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Rapid tumor growth and insufficient blood supply leads to the development of a hypoxic and nutrient deprived microenvironment. To survive, tumor cells need to tolerate these adverse conditions. Here we found the expression of CD39 was enhanced in necrotic regions distant from blood vessels. We speculate that this is a strategy for tumor cells to actively adapt to the hostile environment. Further studies showed that CD39 was induced by nutrient deprivation through the AMPK signalling pathway. We next explored the significance of CD39 for tumor cells. Our results showed that CD39 reduced cellular oxygen consumption, which could be significant for tumor cells if the available oxygen is limited. Metabolomics analysis showed that overexpression of CD39 significantly altered cellular metabolism, and tricarboxylic acid (TCA) cycle was identified as the most impacted metabolic pathway. In order to explore the molecular mechanism, we performed RNA-seq analysis. The results showed that CD39 significantly up-regulated the expression of pyruvate dehydrogenase kinase isozyme 2 (PDK2), thus inhibiting the activity of pyruvate dehydrogenase (PDH) and TCA cycle. Finally, CD39 was shown to protect tumor cells from hypoxia-induced cell death and reduce intratumoral hypoxia levels. CD39 has attracted a great deal of attention as a newly discovered immune checkpoint molecule in recent years. Our results indicate that CD39 not only plays a role in immune regulation, but also enables tumor cells to tolerate hypoxia by inhibiting TCA cycle and reducing cellular oxygen consumption. This study provides evidence that targeting CD39 may be a novel strategy to prevent adaptation of tumor cells in stressed conditions., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Thiamine deficiency is a well-known risk factor for the development of severe encephalopathy, such as Wernicke encephalopathy and Korsakoff syndrome, but the underlying mechanism is still mysterious. This study aims to investigate the expression levels of thiamine metabolism genes in different tissues and their impact on brain susceptibility to thiamine deficiency. The mRNA and protein levels of four genes known to be associated with thiamine metabolism: thiamine pyrophosphokinase-1 ( Tpk ), Solute carrier family 19 member 2 ( Slc19a2 ), Slc19a3 , and Slc25a19 , in the brain, kidney, and liver of mice were examined. Thiamine diphosphate (TDP) levels were measured in these tissues. Mice were subjected to dietary thiamine deprivation plus pyrithiamine (PTD), a specific TPK inhibitor, or pyrithiamine alone to observe the reduction in TDP and associated pathological changes. TPK mRNA and protein expression levels were lowest in the brain compared to the kidney and liver. Correspondingly, TDP levels were also lowest in the brain. Mice treated with PTD or pyrithiamine alone showed an initial reduction in brain TDP levels, followed by reductions in the liver and kidney. PTD treatment caused significant neuron loss, neuroinflammation, and blood-brain barrier disruption, whereas dietary thiamine deprivation alone did not. TPK expression level is the best indicator of thiamine metabolism status. Low TPK expression in the brain appears likely to contribute to brain susceptibility to thiamine deficiency, underscoring a critical role of TPK in maintaining cerebral thiamine metabolism and preventing thiamine deficiency-related brain lesions., (Copyright © 2024 The Author(s). Published by Wolters Kluwer Health, Inc.)

Objective: To explore the application of triplet-primer PCR (TP-PCR) for the genetic testing and prenatal diagnosis in patients with Myotonic dystrophy type 1 (DM1)., Methods: A total of 60 individuals from 48 pedigrees undergoing genetic testing at the Genetic and Prenatal Diagnosis Center of the First Affiliated Hospital of Zhengzhou University from May 2018 to October 2022 were selected as the study subjects. TP-PCR combined with capillary electrophoresis was applied to determine the number of CTG repeats of the DMPK gene, and prenatal testing was provided to four DM1 pedigrees. This study was approved by the First Affiliated Hospital of Zhengzhou University (Ethics No. KS-2018-KY-36)., Results: A total of 52 DM1 patients were detected, mostly with muscle weakness, muscular atrophy and myotonia as the initial symptoms, along with typical myotonic potentials. Some patients also had abnormalities of other systems. The number of abnormal CTG repeats of the DMPK gene was > 50, whilst the number of CTG repeats on the normal allele had ranged from 5 to 18. The number of the most common normal CAG repeats was 6 (30.77%, 16/52). Among the four DM1 pedigrees undergoing prenatal diagnosis, one fetus was healthy, whilst three fetuses were found to have abnormal CTG repeats (> 50 times) and diagnosed with DM1., Conclusion: TP-PCR can diagnose DM1 patients with speed and accuracy. However, this method cannot accurately determine the number of CTG repeats when it exceeds 50.

The integrated stress response (ISR) is a vital signaling pathway initiated by four kinases, PERK, GCN2, HRI and PKR, that ensure cellular resilience and protect cells from challenges. Here, we investigated whether increasing ISR signaling could rescue diabetes-like phenotypes in a mouse model of diet-induced obesity (DIO). We show that the orally available and clinically approved GCN2 activator halofuginone (HF) can activate the ISR in mouse tissues. We found that daily oral administration of HF increases glucose tolerance whilst reducing weight gain, insulin resistance, and serum insulin in DIO mice. Conversely, the ISR inhibitor GSK2656157, used at low doses to optimize its selectivity, aggravates glucose intolerance in DIO mice. Whilst loss of function mutations in mice and humans have revealed that PERK is the essential ISR kinase that protects from diabetes, our work demonstrates the therapeutic value of increasing ISR signaling by activating the related kinase GCN2 to reduce diabetes phenotypes in a DIO mouse model., (© 2024 MRC Laboratory of Molecular Biology.)

Background: In ischemia, acidosis occurs in/around injured tissue and parallels disease progression. Therefore, targeting an acid-sensitive receptor offers unique advantages in achieving the spatial and temporal specificity required for therapeutic interventions. We previously demonstrated that increased expression of GPR68 (G protein-coupled receptor 68), a proton-sensitive G protein-coupled receptor, mitigates ischemic brain injury. Here, we investigated the mechanism underlying GPR68-dependent protection., Methods: We performed biochemical and molecular analyses to examine poststroke signaling. We used in vitro brain slice cultures and in vivo mouse transient middle cerebral artery occlusion (tMCAO) models to investigate ischemia-induced injuries., Results: GPR68 deletion reduced PERK (protein kinase R-like ER kinase) expression in mouse brain. Compared with the wild-type mice, the GPR68-/- (knockout) mice exhibited a faster decline in eIF2α (eukaryotic initiation factor-2α) phosphorylation after tMCAO. Ogerin, a positive modulator of GPR68, stimulated eIF2α phosphorylation at 3 to 6 hours after tMCAO, primarily in the ipsilateral brain tissue. Consistent with the changes in eIF2α phosphorylation, Ogerin enhanced tMCAO-induced reduction in protein synthesis in ipsilateral brain tissue. In organotypic cortical slices, Ogerin reduced pH 6 and oxygen-glucose deprivation-induced neurotoxicity. Following tMCAO, intravenous delivery of Ogerin reduced brain infarction in wild-type but not knockout mice. Coapplication of a PERK inhibitor abolished Ogerin-induced protection. Delayed Ogerin delivery at 5 hours after tMCAO remained protective, and Ogerin has a similar protective effect in females. Correlated with these findings, tMCAO induced GPR68 expression at 6 hours, and Ogerin alters post-tMCAO proinflammatory/anti-inflammatory cytokine/chemokine expression profile., Conclusions: These data demonstrate that GPR68 potentiation leads to neuroprotection, at least in part, through enhancing PERK-eIF2α activation in ischemic tissue but has little impact on healthy tissue., Competing Interests: None.

Mutations in glucokinase (GCK) can either enhance or inhibit insulin secretion, leading to different forms of diabetes, including gestational diabetes. While many glucokinase activators (GKAs) have been explored as treatments, their long-term effectiveness has often been unsatisfactory. However, recent interest has surged with the introduction of dorzagliatin and TTP399. This study investigates the efficacy of four previously studied compounds (Swertiamarin, Apigenin, Mangiferin, and Tatanan A) in activating GCK using computational methods. Initial molecular docking revealed binding affinities ranging from -6.7 to -8.6 kcal/mol. The compounds were then evaluated for drug-likeness and pharmacokinetic properties. Re-docking studies were performed for validation. Based on their favorable binding affinities and compliance with Lipinski's rule and ADMET criteria, three compounds (Swertiamarin, Apigenin, and Tatanan A) were selected for molecular dynamics (MD) simulations. MD simulations demonstrated that Swertiamarin showed excellent stability, as indicated by analyses of RMSD, RMSF, radius of gyration (Rg), hydrogen bonding, and principal component analysis (PCA). These results suggest that Swertiamarin holds promise for further investigation in in vivo and clinical settings to evaluate its potential in enhancing GCK activity and treating diabetes. This study assessed the potential of four compounds as GCK activators using molecular docking, pharmacokinetic profiling, and MD simulations. Swertiamarin, in particular, showed significant stability and adherence to drug-likeness criteria, making it a promising candidate for further research in combating diabetes., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Ciliated muconodular papillary tumor (CMPT) is a rare pulmonary tumor, typically occurring in middle-aged and elderly individuals. The molecular mutation spectrum of CMPT remains insufficiently explored. Commonly known driver gene alterations include KRAS , BRAF , EGFR , and ALK rearrangement. This report details the clinicopathological features of 2 patients presenting with CMPT as pulmonary nodules during clinical examinations. Microscopic analysis revealed tumors with glandular or papillary structures, consisting of mucinous cells, ciliated columnar cells, and basal cells. Notably, both patients exhibited STRN::ALK fusion, a finding not previously associated with CMPT. STRN::ALK fusion serves as a target for therapy in various tumors, including non-small cell lung cancer, thyroid cancer, and colon cancer. Consequently, we conducted a review of relevant literature, summarizing the clinicopathological and molecular characteristics of CMPT to facilitate further research. Our insights enhance the understanding of this uncommon tumor and contribute to the expansion of its molecular alteration spectrum., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Purpose: Despite the generally favourable prognoses observed in patients with ALK-positive non-small cell lung cancer (NSCLC), there remains significant variability in clinical outcomes. The objective of this study is to enhance patient stratification by examining both the specific sites of gene fusion and the presence of co-occurring mutations., Methods: We collected retrospective clinical and pathological data on ALK-positive patients with locally advanced or metastatic disease. ALK fusion variants and concomitant mutations were identified through next-generation sequencing technology. We then assessed treatment efficacy via tumor response and survival metrics., Results: This study included a total of 59 patients, with 49 harboring echinoderm microtubule-associated protein-like 4 (EML4)-ALK fusions and 10 presenting with rare fusions. The median follow-up period was 33 months. Clinical outcomes between non-EML4-ALK and EML4-ALK patients were comparable. Among the EML4-ALK cohort, patients with longer variants (v1, v2, v8) demonstrated superior progression-free survival (PFS) (median PFS: 34 months vs. 11 months; hazard ratio [HR]: 2.28; P = 0.05) compared to those with shorter variants (v3, v5). Furthermore, patients treated with second-generation ALK inhibitors (ALKi) displayed a progression-free survival advantage (median PFS: not reached [NR] vs. 9 months; HR: 5.37; P = 0.013). Baseline TP53 co-mutation were linked with a substantially shorter OS (median OS,37 months vs. NR; HR 2.74; P = 0.047)., Conclusions: In ALK+ NSCLC, longer EML4-ALK variants correlate with improved prognosis and enhanced response to second-generation ALKi, while TP53 co-mutations indicate a negative prognosis., (© 2024. The Author(s), under exclusive licence to Federación de Sociedades Españolas de Oncología (FESEO).)

Retinoblastoma (RB) is the most common intraocular malignancy among children and presents a certain mortality risk, especially in low- and middle-income countries. Clarifying the molecular mechanisms underlying the onset and progression of retinoblastoma is vital for devising effective cancer treatment approaches. PRMT1, a major type I PRMT, plays significant roles in cancer development. However, its expression and role in retinoblastoma are still unclear. Our research revealed a marked increase in PRMT1 levels in both retinoblastoma tissues and Y79 cells. The overexpression of PRMT1 in Y79 cells promoted their growth and cell cycle progression. Conversely, the suppression of PRMT1 hindered the growth of Y79 cells and impeded cell cycle progression. Mechanistically, PRMT1 mediated the growth of Y79 retinoblastoma cells by targeting the p53/p21/CDC2/Cyclin B pathway. Additionally, the ability of PRMT1 knockdown to suppress cell proliferation was also observed in vivo. Overall, PRMT1 could function as a potential target for therapeutic treatment in individuals with retinoblastoma., Competing Interests: Declaration of competing interest The authors disclose that they possess no conflicting interests regarding the content of this work., (Copyright © 2024. Published by Elsevier Ltd.)

Hidrocystoma is thought to be a benign retention cyst of sweat ductal units. The lesion is usually located in the periorbital skin; however, lesions with similar histopathological features are rarely observed in extra-facial sites. Herein, we present four cases of hidrocystoma-like tumours in extra-facial skin sites that harboured a RET or ALK rearrangement. This study features a 67-year-old female with a 10 mm-sized digital tumour (Case 1), a 62-year-old male with an 8 mm-sized clavicular tumour (Case 2), a 61-year-old male with a 19 mm-sized digital tumour (Case 3), and an 11-year-old female with a 10 mm-size lower leg tumour (Case 4) as well as five control cases (Cases 5-9) of classical periorbital hidrocystoma. In Cases 1-4, multicystic tumours comprising a two-cell layer of inner cuboidal ductoglandular (p63- and SOX10+/-) and outer flat myoepithelial (p63+ and SOX10+) cells were observed. The inner ductoglandular tumour cells exhibited micropapillary projections and Roman bridging structures. No apparent atypical cells were observed. NCOA4::RET in Cases 1 and 3, CCDC6::RET in Case 2, and SLC12A2::ALK in Case 4 were revealed by next-generation sequencing or Sanger sequencing. In contrast, control cases of classical hidrocystoma (Cases 5-9) did not show intracystic proliferation, abundant cytoplasm, ALK immunoreactivity, or NCOA4::RET detection in the tumour cells. RET/ALK-rearranged hidrocystoma-like tumours are tumour entities that can be distinguished from classical hidrocystoma. This RET/ALK-rearranged neoplasm is benign and is frequently observed in the digits. Future studies will establish the concept, detailed clinicopathological characteristics, and genetic variations of hidrocystoma-like tumours., (Copyright © 2024 Royal College of Pathologists of Australasia. Published by Elsevier B.V. All rights reserved.)

The DNA dependent protein kinase (DNA-PK) initiates non-homologous recombination (NHEJ), the predominate DNA double-strand break (DSBR) pathway in higher vertebrates. It has been known for decades that the enzymatic activity of DNA-PK [that requires its three component polypeptides, Ku70, Ku80 (that comprise the DNA-end binding Ku heterodimer), and the catalytic subunit (DNA-PKcs)] is present in humans at 10-50 times the level observed in other mammals. Here, we show that the high level of DNA-PKcs protein expression appears evolutionarily in mammals between prosimians and higher primates. Moreover, the RNAs encoding the three component polypeptides of DNA-PK are present at similarly high levels in hominids, new-, and old-world monkeys, but expression of these RNAs in prosimians is ∼5-50 fold less, analogous to the levels observed in other non-primate species. This is reminiscent of the appearance of Alu repeats in primate genomes -- abundant in higher primates, but present at much lower density in prosimians. Alu repeats are well-known for their capacity to promote non-allelic homologous recombination (NAHR) a process known to be inhibited by DNA-PK. Nanopore sequence analyses of cultured cells proficient or deficient in DNA-PK revealed an increase of inter-chromosomal translocations caused by NAHR. Although the high levels of DNA-PK in primates may have many functions, we posit that high levels of DNA-PK may function to restrain deleterious NAHR events between Alu elements., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Pyruvate kinase M2 (PKM2) is a key glycolytic enzyme interacting with the inositol 1,4,5-trisphosphate receptor (IP 3 R). This interaction suppresses IP 3 R-mediated cytosolic [Ca 2+ ] rises. As PKM2 exists in monomeric, dimeric and tetrameric forms displaying different properties including catalytic activity, we investigated the molecular determinants of PKM2 enabling its interaction with IP 3 Rs. Treatment of HeLa cells with TEPP-46, a compound stabilizing the tetrameric form of PKM2, increased both its catalytic activity and the suppression of IP 3 R-mediated Ca 2+ signals. Consistently, in PKM2 knock-out HeLa cells, PKM2 C424L , a tetrameric, highly active PKM2 mutant, but not inactive PKM2 K270M or the less active PKM2 K305Q , suppressed IP 3 R-mediated Ca 2+ release. Surprisingly, however, in vitro assays did not reveal a direct interaction between purified PKM2 and either the purified Fragment 5 of IP 3 R1 (a.a. 1932-2216) or the therein located D5SD peptide (a.a. 2078-2098 of IP 3 R1), the presumed interaction sites of PKM2 on the IP 3 R. Moreover, on-nucleus patch clamp of heterologously expressed IP 3 R1 in DT40 cells devoid of endogenous IP 3 Rs did not reveal any functional effect of purified wild-type PKM2, mutant PKM2 or PKM1 proteins. These results indicate that an additional factor mediates the regulation of the IP 3 R by PKM2 in cellulo. Immunoprecipitation of GRP75 using HeLa cell lysates co-precipitated IP 3 R1, IP 3 R3 and PKM2. Moreover, the D5SD peptide not only disrupted PKM2:IP 3 R, but also PKM2:GRP75 and GRP75:IP 3 R interactions. Our data therefore support a model in which catalytically active, tetrameric PKM2 suppresses Ca 2+ signaling via the IP 3 R through a multiprotein complex involving GRP75., Competing Interests: Declaration of competing interest None., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Competing Interests: Disclosures The authors declare no conflicts of interest.

Medial vascular calcification in chronic kidney disease (CKD) involves pro-inflammatory pathways induced by hyperphosphatemia. Several interleukin 6 family members have been associated with pro-calcific effects in vascular smooth muscle cells (VSMCs) and are considered as therapeutic targets. Therefore, we investigated the role of leukemia inhibitory factor (LIF) during VSMC calcification. LIF expression was found to be increased following phosphate exposure of VSMCs. LIF supplementation aggravated, while silencing of endogenous LIF or LIF receptor (LIFR) ameliorated the pro-calcific effects of phosphate in VSMCs. The soluble LIFR mediated antagonistic effects towards LIF and reduced VSMC calcification. Mechanistically, LIF induced phosphorylation of the non-receptor tyrosine-protein kinase 2 (TYK2) and signal transducer and activator of transcription-3 (STAT3) in VSMCs. TYK2 inhibition by deucravacitinib, a selective, allosteric oral immunosuppressant used in psoriasis treatment, not only blunted the effects of LIF, but also interfered with the pro-calcific effects induced by phosphate. Conversely, TYK2 overexpression aggravated VSMC calcification. Ex vivo calcification of mouse aortic rings was ameliorated by Tyk2 pharmacological inhibition and genetic deficiency. Cholecalciferol-induced vascular calcification in mice was improved by Tyk2 inhibition and in the Tyk2-deficient mice. Similarly, calcification was ameliorated in Abcc6/Tyk2-deficient mice after adenine/high phosphorus-induced CKD. Thus, our observations indicate a role for LIF in CKD-associated vascular calcification. Hence, the effects of LIF identify a central pro-calcific role of TYK2 signaling, which may be a future target to reduce the burden of vascular calcification in CKD., (Copyright © 2024 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Introduction: Biallelic variants in thiamine pyrophosphokinase 1 ( TPK1 ) are known to cause thiamine metabolism dysfunction syndrome 5 (THMD5). This disorder is characterized by neuroregression, ataxia and dystonia with basal ganglia abnormalities on neuroimaging. To date, 27 families have been reported with THMD5 due to variants in TPK1 ., Methods: We ascertained three individuals from three unrelated families. Singleton exome sequencing was performed on all three individuals, followed by in silico mutagenesis of the mutant TPK protein. Additionally, we reviewed the genotypic and phenotypic information of 27 previously reported individuals with THMD5., Results: Singleton exome sequencing revealed a novel homozygous variant c.620A>T p.(Asp207Val) in TPK1 (NM_022445.4) in all three individuals. In silico mutagenesis of the mutant protein revealed a decrease in protein stability and altered interactions with its neighboring residues compared to the wild-type protein. Thus, based on strikingly similar clinical and radiological findings compared to the previously reported individuals and with the support of in silico mutagenesis findings, the above-mentioned variant appears to be the probable cause for the condition observed in the affected individuals in this study., Conclusion: We report a novel homozygous variant in TPK1 , which appears to be recurrent among the Indian population., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

Anaplastic lymphoma kinase-positive histiocytosis, first reported in 2008, is a rare, novel type of neoplasm. To date, no more than 100 cases of anaplastic lymphoma kinase-positive histiocytosis have been reported. In this retrospective study, 12 cases of anaplastic lymphoma kinase-positive histiocytosis, including clinical symptoms, histological features, molecular pathology, treatment, and prognosis, in children were analyzed to gain a deeper understanding of the disease. All patients were Asian children, aged 2 months to 8 years and 2 months (mean 3.1 years), and the male-to-female ratio was 5:7. All patients were followed up closely. One patient died during the follow-up period, seven (case 1-7) had focal anaplastic lymphoma kinase-positive histiocytosis, and five (case 8-12) had multisystem anaplastic lymphoma kinase-positive histiocytosis. In addition, we report the case of a patient who benefited from anaplastic lymphoma kinase-targeted therapy and a patient with the rare EML4-ALK fusion gene. The current study is expected to substantially contribute to increasing the awareness of anaplastic lymphoma kinase-positive histiocytosis., Competing Interests: Declaration of competing interest All the authors have no conflicts of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Calcium/calmodulin dependent protein kinase kinase (CaMKK), a highly conserved protein kinase, is involved in the downstream processes of various biological activities by phosphorylating and activating 5'-AMP-activated protein kinase (AMPK) in response to the increase of cytosolic-free calcium (Ca 2+ ). In the present study, a CaMKKI was identified from Yesso scallop Patinopecten yessoensis. Its mRNA was ubiquitously expressed in haemocytes and all tested tissues with the highest expression level in mantle. The expression level of PyCaMKKI mRNA in adductor muscle was significantly upregulated at 1, 3 and 6 h after high temperature treatment (25 °C), which was 3.43-fold (p < 0.05), 5.25-fold (p < 0.05), and 5.70-fold (p < 0.05) of that in blank group, respectively. At 3 h after high temperature treatment (25 °C), the protein level of PyAMPKα, as well as the phosphorylation level of PyAMPKα at Thr170 in adductor muscle, and the positive co-localized fluorescence signals of PyCaMKKI and PyAMPKα in haemocyte all increased significantly (p < 0.05) compared to blank group (18 °C). The pull-down assay showed that rPyCaMKKI and rPyAMPKα could bind each other in vitro. After PyCaMKKI was silenced by siRNA, the mRNA and protein levels of PyCaMKKI and PyAMPKα, and the phosphorylation level of PyAMPKα at Thr170 in adductor muscle were significantly down-regulated (p < 0.05) compared with the negative control group receiving an injection of siRNA-NC. These results collectively suggested that PyCaMKKI was involved in the activation of PyAMPKα in response to high temperature stress and would be helpful for understanding the function of PyCaMKKI-PyAMPKα pathway in maintaining energy homeostasis under high temperature stress in scallops., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Our previous study showed that pyridoxine 5'-phosphate oxidase (PNPO) is a tissue biomarker of ovarian cancer (OC) and has a prognostic implication but detailed mechanisms remain unclear. The current study focused on PNPO-regulated lysosome/autophagy-mediated cellular processes and the potential role of PNPO in chemoresistance. We found that PNPO was overexpressed in OC cells and was a prognostic factor in OC patients. PNPO significantly promoted cell proliferation via the regulation of cyclin B1 and phosphorylated CDK1 and shortened the G2M phase in a cell cycle. Overexpressed PNPO enhanced the biogenesis and perinuclear distribution of lysosomes, promoting the degradation of autophagosomes and boosting the autophagic flux. Further, an autolysosome marker LAMP2 was upregulated in OC cells. Silencing LAMP2 suppressed cell growth and induced cell apoptosis. LAMP2-siRNA blocked PNPO action in OC cells, indicating that the function of PNPO on cellular processes was mediated by LAMP2. These data suggest the existence of the PNPO-LAMP2 axis. Moreover, silencing PNPO suppressed xenographic tumor formation. Chloroquine counteracted the promotion effect of PNPO on autophagic flux and inhibited OC cell survival, facilitating the inhibitory effect of PNPO-shRNA on tumor growth in vivo. Finally, PNPO was overexpressed in paclitaxel-resistant OC cells. PNPO-siRNA enhanced paclitaxel sensitivity in vitro and in vivo. In conclusion, PNPO has a regulatory effect on lysosomal biogenesis that in turn promotes autophagic flux, leading to OC cell proliferation, and tumor formation, and is a paclitaxel-resistant factor. These data imply a potential application by targeting PNPO to suppress tumor growth and reverse PTX resistance in OC., (© 2024. The Author(s).)

Metastatic spread to the central nervous system (CNS) is frequent in anaplastic lymphoma kinase ( ALK )-rearranged non-small cell lung cancer (NSCLC) and has an important impact on patient prognosis and quality of life. Leptomeningeal involvement may occur in up to 10% of cases of ALK-positive NSCLC. Lorlatinib is a third-generation ALK inhibitor that has excellent CNS penetrability and demonstrated its efficacy both in pretreated and treatment-naive patients. Herein, we present the case of a 34-year-old patient diagnosed with stage IV ALK-rearranged NSCLC who received two lines of ALK inhibitors (crizotinib followed by alectinib) and several courses of brain stereotactic ablative radiotherapy until leptomeningeal involvement was detected. Third-line lorlatinib was then administered, and 2 months later encephalic MRI documented complete regression of the leptomeningeal involvement that is still maintained after 36 months while treatment with lorlatinib is still ongoing with good tolerability. To the best of our knowledge, this is the longer intracranial response reported in the literature, underlining the importance of the most appropriate choice of systemic treatments and their integration with loco-regional approaches to improve outcomes., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

Purpose: This study aimed to elucidate the role of the Cdk1/p53/p21 feedback loop in the pathogenesis of interstitial cystitis (IC)/bladder pain syndrome (BPS)., Materials and Methods: An IC/BPS cell model was established. Cell viability was determined using the CCK-8 assay. Flow cytometry was adopted to assess cell apoptosis rates. ELISA was employed to measure secretion levels of inflammatory factors (IL-6, IL-8, and TNF-α). Gene expressions were assessed using PCR, while protein expressions were analyzed through Western blotting analysis. Epithelial permeability was demonstrated using the phenol red leakage experiment and FITC-dextran permeability assay. The interaction between proteins was determined using co-immunoprecipitation, and protein localization was investigated using immunofluorescence., Results: The CCK-8 assay revealed a significantly reduced viability of IC/BPS cells compared to normal epithelial cells (p < 0.05). Elevated levels of IL-6, IL-8, and TNF-α were detected in IC/BPS cells. Changes in the expressions of E-cadherin and ZO-1 were evident, leading to increased epithelial permeability in IC/BPS cells. Furthermore, within IC/BPS cells, Cdk1 phosphorylated p53 in the nucleus. The Cdk1/p53/p21 feedback loop was established to influence urothelial permeability. Both p21 and Cdk1 inhibitors notably reduced the epithelial permeability in IC/BPS cells., Conclusion: The Cdk1/p53/p21 feedback loop was instrumental in IC/BPS, acting as a regulator of urothelial permeability. This discovery offered a novel therapeutic approach for IC/BPS management., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: This work was supported by the Funding from Young Talent Development Plan of Changzhou Health Commission (CZQM2021004) and The Youth Talent Science and Technology Project of Changzhou Health Commission (QN202307)., (Copyright © 2024. Published by Elsevier B.V.)

Anaplastic lymphoma kinase (ALK)-rearranged mesenchymal neoplasms (non-inflammatory myofibroblastic tumor and non-epithelioid fibrous histiocytoma) have been recently described which tend to occur in the superficial and deep soft tissues. Occurrence as a primary sinonasal neoplasm has not been reported thus far. Herein, we describe the first case of sinonasal ALK-rearranged mesenchymal tumor that harbored remarkable epithelioid and spindle cell morphology. The tumor affected a 40-year-old man who presented with flu-like symptoms and was thought to have influenza A. However, computed tomography demonstrated a nasal polypoid lesion causing curvature of the nasal septum. Histological examination revealed a heterogeneous tumor composed of round to epithelioid cells with foci of spindle cells. The tumor cells exhibited moderate pleomorphism and mitotic activity. By immunohistochemistry, they showed diffuse staining of CD34, S100, ALK (D5F3) and CD30. Fluorescence in situ hybridization analysis demonstrated ALK rearrangement. Subsequent next-generation sequencing (RNA-seq) identified a rare PLEKHH2exon6::ALKexon20 fusion. This study further demonstrates the importance of molecular profiling in identifying kinase fusion-positive soft tissue tumors, particularly for those that arise at unusual sites and display atypical cytomorphology., Competing Interests: Declaration of Conflicting InterestsThe authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

The past year has offered significant advancements in the field of non-small cell lung cancer (NSCLC), both in the early and advanced disease settings. The identification of guideline-recommended actionable targets has provided the foundation for developing multiple new therapeutic agents. There has been a focus on developing drugs designed to overcome acquired resistance, a limitation of tyrosine kinase inhibitor-based therapy in lung cancer. In addition, there is an emerging trend toward combination therapies for patients in the first-line setting with the goal of preventing or delaying resistance. Another promising area of development has been the use of antibody-drug conjugates, where there are the initial reports of central nervous system efficacy and activity in patients with genomic alterations. Over the past year, numerous publications and presentations have highlighted multiple therapeutic advances, offering new treatment options for patients with NSCLC. The focus of this review is to summarize the most impactful findings, emphasizing their significance in the evolving treatment landscape for NSCLC. Several landmark trials in lung cancer with practice-changing clinical implications have been presented and published in 2023. This article reviews a selection of these trials as they relate to early and advanced-stage oncogene-driven lung cancer. The ADAURA and ALINA trials, in which targeted therapy given in the adjuvant setting has demonstrated improved clinical outcomes, are reviewed. In the advanced-stage setting, recent trials in the context of specific oncogene drivers are reviewed, including EGFR, ALK, ROS1, RET, ERBB2 (HER2), BRAF, MET exon 14 skipping (METex14), and KRAS alterations. Also discussed are the results of several trials that have evaluated the use of combination therapies and resistance-mechanism agnostic treatment strategies. PLAIN LANGUAGE SUMMARY: Targeted therapy plays an important role for patients with early and advanced-stage non-small cell lung cancer carrying specific genetic alterations. New strategies that combine multiple therapies are now being studied in randomized clinical trials, with the goal of enhancing the effectiveness of targeted therapy for patients with advanced lung cancer., (© 2024 American Cancer Society.)

Prior studies have emphasized a bioenergetic crisis in the retinal pigment epithelium (RPE) as a critical factor in the development of age-related macular degeneration (AMD). The isoforms Fructose-1,6-bisphosphate aldolase C (ALDOC) and pyruvate kinase M2 (PKM2) have been proposed to play a role in AMD pathogenesis. While PKM2 and ALDOC are crucial for aerobic glycolysis in the neural retina, they are not as essential for the RPE. In this study, we examined the expression and activity of PKM2 and ALDOC in both young and aged RPE cells, as well as in the retina and RPE tissue of mice, including an experimentally induced AMD mouse model. Our findings reveal an upregulation in PKM2 and ALDOC expression, accompanied by increased pyruvate kinase activity, in the aged and AMD mouse RPE. Conversely, there is a decrease in ALDOC expression but an increase in PKM2 expression and pyruvate kinase activity in the aged and AMD retina. Overall, our study indicates that aged and AMD RPE cells tend to favor aerobic glycolysis, while this tendency is diminished in the aged and AMD retina. These results underscore the significance of targeting PKM2 and ALDOC in the RPE as a promising therapeutic approach to address the bioenergetic crisis and prevent vision loss in AMD.

Skeletal muscle is a flexible and adaptable tissue that strongly responds to exercise training. The skeletal muscle responds to exercise by increasing muscle protein synthesis (MPS) when energy is available. One of protein synthesis's major rate-limiting and critical regulatory steps is the translation elongation pathway. The process of translation elongation in skeletal muscle is highly regulated. It requires elongation factors that are intensely affected by various physiological stimuli such as exercise and the total available energy of cells. Studies have shown that exercise involves the elongation pathway by numerous signalling pathways. Since the elongation pathway, has been far less studied than the other translation steps, its comprehensive prospect and quantitative understanding remain in the dark. This study highlights the current understanding of the effect of exercise training on the translation elongation pathway focussing on the molecular factors affecting the pathway, including Ca 2+ , AMPK, PKA, mTORC1/P70S6K, MAPKs, and myostatin. We further discussed the mode and volume of exercise training intervention on the translation elongation pathway. What is the topic of this review? This review summarises the impacts of exercise training on the translation elongation pathway in skeletal muscle focussing on eEF2 and eEF2K. What advances does it highlight? This review highlights mechanisms and factors that profoundly influence the translation elongation pathway and argues that exercise might modulate the response. This review also combines the experimental observations focussing on the regulation of translation elongation during and after exercise. The findings widen our horizon to the notion of mechanisms involved in muscle protein synthesis (MPS) through translation elongation response to exercise training.

Myocardial ischemia-reperfusion (I/R) injury is a prevalent cause of myocardial injury, involving a series of interconnected pathophysiological processes. However, there is currently no clinical therapy for effectively mitigating myocardial I/R injury. Here, we show that p85α protein levels increase in response to I/R injury through a comprehensive analysis of cardiac proteomics, and confirm this in the I/R-injured murine heart and failing human myocardium. Genetic inhibition of p85α in mice activates the Akt-GSK3β/Bcl-x(L) signaling pathway and ameliorates I/R-induced cardiac dysfunction, apoptosis, inflammation, and mitochondrial dysfunction. p85α silencing in cardiomyocytes alleviates hypoxia-reoxygenation (H/R) injury through activating the Akt-GSK3β/Bcl-x(L) signaling pathway, while its overexpression exacerbates the damage. Mechanistically, the interaction between MG53 and p85α triggers the ubiquitination and degradation of p85α, consequently enhancing Akt phosphorylation and ultimately having cardioprotective effects. Collectively, our findings reveal that substantial reduction of p85α and subsequently activated Akt signaling have a protective effect against cardiac I/R injury, representing an important therapeutic strategy for mitigating myocardial damage., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

The aim of this study was to investigate the signaling pathways involved in the proliferation and differentiation of pig Sertoli cells (SCs) mediated by thyroid hormone (T3) to provide a theoretical and practical basis for enhancing pig semen production. The effects of different concentrations of T3 on the proliferation of pig SCs were evaluated using the CCK8 assay. The impact of T3 on the proliferation and differentiation of pig SCs was further examined using RNA-seq, qPCR, and Western Blotting techniques. Additionally, the involvement of the p38 MAPK and NFκB pathways in mediating the effects of T3 on SCs proliferation and differentiation was investigated. Our findings revealed a strong correlation between the dosage of T3 and the inhibition of pig SCs proliferation and promotion of maturation. T3 regulated the activation state of the NFκB signaling pathway by upregulating IKKα, downregulating IKKβ, and promoting IκB phosphorylation. Furthermore, T3 facilitated SCs maturation by upregulating AR and FSHR expression while downregulating KRT-18. In conclusion, T3 inhibits pig SCs proliferation and promote pig SCs maturation through the IKK/NFκB and p38 MAPK pathways. These findings provide valuable insights into the mechanisms by which T3 influences the proliferation and maturation of pig SCs., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Background: The intake of high-fructose corn syrup (HFCS) may increase the risk of colorectal cancer (CRC). This study aimed to explore the potential effects and mechanisms of resistant starch (RS) in HFCS-induced colon tumorigenesis., Methods: The azoxymethane/dextran sodium sulfate (AOM/DSS) and Apc Min/+ mice models were used to investigate the roles of HFCS and RS in CRC in vivo. An immunohistochemistry (IHC) staining analysis was used to detect the expression of proliferation-related proteins in tissues. 16S rRNA sequencing for microbial community, gas chromatography for short-chain fatty acids (SCFAs), and mass spectrometry analysis for glycolysis products in the intestines were performed. Furthermore, lactic acid assay kit was used to detect the glycolysis levels in vitro., Results: RS suppressed HFCS-induced colon tumorigenesis through reshaping the microbial community. Mechanistically, the alteration of the microbial community after RS supplement increased the levels of intestinal SCFAs, especially butyrate, leading to the suppression of glycolysis and CRC cell proliferation by downregulating HK2., Conclusions: Our study identified RS as a candidate of protective factors in CRC and may provide a potential target for HFCS-related CRC treatment., (© 2024. The Author(s).)

Aims: The majority of dermatofibrosarcoma protuberans (DFSP) harbour PDGFB or PDGFD rearrangements. We encountered ALK expression/rearrangement in a PDGFB/D-negative CD34-positive spindle cell neoplasm with features similar to DFSP, prompting evaluation of ALK-rearrangements in DFSP and plaque-like CD34-positive dermal fibroma (P-LDF)., Methods and Results: We searched the archives of academic institutions for cases previously coded as DFSP and P-LDF. NGS-naïve or PDGFB-negative DFSP were screened for ALK (clone D5F3) expression by immunohistochemistry. NGS or ALK FISH was performed on ALK-positive cases. Methylome profiling studies were performed and compared with conventional DFSP. One case of "DFSP" and two "P-LDF" with ALK expression were identified from the archives, while four cases were detected prospectively. These seven cases (6F:1M; 8 months to 76 years) arose in the dermis of the arm (two), scalp, eyelid, thigh, abdomen, and shoulder and ranged from 0.4 to 4.2 cm. Tumours were composed of spindled cells and displayed a storiform growth pattern. Cytologic atypia was absent, and mitotic figures were scarce (0-2/10 HPFs, high power fields). The lesional cells were diffusely positive for CD34 and ALK and negative for S100 protein. By NGS (n = 5), ALK fusion partners included DCTN1 (2), PLEKHH2, and CLIP2 in DFSP-like cases and FLNA in P-LDF-like lesions. ALK FISH was positive in one (of two) cases previously labelled P-LDF. Methylome profiling of two (of three) ALK-rearranged DFSP-like tumours showed clustering with conventional DFSP in the UMAP dimension reduction plot. To date, no tumour has recurred (n = 2; 26, 27 months)., Conclusion: We describe a cohort of novel ALK-rearranged tumours with morphologic features similar to DFSP., (© 2024 The Author(s). Histopathology published by John Wiley & Sons Ltd.)

Here, we report that Caveolin-2 (Cav-2) is a cell cycle regulator in the mitotic clonal expansion (MCE) for adipogenesis. For the G2/M phase transition and re-entry into the G1 phase, dephosphorylated Cav-2 by protein tyrosine phosphatase 1B (PTP1B) controlled epigenetic activation of Ccnb1, Cdk1, and p21 in a lamin A/C-dependent manner, thereby ensuring the survival of preadipocytes. Cav-2, associated with lamin A/C, recruited the repressed promoters of Ccnb1 and Cdk1 for activation, and disengaged the active promoter of p21 from lamin A/C for inactivation through histone H3 modifications at the nuclear periphery. Cav-2 deficiency abrogated the histone H3 modifications and impeded the transactivation of Ccnb1, Cdk1, and p21, leading to a delay in mitotic entry, retardation of re-entry into G1 phase, and the apoptotic cell death of preadipocytes. Re-expression of Cav-2 restored the G2/M phase transition and G1 phase re-entry, preadipocyte survival, and adipogenesis in Cav-2-deficient preadipocytes. Our study uncovers a novel mechanism by which cell cycle transition and apoptotic cell death are controlled for adipocyte hyperplasia., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Two-component systems (TCSs) are prevalent signaling pathways in bacteria. These systems mediate phosphotransfer between histidine kinase and a response regulator, facilitating responses to diverse physical, chemical, and biological stimuli. Advancements in synthetic and structural biology have repurposed TCSs for applications in monitoring heavy metals, disease-associated biomarkers, and the production of bioproducts. However, the utility of many TCS biosensors is hindered by undesired performance due to the lack of effective engineering methods. Here, we briefly discuss the architectures and regulatory mechanisms of TCSs. We also summarize the recent advancements in TCS engineering by experimental or computational-based methods to fine-tune the biosensor functional parameters, such as response curve and specificity. Engineered TCSs have great potential in the medical, environmental, and biorefinery fields, demonstrating a crucial role in a wide area of biotechnology., Competing Interests: Declaration of competing interest The authors declare no competing financial interest., (Copyright © 2023. Published by Elsevier Inc.)

Abstract: Spitz melanocytic neoplasms exhibit frequent chromosomal rearrangements leading to recurring gene fusions, such as ALK fusions. TPM3 and DCTN1 emerge as the predominant fusion partners of ALK , although less common partners such as NPM1 , TPR , CLIP1 , GTF3C2 , MLPH , EEF2 , MYO5A , and KANK1 have also been documented. Although ALK fusions are primarily associated with Spitz nevi or atypical Spitz tumors, instances of Spitz melanoma with ALK fusions documented in the English literature are exceedingly rare. Here, we present a case of Spitz melanoma harboring SLC20A1::ALK fusion, highlighting a novel fusion transcript not previously reported in Spitz melanocytic neoplasms, including Spitz melanomas. In addition, the tumor exhibits multiple aberrant chromosomal alterations characteristic of melanoma, along with a somatic mutation in GRM3 ., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

The liver and intestine play a critical role in nutrient absorption, storage, and metabolism. The aim of this study was to evaluate expression pattern of phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of the rapamycin (mTOR) signaling pathway that included PI3K, AKT1, mTOR, FoxO1, SREBP-1, PPARα, PTEN and FXR in the maternal liver and duodenum. Ovine livers and duodenums were sampled at day 16 of the estrous cycle, and at days 13, 16 and 25 of gestation, and RT-qPCR, western blot and immunohistochemistry analysis were used to detect mRNA and protein expression. The results showed that expression of PI3K, AKT1, p-mTOR, FoxO1, SREBP-1 and PTEN upregulated in the maternal liver, and PPARα upregulated in the duodenum. However, expression of FoxO1, SREBP-1 and PTEN in the duodenum downregulated during early pregnancy. In addition, expression levels of SREBP-1, PTEN and PPARα in the maternal liver, and PI3K in the duodenum peaked at day 13 of pregnancy. In addition, expression levels of PI3K, p-mTOR and FoxO1 in the liver, and AKT1 and p-mTOR in the duodenum peaked at day 16 of pregnancy. Nevertheless, expression levels of FXR both in the maternal liver duodenum downregulated at days 13 and 16 of pregnancy. In conclusion, early pregnancy regulated expression pattern of PI3K/AKT/mTOR signaling pathway in the ovine liver and duodenum in a pregnancy stage-specific and tissue-specific manner, which may be necessary for the adaptations in maternal hepatic nutrient metabolism and intestinal nutrient absorption early pregnancy., Competing Interests: Declaration of competing interest The authors declare no personal conflict of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Green tea polyphenols (GTP), an important phytochemical in the daily human diet, bind to various cellular receptors and exert anti-inflammatory and antioxidant benefits. The environmental contaminant tetrabromobisphenol A (TBBPA) enters the digestive system through multiple pathways, resulting in oxidative stress (OS), gastroenteritis, and mucosal injury. The aim of this study was to explore the molecular mechanisms of TBBPA-induced gastritis in mice treated with GTP in vivo and in an in vitro model. The results showed that exposure to TBBPA increased reactive oxygen species (ROS) levels, activated oxidative stress (OS) induced endoplasmic reticulum stress (ERS), and the expression of endoplasmic reticulum stress-related factors ( e.g. , GRP78, PERK, IRE-1, ATF-6, etc .) increased. The inflammatory pathway NF-κB was activated, and the pro-inflammatory factors TNF-α, IL-1β, and IL-6 increased, while triggering a cascade reaction mediated by caspase-3. However, the addition of GTP could inhibit OS, restore the balance of endoplasmic reticulum homeostasis, and improve the inflammatory infiltration and apoptosis of gastric mucosal epithelial cells. Therefore, GTP alleviated ERS, reduced inflammation and apoptosis, and restored the gastric mucosal barrier by alleviating TBBPA-induced OS in mouse gastric tissues and GES-1 cells. This provides basic information for exploring the antioxidant mechanism of GTP and further investigating the toxic effects of TBBPA on mouse gastric mucosa.

Streptococcus suis serotype 2 (SS2) is an important zoonotic pathogen that induces an NLRP3-dependent cytokine storm. NLRP3 inflammasome activation triggers not only an inflammatory response but also pyroptosis. However, the exact mechanism underlying S. suis-induced macrophage pyroptosis is not clear. Our results showed that SS2 induced the expression of pyroptosis-associated factors, including lactate dehydrogenase (LDH) release, propidium iodide (PI) uptake and GSDMD-N expression, as well as NLRP3 inflammasome activation and IL-1β secretion. However, GSDMD deficiency and NLRP3 inhibition using MCC950 attenuated the SS2-induced expression of pyroptosis-associated factors, suggesting that SS2 induces NLRP3-GSDMD-dependent pyroptosis. Furthermore, RACK1 knockdown also reduced the expression of pyroptosis-associated factors. In addition, RACK1 knockdown downregulated the expression of NLRP3 and Pro-IL-1β as well as the phosphorylation of P65. Surprisingly, the interaction between RACK1 and P65 was detected by co-immunoprecipitation, indicating that RACK1 induces macrophage pyroptosis by mediating the phosphorylation of P65 to promote the transcription of NLRP3 and pro-IL-1β. Similarly, NEK7 knockdown decreased the expression of pyroptosis-associated factors and ASC oligomerization. Moreover, the results of co-immunoprecipitation revealed the interaction of NEK7-RACK1-NLRP3 during SS2 infection, demonstrating that NEK7 mediates SS2-induced pyroptosis via the regulation of NLRP3 inflammasome assembly and activation. These results demonstrate the important role of RACK1 and NEK7 in SS2-induced pyroptosis. Our study provides new insight into SS2-induced cell death., (© 2024. The Author(s).)

Hexokinase is a crucial diagnostic reagent in blood glucose testing, which has high requirements for the enzyme activity and thermal stability. The hexokinases in China mainly rely on imports and are primarily sourced from yeast, with high costs and poor thermal stability, which limit the development of blood glucose diagnostic reagents. Therefore, there is an urgent need for the efficient expression of highly active and thermally stable hexokinases. In this study, an ATP-dependent hexokinase (glucokinase, Glk) from a thermophilic bacterium Glk was heterologously expressed in Escherichia coli BL21(DE3). Glk exhibited high specificity for glucose, dependence on Mg 2+ , and the highest activity at pH 8.5 and 80 ℃. It retained over 90% activity after storage at 30-37 ℃ for 7 days, demonstrating thermal stability as an alkaline glucose kinase. Subsequently, the factors influencing Glk expression, including culture medium, OD 600 , final concentration of the inducer, induction temperature, and induction duration, were systematically optimized. The optimization increased the Glk expression by 4.71 folds Glk compared with non-optimized conditions. After purification, Glk exhibited a specific activity of (43.05±2.00) U/mg and the purity ≥98%. In conclusion, the developed expression and purification method for the highly thermostable hexokinase provides more possibilities for overcoming the shortcomings in the preparation of blood glucose diagnostic reagents in China.

To further standardize lung cancer prevention and treatment measures in China, enhance the quality of diagnosis and treatment, improve patient prognosis, and provide evidence-based medical guidance for clinicians at all levels, the Chinese Medical Association convened experts from respiratory medicine, oncology, thoracic surgery, radiotherapy, imaging, and pathology to develop the Chinese Medical Association's Clinical Diagnosis and Treatment Guidelines for Lung Cancer (2024 edition). This consensus resulted in several updates from the 2023 version. The 2024 guidelines highlight that the risk of lung cancer in smokers remains higher than that of non-smokers even 15 years after quitting. Additionally, a new lung cancer incidence risk model is expected to become a critical tool for screening high-risk groups. In pathology, the guidelines now include pathological evaluation of surgically resected lung cancer specimens following neoadjuvant therapy and suggest that immunohistochemical staining of certain transcription factors may aid in the classification of small cell lung cancer (SCLC). In molecular detection, the guidelines propose simultaneous detection of driver gene variations based on both RNA and DNA from specimens. The new edition also provides detailed descriptions of patient selection and surgical requirements for thoracic sub-lobectomy, aligned with the 9th TNM staging. Moreover, the guidelines expand treatment options, approving more therapies for immunoadjuvant and EGFR-TKI resistant lung cancer patients, as well as additional drug options for advanced non-small cell lung cancer (NSCLC) patients with EGFR mutations, EGFR 20 insertions, ALK fusions, and MET exon 14 skipping. These recommendations are based on state-approved drug applications, international guidelines, and current clinical practices in China, integrating the latest evidence-based medical research in screening, diagnosis, pathology, genetic testing, immune molecular marker detection, treatment methods, and follow-up care. The goal is to provide comprehensive and reasonable recommendations for clinicians, imaging specialists, laboratory technicians, rehabilitation professionals, and other medical staff at all levels.

Therapy targeting the BRAF-MEK cascade created a treatment revolution for patients with BRAF mutant advanced melanoma. Unfortunately, 80% patients treated will progress by 5 years follow-up. Thus, it is imperative we study mechanisms of melanoma progression and therapeutic resistance. We created a scRNA (single cell RNA) atlas of 128,230 cells from 18 tumors across the treatment spectrum, discovering melanoma cells clustered strongly by transcriptome profiles of patients of origins. Our cell-level investigation revealed gains of 1q and 7q as likely early clonal events in metastatic melanomas. By comparing patient tumors and their derivative cell lines, we observed that PD1 responsive tumor fraction disappears when cells are propagated in vitro. We further established three anti-BRAF-MEK treatment resistant cell lines using three BRAF mutant tumors. ALDOA and PGK1 were found to be highly expressed in treatment resistant cell populations and metformin was effective in targeting the resistant cells. Our study suggests that the investigation of patient tumors and their derivative lines is essential for understanding disease progression, treatment response and resistance., (© 2024. The Author(s).)

Hearing loss is the most common congenital sensory deficit worldwide and exhibits high genetic heterogeneity, making molecular diagnoses elusive for most individuals. Detecting novel mutations that contribute to hearing loss is crucial to providing accurate personalized diagnoses, tailored interventions, and improving prognosis. Copy number variants (CNVs) are structural mutations that are understudied, potential contributors to hearing loss. Here, we present the Abnormal Wobbly Gait (AWG) mouse, the first documented mutant exhibiting waltzer-like locomotor dysfunction, hyperactivity, circling behaviour, and profound deafness caused by a spontaneous CNV deletion in cadherin 23 (Cdh23). We were unable to identify the causative mutation through a conventional whole-genome sequencing (WGS) and variant detection pipeline, but instead found a linked variant in hexokinase 1 (Hk1) that was insufficient to recapitulate the AWG phenotype when introduced into C57BL/6J mice using CRISPR-Cas9. Investigating nearby deafness-associated genes revealed a pronounced downregulation of Cdh23 mRNA and a complete absence of full-length CDH23 protein, which is critical for the development and maintenance of inner ear hair cells, in whole head extracts from AWG neonates. Manual inspection of WGS read depth plots of the Cdh23 locus revealed a putative 10.4 kb genomic deletion of exons 11 and 12 that was validated by PCR and Sanger sequencing. This study underscores the imperative to refine variant detection strategies to permit identification of pathogenic CNVs easily missed by conventional variant calling to enhance diagnostic precision and ultimately improve clinical outcomes for individuals with genetically heterogenous disorders such as hearing loss., (© The Author(s) 2024. Published by Oxford University Press.)

Administration of high-dose clindamycin (CLI) and penicillin is recommended for the treatment of streptococcal toxic shock syndrome (STSS). However, CLI-resistant strains have been identified worldwide. In this study, some CLI-resistant strains demonstrated increased extracellular activity of the NAD-glycohydrolase (NADase) exotoxin following CLI treatment. These results support our previous conclusion that CLI-susceptible and CLI-resistant Streptococcus pyogenes strains exhibit CLI-dependent NADase induction. Furthermore, we investigated the mechanism of this phenomenon using 13 types of two-component sensor knockout strains derived from the CLI-susceptible strain 1529 that has a CLI-dependent NADase induction phenotype. Among the knockout strains, only 1529ΔcovS lost the phenotype. Additionally, 1529ΔspeB, 1529Δmga, and 1529Δrgg retained the CLI-dependent NADase induction phenotype. These findings indicate that CovS is related to this phenotype in a SpeB-independent manner.

Doxorubicin (DOX) is an effective chemotherapeutic drug, but its use can lead to cardiomyopathy, which is the leading cause of mortality among cancer patients. Macrophages play a role in DOX-induced cardiomyopathy (DCM), but the mechanisms undlerlying this relationship remain unclear. This study aimed to investigate how IKKα regulates macrophage activation and contributes to DCM in a mouse model. Specifically, the role of macrophage IKKα was evaluated in macrophage-specific IKKα knockout mice that received DOX injections. The findings revealed increased expression of IKKα in heart tissues after DOX administration. In mice lacking macrophage IKKα, myocardial injury, ventricular remodeling, inflammation, and proinflammatory macrophage activation worsened in response to DOX administration. Bone marrow transplant studies confirmed that IKKα deficiency exacerbated cardiac dysfunction. Macrophage IKKα knockout also led to mitochondrial damage and metabolic dysfunction in macrophages, thereby resulting in increased cardiomyocyte injury and oxidative stress. Single-cell sequencing analysis revealed that IKKα directly binds to STAT3, leading to the activation of STAT3 phosphorylation at S727. Interestingly, the inhibition of STAT3-S727 phosphorylation suppressed both DCM and cardiomyocyte injury. In conclusion, the IKKα-STAT3-S727 signaling pathway was found to play a crucial role in DOX-induced cardiomyopathy. Targeting this pathway could be a promising therapeutic strategy for treating DOX-related heart failure., (© 2024. The Author(s).)

Some negative-sense RNA viruses, including measles virus (MeV), share the characteristic that during their infection cycle, cytoplasmic inclusion bodies (IBs) are formed where components of the viral replication machinery are concentrated. As a foci of viral replication, how IBs act to enhance the efficiency of infection by affecting virus-host interactions remains an important topic of investigation. We previously established that upon MeV infection, the epigenetic host protein, WD repeat-containing protein 5 (WDR5), translocates to cytoplasmic viral IBs and facilitates MeV replication. We now show that WDR5 is recruited to IBs by forming a complex with IB-associated MeV phosphoprotein via a conserved binding motif located on the surface of WDR5. Furthermore, we provide evidence that WDR5 promotes viral replication by suppressing a major innate immune response pathway, the double-stranded RNA-mediated activation of protein kinase R and integrated stress response., Importance: MeV is a pathogen that remains a global concern, with an estimated 9 million measles cases and 128,000 measles deaths in 2022 according to the World Health Organization. A large population of the world still has inadequate access to the effective vaccine against the exceptionally transmissible MeV. Measles disease is characterized by a high morbidity in children and in immunocompromised individuals. An important area of research for negative-sense RNA viruses, including MeV, is the characterization of the complex interactome between virus and host occurring at cytoplasmic IBs where viral replication occurs. Despite the progress made in understanding IB structures, little is known regarding the virus-host interactions within IBs and the role of these interactions in promoting viral replication and antagonizing host innate immunity. Herein we provide evidence suggesting a model by which MeV IBs utilize the host protein WDR5 to suppress the protein kinase R-integrated stress response pathway., Competing Interests: The authors declare no conflict of interest.

The IκB Kinase (IKK) complex, containing catalytic IKK2 and noncatalytic NEMO subunits, plays essential roles in the induction of transcription factors of the NF-κB family. Catalytic activation of IKK2 via phosphorylation of its activation loop is promoted upon noncovalent association of linear or K63-linked polyubiquitin chains to NEMO within the IKK complex. The mechanisms of this activation remain speculative. To investigate interaction dynamics within the IKK complex during activation of IKK2, we conducted hydrogen-deuterium exchange coupled with mass spectrometry (HDX-MS) on NEMO and IKK2 proteins in their free and complex-bound states. Altered proton exchange profiles were observed in both IKK2 and NEMO upon complex formation, and changes were consistent with the involvement of distinct regions throughout the entire length of both proteins, including previously uncharacterized segments, in direct or allosteric interactions. Association with linear tetraubiquitin (Ub 4 ) affected multiple regions of the IKK2:NEMO complex, in addition to previously identified interaction sites on NEMO. Intriguingly, observed enhanced solvent accessibility of the IKK2 activation loop within the IKK2:NEMO:Ub 4 complex, coupled with contrasting protection of surrounding segments of the catalytic subunit, suggests an allosteric role for NEMO:Ub 4 in priming IKK2 for phosphorylation-dependent catalytic activation.

Background: Cell-based strategies are being explored as a therapeutic option for muscular dystrophies, using a variety of cell types from different origin and with different characteristics. Primary pericytes are multifunctional cells found in the capillary bed that exhibit stem cell-like and myogenic regenerative properties. This unique combination allows them to be applied systemically, presenting a promising opportunity for body-wide muscle regeneration. We previously reported the successful isolation of ALP + pericytes from skeletal muscle of patients with myotonic dystrophy type 1 (DM1). These pericytes maintained normal growth parameters and myogenic characteristics in vitro despite the presence of nuclear (CUG) n RNA foci, the cellular hallmark of DM1. Here, we examined the behaviour of DM1 pericytes during myogenic differentiation., Methods: DMPK (CTG) n repeat lengths in patient pericytes were assessed using small pool PCR, to be able to relate variation in myogenic properties and disease hallmarks to repeat expansion. Pericytes from unaffected controls and DM1 patients were cultured under differentiating conditions in vitro. In addition, the pericytes were grown in co-cultures with myoblasts to examine their regenerative capacity by forming hybrid myotubes. Finally, the effect of pericyte fusion on DM1 disease hallmarks was investigated., Results: Small pool PCR analysis revealed the presence of somatic mosaicism in pericyte cell pools. Upon differentiation to myotubes, DMPK expression was upregulated, leading to an increase in nuclear foci sequestering MBNL1 protein. Remarkably, despite the manifestation of these disease biomarkers, patient-derived pericytes demonstrated myogenic potential in co-culture experiments comparable to unaffected pericytes and myoblasts. However, only the unaffected pericytes improved the disease hallmarks in hybrid myotubes. From 20% onwards, the fraction of unaffected nuclei in myotubes positively correlated with a reduction of the number of RNA foci and an increase in the amount of free MBNL1., Conclusions: Fusion of only a limited number of unaffected myogenic precursors to DM1 myotubes already ameliorates cellular disease hallmarks, offering promise for the development of cell transplantation strategies to lower disease burden., (© 2024. The Author(s).)

Nucleoside triphosphates are indispensable in numerous biological processes, with enzymes involved in their biogenesis playing pivotal roles in cell proliferation. Pyruvate kinase (PYK), commonly regarded as the terminal glycolytic enzyme that generates ATP in tandem with pyruvate, is also capable of synthesizing a wide range of nucleoside triphosphates from their diphosphate precursors. Despite their substrate promiscuity, some PYKs show preference towards specific nucleotides, suggesting an underlying mechanism for differentiating nucleotide bases. However, the thorough characterization of this mechanism has been hindered by the paucity of nucleotide-bound PYK structures. Here, we present crystal structures of Streptococcus pneumoniae PYK in complex with four different nucleotides. These structures facilitate direct comparison of the protein-nucleotide interactions and offer structural insights into its pronounced selectivity for GTP synthesis. Notably, this selectivity is dependent on a sequence motif in the nucleotide recognition site that is widely present among prokaryotic PYKs, particularly in Firmicutes species. We show that pneumococcal cell growth is significantly impaired when expressing a PYK variant with compromised GTP and UTP synthesis activity, underscoring the importance of PYK in maintaining nucleotide homeostasis. Our findings collectively advance our understanding of PYK biochemistry and prokaryotic metabolism., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Japanese encephalitis is an acute infectious disease of the central nervous system caused by neurotropic Japanese encephalitis virus (JEV). As a member of TAM (Tyro3, Axl and Mertk) family, Mertk has involved in multiple biological processes by engaging with its bridging ligands Gas6 and Protein S, including invasion of pathogens, phagocytosis of apoptotic cells, inflammatory response regulation, and the maintenance of blood brain barrier (BBB) integrity. However, its role in encephalitis caused by JEV infection has not been studied in detail. Here, we found that Mertk -/- mice exhibited higher mortality and more rapid disease progression than wild-type mice after JEV challenge. There were no significant differences in viral load and cytokines expression level in peripheral tissues between Wild type and Mertk -/- mice. Furthermore, the absence of Mertk had little effect on the inflammatory response and immunopathological damage while it can cause an increased viral load in the brain. For the in vitro model of BBB, Mertk was shown to maintain the integrity of the BBB. In vivo, Mertk -/- mice exhibited higher BBB permeability and lower BBB integrity. Taken together, our findings demonstrate that Mertk acts as a protective factor in the development of encephalitis induced by JEV infection, which is mainly associated with its beneficial effect on BBB integrity, rather than its regulation of inflammatory response., (© 2024. The Author(s).)

Reliable gene expression analysis in bone remodeling studies requires an appropriate selection of internal controls, i.e. stable reference genes for the normalization of quantitative real-time PCR (RT-qPCR), the most common method used for quantifying gene expression measurements. Even the most widely used reference genes can have variable expression under different experimental conditions, or in different tissue types or treatment regimes, so selecting appropriate controls is a key step in ensuring reliable results. The aim of this research was to identify the most stable reference gene(s) for the study of olanzapine modulated bone remodeling in rats. RNA was isolated from the maxillary alveolar and femoral bones of olanzapine or placebo-treated Wistar rats and transcribed to cDNA. The expression of 12 candidate reference genes was assessed by RT-qPCR. Their expressions were analysed using GeNorm, NormFinder, BestKeeper and delta Ct algorithms, and by the comprehensive ranking method. PPIA, HRPT1 and PGK1 were the most stably expres sed reference genes and the combination of the three genes was optimal for normalization. This study is the first to identify the optimal reference genes for research in olanzapine-exposed rats, which serve as a pivotal benchmark for enhancing the accuracy and reliability of future RT-qPCR expression in bone studies., (© 2024 Saranda Disha-Ibrahimi et al., published by Sciendo.)

Histidine kinases (HKs) are important sensor proteins in fungi and play an essential role in environmental adaptation. However, the mechanisms by which fungi sense and respond to fungivores attack via HKs are not fully understood. In this study, we utilized Neurospora crassa to investigate the involvement of HKs in responding to fungivores attack. We found that the 11 HKs in N. crassa not only affected the growth and development, but also led to fluctuations in antioxidant production. Ten mutants in the genes encoding HKs (except ∆phy1) showed increased production of reactive oxygen species (ROS), especially upon Sinella curviseta attack. The ROS burst triggered changes in conidia and perithecial beaks formation, as well as accumulation of β-glucan, ergothioneine, ergosterol, and carotenoids. β-glucan was increased in ∆hk9, ∆os1, ∆hcp1, ∆nik2, ∆sln1, ∆phy1 and ∆phy2 mutants compared to the wild-type strain. In parallel, ergothioneine accumulation was improved in ∆phy1 and ∆hk16 mutants and further increased upon attack, except in ∆os1 and ∆hk16 mutants. Additionally, fungivores attack stimulated ergosterol and dehydroergosterol production in ∆hk9 and ∆os1 mutants. Furthermore, deletion of these genes altered carotenoid accumulation, with wild-type strain, ∆hk9, ∆os1, ∆hcp1, ∆sln1, ∆phy2, and ∆dcc1mutants showing an increase in carotenoids upon attack. Taken together, HKs are involved in regulating the production of conidia and antioxidants. Thus, HKs may act as sensors of fungivores attack and effectively improve the adaptive capacity of fungi to environmental stimuli., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)