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Novel biomarkers and non-invasive diagnostic methods are urgently needed for the screening of gastric cancer to reduce its high mortality. We employed quantitative proteomics approach to develop discriminatory biomarker signatures from human saliva for the detection of gastric cancer. Salivary proteins were analyzed and compared between gastric cancer patients and matched control subjects by using tandem mass tags (TMT) technology. More than 500 proteins were identified with quantification, and 48 of them showed significant difference expression (p < 0.05) between normal controls and gastric cancer patients, including 7 up-regulated proteins and 41 down-regulated proteins. Five proteins were selected for initial verification by ELISA and three were successfully verified, namely cystatin B (CSTB), triosephosphate isomerase (TPI1), and deleted in malignant brain tumors 1 protein (DMBT1). All three proteins could differentiate gastric cancer patients from normal control subjects, dramatically (p < 0.05). The combination of these three biomarkers could reach 85% sensitivity and 80% specificity for the detection of gastric cancer with accuracy of 0.93. This study provides the proof of concept of salivary biomarkers for the non-invasive detection of gastric cancer. It is highly encouraging to turn these biomarkers into an applicable clinical test after large scale validation.

Progressive myoclonus epilepsy (PME) of Unverricht–Lundborg type (EPM1) is an autosomal recessive neurodegenerative disorder with the highest incidence of PME worldwide. Mutations in the gene encoding cystatin B (CSTB) are the primary genetic cause of EPM1. Here, we investigate the role of CSTB during neurogenesis in vivo in the developing mouse brain and in vitro in human cerebral organoids (hCOs) derived from EPM1 patients. We find that CSTB (but not one of its pathological variants) is secreted into the mouse cerebral spinal fluid and the conditioned media from hCOs. In embryonic mouse brain, we find that functional CSTB influences progenitors’ proliferation and modulates neuronal distribution by attracting interneurons to the site of secretion via cell‐non‐autonomous mechanisms. Similarly, in patient‐derived hCOs, low levels of functional CSTB result in an alteration of progenitor's proliferation, premature differentiation, and changes in interneurons migration. Secretion and extracellular matrix organization are the biological processes particularly affected as suggested by a proteomic analysis in patients’ hCOs. Overall, our study sheds new light on the cellular mechanisms underlying the development of EPM1. Synopsis Mutations in the cystatin B (CSTB) gene cause EPM1 epilepsy in patients. CSTB secretion induces the recruitment of migrating interneurons and promotes progenitor cells expansion in the mouse cortex and human cerebral organoids (hCOs). Both functions are impaired in EPM1‐derived hCOs. CSTB overexpression induces progenitor cells expansion in hCOs and in the developing mouse cortex. CSTB is secreted and induces recruitment of migrating interneurons. Downregulation of Cstb and R68X overexpression result in decreased number of progenitors and migrating interneurons in the developing mouse cortex. Proliferation is reduced in EPM1‐derived cerebral organoids in a cell non‐autonomous manner. EPM1‐derived cerebral organoids exhibit premature differentiation. Graphical Abstract Mutations in the cystatin B (CSTB) gene cause EPM1 epilepsy in patients. CSTB secretion induces the recruitment of migrating interneurons and promotes progenitor cells expansion in the mouse cortex and human cerebral organoids (hCOs). Both functions are impaired in EPM1‐derived hCOs.

Down syndrome, resulting from trisomy of human chromosome 21, is a common form of chromosomal disorder that results in intellectual disability and altered risk of several medical conditions. Individuals with Down syndrome have a greatly increased risk of Alzheimer’s disease (DSAD), due to the presence of the APP gene on chromosome 21 that encodes the amyloid-β precursor protein (APP). APP can be processed to generate amyloid-β, which accumulates in plaques in the brains of people who have Alzheimer’s disease and is the upstream trigger of disease. Cathepsin B has potential roles in both APP processing and amyloid-β degradation and has been suggested to contribute to amyloid-β accumulation. An endogenous inhibitor of Cathepsin B, Cystatin B ( CSTB ), is encoded on chromosome 21. The abundance of this protein is increased in the brains of individuals with DSAD, which may be associated with a decrease in Cathepsin B activity compared to individuals who have Alzheimer’s disease in the general population. Whether targeting CSTB can modulate Cathepsin B activity in the context of trisomy of chromosome 21 is unclear. Here we test if reducing CSTB can alter Cathepsin B activity in a mouse and a cellular model of trisomy of chromosome 21. We find that reducing CSTB abundance increases Cathepsin B activity in disomic controls but not in the presence of trisomy of chromosome 21. These findings offer new insights into the role of CSTB in regulating Cathepsin B activity.

Lung injury is one of the pathological hallmarks of most respiratory tract diseases including asthma, acute respiratory distress syndrome (ARDS) and chronic obstructive pulmonary disease (COPD). It involves progressive pulmonary tissue damages which are usually irreversible and incurable. Therefore, strategies to facilitate drug development against lung injury are needed. Here, we characterized the zebrafish folate-deficiency (FD) transgenic line that lacks a fully-developed swim bladder. Whole-mount in-situ hybridization revealed comparable distribution patterns of swim bladder tissue markers between wild-type and FD larvae, suggesting a proper development of swim bladder in early embryonic stages. Unexpectedly, neutrophils infiltration was not observed in the defective swim bladder. Microarray analysis revealed a significant increase and decrease of the transcripts for cathepsin L and a cystatin B (CSTB)-like (zCSTB-like) proteins, respectively, in FD larvae. The distribution of cathepsin L and the zCSTB-like transcripts was spatio-temporally specific in developing wild-type embryos and, in appropriate measure, correlated with their potential roles in maintaining swim bladder integrity. Supplementing with 5-formyltetrahydrofolate successfully prevented the swim bladder anomaly and the imbalanced expression of cathepsin L and the zCSTB-like protein induced by folate deficiency. Injecting the purified recombinant zebrafish zCSTB-like protein alleviated FD-induced swim bladder anomaly. We concluded that the imbalanced expression of cathepsin L and the zCSTB-like protein contributed to the swim bladder malformation induced by FD and suggested the potential application of this transgenic line to model the lung injury and ECM remodeling associated with protease/protease inhibitor imbalance.

Domain swapping is the process by which identical monomeric proteins exchange structural elements to generate dimers/oligomers. Although engineered domain swapping is a compelling strategy for protein assembly, its application has been limited due to the lack of simple and reliable design approaches. Here, we demonstrate that the hydrophobic five-residue ‘cystatin motif’ (QVVAG) from the domain-swapping protein Stefin B, when engineered into a solvent-exposed, tight surface loop between two β-strands prevents the loop from folding back upon itself, and drives domain swapping in non-domain-swapping proteins. High-resolution structural studies demonstrate that engineering the QVVAG stretch independently into various surface loops of four structurally distinct non-domain-swapping proteins enabled the design of different modes of domain swapping in these proteins, including single, double and open-ended domain swapping. These results suggest that the introduction of the QVVAG motif can be used as a mutational approach for engineering domain swapping in diverse β-hairpin proteins. Designing interfaces that can induce protein-protein interactions is a challenging problem. Here the authors show that a five amino acid sequence known to mediate domain swapping in cystatins can drive oligomerization when grafted onto functionally and structurally unrelated host proteins, providing a simple approach to the design of protein assemblies.

Cathepsins are involved in a variety of physiological processes including antigen processing and presentation and extracellular matrix degradation. In the present study, we evaluated whether expression levels of cathepsins S and B and their inhibitors cystatins B and C are affected by multiple sclerosis (MS) disease state (relapse and remission) and therapies (interferon‐β[IFN‐β] and the glucocorticoid [GC] methylprednisolone), and whether they are associated with the IFN‐β response phenotype. Real‐time PCR was employed to compare RNA expression levels in peripheral blood leucocytes (PBLs) and ELISA to determine serum protein levels of MS patients and matched healthy individuals. Cathepsin S RNA was higher in MS patients in the relapse state compared to controls (by 74%, P= 3 × 10−5, n= 30 versus n= 18) with a similar increase observed in serum (66%, P= 0.002, n= 18 versus n= 20). GC treatment reduced cathepsin S levels in PBL RNA (by 44%, P= 6 × 10−6, n= 27) and serum proteins (by 27%, P= 1 × 10−5, n= 26), reduced the serum protein levels of pro‐cathepsin B (by 8%, P= 0.0007, n= 23), and in parallel increased the serum levels of their inhibitor cystatin C (by 82%, P= 8 × 10−6, n= 26). IFN‐β therapy significantly elevated the RNA levels (n= 16) of cathepsin B (by 16%, P= 0.03), cystatin B (44%, P= 0.004) and cystatin C (48%, P= 0.011). In the serum, only cathepsin S levels were reduced by IFN‐β (16%, P= 0.006, n= 25). Interestingly, pre‐treatment serum cathepsin S/cystatin C ratio was higher in ‘good responders’ to IFN‐β therapy compared to patients without a good response (by 94%, P= 0.003). These results suggest that cathepsin S and cystatin C may contribute to disease activity in MS, specifically in a subgroup of patients that are responsive to IFN‐β therapy, and that these proteins should be further evaluated as biomarkers in MS.

Cystatin B (CSTB) is a small protease inhibitor protein being involved in cell proliferation and neuronal differentiation. Loss-of-function mutations in CSTB gene cause progressive myoclonic epilepsy 1 (EPM1). We previously demonstrated that CSTB is locally synthesized in synaptic nerve terminals from rat brain and secreted into the media, indicating its role in synaptic plasticity. In this work, we have further investigated the involvement of CSTB in synaptic plasticity, using synaptosomes from human cerebral organoids (hCOs) as well as from rodents’ brain. Our data demonstrate that CSTB is released from synaptosomes in two ways: (i) as a soluble protein and (ii) in extracellular vesicles-mediated pathway. Synaptosomes isolated from hCOs are enriched in pre-synaptic proteins and contain CSTB at all developmental stages analyzed. CSTB presence in the synaptic territories was also confirmed by immunostaining on human neurons in vitro. To investigate if the depletion of CSTB affects synaptic plasticity, we characterized the synaptosomes from EPM1 hCOs. We found that the levels of presynaptic proteins and of an initiation factor linked to local protein synthesis were both reduced in EPM1 hCOs and that the extracellular vesicles trafficking pathway was impaired. Moreover, EPM1 neurons displayed anomalous morphology with longer and more branched neurites bearing higher number of intersections and nodes, suggesting connectivity alterations. In conclusion, our data strengthen the idea that CSTB plays a critical role in the synapse physiology and reveal that pathologically low levels of CSTB may affect synaptic plasticity, leading to synaptopathy and altered neuronal morphology.

Background Both autophagy and glycolysis are essential for pancreatic ductal adenocarcinoma (PDAC) survival due to desmoplasia. We investigated whether targeting a hub gene which participates in both processes could be an efficient strategy for PDAC treatment. Methods The expression pattern of glycolysis signatures (GS) and autophagy signatures (AS) and their correlation with cystatin B (CSTB) in PDAC were analysed. It was discovered how CSTB affected the growth, glycolysis, and autophagy of PDAC cells. We assessed competitive binding to cathepsin B (CTSB) between CSTB and cystatin C (CSTC) via immunoprecipitation (IP) and immunofluorescence (IF). Chromatin immunoprecipitation quantitative polymerase chain reaction (ChIP‐qPCR) and luciferase reporter gene assays were used to unveil the mechanism underlying CSTB upregulation. The expression pattern of CSTB was examined in clinical samples and KrasG12D/+, Trp53R172H/+, Pdx1‐Cre (KPC) mice. Results GS and AS were enriched and closely associated in PDAC tissues. CSTB increased autophagic flux and provided substrates for glycolysis. CSTB knockdown attenuated the proliferation of PDAC cells and patient‐derived xenografts. The liquid chromatography‐tandem mass spectrometry assay indicated CSTB interacted with CTSB and contributed to the proteolytic activity of CTSB in lysosomes. IF and IP assays demonstrated that CSTB competed with CSTC to bind to CTSB. Mutation of the key sites of CSTB abolished the interaction between CSTB and CTSB. CSTB was highly expressed in PDAC due to H3K27acetylation and SP1 expression. High expression of CSTB in PDAC was observed in tissue microarray and patients’ serum samples. Conclusions Our work demonstrated the tumorigenic roles of autophagy and glycolysis in PDAC. CSTB is a key role in orchestrating these processes to ensure energy supply of PDAC cells. Both glycolysis and autophagy processes were obviously active in pancreatic ductal adenocarcinoma (PDAC). Cystatin B (CSTB) promotes autophagic flux by competing with cystatin C to bind to cathepsin B and provides substrates for glycolysis in PDAC. High H3K27ac levels in the promoter region and SP1 upregulation contributed to the high expression of CSTB.

Progressive myoclonus epilepsy of Unverricht-Lundborg type (EPM1) is an autosomal recessively inherited childhood-onset neurodegenerative disorder, characterized by myoclonus, seizures, and ataxia. Mutations in the cystatin B gene ( CSTB ) underlie EPM1. The CSTB-deficient ( Cstb −/− ) mouse model recapitulates key features of EPM1, including myoclonic seizures. The mice show early microglial activation that precedes seizure onset and neuronal loss and leads to neuroinflammation. We here characterized the inflammatory phenotype of Cstb −/− mice in more detail. We found higher concentrations of chemokines and pro-inflammatory cytokines in the serum of Cstb −/− mice and higher CXCL13 expression in activated microglia in Cstb −/− compared to control mouse brains. The elevated chemokine levels were not accompanied by blood-brain barrier disruption, despite increased brain vascularization. Macrophages in the spleen and brain of Cstb −/− mice were predominantly pro-inflammatory. Taken together, these data show that CXCL13 expression is a hallmark of microglial activation in Cstb −/− mice and that the brain inflammation is linked to peripheral inflammatory changes, which might contribute to the disease pathology of EPM1.

Chronic HIV infection leads to the development of cognitive impairments, designated as HIV-associated neurocognitive disorders (HAND). The secretion of soluble neurotoxic factors by HIV-infected macrophages plays a central role in the neuronal dysfunction and cell death associated with HAND. One potentially neurotoxic protein secreted by HIV-1 infected macrophages is cathepsin B. To explore the potential role of cathepsin B in neuronal cell death after HIV infection, we cultured HIV-1(ADA) infected human monocyte-derived macrophages (MDM) and assayed them for expression and activity of cathepsin B and its inhibitors, cystatins B and C. The neurotoxic activity of the secreted cathepsin B was determined by incubating cells from the neuronal cell line SK-N-SH with MDM conditioned media (MCM) from HIV-1 infected cultures. We found that HIV-1 infected MDM secreted significantly higher levels of cathepsin B than did uninfected cells. Moreover, the activity of secreted cathepsin B was significantly increased in HIV-infected MDM at the peak of viral production. Incubation of neuronal cells with supernatants from HIV-infected MDM resulted in a significant increase in the numbers of apoptotic neurons, and this increase was reversed by the addition of either the cathepsin B inhibitor CA-074 or a monoclonal antibody to cathepsin B. In situ proximity ligation assays indicated that the increased neurotoxic activity of the cathepsin B secreted by HIV-infected MDM resulted from decreased interactions between the enzyme and its inhibitors, cystatins B and C. Furthermore, preliminary in vivo studies of human post-mortem brain tissue suggested an upregulation of cathepsin B immunoreactivity in the hippocampus and basal ganglia in individuals with HAND. Our results demonstrate that HIV-1 infection upregulates cathepsin B in macrophages, increases cathepsin B activity, and reduces cystatin-cathepsin interactions, contributing to neuronal apoptosis. These findings provide new evidence for the role of cathepsin B in neuronal cell death induced by HIV-infected macrophages.