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Rvisdiff is an R/Bioconductor package that generates an interactive interface for the interpretation of differential expression results. It creates a local web page that enables the exploration of statistical analysis results through the generation of auto-analytical visualizations. Users can explore the differential expression results and the source expression data interactively in the same view. As input, the package supports the results of popular differential expression packages such as DESeq2, edgeR, and limma. As output, the package generates a local HTML page that can be easily viewed in a web browser. Rvisdiff is freely available at https://bioconductor.org/packages/Rvisdiff/.

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Specificity profiling is a requirement for monoclonal antibodies (mAbs) and antibody-directed biotherapeutics such as CAR-T cells prior to initiating human trials. However, traditional approaches to assess the specificity of mAbs, primarily tissue cross-reactivity studies, have been unreliable, leading to off-target binding going undetected. Here, we review the emergence of cell-based protein arrays as an alternative and improved assessment of mAb specificity. Cell-based protein arrays assess binding across the full human membrane proteome, ~6,000 membrane proteins each individually expressed in their native structural configuration within live or unfixed cells. Our own profiling indicates a surprisingly high off-target rate across the industry, with 33% of lead candidates displaying off-target binding. Moreover, about 20% of therapeutic mAbs in clinical development and currently on the market display off-target binding. Case studies and off-target rates at different phases of biotherapeutic drug approval suggest that off-target binding is likely a major cause of adverse events and drug attrition.

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Primary focal segmental glomerulosclerosis (FSGS) is a disease of the podocytes and glomerulus, leading to nephrotic syndrome and progressive loss of renal function. One of the most serious aspects is its recurrence of disease in over 30% of patients following allogeneic kidney transplantation, leading to early graft loss. This research investigates the individual genetic predispositions and differences in the immune responses leading to recurrence of FSGS after transplantation. We performed exome sequencing on six patients with recurrent FSGS to identify variants in fifty-one genes and found significant variations in the alpha-2-macroglobulin (A2M). Immunoblotting was used to investigate effects of specific gene variants at the protein level. Further expression analysis identified A2M, exophilin 5 (EXPH5) and plectin (PLEC) as specific proteins linked to podocytes, endothelial cells, and the glomerulus. Subsequent protein array screening revealed the presence of non-HLA-specific antibodies, including TRIM21, after transplantation. Using Metascape for pathway and process enrichment analysis, we focused on the IL-17 signaling and chemotaxis pathways. ELISA measurements showed significantly elevated IL-17 levels in patients with recurrent FSGS (32.30 ± 9.12 pg/mL) compared to individuals with other glomerular diseases (23.16 ± 2.49 pg/mL; p < 0.01) and healthy subjects (22.28 ± 0.94 pg/mL; p < 0.01), with no significant difference in plasma CCL2/MCP-1 levels between groups. This study explores the molecular dynamics underlying recurrence of FSGS after transplantation, offering insights into potential biomarkers and therapeutic targets for the future development of individualized treatments for transplant patients.

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We have developed a protein array system, named "Phospho-Totum", which reproduces the phosphorylation state of a sample on the array. The protein array contains 1471 proteins from 273 known signaling pathways. According to the activation degrees of tyrosine kinases in the sample, the corresponding groups of substrate proteins on the array are phosphorylated under the same conditions. In addition to measuring the phosphorylation levels of the 1471 substrates, we have developed and performed the artificial intelligence-assisted tools to further characterize the phosphorylation state and estimate pathway activation, tyrosine kinase activation, and a list of kinase inhibitors that produce phosphorylation states similar to that of the sample. The Phospho-Totum system, which seamlessly links and interrogates the measurements and analyses, has the potential to not only elucidate pathophysiological mechanisms in diseases by reproducing the phosphorylation state of samples, but also be useful for drug discovery, particularly for screening targeted kinases for potential drug kinase inhibitors.

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Epithelial ovarian cancers that are nonhomologous recombination deficient, as well as those that are recurrent and in a platinum-resistant state, have limited therapeutic options. The objectives of this study were to characterize the mechanism of action and investigate the therapeutic potential of a small molecule, VDX-111, against ovarian cancer. We examined the ability of VDX-111 to inhibit the growth of a panel of ovarian cancer cell lines, focusing on BRCA wild-type lines. We found that VDX-111 causes a dose-dependent loss of cell viability across ovarian cancer cell lines. Reverse phase protein array (RPPA) analysis was used to identify changes in cell signaling in response to VDX-111 treatment. An RPPA analysis performed on cells treated with VDX-111 detected changes in cell signaling related to autophagy and necroptosis. Immunoblots of OVCAR3 and SNU8 cells confirmed a dose-dependent increase in LC3A/B and RIPK1. Incucyte live cell imaging was used to measure cell proliferation and death in response to VDX-111 alone and with inhibitors of apoptosis, necroptosis, and autophagy. Annexin/PI assays suggested predominantly nonapoptotic cell death, while real-time kinetic imaging of cell growth indicated the necroptosis inhibitor, necrostatin-1, attenuates VDX-111-induced loss of cell viability, suggesting a necroptosis-dependent mechanism. Furthermore, VDX-111 inhibited tumor growth in patient-derived xenograft and syngeneic murine models. In conclusion, the cytotoxic effects of VDX-111 seen in vitro and in vivo appear to occur in a necroptosis-dependent manner and may promote an antitumor immune response.

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The addition of the proteasome inhibitor bortezomib to standard chemotherapy did not improve survival in pediatric acute myeloid leukemia (AML) when all patients were analyzed as a group in the Children's Oncology Group phase 3 trial AAML1031 (NCT01371981). Proteasome inhibition influences the chromatin landscape and proteostasis, and we hypothesized that baseline proteomic analysis of histone- and chromatin-modifying enzymes (HMEs) would identify AML subgroups that benefitted from bortezomib addition. A proteomic profile of 483 patients treated with AAML1031 chemotherapy was generated using a reverse-phase protein array. A relatively high expression of 16 HME was associated with lower EFS and higher 3-year relapse risk after AML standard treatment compared to low expressions (52% vs. 29%, p = 0.005). The high-HME profile correlated with more transposase-accessible chromatin, as demonstrated via ATAC-sequencing, and the bortezomib addition improved the 3-year overall survival compared with standard therapy (62% vs. 75%, p = 0.033). These data suggest that there are pediatric AML populations that respond well to bortezomib-containing chemotherapy.

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Tick-borne encephalitis is a vaccine-preventable disease of concern for public health in large parts of Europe, with EU notification rates increasing since 2018. It is caused by the orthoflavivirus tick-borne encephalitis virus (TBEV) and a diagnosis of infection is mainly based on serology due to its short viremic phase, often before symptom onset. The interpretation of TBEV serology is hampered by a history of orthoflavivirus vaccination and by previous infections with related orthoflaviviruses. Here, we sought to improve TBEV sero-diagnostics using an antigen combination of in-house expressed NS1 and EDIII in a multiplex, low-specimen-volume set-up for the detection of immune responses to TBEV and other clinically important orthoflaviviruses (i.e., West Nile virus, dengue virus, Japanese encephalitis virus, Usutu virus and Zika virus). We show that the combined use of NS1 and EDIII results in both a specific and sensitive test for the detection of TBEV IgG for patient diagnostics, vaccination responses and in seroprevalence studies. This novel approach potentially allows for a low volume-based, simultaneous analysis of IgG responses to a range of orthoflaviviruses with overlapping geographic circulations and clinical manifestations.

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The last few decades have seen remarkable strides in the field of cancer therapy. Precision oncology coupled with comprehensive genomic profiling has become routine clinical practice for solid tumors, the advent of immune checkpoint inhibitors has transformed the landscape of oncology treatment, and the number of cancer drug approvals has continued to increase. Nevertheless, the application of genomics-driven precision oncology has thus far benefited only 10%-20% of cancer patients, leaving the majority without matched treatment options. This limitation underscores the need to explore alternative avenues with regard to selecting patients for targeted therapies. In contrast with genomics-based approaches, proteomics-based strategies offer a more precise understanding of the intricate biological processes driving cancer pathogenesis. This perspective underscores the importance of integrating complementary proteomic analyses into the next phase of precision oncology to establish robust biomarker-drug associations and surmount challenges related to drug resistance. One promising technology in this regard is the reverse-phase protein array (RPPA), which excels in quantitatively detecting protein modifications, even with limited amounts of sample. Its cost-effectiveness and rapid turnaround time further bolster its appeal for application in clinical settings. Here, we review the current status of genomics-driven precision oncology, as well as its limitations, with an emphasis on drug resistance. Subsequently, we explore the application of RPPA technology as a catalyst for advancing precision oncology. Through illustrative examples drawn from clinical trials, we demonstrate its utility for unraveling the molecular mechanisms underlying drug responses and resistance.

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BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is often treated with FOLFIRINOX, a chemotherapy associated with high toxicity rates and variable efficacy. Therefore, it is crucial to identify patients at risk of early progression during treatment. This study aims to explore the potential of a multi-omics biomarker for predicting early PDAC progression by employing an in-depth mathematical modeling approach. METHODS: Blood samples were collected from 58 PDAC patients undergoing FOLFIRINOX before and after the first cycle. These samples underwent gene (GEP) and inflammatory protein expression profiling (IPEP). We explored the predictive potential of exclusively IPEP through Stepwise (Backward) Multivariate Logistic Regression modeling. Additionally, we integrated GEP and IPEP using Bayesian Kernel Regression modeling, aiming to enhance predictive performance. Ultimately, the FOLFIRINOX IPEP (FFX-IPEP) signature was developed. RESULTS: Our findings revealed that proteins exhibited superior predictive accuracy than genes. Consequently, the FFX-IPEP signature consisted of six proteins: AMN, BANK1, IL1RL2, ITGB6, MYO9B, and PRSS8. The signature effectively identified patients transitioning from disease control to progression early during FOLFIRINOX, achieving remarkable predictive accuracy with an AUC of 0.89 in an independent test set. Importantly, the FFX-IPEP signature outperformed the conventional CA19-9 tumor marker. CONCLUSIONS: Our six-protein FFX-IPEP signature holds solid potential as a liquid biomarker for the early prediction of PDAC progression during toxic FOLFIRINOX chemotherapy. Further validation in an external cohort is crucial to confirm the utility of the FFX-IPEP signature. Future studies should expand to predict progression under different chemotherapies to enhance the guidance of personalized treatment selection in PDAC.

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DNA microarrays have been applied for comprehensive genotyping, but remain a drawback in complicated operations. As a solution, we previously reported the solid-phase collateral cleavage (SPCC) system based on the clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 12 (CRISPR/Cas12). Surface-immobilized Cas12-CRISPR RNA (crRNA) can directly hybridize target double-stranded DNA (dsDNA) and subsequently produce a signal via the cleavage of single-stranded DNA (ssDNA) reporter immobilized on the same spot. Therefore, SPCC-based multiplex dsDNA detection can be performed easily. This study reports the miniaturization of SPCC-based spots patterned by a non-contact printer and its performance in comprehensive genotyping on a massively accumulated array. Initially, printing, immobilization, and washing processes of Cas12-crRNA were established to fabricate the non-contact-patterned SPCC-based sensor array. A target dsDNA concentration response was obtained based on the developed sensor array, even with a spot diameter of 0.64 ± 0.05 mm. Also, the limit of detection was 572 pM, 531 pM, and 3.04 nM with 40, 20, and 10 nL-printing of Cas12-crRNA, respectively. Furthermore, the sensor array specifically detected three dsDNA sequences in one-pot multiplexing; therefore, the feasibility of comprehensive genotyping was confirmed. These results demonstrate that our technology can be miniaturized as a CRISPR/Cas12-based microarray by using non-contact printing. In the future, the non-contact-patterned SPCC-based sensor array can be applied as an alternative tool to DNA microarrays.

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Autoantibodies that recognize self-antigens are believed to have a close relationship with diseases such as autoimmune diseases, cancer, and lifestyle diseases. Analysis of autoantibodies is essential for investigating pathology mechanisms, diagnosis, and therapeutics of these diseases. We developed an autoantibody profiling assay using a cell-free synthesized protein array and high-throughput screening technology. Our assay system can sensitively detect interaction between recombinant antigen protein and autoantibody and efficiently analyze autoantibody profiling in patients' sera.

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OBJECTIVES: To identify and characterize undescribed systemic sclerosis (SSc)-specific autoantibodies targeting nucleolar antigens and to assess their clinical significance. METHODS: We conducted proteome-wide autoantibody screening (PWAS) against serum samples from SSc patients with nucleolar patterned anti-nuclear antibodies (NUC-ANAs) of specific antibodies (Abs) unknown, utilizing wet protein arrays fabricated from in vitro human proteome. Controls included SSc patients with already-known SSc-specific autoantibodies, patients with other connective tissue diseases, and healthy subjects. The selection of nucleolar antigens was performed by database search in the Human Protein Atlas. The Presence of autoantibodies was certified by immunoblots and immunoprecipitations. Indirect immunofluorescence assays on HEp-2 cells were also conducted. Clinical assessment was conducted by retrospective review of electric medical records. RESULTS: PWAS identified three candidate autoantibodies, including anti-nuclear valosin-containing protein-like (NVL) Ab. Additional measurements in disease controls revealed that only anti-NVL Abs are exclusively detected in SSc. Detection of anti-NVL Abs was reproduced by conventional assays such as immunoblotting and immunoprecipitation. Indirect immunofluorescence assays demonstrated homogeneous nucleolar patterns. Anti-NVL Ab-positive cases were characterized by significantly low prevalence of diffuse skin sclerosis and interstitial lung disease, compared with SSc cases with NUC-ANAs other than anti-NVL Abs, such as anti-U3-RNP and anti-Th/To Abs. CONCLUSION: Anti-NVL Ab is an SSc-specific autoantibody associated with a unique combination of clinical features, including limited skin sclerosis and lack of lung involvement.

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AIMS: This study aims to reveal a promising biomarker for Parkinson's disease (PD) based on research with reverse phase protein array (RPPA) technology for the first time and in vivo verification, which gains time for early intervention in PD, thus increasing the effectiveness of treatment and reducing disease morbidity. METHODS AND RESULTS: We employed RPPA technology which can assess both total and post-translationally modified proteins to identify biomarker candidates of PD in a cellular PD model. As a result, the phosphorylation (pY-1248) of the epidermal growth factor receptor (EGFR) ErbB2 is a promising biomarker candidate for PD. In addition, lapatinib, an ErbB2 tyrosine kinase inhibitor, was used to verify this PD biomarker candidate in vivo. We found that lapatinib-attenuated dopaminergic neuron loss and PD-like behavior in the zebrafish PD model. Accordingly, the expression of ErbB2pY-1248 significantly increased in the MPTP-induced mouse PD model. Our results suggest that ErbB2pY-1248 is a predictive biomarker for PD. CONCLUSIONS: In this study, we found that ErbB2pY-1248 is a predictive biomarker of PD by using RPPA technology and in vivo verification. It offers a new perspective on PD diagnosing and treatment, which will be essential in identifying individuals at risk of PD. In addition, this study provides new ideas for digging into biomarkers of other neurodegenerative diseases.

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IMPORTANCE: This manuscript explores the host humoral response to selected antigens of the syphilis agent during infection to evaluate their potential use as diagnostic tests and markers for treatment.

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ANCA-associated vasculitides (AAV) are rare autoimmune diseases causing inflammation and damage to small blood vessels. New autoantibody biomarkers are needed to improve the diagnosis and treatment of AAV patients. In this study, we aimed to profile the autoantibody repertoire of AAV patients using in-house developed antigen arrays to identify previously unreported antibodies linked to the disease per se, clinical subgroups, or clinical activity. A total of 1743 protein fragments representing 1561 unique proteins were screened in 229 serum samples collected from 137 AAV patients at presentation, remission, and relapse. Additionally, serum samples from healthy individuals and patients with other type of vasculitis and autoimmune-inflammatory conditions were included to evaluate the specificity of the autoantibodies identified in AAV. Autoreactivity against members of the kinesin protein family were identified in AAV patients, healthy volunteers, and disease controls. Anti-KIF4A antibodies were significantly more prevalent in AAV. We also observed possible associations between anti-kinesin antibodies and clinically relevant features within AAV patients. Further verification studies will be needed to confirm these findings.

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Protein lattices that shift the structure and shape anisotropy in response to environmental cues are closely coupled to potential functionality. However, to design and construct shape-anisotropic protein arrays from the same building blocks in response to different external stimuli remains challenging. Here, by a combination of the multiple, symmetric interaction sites on the outer surface of protein nanocages and the tunable features of phenylalanine-phenylalanine interactions, a protein engineering approach is reported to construct a variety of superstructures with shape anisotropy, including 3D cubic, 2D hexagonal layered, and 1D rod-like crystalline protein nanocage arrays by using one single protein building block. Notably, the assembly of these crystalline protein arrays is reversible, which can be tuned by external stimuli (pH and ionic strength). The anisotropic morphologies of the fabricated macroscopic crystals can be correlated with the Å-to-nm scale protein arrangement details by crystallographic elucidation. These results enhance the understanding of the freedom offered by an object's symmetry and inter-object π-π stacking interactions for protein building blocks to assemble into direction- and shape-anisotropic biomaterials.

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BACKGROUND: Neuropsychiatric systemic lupus erythematosus (NPSLE), with various morbidities and multiple manifestations in the central nervous system, remains a limited standard for diagnosis. Our study was to discover novel biomarkers for improving the diagnostic efficiency for NPSLE. METHODS: We performed a quantitative planar protein antibody microarray to screen 1000 proteins in cerebrospinal fluid from controls, systemic lupus erythematosus (SLE, non-NPSLE) patients, and NPSLE patients. Differentially expressed proteins (DEPs) as candidate biomarkers were developed into a custom multiplexed protein antibody array for further validation in an independent larger cohort. Subsequently, we used least absolute shrinkage and selection operator regression (LASSO) analysis and multivariable logistic regression analysis for optimizing feature selection and constructing a diagnostic model. A receiver operating characteristic curve (ROC) was generated to assess the effectiveness of the models. RESULTS: The expression of 29 proteins in CSF was significantly altered in the comparison of the three groups. We selected 17 proteins as candidate biomarkers in accordance with protein interaction analysis. In the larger cohort, we identified 5 DEPs as biomarkers for NPSLE, including TCN2, CST6, KLK5, L-selectin, and Trappin-2. The diagnostic model included 3 hub proteins (CST6, TCN2, KLK5) and was best at discriminating NPSLE from SLE patients. These CSF biomarkers were also highly associated with disease activity. In addition, there were 6 molecules with remarkable changes in NPSLE CSF and hippocampus, which indicated the consistency of the environment in the brain and the promising molecular targets in the pathogenesis of NPSLE. CONCLUSIONS: The dual-chips screening strategy demonstrated KLK5, L-selectin, Trappin-2, TCN2, and CST6 as CSF biomarkers for diagnosing NPSLE.

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Bone marrow stromal cells (BMSCs) have emerged as a potential therapy for sepsis, yet the underlying mechanisms remain unclear. In this study, we investigated the effects of BMSCs on serum inflammatory cytokines in a rat model of lipopolysaccharide (LPS)-induced sepsis. Sepsis was induced by intravenous injection of LPS, followed by transplantation of BMSCs. We monitored survival rates for 72 h and evaluated organ functions, histopathological changes, and cytokines expression. Sepsis rats showed decreased levels of white blood cells, platelets, lymphocyte ratio, and oxygen partial pressure, along with increased levels of neutrophil ratio, carbon dioxide partial pressure, lactic acid, alanine aminotransferase, and aspartate aminotransferase. Histologically, lung, intestine, and liver tissues exhibited congestion, edema, and infiltration of inflammatory cells. However, after BMSCs treatment, there was improvement in organ functions, histopathological injuries, and survival rates. Protein microarray analysis revealed significant changes in the expression of 12 out of 34 inflammatory cytokines. These findings were confirmed by enzyme-linked immunosorbent assay. Pro-inflammatory factors, such as interleukin-1ß (IL-1ß), IL-1α, tumor necrosis factor-α (TNF-α), tissue inhibitor of metal protease 1 (TIMP-1), matrix metalloproteinase 8 (MMP-8), Leptin, and L-selectin were upregulated in sepsis, whereas anti-inflammatory and growth factors, including IL-4, ß-nerve growth factor (ß-NGF), ciliary neurotrophic factor (CNTF), interferon γ (IFN-γ), and Activin A were downregulated. BMSCs transplantation led to a decrease in pro-inflammatory cytokines and an increase in anti-inflammatory and growth factors. We summarized relevant molecular signaling pathways that resulted from cytokines in BMSCs for treating sepsis. Our results illustrated that BMSCs could promote tissue repair and improve organ functions and survival rates in sepsis through modulating cytokine networks.

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The construction of three-dimensional (3D) array materials from nanoscale building blocks has drawn significant interest because of their potential to exhibit collective properties and functions arising from the interactions between individual building blocks. Protein cages such as virus-like particles (VLPs) have distinct advantages as building blocks for higher-order assemblies because they are extremely homogeneous in size and can be engineered with new functionalities by chemical and/or genetic modification. In this chapter, we describe a protocol for constructing a new class of protein-based superlattices, called protein macromolecular frameworks (PMFs). We also describe an exemplary method to evaluate the catalytic activity of enzyme-enclosed PMFs, which exhibit enhanced catalytic activity due to the preferential partitioning of charged substrates into the PMF.

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Introduction: The development and maintenance of neural circuits is highly sensitive to neural activity. General anesthetics have profound effects on neural activity and, as such, there is concern that these agents may alter cellular integrity and interfere with brain wiring, such as when exposure occurs during the vulnerable period of brain development. Under those conditions, exposure to anesthetics in clinical use today causes changes in synaptic strength and number, widespread apoptosis, and long-lasting cognitive impairment in a variety of animal models. Remarkably, most anesthetics produce these effects despite having differing receptor mechanisms of action. We hypothesized that anesthetic agents mediate these effects by inducing a shared signaling pathway. Methods: We exposed cultured cortical cells to propofol, etomidate, or dexmedetomidine and assessed the protein levels of dozens of signaling molecules and post-translational modifications using reverse phase protein arrays. To probe the role of neural activity, we performed separate control experiments to alter neural activity with non-anesthetics. Having identified anesthetic-induced changes in vitro, we investigated expression of the target proteins in the cortex of sevoflurane anesthetized postnatal day 7 mice by Western blotting. Results: All the anesthetic agents tested in vitro reduced phosphorylation of the ribosomal protein S6, an important member of the mTOR signaling pathway. We found a comparable decrease in cortical S6 phosphorylation by Western blotting in sevoflurane anesthetized neonatal mice. Using a systems approach, we determined that propofol, etomidate, dexmedetomidine, and APV/TTX all similarly modulate a signaling module that includes pS6 and other cell mediators of the mTOR-signaling pathway. Discussion: Reduction in S6 phosphorylation and subsequent suppression of the mTOR pathway may be a common and novel signaling event that mediates the impact of general anesthetics on neural circuit development.