biomarker testing, outline best practices and guidance for achieving consensus in HER2 IHC scoring for BC. Methods and results: The authors describe current knowledge and challenges of IHC testing and scoring of HER2‐low expressing BC and provide best practices and guidance for accurate identification of BCs expressing low levels of HER2. These expert pathologists propose an algorithm for assessing HER2 expression with validated IHC assays and incorporate the 2023 American Society of Clinical Oncology and College of American Pathologist guideline update. The authors also provide guidance on when to seek consensus for HER2 IHC scoring, how to incorporate HER2‐low into IHC reporting and present examples of HER2 IHC staining, including challenging cases. Conclusions: Awareness of BC cases that are negative for HER protein overexpression/gene amplification and the related clinical relevance for targeted therapy highlight the importance of accurate HER2 IHC scoring for optimal treatment selection. [ABSTRACT FROM AUTHOR]

To explore the correlation of endoplasmic reticulum stress (ERS) and oxidative stress (OS), and the protective effect of Sestrin2 (SESN2) on human lens epithelial cells (HLECs). Tunicamycin (TM) was used to induce ERS in HLECs. 4-Phenylbutyric acid (4-PBA) was used to inhibit ERS. Eupatilin applied to HLECs as SESN2 agonist. SESN2 expression was knocked down via si-RNA in HLECs. The morphological changes of HLECs were observed by microscope. ER-tracker to evaluate ERS, ROS production assay to measure ROS, flow cytometry to calculate cell apoptosis rate. Immunofluorescence to observe Nrf2 translocation, and effects of TM or EUP on SESN2. Western blot and qPCR were used to evaluate the expression of GRP78, PERK, ATF4, CHOP, Nrf2, and SESN2 expression in HLECs with different treatment groups. ERS can elevate the expression of ROS and Nrf2 to induce OS. Upregulation of SESN2 was observed in ERS-mediate OS. Overexpression of SESN2 can reduce the overexpression of ERS-related protein GRP78, PERK, ATF4, proapoptotic protein CHOP, OS-related protein Nrf2, as well as ROS, and alleviate ERS injury at the same time. Whereas knockdown of SESN2 can upregulate the expression of GRP78, PERK, ATF4, CHOP, Nrf2, ROS, and deteriorate ERS damage. ERS can induce OS, they form a vicious cycle to induce apoptosis in HLECs, which may contribute to cataract formation. SESN2 could protect HLECs against the apoptosis by regulating the vicious cycle between ERS and OS. [ABSTRACT FROM AUTHOR]

Ribosome-inactivating proteins (RIPs) are plant enzymes that target the rRNA. The cytoplasmic RIP, called OsRIP1, plays a crucial role in regulating jasmonate, a key plant hormone. Understanding the role of OsRIP1 can provide insights into enhancing stress tolerance and optimizing growth of rice. Transcription profiling by mRNA sequencing was employed to measure the changes in gene expression in rice plants in response to MeJA treatment. Compared to wild type (WT) plants, OsRIP1 overexpressing rice plants showed a lower increase in mRNA transcripts for genes related to jasmonate responses when exposed to MeJA treatment for 3 h. After 24 h of MeJA exposure, the mRNA transcripts associated with the gibberellin pathway occurred in lower levels in OsRIP1 overexpressing plants compared to WT plants. We hypothesize that the mechanism underlying OsRIP1 antagonization of MeJA-induced shoot growth inhibition involves cytokininmediated leaf senescence and positive regulation of cell cycle processes, probably via OsRIP1 interaction with 40S ribosomal protein S5 and α-tubulin. Moreover, the photosystem II 10kDa polypeptide was identified to favorably bind to OsRIP1, and its involvement may be attributed to the reduction of photosynthesis in OsRIP1-overexpressing plants subjected to MeJA at the early timepoint (3 h). [ABSTRACT FROM AUTHOR]

In many countries, hormone receptor status assessment of ductal carcinoma in situ (DCIS) is routinely performed, as hormone receptor-positive DCIS patients are eligible for adjuvant anti-hormonal treatment, aiming to reduce the ipsilateral and contralateral breast cancer risk. Although HER2 gene amplification and its associated HER2 protein overexpression constitute a major prognostic and predictive marker in invasive breast carcinoma, its use in the diagnosis and treatment of DCIS is less straightforward. HER2 immunohistochemistry is not routinely performed yet, as the role of HER2-positivity in DCIS biology is unclear. Nonetheless, recent data challenge this practice. Here, we discuss the value of routine HER2 assessment for DCIS. HER2-positivity correlates strongly with DCIS grade: around four in five HER2-positive DCIS show high grade atypia. As morphological DCIS grading is prone to interobserver variability, HER2 immunohistochemistry could render grading more robust. Several studies showed an association between HER2-positive DCIS and ipsilateral recurrence risk, albeit currently unclear whether this is for overall, in situ or invasive recurrence. HER2-positive DCIS tends to be larger, with a higher risk of involved surgical margins. HER2-positive DCIS patients benefit more from adjuvant radiotherapy: it substantially decreases the local recurrence risk after lumpectomy, without impact on overall survival. HER2-positivity in pure biopsy-diagnosed DCIS is associated with increased upstaging to invasive carcinoma after surgery. HER2 immunohistochemistry on preoperative biopsies might therefore provide useful information to surgeons, favoring wider excisions. The time seems right to consider DCIS subtype-dependent treatment, comprising appropriate local treatment for HER2-positive DCIS patients and de-escalation for hormone receptor-positive, HER2-negative DCIS patients. [ABSTRACT FROM AUTHOR]

In this study, we demonstrated that both the expression of most ribosomal protein genes and the amount of ribosomes were decreased in the Δaa3 mutant of Mycobacterium smegmatis, in which the major terminal oxidase (aa3 cytochrome c oxidase) of the respiratory electron transport chain (ETC) is inactivated, compared to those in the wild-type strain. Deletion of the rel gene encoding the major (p)ppGpp synthetase in the background of the Δaa3 mutant restored the reduced expression of ribosomal protein genes, suggesting that inhibition of the respiratory ETC leads to the Rel-dependent stringent response (SR) in this bacterium. Both a decrease in the expression of ribosomal protein genes by overexpression of rel and the increased expression of rel in the Δaa3 mutant relative to the wild-type strain support the Rel-dependent induction of SR in the Δaa3 mutant. We also demonstrated that the expression of ribosomal protein genes was decreased in M. smegmatis exposed to respiration-inhibitory conditions, such as KCN and bedaquiline treatment, null mutation of the cytochrome bcc1 complex, and hypoxia. The MprBA-SigE-SigB regulatory pathway was implicated in both the increased expression of rel and the decreased expression of ribosomal protein genes in the Δaa3 mutant of M. smegmatis. [ABSTRACT FROM AUTHOR]

Plants respond to pathogen exposure by activating the expression of a group of defense-related proteins known as Pathogenesis-Related (PR) proteins, initially discovered in the 1970s. These PR proteins are categorized into 17 distinct families, denoted as PR1-PR17. Predominantly secreted, most of these proteins execute their defensive roles within the apoplastic space. Several PR proteins possess well-defined enzymatic functions, such as β-glucanase (PR2), chitinases (PR3, 4, 8, 11), proteinase (PR7), or RNase (PR10). Enhanced resistance against pathogens is observed upon PR protein overexpression, while their downregulation renders plants more susceptible to pathogen infections. Many of these proteins exhibit antimicrobial activity in vitro, and due to their compact size, some are classified as antimicrobial peptides. Recent research has unveiled that phytopathogens, including nematodes, fungi, and phytophthora, employ analogous proteins to bolster their virulence and suppress plant immunity. This raises a fundamental question: how can these conserved proteins act as antimicrobial agents when produced by the host plant but simultaneously suppress plant immunity when generated by the pathogen? In this hypothesis, we investigate PR proteins produced by pathogens, which we term "PR-like proteins," and explore potential mechanisms by which this class of virulence factors operate. Preliminary data suggests that these proteins may form complexes with the host's own PR proteins, thereby interfering with their defense-related functions. This analysis sheds light on the intriguing interplay between plant and pathogen-derived PR-like proteins, providing fresh insights into the intricate mechanisms governing plant-pathogen interactions. [ABSTRACT FROM AUTHOR]

Overexpression of the agouti-signaling protein (asip1), an endogenous melanocortin antagonist, under the control of a constitutive promoter in zebrafish [Tg(Xla.Eef1a1:Cau.Asip1]iim4] (asip1-Tg) increases food intake by reducing sensitivity of the central satiety systems and abolish circadian activity rhythms. The phenotype also shows increased linear growth and body weight, yet no enhanced aggressiveness in dyadic fights is observed. In fact, asip1-Tg animals choose to flee to safer areas rather than face a potential threat, thus suggesting a potential anxiety-like behavior (ALB). Standard behavioral tests, i.e., the open field test (OFT), the novel object test (NOT), and the novel tank dive test (NTDT), were used to investigate thigmotaxis and ALB in male and female zebrafish. Results showed that the asip1-Tg strain exhibited severe ALB in every test, mainly characterized by pronounced freezing behavior and increased linear and angular swimming velocities. asip1-Tg animals exhibited low central serotonin (5-HT) and dopamine (DA) levels and high turnover rates, thus suggesting that central monoaminergic pathways might mediate melanocortin antagonist-induced ALB. Accordingly, the treatment of asip1-Tg animals with fluoxetine, a selective serotonin reuptake inhibitor (SSRI), reversed the ALB phenotype in NTDT as well as 5-HT turnover. Genomic and anatomical data further supported neuronal interaction between melanocortinergic and serotonergicsystems. These results suggest that inhibition of the melanocortin system by ubiquitous overexpression of endogenous antagonist has an anxiogenic effect mediated by serotonergic transmission. [ABSTRACT FROM AUTHOR]

The northward shift of Pyropia yezoensis aquaculture required the breeding of germplasms with tolerance to the oxidative stress due to the high light conditions of the North Yellow Sea area. The MPV17/PMP22 family proteins were identified as a molecule related to reactive oxygen species (ROS) metabolism. Here, one of the MPV17 homolog genes designated as PyM‐LP2 was selected for functional identification by introducing the encoding sequence region/reverse complementary fragment into the Py. yezoensis genome. Although the photosynthetic activity, the respiratory rate, and the ROS level in wild type (WT) and different gene‐transformed algal strains showed similar levels under normal conditions, the overexpression (OE) strain exhibited higher values of photosynthesis, respiration, and reducing equivalents pool size but lower intracellular ROS production under stress conditions compared with the WT. Conversely, all the above parameters showed opposite variation trends in RNAi strain as those in the OE strain. This implied that the PyM‐LP2 protein was involved in the mitigation of the oxidative stress. Sequence analysis revealed that this PyM‐LP2 protein was assorted to peroxisomes and might serve as a poring channel for transferring malate (Mal) to peroxisomes. By overexpressing PyM‐LP2, the transfer of Mal from chloroplasts to peroxisomes was enhanced under stress conditions, which promoted photorespiration and ultimately alleviated excessive reduction of the photosynthetic electron chain. This research lays the groundwork for the breeding of algae with enhanced resistance to oxidative stresses. [ABSTRACT FROM AUTHOR]

Background: The most important factors contributing to multi-drug resistance in oral cancer include overexpression of the EGFR protein and the downstream malignancy regulators that are associated with it. This study investigates the impact of solanine on inflammation, proliferation, and angiogenesis inhibition in multidrug-resistant oral cancer KB-Chr-8-5 cells through inhibition of the EGFR/PI3K/Akt/NF-κB signaling pathway. Methods: Cell viability was assessed using an MTT assay to evaluate cytotoxic effects. Production of reactive oxygen species (ROS), mitochondrial membrane potential (ΔΨM), and AO/EtBr staining were analyzed to assess apoptosis and mitochondrial dysfunction. Western blotting was employed to examine protein expression related to angiogenesis, apoptosis, and signaling pathways. Experiments were conducted in triplicate. Results: Solanine treatment at concentrations of 10, 20, and 30 μM significantly increased ROS production, which is indicative of its antioxidant properties. This increase was associated with decreased mitochondrial membrane potential (ΔΨM) with p < 0.05, suggesting mitochondrial dysfunction. Inhibition of EGFR led to reduced activity of PI3K, Akt, and NF-κB, resulting in decreased expression of iNOS, IL-6, Cyclin D1, PCNA, VEGF, Mcl-1, and HIF-1α and increased levels of the apoptotic proteins Bax, caspase-9, and caspase-3. These changes collectively inhibited the growth of multidrug-resistant (MDR) cancer cells. Conclusions: Solanine acts as a potent disruptor of cellular processes by inhibiting the EGFR-mediated PI3K/Akt/NF-κB signaling pathway. These results suggest that solanine holds promise as a potential preventive or therapeutic agent against multidrug-resistant cancers. [ABSTRACT FROM AUTHOR]

We predicted the protein therapeutic targets specific to a Ru-based potential drug and its combination with pristine and N-doped carbon dot drug delivery systems, denoted as RuCN/CDs and RuCN/N-CDs. Synchrotron-based FTIR microspectroscopy (µFTIR) in addition to bioinformatics data on drug structures and protein sequences were applied to assess changes in the protein secondary structure of A2780 cancer cells. µFTIR revealed the moieties of the target proteins' secondary structure changes only after the treatment with RuCN and RuCN/N-CDs. A higher content of α-helices and a lower content of β-sheets appeared in A2780 cells after RuCN treatment. Treatment with RuCN/N-CDs caused a substantial increase in parallel β-sheet numbers, random coil content, and tyrosine residue numbers. The results obtained suggest that the mitochondrion-related proteins NDUFA1 and NDUFB5 are affected by RuCN either via overexpression or stabilisation of helical structures. RuCN/N-CDs either induce overexpression of the β-sheet-rich protein NDUFS1 and affect its random coil structure or interact and stabilise its structure via hydrogen bonding between -NH2 groups from N-CDs with protein C=O groups and –OH groups of serine, threonine, and tyrosine residues. The N-CD nanocarrier tunes this drug's action by directing it toward a specific protein target, changing this drug's coordination ability and inducing changes in the protein's secondary structures and function. [ABSTRACT FROM AUTHOR]

Most cases of human prion disease arise due to spontaneous misfolding of WT or mutant prion protein, yet recapitulating this event in animal models has proven challenging. It remains unclear whether spontaneous prion generation can occur within the mouse lifespan in the absence of protein overexpression and how disease-causing mutations affect prion strain properties. To address these issues, we generated knockin mice that express the misfolding-prone bank vole prion protein (BVPrP). While mice expressing WT BVPrP (I109 variant) remained free from neurological disease, a subset of mice expressing BVPrP with mutations (D178N or E200K) causing genetic prion disease developed progressive neurological illness. Brains from spontaneously ill knockin mice contained prion disease--specific neuropathological changes as well as atypical proteaseresistant BVPrP. Moreover, brain extracts from spontaneously ill D178N- or E200K-mutant BVPrP--knockin mice exhibited prion seeding activity and transmitted disease to mice expressing WT BVPrP. Surprisingly, the properties of the D178N- and E200K-mutant prions appeared identical before and after transmission, suggesting that both mutations guide the formation of a similar atypical prion strain. These findings imply that knockin mice expressing mutant BVPrP spontaneously develop a bona fide prion disease and that mutations causing prion diseases may share a uniform initial mechanism of action. [ABSTRACT FROM AUTHOR]

Atherosclerosis (AS) is the major cause of multiple cardiovascular diseases. In addition, the lipid accumulation of human vascular smooth muscle cells (HVSMCs) can cause the occurrence of AS. Secreted frizzled-related protein 5 (Sfrp5) was known to be downregulated in AS; however, the detailed function of Sfrp5 in HVSMCs remains unclear. Specifically, we found that Sfrp5 expression in oxLDL-treated HVSMCs was downregulated. Sfrp5 overexpression inhibited the viability of HVSMCs induced by oxLDL. In addition, oxLDL-induced proliferation and migration in HVSMCs were abolished by Sfrp5 overexpression. Sfrp5 overexpression reduced oxLDL-caused oxidative stress, lipid accumulation, and inflammation in HVSMCs. Meanwhile, oxLDL treatment increased the expressions of Wnt5a, c-Myc, and β-catenin in HVSMCs, while this phenomenon was rescued by Sfrp5 overexpression. Furthermore, the inhibitory effect of Sfrp5 upregulation on the viability and migration of HVSMCs was reversed by R-spondin 1. These results indicate that Sfrp5 overexpression could reverse oxLDL-induced lipid accumulation in HVSMCs through inactivating Wnt5a/β-catenin signaling pathway. [ABSTRACT FROM AUTHOR]

Background: Diabetes-induced cardiac fibrosis is one of the main mechanisms of diabetic cardiomyopathy. As a common histone methyltransferase, enhancer of zeste homolog 2 (EZH2) has been implicated in fibrosis progression in multiple organs. However, the mechanism of EZH2 in diabetic myocardial fibrosis has not been clarified. Methods: In the current study, rat and mouse diabetic model were established, the left ventricular function of rat and mouse were evaluated by echocardiography and the fibrosis of rat ventricle was evaluated by Masson staining. Primary rat ventricular fibroblasts were cultured and stimulated with high glucose (HG) in vitro. The expression of histone H3 lysine 27 (H3K27) trimethylation, EZH2, and myocardial fibrosis proteins were assayed. Results: In STZ-induced diabetic ventricular tissues and HG-induced primary ventricular fibroblasts in vitro, H3K27 trimethylation was increased and the phosphorylation of EZH2 was reduced. Inhibition of EZH2 with GSK126 suppressed the activation, differentiation, and migration of cardiac fibroblasts as well as the overexpression of the fibrotic proteins induced by HG. Mechanical study demonstrated that HG reduced phosphorylation of EZH2 on Thr311 by inactivating AMP-activated protein kinase (AMPK), which transcriptionally inhibited peroxisome proliferator-activated receptor γ (PPAR-γ) expression to promote the fibroblasts activation and differentiation. Conclusion: Our data revealed an AMPK/EZH2/PPAR-γ signal pathway is involved in HG-induced cardiac fibrosis. [ABSTRACT FROM AUTHOR]

Mini-G proteins are engineered, thermostable variants of Gα subunits designed to stabilize G protein–coupled receptors (GPCRs) in their active conformations. Because of their small size and ease of use, they are popular tools for assessing GPCR behaviors in cells, both as reporters of receptor coupling to Gα subtypes and for cellular assays to quantify compartmentalized signaling at various subcellular locations. Here, we report that overexpression of mini-G proteins with their cognate GPCRs disrupted GPCR endocytic trafficking and associated intracellular signaling. In cells expressing the Gαs-coupled GPCR glucagon-like peptide 1 receptor (GLP-1R), coexpression of mini-Gs, a mini-G protein derived from Gαs, blocked β-arrestin 2 recruitment and receptor internalization and disrupted endosomal GLP-1R signaling. These effects did not involve changes in receptor phosphorylation or lipid nanodomain segregation. Moreover, we found that mini-G proteins derived from Gαi and Gαq also inhibited the internalization of GPCRs that couple to them. Finally, we developed an alternative intracellular signaling assay for GLP-1R using a nanobody specific for active Gαs:GPCR complexes (Nb37) that did not affect GLP-1R internalization. Our results have important implications for designing methods to assess intracellular GPCR signaling. Editor's summary: Mini-G proteins are engineered forms of Gα subunits that stably interact with activated G protein–coupled receptors (GPCRs) and are often used to investigate GPCR signaling from specific subcellular compartments. Manchanda et al. found that mini-G proteins interfered with GPCR internalization and compartmentalized signaling. Overexpression of a mini-G protein derived from Gαs prevented agonist-induced β-arrestin 2 recruitment to the glucagon-like peptide 1 receptor (GLP-1R), which inhibited receptor internalization and signaling at endosomes. Other GPCRs also showed reduced internalization when the mini-G versions of their cognate Gα subunits were overexpressed. These potentially confounding effects indicate that results from experiments using mini-G proteins to measure compartmentalized GPCR signaling should be interpreted cautiously. —Annalisa M. VanHook [ABSTRACT FROM AUTHOR]

Context: Non-small cell lung cancer (NSCLC) is the most common type of lung cancer. Gefitinib is a first-line treatment for NSCLC. however, its effectiveness is hindered by the development of drug resistance. at present, shenqi Fuzheng injection (SFI) is widely accepted as an adjuvant therapy in NSCLC. Objective: this study investigates the molecular mechanism of SFI when combined with gefitinib in regulating cell progression among EGFR-TKI-resistant NSCLC. Materials and methods: We established gefitinib-resistant PC9-GR cells by exposing gefitinib escalation from 10 nM with the indicated concentrations of SFI in PC9 cells (1, 4, and 8 mg/ml). Quantitative real-time polymerase chain reaction was performed to assess gene expression. PC9/GR and H1975 cells were treated with 50 ng/ml of interleukin (IL)-22 alone or in combination with 10 mg/ml of SFI. STAT3, p-STAT3, AKT, and p-AKT expression were evaluated using Western blot. the effects on cell proliferation, clonogenicity, and apoptosis in Nsclc cells were assessed by 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide (MTT), colony formation and flow cytometry assays. Results: SFI treatment alleviated the development of gefitinib resistance in NSCLC. PC9/GR and H1975 cells treated with SFI significantly exhibited a reduction in IL-22 protein and mRNa overexpression levels. sFi effectively counteracted the activation of the stat3/aKt signaling pathway induced by adding exogenous IL-22 to Pc9/GR and H1975 cells. Moreover, IL-22 combined with gefitinib markedly increased cell viability while reducing apoptosis. in contrast, combining SFI with gefitinib and the concurrent treatment of SFI with gefitinib and IL-22 demonstrated the opposite effect. Discussion and Conclusion: sFi can be a valuable therapeutic option to address gefitinib resistance in NSCLC by suppressing the IL-22/STAT3/AKT pathway. [ABSTRACT FROM AUTHOR]

The relative amount of AMPA receptors expressed at the surface of neurons can be measured using superecliptic pHluorin (SEP) labeling at their N-terminus. However, the high signal variability resulting from protein overexpression in neurons and the low signal observed in intracellular vesicles make quantitative characterization of receptor trafficking difficult. Here, we establish a real-time live-cell assay of AMPAR trafficking based on fluorescence lifetime imaging (FLIM), which allows for simultaneous visualization of both surface and intracellular receptors. Using this assay, we found that elevating amyloid-beta (Ab) levels leads to a strong increase in intracellular GluA1 and GluA2-containing receptors, indicating that Aβ triggers the endocytosis of these AMPARs. In APP/PS1 Alzheimer's disease model mouse neurons, FLIM revealed strikingly different AMPAR trafficking properties for GluA1- and GluA3- containing receptors, suggesting that chronic Aβ exposure triggered the loss of both surface and intracellular GluA3-containing receptors. Interestingly, overexpression of protein phosphatase 1 (PP1) also resulted inGluA1 endocytosis as well as depressed synaptic transmission, confirming the important role of phosphorylation in regulating AMPAR trafficking. This new approach allows for the quantitative measurement of extracellular pH, small changes in receptor trafficking, as well as simultaneous measurement of surface and internalized AMPARs in living neurons, and could therefore be applied to several different studies in the future. [ABSTRACT FROM AUTHOR]

Background: Globally, breast cancer in women is the fifth leading cause of cancer death. There is an urgent need to explore the molecular mechanism of breast cancer proliferation and metastasis. Method: TCGA database analysis was used to analyze genes expression in breast cancer and normal samples and the association between gene expression and prognosis. Immunohistochemical staining, qPCR and western blotting was sued to detected gene expression. The cell function tests were conducted to investigate the effects of TEX19 and CDK4 with abnormal expression on cell proliferation, migration, apoptosis, cell cycle, and colony formation. Bioinformatics analysis methods combined with CHX tracking experiment and Co-IP experiment were performed to screen and verify the downstream molecule and regulatory mechanism of TEX19. Besides, subcutaneous tumorigenesis model in nude mice was constructed. Results: TEX19 was significantly upregulated in breast cancer, and the TEX19 level was related to tumor invasion and prognosis. TEX19 knockdown inhibited the proliferation and migration of breast cancer cells, increased cell apoptosis, and blocked the cell cycle in the G2 phase. Besides, TEX19 suppressed the growth of tumors in the body. Mechanically, TEX19 upregulated the level of CDK4 protein, which depended on the E3 ubiquitin ligase SKP2. Specifically, TEX19 knockdown and SKP2 protein overexpression destroyed the stability of CDK4 protein and enhanced the ubiquitination of CDK4 protein. Additionally, CDK4 knockdown inhibited the proliferation, migration, and colony formation of breast cancer cells, and alleviated the promotion of TEX19 overexpression on the proliferation and migration of breast cancer cell. Conclusion: TEX19 and CDK4 were upregulated in breast cancer, and TEX19 increased the level of CDK4 protein by influencing SKP2-mediated ubiquitination of CDK4, thereby promoting the progression of breast cancer. [ABSTRACT FROM AUTHOR]

Approximately 50% of patients with hematologic malignancies relapse after chimeric antigen receptor (CAR) T cell treatment; mechanisms of failure include loss of CAR T persistence and tumor resistance to apoptosis. We hypothesized that both of these challenges could potentially be overcome by overexpressing one or more of the Bcl-2 family proteins in CAR T cells to reduce their susceptibility to apoptosis, both alone and in the presence of BH3 mimetics, which can be used to activate apoptotic machinery in malignant cells. We comprehensively investigated overexpression of different Bcl-2 family proteins in CAR T cells with different signaling domains as well as in different tumor types. We found that Bcl-xL and Bcl-2 overexpression in CAR T cells bearing a 4-1BB costimulatory domain resulted in increased expansion and antitumor activity, reduced exhaustion, and decreased apoptotic priming. In addition, CAR T cells expressing either Bcl-xL or a venetoclax-resistant Bcl-2 variant led to enhanced antitumor efficacy and survival in murine xenograft models of lymphoma and leukemia in the presence or absence of the BH3 mimetic venetoclax, a clinically approved BH3 mimetic. In this setting, Bcl-xL overexpression had stronger effects than overexpression of Bcl-2 or the Bcl-2(G101V) variant. These findings suggest that CAR T cells could be optimally engineered by overexpressing Bcl-xL to enhance their persistence while opening a therapeutic window for combination with BH3 mimetics to prime tumors for apoptosis. Editor's summary: A major issue with chimeric antigen receptor (CAR) T cell therapy is poor persistence of the CAR T cells. To address this, Korell et al. developed CAR T cells overexpressing members of the Bcl-2 family, proteins involved in protection from apoptosis. The authors found that overexpression of Bcl-xL in particular improved CAR T cell persistence and function in vivo and could be combined with the BH3 mimetic venetoclax to further improve tumor control in mice. Together, these data highlight a potential strategy to improve CAR T cell therapies as well as suggest a combination therapy with BH3 mimetics. —Courtney Malo [ABSTRACT FROM AUTHOR]

Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease marked by inflammatory cell infiltration and joint damage. The Chinese government has approved the prescription medication sinomenine (SIN), an effective anti-inflammation drug, for treating RA. This study evaluated the possible anti-inflammatory actions of SIN in RA based on bioinformatics analysis and experiments. Six microarray datasets were acquired from the gene expression omnibus (GEO) database. We used R software to identify differentially expressed genes (DEGs) and perform function evaluations. The CIBERSORT was used to calculate the abundance of 22 infiltrating immune cells. The weighted gene co-expression network analysis (WGCNA) was used to discover genes associated with M1 macrophages. Four public datasets were used to predict the genes of SIN. Following that, function enrichment analysis for hub genes was performed. The cytoHubba and least absolute shrinkage and selection operator (LASSO) were employed to select hub genes, and their diagnostic effectiveness was predicted using the receiver operator characteristic (ROC) curve. Molecular docking was undertaken to confirm the affinity between the SIN and hub gene. Furthermore, the therapeutic efficacy of SIN was validated in LPS-induced RAW264.7 cells line using Western blot and Enzyme-linked immunosorbent assay (ELISA). The matrix metalloproteinase 9 (MMP9) was identified as the hub M1 macrophages-related biomarker in RA using bioinformatic analysis and molecular docking. Our study indicated that MMP9 took part in IL-17 and TNF signaling pathways. Furthermore, we found that SIN suppresses the MMP9 protein overexpression and pro-inflammatory cytokines, including tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in the LPS-induced RAW264.7 cell line. In conclusion, our work sheds new light on the pathophysiology of RA and identifies MMP9 as a possible RA key gene. In conclusion, the above findings demonstrate that SIN, from an emerging research perspective, might be a potential cost-effective anti-inflammatory medication for treating RA. [ABSTRACT FROM AUTHOR]

In the United States, it is now estimated that 6.7 million people over the age of 65 are afflicted by Alzheimer's disease (AD), over 1 million people are living with Parkinson's disease (PD), and over 200 000 have or are at risk for developing Huntington's disease (HD). All three of these neurodegenerative diseases result in the ultimate death of distinct neuronal subtypes, and it is widely thought that age-related damage is the single biggest contributing factor to this neuronal death. However, recent studies are now suggesting that developmental defects during early neurogenesis could also play a role in the pathology of neurodegenerative diseases. Loss or overexpression of proteins associated with HD, PD, and AD also result in embryonic phenotypes but whether these developmental defects slowly unmask over time and contribute to age-related neurodegeneration remains highly debated. Here, we discuss known links between embryonic neurogenesis and neurodegenerative disorders (including common signaling pathways), potential compensatory mechanisms that could delay presentation of neurodegenerative disorders, and the types of model systems that could be used to study these links in vivo. [ABSTRACT FROM AUTHOR]

Glioblastoma multiform (GBM) is considered the deadliest brain cancer. Conventional therapies are followed by poor patient survival outcomes, so novel and more efficacious therapeutic strategies are imperative to tackle this scourge. Gene therapy has emerged as an exciting and innovative tool in cancer therapy. Its combination with chemotherapy has significantly improved therapeutic outcomes. In line with this, our team has developed temozolomide–transferrin (Tf) peptide (WRAP5)/p53 gene nanometric complexes that were revealed to be biocompatible with non-cancerous cells and in a zebrafish model and were able to efficiently target and internalize into SNB19 and U373 glioma cell lines. The transfection of these cells, mediated by the formulated peptide-drug/gene complexes, resulted in p53 expression. The combined action of the anticancer drug with p53 supplementation in cancer cells enhances cytotoxicity, which was correlated to apoptosis activation through quantification of caspase-3 activity. In addition, increased caspase-9 levels revealed that the intrinsic or mitochondrial pathway of apoptosis was implicated. This assumption was further evidenced by the presence, in glioma cells, of Bax protein overexpression—a core regulator of this apoptotic pathway. Our findings demonstrated the great potential of peptide TMZ/p53 co-delivery complexes for cellular transfection, p53 expression, and apoptosis induction, holding promising therapeutic value toward glioblastoma. [ABSTRACT FROM AUTHOR]

Adenoid cystic carcinoma (AdCC) of the Bartholin's gland (AdCC-BG) is a very rare gynecologic vulvar malignancy. AdCC-BGs are slow-growing but locally aggressive and are associated with high recurrence rates. Here we sought to characterize the molecular underpinning of AdCC-BGs. AdCC-BGs (n = 6) were subjected to a combination of RNA-sequencing, targeted DNA-sequencing, reverse-transcription PCR, fluorescence in situ hybridization (FISH) and MYB immunohistochemistry (IHC). Clinicopathologic variables, somatic mutations, copy number alterations and chimeric transcripts were assessed. All six AdCC-BGs were biphasic, composed of ductal and myoepithelial cells. Akin to salivary gland and breast AdCCs, three AdCC-BGs had the MYB :: NFIB fusion gene with varying breakpoints, all of which were associated with MYB overexpression by IHC. Two AdCC-BGs were underpinned by MYBL1 fusion genes with different gene partners, including MYBL1 :: RAD51B and MYBL1 :: EWSR1 gene fusions, and showed MYB protein expression. Although the final AdCC-BG studied had MYB protein overexpression, no gene fusion was identified. AdCC-BGs harbored few additional somatic genetic alterations, and only few mutations in cancer-related genes were identified, including GNAQ , GNAS , KDM6A , AKT1 and BCL2 , none of which were recurrent. Two AdCC-BGs, both with a MYB :: NFIB fusion gene, developed metastatic disease. AdCC-BGs constitute a convergent phenotype, whereby activation of MYB or MYBL1 can be driven by the MYB :: NFIB fusion gene or MYBL1 rearrangements. Our observations further support the notion that AdCCs, irrespective of organ site, constitute a genotypic–phenotypic correlation. Assessment of MYB or MYBL1 rearrangements may be used as an ancillary marker for the diagnosis of AdCC-BGs. • Adenoid cystic carcinoma of the Bartholin's gland (AdCC-BG) is underpinned by MYB or MYBL1 fusion genes. • AdCC-BGs have few somatic genetic alterations in addition to the MYB/ MYBL1 fusion gene. • AdCC-BG constitutes a genotypic–phenotypic correlation. • MYB/ MYBL1 rearrangement assessment may be used as an ancillary marker for AdCC-BG diagnosis. [ABSTRACT FROM AUTHOR]

Alzheimer's disease (AD) is a progressive neurodegenerative disease with no effective treatments, not least due to the lack of authentic animal models. Typically, rodent models recapitulate the effects but not causes of AD, such as cholinergic neuron loss: lesioning of cholinergic neurons mimics the cognitive decline reminiscent of AD but not its neuropathology. Alternative models rely on the overexpression of genes associated with familial AD, such as amyloid precursor protein, or have genetically amplified expression of mutant tau. Yet transgenic rodent models poorly replicate the neuropathogenesis and protein overexpression patterns of sporadic AD. Seeding rodents with amyloid or tau facilitates the formation of these pathologies but cannot account for their initial accumulation. Intracerebral infusion of proinflammatory agents offer an alternative model, but these fail to replicate the cause of AD. A novel model is therefore needed, perhaps similar to those used for Parkinson's disease, namely adult wildtype rodents with neuron-specific (dopaminergic) lesions within the same vulnerable brainstem nuclei, 'the isodendritic core', which are the first to degenerate in AD. Site-selective targeting of these nuclei in adult rodents may recapitulate the initial neurodegenerative processes in AD to faithfully mimic its pathogenesis and progression, ultimately leading to presymptomatic biomarkers and preventative therapies. [ABSTRACT FROM AUTHOR]

Plasmodium knowlesi is the only Plasmodium that causes zoonotic disease among the Plasmodium that cause infection in humans. It is fatal due to its short asexual growth cycle within 24 h. Lactate dehydrogenase (LDH), an enzyme that catalyzes the final step of glycolysis, is a biomarker for diagnosing infection by Plasmodium spp. parasite. Therefore, this study aimed to efficiently produce the soluble form of P. knowlesi LDH (PkLDH) using a bacterial expression system for studying malaria caused by P. knowlesi. Recombinant pET-21a(+)-PkLDH plasmid was constructed by inserting the PkLDH gene into a pET-21a(+) expression vector. Subsequently, the recombinant plasmid was inserted into the protein-expressing Escherichia coli Rosetta(DE3) strain, and the optimal conditions for overexpression of the PkLDH protein were established using this strain. We obtained a yield of 52.0 mg/L PkLDH from the Rosetta(DE3) strain and confirmed an activity of 483.9 U/mg through experiments. This methodology for high-efficiency PkLDH production can be utilized for the development of diagnostic methods and drug candidates for distinguishing malaria caused by P. knowlesi. [ABSTRACT FROM AUTHOR]

Verticillium dahliae is a xylem‐invading fungal pathogen that causes vascular wilt in a wide range of angiosperms. The pathogen uses a variety of virulence factors to invade and colonize its hosts. Here, we report that VdNEP, an NLP (Necrosis and ethylene inducing peptide 1‐Like Protein), functions as one such factor in multiple hosts. Eggplant leaves treated with VdNEP developed necrotic symptoms. Overexpression of VdNEP by incorporating extra copies of the VdNEP gene increased virulence to cotton, eggplant and tomato plants, suggesting its role as a virulence factor in diverse plants. Increased expression of VdNEP among the transformants did not correlate with the number of VdNEP inserts, suggesting that its expression was affected by the genomic context of the insertion sites. Interestingly, a transformant derived from a defoliating strain with high VdNEP transcript levels caused disease symptoms in tomato plants, whereas the corresponding wild‐type strain did not cause visible symptoms. The amount of V. dahliae DNA in plants infected with this VdNEP‐overexpressing transformant was 22 times higher than that in plants infected with the wild‐type isolate, further supporting the critical role of VdNEP in infection. A VdNEP‐EGFP fusion was constructed to follow its localization in fungal cells and during infection. [ABSTRACT FROM AUTHOR]

Amelogenesis imperfecta (AI) is a diverse group of inherited diseases featured by various presentations of enamel malformations that are caused by disturbances at different stages of enamel formation. While hypoplastic AI suggests a thickness defect of enamel resulting from aberrations during the secretory stage of amelogenesis, hypomaturation AI indicates a deficiency of enamel mineralization and hardness established at the maturation stage. Mutations in ENAM, which encodes the largest enamel matrix protein, enamelin, have been demonstrated to cause generalized or local hypoplastic AI. Here, we characterized 2 AI families with disparate hypoplastic and hypomaturation enamel defects and identified 2 distinct indel mutations at the same location of ENAM, c588+1del and c.588+1dup. Minigene splicing assays demonstrated that they caused frameshifts and truncation of ENAM proteins, p.Asn197Ilefs*81 and p.Asn197Glufs*25, respectively. In situ hybridization of Enam on mouse mandibular incisors confirmed its restricted expression in secretory stage ameloblasts and suggested an indirect pathogenic mechanism underlying hypomaturation AI. In silico analyses indicated that these 2 truncated ENAMs might form amyloid structures and cause protein aggregation with themselves and with wild-type protein through the added aberrant region at their C-termini. Consistently, protein secretion assays demonstrated that the truncated proteins cannot be properly secreted and impede secretion of wild-type ENAM. Moreover, compared to the wild-type, overexpression of the mutant proteins significantly increased endoplasmic reticulum stress and upregulated the expression of unfolded protein response (UPR)–related genes and TNFRSF10B, a UPR-controlled proapoptotic gene. Caspase, terminal deoxynucleotidyl transferase UTP nick-end labeling (TUNEL), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assays further revealed that both truncated proteins, especially p.Asn197Ilefs*81, induced cell apoptosis and decreased cell survival, suggesting that the 2 ENAM mutations cause AI through ameloblast cell pathology and death rather than through a simple loss of function. This study demonstrates that an ENAM mutation can lead to generalized hypomaturation enamel defects and suggests proteinopathy as a potential pathogenesis for ENAM -associated AI. [ABSTRACT FROM AUTHOR]

Inclusion bodies (IBs) are protein aggregates formed as a result of overexpression of recombinant protein in E. coli. The formation of IBs is a valuable strategy of recombinant protein production despite the need for additional processing steps, i.e., isolation, solubilization and refolding. Industrial process development of protein refolding is a labor-intensive task based largely on empirical approaches rather than knowledge-driven strategies. A prerequisite for knowledge-driven process development is a reliable monitoring strategy. This work explores the potential of intrinsic tryptophan and tyrosine fluorescence for real-time and in situ monitoring of protein refolding. In contrast to commonly established process analytical technology (PAT), this technique showed high sensitivity with reproducible measurements for protein concentrations down to 0.01 g L - 1 . The change of protein conformation during refolding is reflected as a shift in the position of the maxima of the tryptophan and tyrosine fluorescence spectra as well as change in the signal intensity. The shift in the peak position, expressed as average emission wavelength of a spectrum, was correlated to the amount of folding intermediates whereas the intensity integral correlates to the extent of aggregation. These correlations were implemented as an observation function into a mechanistic model. The versatility and transferability of the technique were demonstrated on the refolding of three different proteins with varying structural complexity. The technique was also successfully applied to detect the effect of additives and process mode on the refolding process efficiency. Thus, the methodology presented poses a generic and reliable PAT tool enabling real-time process monitoring of protein refolding. Real-time intrinsic fluorescence monitoring in protein refolding [ABSTRACT FROM AUTHOR]

Multidrug resistance (MDR) is frequently induced after long-term exposure to reduce the therapeutic effect of chemotherapeutic drugs, which is always associated with the overexpression of efflux proteins, such as P-glycoprotein (P-gp). Nano-delivery technology can be used as an efficient strategy to overcome tumor MDR. In this study, mesoporous silica nanoparticles (MSNs) were synthesized and linked with a disulfide bond and then coated with lipid bilayers. The functionalized shell/core delivery systems (HT-LMSNs-SS@DOX) were developed by loading drugs inside the pores of MSNs and conjugating with D-α-tocopherol polyethylene glycol 1000 succinate (TPGS) and hyaluronic acid (HA) on the outer lipid surface. HT-LMSNs-SS and other carriers were characterized and assessed in terms of various characteristics. HT-LMSNs-SS@DOX exhibited a dual pH/reduction responsive drug release. The results also showed that modified LMSNs had good dispersity, biocompatibility, and drug-loading capacity. In vitro experiment results demonstrated that HT-LMSNs-SS were internalized by cells and mainly by clathrin-mediated endocytosis, with higher uptake efficiency than other carriers. Furthermore, HT-LMSNs-SS@DOX could effectively inhibit the expression of P-gp, increase the apoptosis ratios of MCF-7/ADR cells, and arrest cell cycle at the G0/G1 phase, with enhanced ability to induce excessive reactive oxygen species (ROS) production in cells. In tumor-bearing model mice, HT-LMSNs-SS@DOX similarly exhibited the highest inhibition activity against tumor growth, with good biosafety, among all of the treatment groups. Therefore, the nano-delivery systems developed herein achieve enhanced efficacy towards resistant tumors through targeted delivery and redox-responsive drug release, with broad application prospects. [ABSTRACT FROM AUTHOR]

Aim: Chronic heart failure (CHF) is often linked to liver malfunction and systemic endothelial dysfunction. However, whether cardio‐hepatic interactions in heart failure involve dysfunction of liver sinusoidal endothelial cells (LSECs) is not known. Here we characterize LSECs phenotype in early and end stages of chronic heart failure in a murine model. Methods: Right ventricle (RV) function, features of congestive hepatopathy, and the phenotype of primary LSECs were characterized in Tgαq*44 mice, with cardiomyocyte‐specific overexpression of the Gαq protein, at the age of 4‐ and 12‐month representative for early and end‐stage phases of CHF, respectively. Results: 4‐ and 12‐month‐old Tgαq*44 mice displayed progressive impairment of RV function and alterations in hepatic blood flow velocity resulting in hepatic congestion with elevated GGT and bilirubin plasma levels and decreased albumin concentration without gross liver pathology. LSECs isolated from 4‐ and 12‐month‐old Tgαq*44 mice displayed significant loss of fenestrae with impaired functional response to cytochalasin B, significant changes in proteome related to cytoskeleton remodeling, and altered vasoprotective function. However, LSECs barrier function and bioenergetics were largely preserved. In 4‐ and 12‐month‐old Tgαq*44 mice, LSECs defenestration was associated with prolonged postprandial hypertriglyceridemia and in 12‐month‐old Tgαq*44 mice with proteomic changes of hepatocytes indicative of altered lipid metabolism. Conclusion: Tgαq*44 mice displayed right‐sided HF and altered hepatic blood flow leading to LSECs dysfunction involving defenestration, shift in eicosanoid profile, and proteomic changes. LSECs dysfunction appears as an early and persistent event in CHF, preceding congestive hepatopathy and contributing to alterations in lipoprotein transport and CHF pathophysiology. [ABSTRACT FROM AUTHOR]

Long non-coding RNAs (lncRNAs) play crucial roles in the tumorigenesis and progression of lung adenocarcinoma (LUAD). However, little was known about the role of lncRNAs in high-risk LUAD subtypes: micropapillary-predominant adenocarcinoma (MPA) and solid-predominant adenocarcinoma (SPA). In this study, we conducted a systematic screening of differentially expressed lncRNAs using RNA sequencing in 10 paired MPA/SPA tumor tissues and adjacent normal tissues. Consequently, 110 significantly up-regulated lncRNAs and 288 aberrantly down-regulated lncRNAs were identified (|Log2 Foldchange| ≥ 1 and corrected P < 0.05). The top 10 lncRNAs were further analyzed in 89 MPA/SPA tumor tissues and 59 normal tissues from The Cancer Genome Atlas database. Among them, CARD8-AS1 showed the most significant differential expression, and decreased expression of CARD8-AS1 was significantly associated with a poorer prognosis. Functionally, CARD8-AS1 overexpression remarkably suppressed the proliferation, migration and invasion of LUAD cells both in vitro and in vivo. Conversely, inhibition of CARD8-AS1 yielded opposite effects. Mechanistically, CARD8-AS1 acted as a scaffold to facilitate the interaction between TXNRD1 and E3 ubiquitin ligase TRIM25, thereby promoting the degradation of TXNRD1 through the ubiquitin–proteasome pathway. Additionally, TXNRD1 was found to promote LUAD cell proliferation, migration and invasion in vitro. Furthermore, the suppressed progression of LUAD cells resulting from CARD8-AS1 overexpression could be significantly reversed by simultaneous overexpression of TXNRD1. In conclusion, this study revealed that the lncRNA CARD8-AS1 played a suppressive role in the progression of LUAD by enhancing TRIM25-mediated ubiquitination of TXNRD1. The CARD8-AS1-TRIM25-TXNRD1 axis may represent a promising therapeutic target for LUAD. [ABSTRACT FROM AUTHOR]

Activation of the Wnt-β-catenin signaling pathway by CHIR99021, a specific inhibitor of GSK3β, induces Tcf7l1 protein degradation, which facilitates the maintenance of an undifferentiated state in mouse embryonic stem cells (mESCs); however, the precise mechanism is still unclear. Here, we showed that the overexpression of transducin-β-like protein 1 (Tbl1, also known as Tbl1x) or its family member Tblr1 (also known as Tbl1xr1) can decrease Tcf7l1 protein levels, whereas knockdown of each gene increases Tcf7l1 levels without affecting Tcf7l1 transcription. Interestingly, only Tbl1, and not Tblr1, interacts with Tcf7l1.Mechanistically, Tbl1 translocates from the cytoplasm into the nucleus in association with β-catenin (CTNNB1) after the addition of CHIR99021 and functions as an adaptor to promote ubiquitylation of the Tcf7l1 protein. Functional assays further revealed that enforced expression of Tbl1 is capable of delaying mESC differentiation. In contrast, knockdown of Tbl1 attenuates the effect of CHIR99021 on Tcf7l1 protein stability and mESC self-renewal. Our results provide insight into the regulatory network of the Wnt-β-catenin signaling pathway involved in promoting the maintenance of naïve pluripotency. [ABSTRACT FROM AUTHOR]

Thioredoxin (Trx) is a small redox mediator protein involved in the regulation of various chloroplast functions by modulating the redox state of Trx target proteins in ever-changing light environments. Using reducing equivalents produced by the photosynthetic electron transport chain, Trx reduces the disulfide bonds on target proteins and generally turns on their activities. While the details of the protein-reduction mechanism by Trx have been well investigated, the oxidation mechanism that counteracts it has long been unclear. We have recently demonstrated that Trx-like proteins such as Trx-like2 and atypical Cys His-rich Trx (ACHT) can function as protein oxidation factors in chloroplasts. Our latest study on transgenic Arabidopsis plants indicated that the ACHT isoform ACHT2 is involved in regulating the thermal dissipation of light energy. To understand the role of ACHT2 in vivo, we characterized phenotypic changes specifically caused by ACHT2 overexpression in Arabidopsis. ACHT2-overexpressing plants showed growth defects, especially under high light conditions. This growth phenotype was accompanied with the impaired reductive activation of Calvin–Benson cycle enzymes, enhanced thermal dissipation of light energy, and decreased photosystem II activity. Overall, ACHT2 overexpression promoted protein oxidation that led to the inadequate activation of Calvin–Benson cycle enzymes in light and consequently induced negative feedback control of the photosynthetic electron transport chain. This study highlights the importance of the balance between protein reduction and oxidation in chloroplasts for optimal photosynthetic performance and plant growth. [ABSTRACT FROM AUTHOR]

Overexpression of the 14-3-3ε protein is associated with suppression of apoptosis in cutaneous squamous cell carcinoma (cSCC). This antiapoptotic activity of 14-3-3ε is dependent on its binding to CDC25A; thus, inhibiting 14-3-3ε – CDC25A interaction is an attractive therapeutic approach to promote apoptosis in cSCC. In this regard, designing peptide inhibitors of 14-3-3ε – CDC25A interactions is of great interest. This work reports the rational design of peptide analogs of pS, a CDC25A-derived peptide that has been shown to inhibit 14-3-3ε–CDC25A interaction and promote apoptosis in cSCC with micromolar IC50. We designed new peptide analogs in silico by shortening the parent pS peptide from 14 to 9 amino acid residues; then, based on binding motifs of 14-3-3 proteins, we introduced modifications in the pS(174–182) peptide. We studied the binding of the peptides using conventional molecular dynamics (MD) and steered MD simulations, as well as biophysical methods. Our results showed that shortening the pS peptide from 14 to 9 amino acids reduced the affinity of the peptide. However, substituting Gln176 with either Phe or Tyr amino acids rescued the binding of the peptide. The optimized peptides obtained in this work can be candidates for inhibition of 14-3-3ε – CDC25A interactions in cSCC. [ABSTRACT FROM AUTHOR]

[Display omitted] • HMGB1/STAT3/p65 axis mediated microglial activation and autophagy in mPFC correlated with MDD were first identified, and HMGB1 was found to be a potential biomarker for MDD. • Increased HMGB1 expression was mainly observed in microglia from the medial prefrontal cortex of MDD model mice. • Specific knockdown of HMGB1 rescues chronic stress-induced depressive-related phenotypes, microglial activation, and autophagy in mice. • The effects induced by chronic stress were mimicked by exogenous administration of recombinant HMGB1 or specific overexpression of HMGB1, while blocked by STAT3 specific inhibitor or p65 knockdown. • Inhibition of HMGB1/STAT3/p65 axis prevented LPS-induced microglial activation and autophagy in vitro , which was reversed by recombinant HMGB1 protein overexpression. • Microglial HMGB1/STAT3/p65 axis within the mPFC is a novel therapeutic target in the treatment of MDD associated with stress. Neuroinflammation and autophagy are implicated in stress-related major depressive disorder (MDD), but the underlying molecular mechanisms remain largely unknown. Here, we identified that MDD regulated by HMGB1/STAT3/p65 axis mediated microglial activation and autophagy for the first time. Further investigations were performed to uncover the effects of this axis on MDD in vivo and in vitro. Bioinformatics analyses were used to re-analysis the transcriptome data from the dorsolateral prefrontal cortex (dlPFC) of post-mortem male MDD patients. The expression level of HMGB1 and its correlation with depression symptoms were explored in MDD clinical patients and chronic social defeat stress (CSDS)-induced depression model mice. Specific adeno-associated virus and recombinant (r)HMGB1 injection into the medial PFC (mPFC) of mice, and pharmacological inhibitors with rHMGB1 in two microglial cell lines exposed to lipopolysaccharide were used to analyze the effects of HMGB1/STAT3/p65 axis on MDD. The differential expression of genes from MDD patients implicated in microglial activation and autophagy regulated by HMGB1/STAT3/p65 axis. Serum HMGB1 level was elevated in MDD patients and positively correlated with symptom severity. CSDS not only induced depression-like states in mice, but also enhanced microglial reactivity, autophagy as well as activation of the HMGB1/STAT3/p65 axis in mPFC. The expression level of HMGB1 was mainly increased in the microglial cells of CSDS-susceptible mice, which also correlated with depressive-like behaviors. Specific HMGB1 knockdown produced a depression-resilient phenotype and suppressed the associated microglial activation and autophagy effects of CSDS-induced. The effects induced by CSDS were mimicked by exogenous administration of rHMGB1 or specific overexpression of HMGB1, while blocked by STAT3 inhibitor or p65 knockdown. In vitro , inhibition of HMGB1/STAT3/p65 axis prevented lipopolysaccharide-induced microglial activation and autophagy, while rHMGB1 reversed these changes. Our study established the role of microglial HMGB1/STAT3/p65 axis in mPFC in mediating microglial activation and autophagy in MDD. [ABSTRACT FROM AUTHOR]

CBL-interacting protein kinases (CIPKs) play important regulatory roles in plant growth development and abiotic stress tolerance. However, the biological roles of these genes in response to low-nitrate (LN) stress in potato plants have not been determined. Here, we reported that StCIPK23 was expressed mainly in roots and leaves. StCIPK23 was located mainly in the cell membrane, nucleus, and cytoplasm. Further research suggested that, compared with wild-type (WT) plants, StCIPK23-overexpressing plants were taller and had significantly greater nitrate and ammonium nitrogen contents under LN stress. StCIPK23 overexpression can increase StAT, StNRT2.1, StNR, StGS1-3, and StGOGAT expression levels in StCIPK23 transgenic seedlings compared to those in WT plants under LN stress. The results of yeast two-hybrid and luciferase complementation imaging experiments suggested that StCIPK23 could interact with StCBL3. Real-time reverse transcription–PCR revealed the StCIPK23 expression level peaked at 6 h and the StCBL3 expression level peaked at 9 h in the roots under LN stress. In conclusion, we found that StCIPK23 and StCBL3 form a complex to regulate the expression of key genes in the nitrogen metabolism pathway to improve LN tolerance in potato plants. [ABSTRACT FROM AUTHOR]