Efficacy of immune checkpoint inhibitors along with chemotherapy in non-small cell lung cancer and the impact on adverse reactions and serum tumor markers.

OBJECTIVE: To examine the efficacy of immune checkpoint inhibitors along with chemotherapy in non-small cell lung cancer (NSCLC) and the effect on adverse reactions and serum tumor markers. METHODS: Data of 112 NSCLC patients admitted to Geriatric respiratory department, Xi'an International Medical Center Hospital from February 2018 to March 2021 were analyzed retrospectively. Among them, 54 patients treated with concurrent chemotherapy were labeled as the control group (CG), and 58 patients treated with PD-1/PD-L1 inhibitors in addition to chemotherapy were the observation group (OG). The two groups were compared in terms of immune function indexes, therapeutic efficacy, incidence of adverse reactions, 1-year survival rate, serum tumor markers before and after treatment, and independent risk factors affecting patients' prognosis. RESULTS: Compared to the CG, the OG exhibited significantly better therapeutic efficacy. The levels of IgG, IgA and IgM 6 months after treatment were significantly higher in both groups than those before treatment, and the elevations in the OG were more evident than those in the CG, and the OG demonstrated markedly lower Recombinant Cytokeratin Fragment Antigen 21-1 (CYFRA21-1), Carcinoembryonic antigen (CEA) and Carbohydrate antigen 125 (CA125) levels after treatment than the CG did. Between the two groups, there was no significant difference identified in the incidence of adverse reactions, but the OG was observed to have much higher 1-year survival rate. The pathological stage, differentiation and treatment regimen were independent risk factors affecting patients' prognosis. CONCLUSION: For NSCLC patients, the adoption of PD-1/PD-L1 inhibitors following chemoradiotherapy shows potential in enhancing clinical efficacy, boosting patients' immune function, and improving long-term survival rates, with premising safety profile.

Screening and epitope characterization of diagnostic nanobody against total and activated Bacteroides fragilis toxin.

Enterotoxigenic Bacteroides fragilis (ETBF) can rapidly secrete an enterotoxin termed B. fragilis toxin (BFT), which is thought to be the only recognized virulence factor in ETBF. ETBF can cause acute diarrhea, inflammatory bowel disease (IBD), colorectal cancer, and breast cancer. BFT is divided into three subtypes, BFT1, BFT2, and BFT3. BFT1 is the most widely distributed in human B. fragilis isolates. BFT can be used as a biomarker for predicting the inflammation-cancer transformation of intestine and breast. Nanobodies have the advantages of small structure, complete antigen recognition capacity, rapid selection via phage display technology, and can be massively produced in microbial expression systems. Nanobodies have become a powerful tool for medical diagnosis and treatment. This study focuses on screening and structural characterization of nanobodies targeting full length and active BFT. By constructing prokaryotic expression systems to obtain recombinant BFT1 protein, high purity BFT1 protein was used to immunize alpacas. Phage display technology was used to construct a phage display library. The positive clones were selected by bio-panning, and the isothermal titration calorimetry was used to select high-affinity nanobodies. Then the three-dimensional structures of BFT1:Nb2.82 and BFT1:Nb3.27 were solved by crystal X-ray diffraction. We got two kinds of nanobodies, Nb2.82 targeting the BFT1 prodomain and Nb3.27 recognizing the BFT1 catalytic domain. This study provides a new strategy for the early diagnosis of ETBF and the possibility for BFT as a biomarker for diagnosing diseases.

Prostate-specific oncogene OTUD6A promotes prostatic tumorigenesis via deubiquitinating and stabilizing c-Myc.

MYC drives the tumorigenesis of human cancers, including prostate cancer (PrCa), thus deubiquitinase (DUB) that maintains high level of c-Myc oncoprotein is a rational therapeutic target. Several ubiquitin-specific protease (USP) family members of DUB have been reported to deubiquitinate c-Myc, but none of them is the physiological DUB for c-Myc in PrCa. By screening all the DUBs, here we reveal that OTUD6A is exclusively amplified and overexpressed in PrCa but not in other cancers, eliciting a prostatic-specific oncogenic role through deubiquitinating and stabilizing c-Myc oncoprotein. Moreover, genetic ablation of OTUD6A efficiently represses prostatic tumorigenesis of both human PrCa cells and the Hi-Myc transgenic PrCa mice, via reversing the metabolic remodeling caused by c-Myc overexpression in PrCa. These results indicate that OTUD6A is a physiological DUB for c-Myc in PrCa setting and specifically promotes prostatic tumorigenesis through stabilizing c-Myc oncoprotein, suggesting that OTUD6A could be a unique therapeutic target for Myc-driven PrCa.

Non-canonical phosphorylation of Bmf by p38 MAPK promotes its apoptotic activity in anoikis.

Bmf contributes to the onset of anoikis by translocating from cytoskeleton to mitochondria when cells lose attachment to the extracellular matrix. However, the structural details of Bmf cytoskeleton tethering and the control of Bmf release upon loss of anchorage remained unknown. Here we showed that cell detachment induced rapid and sustained activation of p38 MAPK in mammary epithelial cell lines. Inhibition of p38 signaling or Bmf knockdown rescued anoikis. Activated p38 MAPK could directly phosphorylate Bmf at multiple sites including a non-proline-directed site threonine 72 (T72). Crystallographic studies revealed that Bmf T72 directly participated in DLC2 binding and its phosphorylation would block Bmf/DLC2 interaction through steric hindrance. Finally, we showed that phosphomimetic mutation of T72 enhanced Bmf apoptotic activity in vitro and in a knock-in mouse model. This work unraveled a novel regulatory mechanism of Bmf activity during anoikis and provided structural basis for Bmf cytoskeleton tethering and dissociation.

A genetically encoded fluorescent biosensor for extracellular L-lactate.

L-Lactate, traditionally considered a metabolic waste product, is increasingly recognized as an important intercellular energy currency in mammals. To enable investigations of the emerging roles of intercellular shuttling of L-lactate, we now report an intensiometric green fluorescent genetically encoded biosensor for extracellular L-lactate. This biosensor, designated eLACCO1.1, enables cellular resolution imaging of extracellular L-lactate in cultured mammalian cells and brain tissue.

CIC Is a Mediator of the ERK1/2-DUSP6 Negative Feedback Loop.

DUSP6 functions as an important negative feedback component of the MAPK/ERK signaling pathway. Although DUSP6 expression is tightly regulated by ERK1/2 signaling, the molecular mechanism of this regulation remains partially understood. In this work, we show that the transcriptional repressor CIC functions downstream of the ERK1/2 signaling to negatively regulate DUSP6 expression. CIC directly represses DUSP6 transcription by binding to three cis-regulatory elements (CREs) in DUSP6 promoter. p90RSK, a downstream target of ERK1/2, phosphorylates CIC at S173 and S301 sites, which creates a 14-3-3 recognition motif, resulting in 14-3-3-mediated nuclear export of CIC and derepression of DUSP6. Finally, we demonstrate that the oncogenic CIC-DUX4 fusion protein acts as a transcriptional activator of DUSP6 and its nuclear/cytoplasmic distribution remains regulated by ERK1/2 signaling. These results complete an ERK1/2/p90RSK/CIC/DUSP6 negative feedback circuit and elucidate the molecular mechanism of how RTK/MAPK signaling harnesses the transcriptional repressor activity of CIC in mammalian cells.

NIRF-Molecular Imaging with Synovial Macrophages-Targeting Vsig4 Nanobody for Disease Monitoring in a Mouse Model of Arthritis.

Nanobody against V-set and Ig domain-containing 4 (Vsig4) on tissue macrophages, such as synovial macrophages, could visualize joint inflammation in multiple experimental arthritis models via single-photon emission computed tomography imaging. Here, we further addressed the specificity and assessed the potential for arthritis monitoring using near-infrared fluorescence (NIRF) Cy7-labeled Vsig4 nanobody (Cy7-Nb119). In vivo NIRF-imaging of collagen-induced arthritis (CIA) was performed using Cy7-Nb119. Signals obtained with Cy7-Nb119 or isotope control Cy7-NbBCII10 were compared in joints of naive mice versus CIA mice. In addition, pathological microscopy and fluorescence microscopy were used to validate the arthritis development in CIA. Cy7-Nb119 accumulated in inflamed joints of CIA mice, but not the naive mice. Development of symptoms in CIA was reflected in increased joint accumulation of Cy7-Nb119, which correlated with the conventional measurements of disease. Vsig4 is co-expressed with F4/80, indicating targeting of the increasing number of synovial macrophages associated with the severity of inflammation by the Vsig4 nanobody. NIRF imaging with Cy7-Nb119 allows specific assessment of inflammation in experimental arthritis and provides complementary information to clinical scoring for quantitative, non-invasive and economical monitoring of the pathological process. Nanobody labelled with fluorescence can also be used for ex vivo validation experiments using flow cytometry and fluorescence microscopy.

A tetrameric protein scaffold as a nano-carrier of antitumor peptides for cancer therapy.

A major pharmacological barrier to peptide therapeutics is their susceptibility to proteolytic degradation and poor membrane permeability, which, in principle, can be overcome by nanoparticle-based delivery technologies. Proteins, by definition, are nano materials and have been clinically proven as an efficient delivery vehicle for small molecule drugs. Here we describe the design of a protein-based peptide drug carrier derived from the tetramerization domain of the chimeric oncogenic protein Bcr/Abl of chronic myeloid leukemia. A dodecameric peptide inhibitor of the p53-MDM2/MDMX interaction, termed PMI, was grafted to the N-terminal helical region of Bcr/Abl tetramer. To antagonize intracellular MDM2/MDMX for p53 activation, we extended this protein, PMIBcr/Abl, by a C-terminal Arg-repeating hexapeptide to facilitate its cellular uptake. The resultant tetrameric protein PMIBcr/Abl-R6 adopted an alpha-helical conformation in solution and bound to MDM2 at an affinity of 32 nM. PMIBcr/Abl-R6 effectively induced apoptosis of HCT116 p53+/+ cells in vitro in a p53-dependent manner and potently inhibited tumor growth in a nude mouse xenograft model by stabilizing p53 in vivo. Our protein-based delivery strategy thus provides a clinically viable solution to p53-inspired anticancer therapy and is likely applicable to the development of many other peptide therapeutics to target a great variety of intracellular protein-protein interactions responsible for disease initiation and progression.

Development and optimization of a tumor targeting system based on microbial synthesized PHA biopolymers and PhaP mediated functional modification.

Polyhydroxyalkanoate (PHA) is a class of microbial synthesized biodegradable and biocompatible aliphatic polymer which has been developed into nanoparticles (NPs) for sustained release of hydrophobic compounds. Taking advantage of the natural PHA binding protein PhaP which could be steadily adsorbed onto PHA NPs through hydrophobic interaction, a tumor targeting system was developed in this study by presenting an epidermal growth factor receptor (EGFR)-targeting peptide (ETP) on the surface of PHA NPs, via PhaP mediated adsorption. To reveal the effects of residual emulsifiers on PhaP mediated ETP modification and optimize the tumor targeting capacity of the system, a novel emulsifier-free PHA NPs (EF-NPs) was fabricated together with other two kinds of conventional emulsifier-required PHA NPs (PVA-NPs and P68-NPs, which were prepared with poly(vinyl alcohol) (PVA) and Pluronic F68 as emulsifiers, respectively). By analyzing the surface hydrophobicity, the amount of adsorbed fusion protein, and the cellular uptake of all kinds of PHA NPs, our results demonstrated that EF-NPs with stronger surface hydrophobicity were the most proper formulation for further PhaP mediated ETP functionalization. The residual PVA and Pluronic F68 affected the modification efficiency and secondary structure of ETP-PhaP fusion protein, and finally obstructed the targeting effect of ETP-PhaP modified PVA-NPs and P68-NPs to EGFR over-expressed tumor cells. The animal experiment further confirmed the effectiveness and feasibility of in vivo application of ETP-PhaP functionalized EF-NPs, indicating that it could be served as a promising tumor targeting system with satisfactory EGFR targeting ability. This PhaP mediated bio-modification process also opens a wide way for developing various PHA-based targeting systems by presenting different tumor or other tissue-specific targeting peptides.

Bioinformatics analysis of genetic variants of endoplasmic reticulum aminopeptidase 1 in ankylosing spondylitis.

According to the results of the first genome-wide association study of ankylosing spondylitis (AS), endoplasmic reticulum aminopeptidase 1 (ERAP1) may serve an important role. However, a number of case-control studies have not been able to replicate this result using the same genetic markers. In the present study, the role of common genetic variants of ERAP1 in AS was investigated using two-stage bioinformatics analysis. In the first stage, a classical meta-analysis was performed to assess AS susceptibility markers in ERAP1 using data from available published case-control association studies. The summary odds ratios for 10 single nucleotide polymorphisms (SNPs) were observed to be statistically significant in different studies. In the second stage, the functional effects of these genetic ERAP1 variants were investigated using prediction tools and structural analyses. The K528R (rs30187) substitution SNP in ERAP1 was termed as likely damaging by PolyPhen-2 software, was observed to be located close to the entrance of the substrate pocket, and was predicted to contribute to reduced ERAP1 aminopeptidase activity. In addition, the R725Q (rs17482078) SNP, which was an additional potentially damaging substitution, was suggested to decrease the enzymatic activity of ERAP1, as this substitution may lead to the loss of two hydrogen bonds between R725 and D766 and affect the stability of the C-terminus of ERAP1. In conclusion, the results of the two-stage bioinformatics analysis supported the hypothesis that ERAP1 may present an important susceptibility gene for AS. In addition, the results revealed that two functional SNPs (rs30187 and rs17482078) demonstrated the potential to decrease the enzymatic activity of ERAP1 by affecting its protein structure. Further protein structure-guided studies of the specificity and activity of these ERAP1 variants are therefore warranted.

miR-143 is associated with proliferation and apoptosis involving ERK5 in HeLa cells.

Inappropriate expression of microRNA (miR) is strongly associated with carcinogenesis. miR-143 was reported to be one of the most prominent miRs implicated in the genesis and progression of human cancer. However, its correlation with cell proliferation and apoptosis in cervical cancer remains to be fully elucidated. In the present study, it was demonstrated that miR-143 is able to suppress the proliferation of cervical cancer HeLa cells and induce cell apoptosis in a time- and dose-dependent manner. The present study also investigated the potential targets of miR-143, extracellular-signal-regulated kinase 5 (ERK5) and its downstream substrate oncoprotein c-Fos, both of which are involved in cell proliferation and apoptosis. Upon increasing the miR-143 level, the ERK5 and c-Fos protein expression was significantly decreased without the effect of ERK5 transcription. Therefore, miR-143 is able to suppress cell proliferation and induce apoptosis in HeLa cells, potentially through negative regulation of ERK5 at its post-transcriptional stage.

ATP and autophosphorylation driven conformational changes of HipA kinase revealed by ion mobility and crosslinking mass spectrometry.

Toxin-antitoxin systems are genetic modules involved in a broad range of bacterial cellular processes including persistence, multidrug resistance and tolerance, biofilm formation, and pathogenesis. In type II toxin-antitoxin systems, both the toxin and antitoxin are proteins. In the prototypic Escherichia coli HipA-HipB module, the antitoxin HipB forms a complex with the protein kinase HipA and sequesters it in the nucleoid. HipA is then no longer able to phosphorylate glutamyl-tRNA-synthetase and this prevents the initiation of the forthcoming stringent response. Here we investigated the assembly of the Shewanella oneidensis MR-1 HipA-HipB complex using native electrospray ion mobility-mass spectrometry and chemical crosslinking combined with mass spectrometry. We revealed that the HipA autophosphorylation was accompanied by a large conformational change, and confirmed structural evidence that S. oneidensis MR-1 HipA-HipB assembly was distinct from the prototypic E. coli HipA-HipB complex. Graphical abstract Ion mobility mass spectrometry shows a two phase transition from unstructured HipA to a compact folded phosphorylated protein.

Structural basis of IL-23 antagonism by an Alphabody protein scaffold.

Protein scaffolds can provide a promising alternative to antibodies for various biomedical and biotechnological applications, including therapeutics. Here we describe the design and development of the Alphabody, a protein scaffold featuring a single-chain antiparallel triple-helix coiled-coil fold. We report affinity-matured Alphabodies with favourable physicochemical properties that can specifically neutralize human interleukin (IL)-23, a pivotal therapeutic target in autoimmune inflammatory diseases such as psoriasis and multiple sclerosis. The crystal structure of human IL-23 in complex with an affinity-matured Alphabody reveals how the variable interhelical groove of the scaffold uniquely targets a large epitope on the p19 subunit of IL-23 to harness fully the hydrophobic and hydrogen-bonding potential of tryptophan and tyrosine residues contributed by p19 and the Alphabody, respectively. Thus, Alphabodies are suitable for targeting protein-protein interfaces of therapeutic importance and can be tailored to interrogate desired design and binding-mode principles via efficient selection and affinity-maturation strategies.

Human IL-34 and CSF-1 establish structurally similar extracellular assemblies with their common hematopoietic receptor.

The discovery that hematopoietic human colony stimulating factor-1 receptor (CSF-1R) can be activated by two distinct cognate cytokines, colony stimulating factor-1 (CSF-1) and interleukin-34 (IL-34), created puzzling scenarios for the two possible signaling complexes. We here employ a hybrid structural approach based on small-angle X-ray scattering (SAXS) and negative-stain EM to reveal that bivalent binding of human IL-34 to CSF-1R leads to an extracellular assembly hallmarked by striking similarities to the CSF-1:CSF-1R complex, including homotypic receptor-receptor interactions. Thus, IL-34 and CSF-1 have evolved to exploit the geometric requirements of CSF-1R activation. Our models include N-linked oligomannose glycans derived from a systematic approach resulting in the accurate fitting of glycosylated models to the SAXS data. We further show that the C-terminal region of IL-34 is heavily glycosylated and that it can be proteolytically cleaved from the IL-34:hCSF-1R complex, providing insights into its role in the functional nonredundancy of IL-34 and CSF-1.

Methotrexate ameliorates pristane-induced arthritis by decreasing IFN-γ and IL-17A expressions.

OBJECTIVE: This study was carried out to test the effects of methotrexate (MTX) and black seed oil (BSO) on pristane-induced arthritis (PIA) in rats. METHODS: Inbred dark agouti (DA) rats were induced by a single subcutaneous injection of pristane, and then treated with MTX or BSO. Arthritis severity was evaluated macroscopically and microscopically. Plasma nitric oxide (NO) concentration was determined by the Griess method and cytokine mRNA expression in the spleen was detected by the real-time reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: The clinical arthritis severity was decreased after MTX treatment, while the BSO groups did not show significant changes compared with the disease group. The plasma NO level of the MTX group was significantly decreased compared with the disease group, but the BSO groups showed no difference from the disease group in plasma NO levels. The interferon-γ (IFN-γ) and interleukin-17A (IL-17A) mRNA expressions in the spleens were significantly decreased in the MTX group, but only showed a declining trend in the BSO groups compared with the disease group. Neither MTX nor BSO had an effect on the mRNA expressions of IL-4, transforming growth factor ß (TGF-ß), and tumor necrosis factor-α (TNF-α) in the spleen. CONCLUSIONS: MTX, but not BSO, can reduce the arthritis severity and decrease the mRNA expressions of IFN-γ and IL-17A in pristane-induced arthritis of rats.