Optimizing perioperative treatment for potentially resectable stage III squamous cell lung carcinoma: promising results of a condensed four-cycle regimen with tislelizumaband chemotherapy.

BACKGROUND: The standard care for resectable non-small cell lung cancer (NSCLC) involves perioperative therapy combining chemotherapy and immune checkpoint inhibitors, typically lasting 6 to 12 months. However, the optimal treatment strategies for potentially resectable squamous cell lung carcinoma (SCC) remain unclear. This Phase 2 trial aimed to assess the efficacy and safety of a condensed four-cycle perioperative treatment regimen with tislelizumab combined with chemotherapy in patients with potentially resectable stage III SCC. METHODS: Patients with potentially resectable stage IIIA-IIIB (N2) SCC received intravenous tislelizumab, albumin-bound paclitaxel, and carboplatin for up to four cycles. The primary endpoints were major pathologic response (MPR) and incidence of treatment-related adverse events. Safety and potential biomarkers for efficacy prediction were also assessed. RESULTS: Among 35 enrolled patients, 32 underwent surgery with R0 resection achieved in all cases. MPR was achieved in 24 patients and pathological complete response (pCR) in 14 patients. Radiographic objective response was observed in 31 patients. The 12-month and 24-month event-free survival rate was 85.7 and 61.0%, respectively. Four patients experienced grade 3 or 4 adverse events. Tumor tissue based next-generation sequencing revealed the potential associations between several biomarkers and pathological response, including tumor neoantigen burden score, 18-gene expression profile score, CD8 + T cells, M1/M2 macrophages ratio and interferon-gamma expression level. Besides, circulating tumor DNA (ctDNA) dynamics and concentration were also associated with pathological response and the presence of ctDNA at postoperative month 1 was a strong predictor for disease relapse. Furthermore, metagenomic sequencing in bronchoalveolar lavage fluid demonstrated Streptococcus was the most abundant genus in the pCR group. CONCLUSIONS: A condensed four-cycle perioperative treatment regimen of tislelizumab combined with chemotherapy demonstrated promising efficacy and manageable toxicities in potentially resectable stage III SCC. Specific biomarkers showed potential for predicting treatment efficacy and the mechanism of superior antitumor response of pCR patients was preliminarily and indirectly explored. TRIAL REGISTRATION: ClinicalTrials.gov, NCT05024266. Registered August 27, 2021.

Ars2 links the nuclear cap-binding complex to RNA interference and cell proliferation.

Here we identify a component of the nuclear RNA cap-binding complex (CBC), Ars2, that is important for miRNA biogenesis and critical for cell proliferation. Unlike other components of the CBC, Ars2 expression is linked to the proliferative state of the cell. Deletion of Ars2 is developmentally lethal, and deletion in adult mice led to bone marrow failure whereas parenchymal organs composed of nonproliferating cells were unaffected. Depletion of Ars2 or CBP80 from proliferating cells impaired miRNA-mediated repression and led to alterations in primary miRNA processing in the nucleus. Ars2 depletion also reduced the levels of several miRNAs, including miR-21, let-7, and miR-155, that are implicated in cellular transformation. These findings provide evidence for a role for Ars2 in RNA interference regulation during cell proliferation.

IKKß promotes metabolic adaptation to glutamine deprivation via phosphorylation and inhibition of PFKFB3.

Glutamine is an essential nutrient for cancer cell survival and proliferation. Enhanced utilization of glutamine often depletes its local supply, yet how cancer cells adapt to low glutamine conditions is largely unknown. Here, we report that IκB kinase ß (IKKß) is activated upon glutamine deprivation and is required for cell survival independently of NF-κB transcription. We demonstrate that IKKß directly interacts with and phosphorylates 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase isoform 3 (PFKFB3), a major driver of aerobic glycolysis, at Ser269 upon glutamine deprivation to inhibit its activity, thereby down-regulating aerobic glycolysis when glutamine levels are low. Thus, due to lack of inhibition of PFKFB3, IKKß-deficient cells exhibit elevated aerobic glycolysis and lactate production, leading to less glucose carbons contributing to tricarboxylic acid (TCA) cycle intermediates and the pentose phosphate pathway, which results in increased glutamine dependence for both TCA cycle intermediates and reactive oxygen species suppression. Therefore, coinhibition of IKKß and glutamine metabolism results in dramatic synergistic killing of cancer cells both in vitro and in vivo. In all, our results uncover a previously unidentified role of IKKß in regulating glycolysis, sensing low-glutamine-induced metabolic stress, and promoting cellular adaptation to nutrient availability.

An emerging link between lncRNAs and cancer sex dimorphism.

The prevalence and progression of cancer differ in males and females, and thus, sexual dimorphism in tumor development directly impacts clinical research and medicine. Long non-coding RNAs (lncRNAs) are increasingly recognized as important players in gene expression and various cellular processes, including cancer development and progression. In recent years, lncRNAs have been implicated in the differences observed in cancer incidence, progression, and treatment responses between men and women. Here, we present a brief overview of the current knowledge regarding the role of lncRNAs in cancer sex dimorphism, focusing on how they affect epigenetic processes in male and female mammalian cells. We discuss the potential mechanisms by which lncRNAs may contribute to sex differences in cancer, including transcriptional control of sex chromosomes, hormonal signaling pathways, and immune responses. We also propose strategies for studying lncRNA functions in cancer sex dimorphism. Furthermore, we emphasize the importance of considering sex as a biological variable in cancer research and the need to investigate the role lncRNAs play in mediating these sex differences. In summary, we highlight the emerging link between lncRNAs and cancer sex dimorphism and their potential as therapeutic targets.

A Novel Assessment of Metabolic Pathways in Peritoneal Metastases from Low-Grade Appendiceal Mucinous Neoplasms.

BACKGROUND: There is a paucity of targeted therapies for patients with pseudomyxoma peritonei (PMP) secondary to low-grade appendiceal mucinous neoplasms (LAMNs). Dysregulated metabolism has emerged as a hallmark of cancer, and the relationship of metabolomics and cancer is an area of active scientific exploration. We sought to characterize phenotypic differences found in peritoneal metastases (PM) derived from LAMN versus adenocarcinoma. METHODS: Tumors were washed with phosphate-buffered saline (PBS), microdissected, then dissociated in ice-cold methanol dried and reconstituted in pyridine. Samples were derivatized in tert-butyldimethylsilyl (TBDMS) and subjected to gas chromatography-coupled mass spectrometry. Metabolites were assessed based on a standard library. RNA sequencing was performed, with pathway and network analyses on differentially expressed genes. RESULTS: Eight peritoneal tumor samples were obtained and analyzed: LAMNs (4), and moderate to poorly differentiated adenocarcinoma (colon [1], appendix [3]). Decreases in pyroglutamate, fumarate, and cysteine in PM from LAMNs were found compared with adenocarcinoma. Analyses showed the differential gene expression was dominated by the prevalence of metabolic pathways, particularly lipid metabolism. The gene retinol saturase (RETSAT), downregulated by LAMN, was involved in the multiple metabolic pathways that involve lipids. Using network mapping, we found IL1B signaling to be a potential top-level modulation candidate. CONCLUSIONS: Distinct metabolic signatures may exist for PM from LAMN versus adenocarcinoma. A multitude of genes are differentially regulated, many of which are involved in metabolic pathways. Additional research is needed to identify the significance and applicability of targeting metabolic pathways in the potential development of novel therapeutics for these challenging tumors.

Effects of Dietary Phytochemicals on DNA Damage in Cancer Cells.

With the increasing incidence of cancer worldwide, the prevention and treatment of cancer have garnered considerable scientific attention. Traditional chemotherapeutic drugs are highly toxic and associated with substantial side effects; therefore, there is an urgent need for developing new therapeutic agents. Dietary phytochemicals are important in tumor prevention and treatment because of their low toxicity and side effects at low concentrations; however, their exact mechanisms of action remain obscure. DNA damage is mainly caused by physical or chemical factors in the environment, such as ultraviolet light, alkylating agents and reactive oxygen species that cause changes in the DNA structure of cells. Several phytochemicals have been shown inhibit the occurrence and development of tumors by inducing DNA damage. This article reviews the advances in phytochemical research; particularly regarding the mechanisms related to DNA damage and provide a theoretical basis for future chemoprophylaxis research.

The B56α subunit of PP2A is necessary for mesenchymal stem cell commitment to adipocyte.

Adipose tissue plays a major role in maintaining organismal metabolic equilibrium. Control over the fate decision from mesenchymal stem cells (MSCs) to adipocyte differentiation involves coordinated command of phosphorylation. Protein phosphatase 2A plays an important role in Wnt pathway and adipocyte development, yet how PP2A complexes actively respond to adipocyte differentiation signals and acquire specificity in the face of the promiscuous activity of its catalytic subunit remains unknown. Here, we report the PP2A phosphatase B subunit B56α is specifically induced during adipocyte differentiation and mediates PP2A to dephosphorylate GSK3ß, thereby blocking Wnt activity and driving adipocyte differentiation. Using an inducible B56α knock-out mouse, we further demonstrate that B56α is essential for gonadal adipose tissue development in vivo and required for the fate decision of adipocytes over osteoblasts. Moreover, we show B56α expression is driven by the adipocyte transcription factor PPARγ thereby establishing a novel link between PPARγ signaling and Wnt blockade. Overall, our results reveal B56α is a necessary part of the machinery dictating the transition from pre-adipocyte to mature adipocyte and provide fundamental insights into how PP2A complex specifically and actively regulates unique signaling pathway in biology.

MicroRNA-23a-3p overexpression represses proliferation and accelerates apoptosis of granular cells in polycystic ovarian syndrome by targeting HMGA2.

OBJECTIVE: Granular cells (GCs) are involved in polycystic ovarian syndrome (PCOS) progression. MicroRNA (miR)-23a downregulation is linked to PCOS development. Therefore, this research explored the influences of miR-23a-3p on GC proliferation and apoptosis in PCOS. METHODS: Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blotting were conducted to examine miR-23a-3p and HMGA2 expression in GCs of patients with PCOS. Then, miR-23a-3p and/or HMGA2 expression was altered in GCs (KGN and SVOG), after which miR-23a-3p, HMGA2, Wnt2, and ß-catenin expression, GC viability, and GC apoptosis were measured by RT-qPCR and western blotting, MTT assay, and flow cytometry, respectively. A dual-luciferase reporter gene assay was utilized to assess the targeting relationship between miR-23a-3p and HMGA2. Finally, GC viability and apoptosis were tested after the combined treatment of miR-23a-3p mimic and pcDNA3.1-HMGA2. RESULTS: miR-23a-3p was poorly expressed but HMGA2 was overexpressed in GCs of patients with PCOS. Mechanistically, HMGA2 was negatively targeted by miR-23a-3p in GCs. Furthermore, miR-23a-3p inhibition or HMGA2 upregulation elevated viability and reduced apoptosis of KGN and SVOG cells, along with increased Wnt2 and ß-catenin expression. In KNG cells, HMGA2 overexpression abrogated the impacts of miR-23a-3p overexpression on GC viability and apoptosis. CONCLUSIONS: Collectively, miR-23a-3p decreased HMGA2 expression to block the Wnt/ß-catenin pathway, thereby depressing viability and facilitating apoptosis of GCs.

Protein interactions network of goat innate immune signalling pathway proteins and Haemonchus contortus excretory-secretory proteins.

Haemonchus contortus (H. contortus) has caused a huge impact on the animal husbandry economy in the world's tropical and subtropical regions. Innate immunity is the first-line of host defence. The host recognizes pathogen-associated molecular patterns (PAMPs) through a variety of pattern recognition receptors (PRRs) and activates downstream signalling pathways to resist pathogens invasion. Therefore, elucidating the immune interaction between host and pathogen is key to understanding how the host resists the pathogen. We identified 1516 protein-protein interactions (PPIs) between goat innate immune signal pathway proteins and H. contortus excretory-secretory proteins (ESPs) by Recombination-based "Library vs. Library" yeast two-hybrid system (RLL-Y2H) and constructed the PPIs network. Among them, the NLR and IL-17 signalling pathways have the most protein interactions. And there were more interaction proteins between NOD1 and MUC5AC proteins in the pathways. Combined with the differentially expressed genes (DEGs) of susceptible and resistant goats identified in the preliminary work of our laboratory, we selected the intersection genes to construct the PPIs network, and TRAF2 appeared as a key protein of goat innate immune signalling pathway. We initially studied the PPIs between goat and H. contortus ESPs, which provides valuable information for better understanding the immune interaction between the goats and the H. contortus.

Optimization of the moving averaging-moving differential algorithm for Φ-OTDR.

The moving averaging-moving differential (MA-MD) algorithm is widely adopted for event recognition and positioning in phase-sensitive optical time-domain reflectometry (Φ-OTDR) vibration sensing. In this paper, two theorems about the two critical parameters of this algorithm, the MA window width and the MD interval, are found, proved, and verified. According to them we can not only obtain an optimal signal-to-noise ratio and avoid missing the vibration signal but also greatly improve the signal-searching efficiency. Work in this paper is of essential importance for understanding the characteristics of the MA-MD algorithm and will remarkably benefit its practical applications.

A novel genomic classification system of gastric cancer via integrating multidimensional genomic characteristics.

BACKGROUND: Gastric cancer (GC) is one of the leading causes of cancer deaths with high heterogeneity. There is currently a paucity of clinically applicable molecular classification system to guide precise medicine. METHODS: A total of 70 Chinese patients with GC were included in this study and whole-exome sequencing was performed. Unsupervised clustering was undertaken to identify genomic subgroups, based on mutational signature, copy number variation, neoantigen, clonality, and essential genomic alterations. Subgroups were characterized by clinicopathological factors, molecular features, and prognosis. RESULTS: We identified 32 significantly mutated genes (SMGs), including TP53, ARID1A, PIK3CA, CDH1, and RHOA. Of these, PREX2, PIEZO1, and FSIP2 have not been previously reported in GC. Using a novel genome-based classification method that integrated multidimensional genomic features, we categorized GC into four subtypes with distinct clinical phenotypes and prognosis. Subtype 1, which was predominantly Lauren intestinal type, harbored recurrent TP53 mutation and ERBB2 amplification, high tumor mutation burden (TMB)/tumor neoantigen burden (TNB), and intratumoral heterogeneity, with a liver metastasis tendency. Subtype 2 tended to occur at an elder age, accompanying with frequent TP53 and SYNE1 mutations, high TMB/TNB, and was associated with poor prognosis. Subtype 3 and subtype 4 included patients with mainly diffuse/mixed type tumors, high frequency of peritoneal metastasis, and genomical stability, whereas subtype 4 was associated with a favorable prognosis. CONCLUSIONS: By integrating multidimensional genomic characteristics, we proposed a novel genomic classification system of GC associated with clinical phenotypes and provided a new insight to facilitate genome-guided risk stratification and disease management.

The B55α subunit of PP2A drives a p53-dependent metabolic adaptation to glutamine deprivation.

Glutamine is an essential nutrient for cancer cell survival and proliferation, yet the signaling pathways that sense glutamine levels remain uncharacterized. Here, we report that the protein phosphatase 2A (PP2A)-associated protein, α4, plays a conserved role in glutamine sensing. α4 promotes assembly of an adaptive PP2A complex containing the B55α regulatory subunit via providing the catalytic subunit upon glutamine deprivation. Moreover, B55α is specifically induced upon glutamine deprivation in a ROS-dependent manner to activate p53 and promote cell survival. B55α activates p53 through direct interaction and dephosphorylation of EDD, a negative regulator of p53. Importantly, the B55α-EDD-p53 pathway is essential for cancer cell survival and tumor growth under low glutamine conditions in vitro and in vivo. This study delineates a previously unidentified signaling pathway that senses glutamine levels as well as provides important evidence that protein phosphatase complexes are actively involved in signal transduction.

Using DFB laser self-injection locked to an optical waveguide ring resonator as a light source of Φ-OTDR.

A distributed feedback (DFB) semiconductor laser self-injection locked to an optical waveguide ring resonator (OWRR) is used in a phase-sensitive optical time-domain reflectometry (Φ-OTDR) vibration sensing system as its light source. A frequency-hopping-free period of more than 100 s is realized. A spatial resolution of 13 m at 4700 m and simultaneous measurement of two vibration sources are realized. The measurable vibration frequency is from 8 Hz to the upper limit of the sampling theorem. These satisfactory performances demonstrate that the DFB laser locked to an OWRR is not only cost effective, but also stable and reliable for Φ-OTDR sensing.

Glutamine deficiency induces DNA alkylation damage and sensitizes cancer cells to alkylating agents through inhibition of ALKBH enzymes.

Driven by oncogenic signaling, glutamine addiction exhibited by cancer cells often leads to severe glutamine depletion in solid tumors. Despite this nutritional environment that tumor cells often experience, the effect of glutamine deficiency on cellular responses to DNA damage and chemotherapeutic treatment remains unclear. Here, we show that glutamine deficiency, through the reduction of alpha-ketoglutarate, inhibits the AlkB homolog (ALKBH) enzymes activity and induces DNA alkylation damage. As a result, glutamine deprivation or glutaminase inhibitor treatment triggers DNA damage accumulation independent of cell death. In addition, low glutamine-induced DNA damage is abolished in ALKBH deficient cells. Importantly, we show that glutaminase inhibitors, 6-Diazo-5-oxo-L-norleucine (DON) or CB-839, hypersensitize cancer cells to alkylating agents both in vitro and in vivo. Together, the crosstalk between glutamine metabolism and the DNA repair pathway identified in this study highlights a potential role of metabolic stress in genomic instability and therapeutic response in cancer.

CARM1 suppresses de novo serine synthesis by promoting PKM2 activity.

Glucose is a critical nutrient for cell proliferation. However, the molecular pathways that regulate glucose metabolism are still elusive. We discovered that co-activator-associated arginine methyltransferase 1 (CARM1) suppresses glucose metabolism toward serine biosynthesis. By tracing the 13C-labeled glucose, we found that Carm1 knockout mouse embryonic fibroblasts exhibit significantly increased de novo serine synthesis than WT cells. This is caused, at least in part, by the reduced pyruvate kinase (PK) activity in these cells. The M2 isoform of PK (PKM2) is arginine-methylated by CARM1, and methylation enhances its activity. Mechanistically, CARM1 methylates PKM2 at arginines 445 and 447, which enhances PKM2 tetramer formation. Consequently, Carm1 knockout cells exhibit significant survival advantages over WT cells when extracellular serine is limited, likely due to their enhanced de novo serine synthesis capacity. Altogether, we identified CARM1 as an important regulator of glucose metabolism and serine synthesis.

A genomic and clinicopathological study of non-small-cell lung cancers with discordant ROS1 gene status by fluorescence in-situ hybridisation and immunohistochemical analysis.

AIMS: ROS1 immunohistochemistry (IHC) using D4D6 antibody is a useful tool for screening patients with non-small-cell lung cancer (NSCLC) who may be suitable for targeted therapy. Many studies and our data have identified cases that express the ROS1 protein strongly but are negative for ROS1 by fluorescence in-situ hybridisation (FISH). The present study investigated the driver mutation and clinicopathological characteristics of 26 discordant cases (ROS1 IHC-positive but FISH-negative) to find new clues for distinguishing real ROS1-rearranged cases. METHODS AND RESULTS: Tumours from 26 discordant cases were analysed for clinicopathological characteristics, mutations in EGFR, KRAS, ERBB2, BRAF and PIK3CA; fusions in ALK and RET; and amplifications in MET, ERBB2 and ROS1. ROS1-rearranged NSCLCs were significantly more likely to be found in younger patients and at an advanced stage; they showed cribriform features, extracellular mucus and psammoma bodies, whereas ROS1-discordant cases were found in older patients at a relatively early tumour-node-metastasis (TNM) stage and showed a lepidic growth pattern (all P < 0.001). Most ROS1-rearranged NSCLCs had no concurrent mutation, whereas 73% of discordant cases harboured genetic aberrations, including EGFR and ERBB2. Compared with general lung adenocarcinomas, ERBB-2 abnormality was disproportionately high in ROS1-discordant cases. Moreover, we optimised the scoring criteria for ROS1 IHC as 'H score > 150 and no concurrent mutations'; the specificity was then increased to 81.6%. CONCLUSIONS: Compared with ROS1-rearranged cases, ROS1-discordant patients showed distinct clinical and morphological features and often harboured another oncogenic driver alteration. The use of optimised screening criteria will increase the specificity of ROS1 antibody.

Alpha4 is an essential regulator of PP2A phosphatase activity.

The activity and specificity of serine/threonine phosphatases are governed largely by their associated proteins. alpha4 is an evolutionarily conserved noncatalytic subunit for PP2A-like phosphatases. Though alpha4 binds to only a minority of PP2A-related catalytic subunits, alpha4 deletion leads to progressive loss of all PP2A, PP4, and PP6 phosphatase complexes. In healthy cells, association with alpha4 renders catalytic (C) subunits enzymatically inactive while protecting them from proteasomal degradation until they are assembled into a functional phosphatase complex. During cellular stress, existing PP2A complexes can become unstable. Under such conditions, alpha4 sequesters released C subunits and is required for the adaptive increase in targeted PP2A activity that can dephosphorylate stress-induced phosphorylated substrates. Consistent with this, overexpression of alpha4 protects cells from a variety of stress stimuli, including DNA damage and nutrient limitation. These findings demonstrate that alpha4 plays a required role in regulating the assembly and maintenance of adaptive PP2A phosphatase complexes.

[Changes of perimysial junctional plates induced by excessive eccentric training and the effects of acupuncture intervention].

This study aimed to investigate the effects of acupuncture intervention on excessive eccentric training-induced changes of perimysial junctional plates (PJPs) domain. Thirty healthy male Wistar rats were randomly assigned to 5 groups: control group, four-week training group, four-week training + 1-week recovery group and four-week training + 1-week acupuncture group. Rats were subjected to continuous excessive eccentric training for 4 weeks (incline -16°, speed 16-20 m/min, 60-90 min/d, 5 day per week), and then were subjected to one-week spontaneous recovery or one-week recovery with acupuncture intervention (a piece of filiform needle for 4 min every day). The PJPs domain changes were observed under transmission electron microscopy, and the perimysial collagen network structural changes were examined by scanning electron microscopy with or without a digestion technique (NaOH). The following results were obtained: (1) Compared with control group, PJPs domain of four-week training group showed excessive shortening of sarcomere (P < 0.001), serious damage of sarcomere structure, and altered mitochondria morphology in intermyofibria and subsarcolemma; 54% degradation of sarcolemma, and increased number of caveolae (P < 0.01); reduced number of PJPs (P < 0.001). (2) In comparison with four-week training group, PJPs domain was slightly changed in four-week training + 1-week recovery group, i.e., partial recovery of sarcomere length and structure (accounting for 85.23% of control group), and recovery of intermyofibrial and subsarcolemmal mitochondria morphology; decreased sarcolemmal degradation (P < 0.001), and increased number of caveolae (P < 0.05); increased PJPs number (P < 0.001). (3) PJPs domain changed in four-week training + 1-week acupuncture group compared with four-week training + 1-week recovery group, which were substantial recovery of sarcomere length (accounting for 94.51% of control group), increased subsarcolemmal mitochondrial fusion (P < 0.001), decreased caveolae number (P < 0.001), and decreased PJPs number (P < 0.001). The results indicated that excessive eccentric training resulted in excessively reduced number of PJPs with altered PJPs domain homeostasis, thus impeding the adaptability to eccentric training. After 1 week of natural recovery, the number of PJPs was excessively increased, hindering muscle damage repair. Acupuncture intervention helped to recover PJPs number and PJPs domain homeostasis, thus significantly relieving overuse injuries.

Vemurafenib resistance reprograms melanoma cells towards glutamine dependence.

BACKGROUND: (V600) BRAF mutations drive approximately 50% of metastatic melanoma which can be therapeutically targeted by BRAF inhibitors (BRAFi) and, based on resistance mechanisms, the combination of BRAF and MEK inhibitors (BRAFi + MEKi). Although the combination therapy has been shown to provide superior clinical benefits, acquired resistance is still prevalent and limits the overall survival benefits. Recent work has shown that oncogenic changes can lead to alterations in tumor cell metabolism rendering cells addicted to nutrients, such as the amino acid glutamine. Here, we evaluated whether melanoma cells with acquired resistance display glutamine dependence and whether glutamine metabolism can be a potential molecular target to treat resistant cells. METHODS: Isogenic BRAFi sensitive parental (V600) BRAF mutant melanoma cell lines and resistant (derived by chronic treatment with vemurafenib) sub-lines were used to assess differences in the glutamine uptake and sensitivity to glutamine deprivation. To evaluate a broader range of resistance mechanisms, isogenic pairs where the sub-lines were resistant to BRAFi + MEKi were also studied. Since resistant cells demonstrated increased sensitivity to glutamine deficiency, we used glutaminase inhibitors BPTES [bis-2-(5 phenylacetamido-1, 2, 4-thiadiazol-2-yl) ethyl sulfide] and L-L-DON (6-Diazo-5-oxo-L-norleucine) to treat MAPK pathway inhibitor (MAPKi) resistant cell populations both in vitro and in vivo. RESULTS: We demonstrated that MAPKi-acquired resistant cells uptook greater amounts of glutamine and have increased sensitivity to glutamine deprivation than their MAPKi-sensitive counterparts. In addition, it was found that both BPTES and L-DON were more effective at decreasing cell survival of MAPKi-resistant sub-lines than parental cell populations in vitro. We also showed that mutant NRAS was critical for glutamine addiction in mutant NRAS driven resistance. When tested in vivo, we found that xenografts derived from resistant cells were more sensitive to BPTES or L-DON treatment than those derived from parental cells. CONCLUSION: Our study is a proof-of-concept for the potential of targeting glutamine metabolism as an alternative strategy to suppress acquired MAPKi-resistance in melanoma.

Design and Evaluation of a Multi-Epitope Peptide of Human Metapneumovirus.

OBJECTIVES: No licensed vaccines or therapeutic agents for human metapneumovirus (hMPV) infection exist to date. We aimed to construct a multi-epitope peptide (MEP) of hMPV to show promising results for epitope-based vaccine development. METHODS: Six independent algorithms were screened to predict B-cell epitopes of hMPV, and three algorithms were used to predict cytotoxic T lymphocyte and T helper (Th) lymphocyte epitopes. Predicted epitopes were assembled in series with the spacers GPGPG and KK introduced, termed MEP. Recombinant mep genes were inserted into pET32a(+) plasmid and expressed in Escherichia coli strain BL21 (DE3). BALB/c mice were immunized with MEP with different adjuvants. Antibody titer, lymphocyte proliferation, cytotoxic T lymphocyte (CTL) activity and splenocyte cytokines were detected 2 weeks later after the last immunization. Microneutralization assay was used to detect neutralizing antibodies. RESULTS: Six B-cell epitopes, four CTL epitopes and two Th epitopes were screened to construct the mep gene. Expressed MEP induced >104 antibodies in BALB/c mice, and produced anti-MEP antibody reacting with hMPV strains specifically as detected in indirect fluorescent assay (the titer was 160). The lymphocyte proliferation index, CTL activity and splenocyte cytokines of the MEP immunization groups were higher than in the control group (p < 0.05). Both IgG1 and IgG2a antibodies could be detected in the different groups, and balanced Th1/Th2 cytokines were secreted by splenocytes in these groups. The mean neutralizing titers of the MEP+CpG ODN, MEP+Alum and MEP+Alum+ CpG ODN groups were 87 (95% CI 50-126), 93 (95% CI 67-121) and 96 (95% CI 69-147), respectively. CONCLUSION: MEP of hMPV elicited both strong humoral immunity and cell-mediated immunity in mice. The anti-MEP serum could neutralize hMPV infection in vitro. Joint use of CpG ODN and aluminum hydroxide adjuvants obtained the best immune effects. This study may contribute to hMPV epitope-based vaccine development.