A conserved signaling pathway activates bacterial CBASS immune signaling in response to DNA damage.

To protect themselves from the constant threat of bacteriophage (phage) infection, bacteria have evolved diverse immune systems including restriction-modification, CRISPR-Cas, and many others. Here, we describe the discovery of a two-protein transcriptional regulator module associated with hundreds of CBASS immune systems and demonstrate that this module drives the expression of its associated CBASS system in response to DNA damage. We show that the helix-turn-helix transcriptional repressor CapH binds the promoter region of its associated CBASS system to repress transcription until it is cleaved by the metallopeptidase CapP. CapP is activated in vitro by single-stranded DNA, and in cells by DNA-damaging drugs. Together, CapH and CapP drive increased expression of their associated CBASS system in response to DNA damage. We identify CapH- and CapP-related proteins associated with diverse known and putative bacterial immune systems including DISARM and Pycsar antiphage operons. Overall, our data highlight a mechanism by which bacterial immune systems can sense and respond to a universal signal of cell stress, potentially enabling multiple immune systems to mount a coordinated defensive response against an invading pathogen.

Phagocytosis of aggrecan-positive perineuronal nets surrounding motor neurons by reactive microglia expressing MMP-9 in TDP-43Q331K ALS model mice.

Perineuronal nets (PNNs) are extracellular matrix structures that surround excitable neurons and their proximal dendrites. PNNs play an important role in neuroprotection against oxidative stress. Oxidative stress within motor neurons can act as a trigger for neuronal death, and this has been implicated in motor neuron degeneration in amyotrophic lateral sclerosis (ALS). We therefore characterised PNNs around alpha motor neurons and the possible contributing cellular factors in the mutant TDP-43Q331K transgenic mouse, a slow onset ALS mouse model. PNNs around alpha motor neurons showed significant loss at mid-stage disease in TDP-43Q331K mice compared to wild type strain control mice. PNN loss coincided with an increased expression of matrix metallopeptidase-9 (MMP-9), an endopeptidase known to cleave PNNs, within the ventral horn. During mid-stage disease, increased numbers of microglia and astrocytes expressing MMP-9 were present in the ventral horn of TDP-43Q331K mice. In addition, TDP-43Q331K mice showed increased levels of aggrecan, a PNN component, in the ventral horn by microglia and astrocytes during this period. Elevated aggrecan levels within glia were accompanied by an increase in fractalkine expression, a chemotaxic protein responsible for the recruitment of microglia, in alpha motor neurons of onset and mid-stage TDP-43Q331K mice. Following PNN loss, alpha motor neurons in mid-stage TDP-43Q331K mice showed increased 3-nitrotyrosine expression, an indicator of protein oxidation. Together, our observations along with previous PNN research provide suggests a possible model whereby microglia and astrocytes expressing MMP-9 degrade PNNs surrounding alpha motor neurons in the TDP-43Q331K mouse. This loss of nets may expose alpha-motor neurons to oxidative damage leading to degeneration of the alpha motor neurons in the TDP-43Q331K ALS mouse model.

Lonicera japonica Thunb. Ethanol Extract Exerts a Protective Effect on Normal Human Gastric Epithelial Cells by Modulating the Activity of Tumor-Necrosis-Factor-α-Induced Inflammatory Cyclooxygenase 2/Prostaglandin E2 and Matrix Metalloproteinase 9.

Gastric inflammation-related disorders are commonly observed digestive system illnesses characterized by the activation of proinflammatory cytokines, particularly tumor necrosis factor-α (TNF-α). This results in the induction of cyclooxygenase-2 (COX-2)/prostaglandin E2 (PEG2) and matrix metallopeptidase-9 (MMP-9). These factors contribute to the pathogenesis of gastric inflammation disorders. We examined the preventive effects of Lonicera japonica Thunb. ethanol extract (Lj-EtOH) on gastric inflammation induced by TNF-α in normal human gastric mucosa epithelial cells (GES-1). The GES-1 cell line was used to establish a model that simulated the overexpression of COX-2/PGE2 and MMP-9 proteins induced by TNF-α to examine the anti-inflammatory properties of Lj extracts. The results indicated that Lj-EtOH exhibits significant inhibitory effects on COX-2/PEG2 and MMP-9 activity, attenuates cell migration, and provides protection against TNF-α-induced gastric inflammation. The protective effects of Lj-EtOH are associated with the modulation of COX-2/PEG2 and MMP-9 through the activation of TNFR-ERK 1/2 signaling pathways as well as the involvement of c-Fos and nuclear factor kappa B (NF-κB) signaling pathways. Based on our findings, Lj-EtOH exhibits a preventive effect on human gastric epithelial cells. Consequently, it may represent a novel treatment for the management of gastric inflammation.

Perineuronal nets are phagocytosed by MMP-9 expressing microglia and astrocytes in the SOD1G93A ALS mouse model.

AIMS: Perineuronal nets (PNNs) are an extracellular matrix structure that encases excitable neurons. PNNs play a role in neuroprotection against oxidative stress. Oxidative stress within motor neurons can trigger neuronal death, which has been implicated in amyotrophic lateral sclerosis (ALS). We investigated the spatio-temporal timeline of PNN breakdown and the contributing cellular factors in the SOD1G93A strain, a fast-onset ALS mouse model. METHODS: This was conducted at the presymptomatic (P30), onset (P70), mid-stage (P130), and end-stage disease (P150) using immunofluorescent microscopy, as this characterisation has not been conducted in the SOD1G93A strain. RESULTS: We observed a significant breakdown of PNNs around α-motor neurons in the ventral horn of onset and mid-stage disease SOD1G93A mice compared with wild-type controls. This was observed with increased numbers of microglia expressing matrix metallopeptidase-9 (MMP-9), an endopeptidase that degrades PNNs. Microglia also engulfed PNN components in the SOD1G93A mouse. Further increases in microglia and astrocyte number, MMP-9 expression, and engulfment of PNN components by glia were observed in mid-stage SOD1G93A mice. This was observed with increased expression of fractalkine, a signal for microglia engulfment, within α-motor neurons of SOD1G93A mice. Following PNN breakdown, α-motor neurons of onset and mid-stage SOD1G93A mice showed increased expression of 3-nitrotyrosine, a marker for protein oxidation, which could render them vulnerable to death. CONCLUSIONS: Our observations suggest that increased numbers of MMP-9 expressing glia and their subsequent engulfment of PNNs around α-motor neurons render these neurons sensitive to oxidative damage and eventual death in the SOD1G93A ALS model mouse.

Recombinant protein production and functional analysis of a M60-like-2 metallopeptidase enzyme from the carcinogenic liver fluke Opisthorchis viverrini.

Mucin plays a crucial role in safeguarding mucosal tissues by obstructing the translocation of microorganisms. Mucosal tissue-dwelling parasites must devise a strategy to surmount this mucin barrier in order to establish colonization. In a recent discovery, it was observed that the liver fluke Opisthorchis viverrini secretes two mucinases, namely Ov-M60-like-1 and Ov-M60-like-2. Ov-M60-like-1 was previously characterized. Here, we study the Ov-M60-like-2 by utilizing the wheat germ expression system to produce recombinant proteins and conducted a functional analysis of its enzymatic activity on bovine submaxillary mucin (BSM). Subsequently, we delved deeper into understanding the role of this enzyme in host-parasite interactions by evaluating its mucinase activity on mucins from the bile duct of O. viverrini-infected hamsters. Through successful production of recombinant proteins using the wheat germ expression system, we observed that this enzyme displayed mucinase activity over a wide pH range (pH 2 to pH 10) against BSM. Our investigations revealed it ability to digest mucin from the bile duct. These findings suggest that Ov-M60-like-2 possess a mucinase activity, together with Ov-M60-like-1, enabling the liver fluke to successful colonization of the host's bile duct.

The Secretome of Adult Murine Hookworms Is Shaped by Host Expression of STAT6.

Co-evolutionary adaptation of hookworms with their mammalian hosts has been selected for immunoregulatory excretory/secretory (E/S) products. However, it is not known whether, or if so, how host immunological status impacts the secreted profile of hematophagous adult worms. This study interrogated the impact of host Signal transducer and activator of transcription 6 (STAT6) expression during the experimental evolution of hookworms through the sequential passage of the life cycle in either STAT6 deficient or WT C57BL/6 mice. Proteomic analysis of E/S products by LC-MS showed increased abundance of 15 proteins, including myosin-3, related to muscle function, and aconitate hydratase, related to iron homeostasis. However, most E/S proteins (174 of 337 unique identities) were decreased, including those in the Ancylostoma-secreted protein (ASP) category, and metallopeptidases. Several identified proteins are established immune-modulators such as fatty acid-binding protein homologue, cystatin, and acetylcholinesterase. Enrichment analysis of InterPro functional categories showed down-regulation of Cysteine-rich secretory proteins, Antigen 5, and Pathogenesis-related 1 proteins (CAP), Astacin-like metallopeptidase, Glycoside hydrolase, and Transthyretin-like protein groups in STAT6 KO-adapted worms. Taken together, these data indicate that in an environment lacking Type 2 immunity, hookworms alter their secretome by reducing immune evasion proteins- and increasing locomotor- and feeding-associated proteins.

The extent of damage to the blood-brain barrier in the hypercholesterolemic LDLR-/-/Apo E-/- double knockout mice depends on the animal's age, duration of pathology and brain area.

One of the effects of hypercholesterolemia (Hch) exerted on the central nervous system (CNS) is damage to the blood-brain barrier (BBB). Increased permeability of BBB results from structural changes in the vascular wall, loss of the tight junctions and barrier function, as well as alterations in the concentration of proteins located in the layers of the vascular wall. These changes occur in the course of metabolic and neurodegenerative diseases. The important role in the course of these processes is attributed to agrin, matrix metalloproteinase-9, and aquaporin-4. In this study, we aimed to determine: 1) the extent of Hch-induced damage to the BBB during maturation, and 2) the distribution of the above-mentioned markers in the vascular wall. Immunohistochemical staining and confocal microscopy were used for vascular wall protein assessment. The size of BBB damage was studied based on perivascular leakage of fluorescently labeled dextran. Three- and twelve-month-old male LDLR-/-/Apo E-/- double knockout mice (EX) developing Hch were used in the study. Age-matched male wild-type (WT) C57BL/6 mice were used as a control group. Differences in the concentration of studied markers coexisted with BBB disintegration, especially in younger mice. A relationship between the maturation of the vascular system and reduction of the BBB damage was also observed. We conclude that the extent of BBB permeability depends on animal age, duration of Hch, and brain region. These may explain different susceptibility of various brain areas to Hch, and different presentation of this pathology depending on age and its duration.

The crystal structure of a 250-kDa heterotetrameric particle explains inhibition of sheddase meprin ß by endogenous fetuin-B.

Meprin ß (Mß) is a multidomain type-I membrane metallopeptidase that sheds membrane-anchored substrates, releasing their soluble forms. Fetuin-B (FB) is its only known endogenous protein inhibitor. Herein, we analyzed the interaction between the ectodomain of Mß (MßΔC) and FB, which stabilizes the enzyme and inhibits it with subnanomolar affinity. The MßΔC:FB crystal structure reveals a ∼250-kDa, ∼160-Å polyglycosylated heterotetrameric particle with a remarkable glycan structure. Two FB moieties insert like wedges through a "CPDCP trunk" and two hairpins into the respective peptidase catalytic domains, blocking the catalytic zinc ions through an "aspartate switch" mechanism. Uniquely, the active site clefts are obstructed from subsites S4 to S10', but S1 and S1' are spared, which prevents cleavage. Modeling of full-length Mß reveals an EGF-like domain between MßΔC and the transmembrane segment that likely serves as a hinge to transit between membrane-distal and membrane-proximal conformations for inhibition and catalysis, respectively.

A previously uncharacterized O-glycopeptidase from Akkermansia muciniphila requires the Tn-antigen for cleavage of the peptide bond.

Akkermansia muciniphila is key member of the human gut microbiota that impacts many features of host health. A major characteristic of this bacterium is its interaction with host mucin, which is abundant in the gut environment, and its ability to metabolize mucin as a nutrient source. The machinery deployed by A. muciniphila to enable this interaction appears to be extensive and sophisticated, yet it is incompletely defined. The uncharacterized protein AMUC_1438 is encoded by a gene that was previously shown to be upregulated when the bacterium is grown on mucin. This uncharacterized protein has features suggestive of carbohydrate-recognition and peptidase activity, which led us to hypothesize that it has a role in mucin depolymerization. Here, we provide structural and functional support for the assignment of AMUC_1438 as a unique O-glycopeptidase with mucin-degrading capacity. O-glycopeptidase enzymes recognize glycans but hydrolyze the peptide backbone and are common in host-adapted microbes that colonize or invade mucus layers. Structural, kinetic, and mutagenic analyses point to a metzincin metalloprotease catalytic motif but with an active site that specifically recognizes a GalNAc residue α-linked to serine or threonine (i.e., the Tn-antigen). The enzyme catalyzes hydrolysis of the bond immediately N-terminal to the glycosylated residue. Additional modeling analyses suggest the presence of a carbohydrate-binding module that may assist in substrate recognition. We anticipate that these results will be fundamental to a wider understanding of the O-glycopeptidase class of enzymes and how they may contribute to host adaptation.

The Potential Inhibitory Role of Acetyl-L-Carnitine on Proliferation, Migration, and Gene Expression in HepG2 and HT29 Human Adenocarcinoma Cell Lines.

Malignancies of the liver and colon are the most prevalent forms of digestive system cancer globally. Chemotherapy, one of the most significant treatments, has severe side effects. Chemoprevention using natural or synthetic medications can potentially reduce cancer severity. Acetyl-L-carnitine (ALC) is an acetylated derivative of carnitine essential for intermediate metabolism in most tissues. This study aimed to investigate the effects of ALC on the proliferation, migration, and gene expression of human liver (HepG2) and colorectal (HT29) adenocarcinoma cell lines. The cell viability and half maximal inhibitory concentration of both cancer cell lines were determined using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Wound healing after treatment was assessed using a migration assay. Morphological changes were imaged using brightfield and fluorescence microscopy. Post treatment, apoptotic DNA was detected using a DNA fragmentation assay. The relative mRNA expressions of matrix metallopeptidase 9 (MMP9) and vascular endothelial growth factor (VEGF) were evaluated using RT-PCR. The results showed that ALC treatment affects the wound-healing ability of HepG2 and HT29 cell lines. Changes in nuclear morphology were detected under fluorescent microscopy. ALC also downregulates the expression levels of MMP9 and VEGF in HepG2 and HT29 cell lines. Our results indicate that the anticancer action of ALC is likely mediated by a decrease in adhesion, migration, and invasion.

Enzyme-Triggered Transforming of Assembly Peptide-Modified Magnetic Resonance-Tuned Probe for Highly Sensitive Imaging of Bacterial Infection In Vivo.

Confirming bacterial infection at an early stage and distinguishing between sterile inflammation and bacterial infection is still highly needed for efficient treatment. Here, in situ highly sensitive magnetic resonance imaging (MRI) bacterial infection in vivo based on a peptide-modified magnetic resonance tuning (MRET) probe (MPD-1) that responds to matrix metallopeptidase 2 (MMP-2) highly expressed in bacteria-infected microenvironments is achieved. MPD-1 is an assembly of magnetic nanoparticle (MNP) bearing with gadolinium ion (Gd3+ ) modified MMP-2-cleavable self-assembled peptide (P1 ) and bacteria-targeting peptide (P), and it shows T2 -weighted signal due to the assemble of MNP and MRET ON phenomenon between MNP assembly and Gd3+ . Once MPD-1 accumulates at the bacterially infected site, P1 included in MPD-1 is cleaved explicitly by MMP-2, which triggers the T2 contrast agent of MPD-1 to disassemble into the monomer of MNP, leading the recovery of T1 -weighted signal. Simultaneously, Gd3+ detaches from MNP, further enhancing the T1 -weighted signal due to MRET OFF. The sensitive MRI of Staphylococcus aureus (low to 104 CFU) at the myositis site and accurate differentiation between sterile inflammation and bacterial infection based on the proposed MPD-1 probe suggests that this novel probe would be a promising candidate for efficiently detecting bacterial infection in vivo.

Initial detachment of the mouse oocyte from the zona pellucida is mediated by metallopeptidase activity†.

The fully grown mammalian oocyte is tightly attached to its extracellular matrix shell, the zona pellucida (ZP), but the oocyte detaches from the ZP shortly after ovulation is signaled. The mechanism by which the oocyte detaches from the ZP is unknown. Because ZP proteins are initially secreted as transmembrane proteins, we hypothesized that attachment of the oocyte to the ZP is mediated by transmembrane ZP proteins and that detachment occurs when these proteins are cleaved by peptidases. To identify potential candidates for the type of peptidase, we used mouse oocyte transcriptome data sets to identify candidate peptidases localized to the exterior of the oocyte. Screening with a set of small molecule inhibitors that broadly target the families of peptidases represented by the candidates, we found that only inhibitors of the M10 and M12 families of metallopeptidases prevented detachment. Using more selective inhibitors indicated that detachment was prevented by an inhibitor, GI254023X, developed to be selective for ADAM10 in the M12 family but not by those considered selective for the M10 family or for other M12 metallopeptidases expressed in oocytes. Using an antibody that binds to an epitope just distal to the likely cleavage site of murine ZP3 showed that this site was gradually lost from the oocyte surface during the period when detachment occurs and that inhibiting metallopeptidase activity prevented the loss of this epitope. Taken together, these results indicate that detachment of the oocyte from the ZP is mediated by a metallopeptidase.

Lab-Attenuated Rabies Virus Facilitates Opening of the Blood-Brain Barrier by Inducing Matrix Metallopeptidase 8.

Infection with laboratory-attenuated rabies virus (RABV), but not wild-type (wt) RABV, can enhance the permeability of the blood-brain barrier (BBB), which is considered a key determinant for RABV pathogenicity. A previous study showed that the enhancement of BBB permeability is directly due not to RABV infection but to virus-induced inflammatory molecules. In this study, the effect of the matrix metallopeptidase (MMP) family on the permeability of the BBB during RABV infection was evaluated. We found that the expression level of MMP8 was upregulated in mice infected with lab-attenuated RABV but not with wt RABV. Lab-attenuated RABV rather than wt RABV activates inflammatory signaling pathways mediated by the nuclear factor κB (NF-κB) and mitogen-activated protein kinase (MAPK) pathways. Activated NF-κB (p65) and AP-1 (c-Fos) bind to the MMP8 promoter, resulting in upregulation of its transcription. Analysis of mouse brains infected with the recombinant RABV expressing MMP8 indicated that MMP8 enhanced BBB permeability, leading to infiltration of inflammatory cells into the central nervous system (CNS). In brain-derived endothelial cells, treatment with MMP8 recombinant protein caused the degradation of tight junction (TJ) proteins, and the application of an MMP8 inhibitor inhibited the degradation of TJ proteins after RABV infection. Furthermore, an in vivo experiment using an MMP8 inhibitor during RABV infection demonstrated that BBB opening was diminished. In summary, our data suggest that the infection of lab-attenuated RABV enhances the BBB opening by upregulating MMP8. IMPORTANCE The ability to change BBB permeability was associated with the pathogenicity of RABV. BBB permeability was enhanced by infection with lab-attenuated RABV instead of wt RABV, allowing immune cells to infiltrate into the CNS. We found that MMP8 plays an important role in enhancing BBB permeability by degradation of TJ proteins during RABV infection. Using an MMP8 selective inhibitor restores the reduction of TJ proteins. We reveal that MMP8 is upregulated via the MAPK and NF-κB inflammatory pathways, activated by lab-attenuated RABV infection but not wt RABV. Our findings suggest that MMP8 has a critical role in modulating the opening of the BBB during RABV infection, which provides fresh insight into developing effective therapeutics for rabies and infection with other neurotropic viruses.

The cell volume-regulatory glycine transporter GLYT1 is activated following metallopeptidase-mediated detachment of the oocyte from the zona pellucida.

Independent cell volume regulation is first acquired by the oocyte in two steps that occur during meiotic maturation: (1) activation of the glycine transporter GLYT1 (Slc6a9) that mediates the intracellular accumulation of glycine to provide osmotic support in the mature egg and early preimplantation embryo, and (2) release of the oocyte from the strong attachment to its rigid extracellular matrix shell, the zona pellucida (ZP). It was recently shown that oocyte-ZP detachment requires metallopeptidase activity that is proposed to cleave transmembrane ZP proteins connecting the oocyte to the ZP. It is unknown, however, how GLYT1 is activated. We hypothesized that oocyte-ZP detachment precedes and may be required for GLYT1 activation. In identically treated pools of oocytes, oocyte-ZP detachment occurred ~20 min before GLYT1 activation. In individual oocytes, GLYT1 activity was detected only in those that were mostly or fully detached. Blocking detachment using previously validated small molecule metallopeptidase inhibitors partly suppressed GLYT1 activation. However, removal of the ZP did not accelerate GLYT1 activation. This indicates that oocyte-ZP detachment or cleavage of transmembrane ZP proteins may be required for GLYT1 to become fully activated, or alternatively that metallopeptidase activity independently affects both detachment and GLYT1 activation.

Jojan: a novel virus that lyses Stenotrophomonas maltophilia from dog.

Stenotrophomonas maltophilia is a Gram-negative bacterium widely distributed in the environment and associated with nosocomial infections, pneumonia, and bacteremia in humans and other mammals. We have isolated and sequenced a new virus that lyses the S. maltophilia strain from a dog skin. The virus has a siphovirus-like morphology and a linear dsDNA genome 60,804 pb in length with terminal repeats, four tRNA genes, and 111 putative proteins. The annotated genes resemble the corresponding genes of some siphoviruses, but the unique genome arrangement and limited similarity of the encoded proteins suggest that this virus does not belong to any known genus. The virus uses zinc metallopeptidase for lysis of its host. This enzyme is active in the presence of Zn2+ or Mg2+ ions and maintains its bactericidal activity up to 50 °C. Both the virus itself and the endolysin specifically degrade only the host bacterial strain.

Reversible autoinhibitory regulation of Escherichia coli metallopeptidase BepA for selective ß-barrel protein degradation.

Escherichia coli periplasmic zinc-metallopeptidase BepA normally functions by promoting maturation of LptD, a ß-barrel outer-membrane protein involved in biogenesis of lipopolysaccharides, but degrades it when its membrane assembly is hampered. These processes should be properly regulated to ensure normal biogenesis of LptD. The underlying mechanism of regulation, however, remains to be elucidated. A recently solved BepA structure has revealed unique features: In particular, the active site is buried in the protease domain and conceivably inaccessible for substrate degradation. Additionally, the His-246 residue in the loop region containing helix α9 (α9/H246 loop), which has potential flexibility and covers the active site, coordinates the zinc ion as the fourth ligand to exclude a catalytic water molecule, thereby suggesting that the crystal structure of BepA represents a latent form. To examine the roles of the α9/H246 loop in the regulation of BepA activity, we constructed BepA mutants with a His-246 mutation or a deletion of the α9/H246 loop and analyzed their activities in vivo and in vitro. These mutants exhibited an elevated protease activity and, unlike the wild-type BepA, degraded LptD that is in the normal assembly pathway. In contrast, tethering of the α9/H246 loop repressed the LptD degradation, which suggests that the flexibility of this loop is important to the exhibition of protease activity. Based on these results, we propose that the α9/H246 loop undergoes a reversible structural change that enables His-246-mediated switching (histidine switch) of its protease activity, which is important for regulated degradation of stalled/misassembled LptD.

Matrix Metallopeptidase-Gene Signature Predicts Stage I Lung Adenocarcinoma Survival Outcomes.

Tumor recurrence poses a significant challenge to the clinical management of stage I lung adenocarcinoma after curative surgical resection. Matrix metalloproteinases (MMPs) increase expression and correlate with recurrence and metastasis in surgically resected non-small cell lung cancer. However, the impact of MMPs on survival outcome varies, and their roles in patients with stage I lung adenocarcinoma remain unclear. In two discovery cohorts, we first analyzed 226 stage I-II lung adenocarcinoma cases in the GSE31210 cohort using a clustering-based method and identified a 150-gene MMP cluster with increased expression in tumors associated with worse survival outcomes. A similar analysis was performed on 517 lung adenocarcinoma cases in the Cancer Genome Atlas cohort. A 185-gene MMP cluster was identified, which also showed increased expression in tumors and correlated with poor survival outcomes. We further streamlined from the discovery cohorts a 36-gene MMP signature significantly associated with recurrence and worse overall survival in patients with stage I lung adenocarcinoma after surgical resection. After adjusting for covariates, the high MMP-gene signature expression remained an independent risk factor. In addition, the MMP-gene signature showed enrichment in epidermal growth factor receptor wild-type lung tumors, especially for those with Kirsten rat sarcoma virus mutations. Using an independent validation cohort, we further validated the MMP-gene signature in 70 stage I lung adenocarcinoma cases. In conclusion, MMP-gene signature is a potential predictive and prognostic biomarker to stratify patients with stage I lung adenocarcinoma into subgroups based on their risk of recurrence for aiding physicians in deciding the personalized adjuvant therapeutics.

Silencing GMPPB Inhibits the Proliferation and Invasion of GBM via Hippo/MMP3 Pathways.

Glioblastoma multiforme (GBM) is a highly aggressive malignancy and represents the most common brain tumor in adults. To better understand its biology for new and effective therapies, we examined the role of GDP-mannose pyrophosphorylase B (GMPPB), a key unit of the GDP-mannose pyrophosphorylase (GDP-MP) that catalyzes the formation of GDP-mannose. Impaired GMPPB function will reduce the amount of GDP-mannose available for O-mannosylation. Abnormal O-mannosylation of alpha dystroglycan (α-DG) has been reported to be involved in cancer metastasis and arenavirus entry. Here, we found that GMPPB is highly expressed in a panel of GBM cell lines and clinical samples and that expression of GMPPB is positively correlated with the WHO grade of gliomas. Additionally, expression of GMPPB was negatively correlated with the prognosis of GBM patients. We demonstrate that silencing GMPPB inhibits the proliferation, migration, and invasion of GBM cells both in vitro and in vivo and that overexpression of GMPPB exhibits the opposite effects. Consequently, targeting GMPPB in GBM cells results in impaired GBM tumor growth and invasion. Finally, we identify that the Hippo/MMP3 axis is essential for GMPPB-promoted GBM aggressiveness. These findings indicate that GMPPB represents a potential novel target for GBM treatment.

Neurotherapeutic efficacy of loaded sulforaphane on iron oxide nanoparticles against cuprizone-induced neurotoxicity: role of MMP-9 and S100ß.

Cuprizone (CUP) induces neurotoxicity and demyelination in animal models by provoking the activation of glial cells and the generation of reactive oxygen species (ROS). Sulforaphane (SF) is a phytochemical that exhibits a neuroprotective potential. In this study, we investigated the neurotherapeutic and pro-remyelinating activities of SF and SF-loaded within iron oxide nanoparticles (IONP-SF) in CUP-exposed rats. Magnetite iron oxide nanoparticles (IONPs) were prepared using the hydrothermal method that was further loaded with SF (IONP-SF). The loading of SF within the magnetite nanoparticles was assessed using FTIR, TEM, DLS, Zetasizer, and XPS. For the in vivo investigations, adult male Wistar rats (n = 40) were administrated either on a regular diet or a diet with CUP (0.2%) for 5 weeks. The rats were divided into four groups: negative control, CUP-induced, CUP + SF, and CUP + IONP-SF. CUP-exposed brains exhibited a marked elevation in lipid peroxidation, along with a significant decrease in the activities of glutathione peroxidase (GPx), and catalase (CAT). In addition, CUP intoxication downregulated the expression of myelin basic protein (MBP) and myelin proteolipid protein (PLP), upregulated the expression of Matrix metallopeptidase-9 (MMP-9) and S100ß, and increased caspase-3 immunoexpression, these results were supported histopathologically in the cerebral cortexes. Treatment of CUP-rats with either SF or IONP-SF demonstrated remyelinating and neurotherapeutic activities. We could conclude that IONP-SF was more effective than free SF in mitigating the CUP-induced downregulation of MBP, upregulation of S100ß, and caspase-3 immunoexpression.

Crystallization, X-ray diffraction analysis and structure of ICMP from Pseudomonasaeruginosa.

Insulin-cleaving membrane protease (ICMP), an outmember protein of Pseudomonas aeruginosa (P. aeruginosa), plays a critical role in the pathogenesis of the bacterium. ICMP has been reported to be involved in the process of iron uptake. In this study, we report the high-resolution structure of ICMP determined by single-wavelength anomalous diffraction (SAD), which shows an atypical HxxE motif that differs from the canonical zinc dependent M75 peptidases and a "V-shaped" cleft that is observed to coordinate the metal ion for the first time. Crystals from the selenomethionine-substituted ICMP(Se-Met ICMP) diffract to 1.9 Å resolution and belong to space group P21, with unit-cell parameters a = 87.93, b = 78.14, c = 9.92 Å, α = 90°, ß = 113.5°, γ = 90°. ICMP consists of two up-and-down helix bundles, which are arranged into an inverted "V" shape. Unexpectedly, no electron densities of metal ions are observed around the ICMP HxxE motif, which is shown to be involved in metal coordination in zinc-dependent M75 peptidases. In contrast, we find a metal ion at the opening cleft of the V-shaped structure of ICMP, where the ICMP residues Asp211, Glu316, Cys319, Asp322, and Asp397 are observed to coordinate the metal via hydrogen-bond interactions. Such observations might imply new potential substrate-binding and catalytic sites. The current work therefore provides novel insights into the diversity of the HxxE-motif-containing peptidase and paves the way for future studies aiming to delineate the mechanism of ICMP catalysis.