Eye Drop with Fas-Blocking Peptide Attenuates Age-Related Macular Degeneration.

Age-related macular degeneration (AMD), characterized by macular retinal degeneration, poses a significant health concern due to the lack of effective treatments for prevalent dry AMD. The progression of AMD is closely linked to reactive oxygen species and Fas signaling, emphasizing the need for targeted interventions. In this study, we utilized a NaIO3-induced retinal degeneration mouse model to assess the efficacy of Fas-blocking peptide (FBP). Intravitreal administration of FBP successfully suppressed Fas-mediated inflammation and apoptosis, effectively arresting AMD progression in mice. We developed a 6R-conjugated FBP (6R-FBP) for eye drop administration. 6R-FBP, administered as an eye drop, reached the retinal region, attenuating degeneration by modulating the expression of inflammatory cytokines and blocking Fas-mediated apoptosis in rodent and rabbit NaIO3-induced retinal degeneration models to address practical concerns. Intravitreal FBP and 6R-FBP eye drops effectively reduced retinal degeneration and improved retinal thickness in rodent and rabbit models. This study highlights the therapeutic potential of FBP, particularly 6R-FBP as an eye drop, in inhibiting Fas-mediated cell signaling and protecting against retinal cell death and inflammation in dry AMD. Future investigations should explore the translational prospects of this approach in primates with eye structures comparable to those of humans.

A Phase II Open-Label Randomized Clinical Trial of Preoperative Durvalumab or Durvalumab plus Tremelimumab in Resectable Head and Neck Squamous Cell Carcinoma.

PURPOSE: Clinical implications of neoadjuvant immunotherapy in patients with locally advanced but resectable head and neck squamous cell carcinoma (HNSCC) remain largely unexplored. PATIENTS AND METHODS: Patients with resectable HNSCC were randomized to receive a single dose of preoperative durvalumab (D) with or without tremelimumab (T) before resection, followed by postoperative (chemo)radiotherapy based on multidisciplinary discretion and 1-year D treatment. Artificial intelligence (AI)-powered spatial distribution analysis of tumor-infiltrating lymphocytes and high-dimensional profiling of circulating immune cells tracked dynamic intratumoral and systemic immune responses. RESULTS: Of the 48 patients enrolled (D, 24 patients; D+T, 24 patients), 45 underwent surgical resection per protocol (D, 21 patients; D+T, 24 patients). D±T had a favorable safety profile and did not delay surgery. Distant recurrence-free survival (DRFS) was significantly better in patients treated with D+T than in those treated with D monotherapy. AI-powered whole-slide image analysis demonstrated that D+T significantly reshaped the tumor microenvironment toward immune-inflamed phenotypes, in contrast with the D monotherapy or cytotoxic chemotherapy. High-dimensional profiling of circulating immune cells revealed a significant expansion of T-cell subsets characterized by proliferation and activation in response to D+T therapy, which was rare following D monotherapy. Importantly, expansion of specific clusters in CD8+ T cells and non-regulatory CD4+ T cells with activation and exhaustion programs was associated with prolonged DRFS in patients treated with D+T. CONCLUSIONS: Preoperative D±T is feasible and may benefit patients with resectable HNSCC. Distinct changes in the tumor microenvironment and circulating immune cells were induced by each treatment regimen, warranting further investigation.

Investigation of daily patterns for smartphone keystroke dynamics based on loneliness and social isolation.

This study examined the relationship between loneliness levels and daily patterns of mobile keystroke dynamics in healthy individuals. Sixty-six young healthy Koreans participated in the experiment. Over five weeks, the participants used a custom Android keyboard. We divided the participants into four groups based on their level of loneliness (no loneliness, moderate loneliness, severe loneliness, and very severe loneliness). The very severe loneliness group demonstrated significantly higher typing counts during sleep time than the other three groups (one-way ANOVA, F = 3.75, p < 0.05). In addition, the average cosine similarity value of weekday and weekend typing patterns in the very severe loneliness group was higher than that in the no loneliness group (Welch's t-test, t = 2.27, p < 0.05). This meant that the no loneliness group's weekday and weekend typing patterns varied, whereas the very severe loneliness group's weekday and weekend typing patterns did not. Our results indicated that individuals with very high levels of loneliness tended to use mobile keyboards during late-night hours and did not significantly change their smartphone usage behavior between weekdays and weekends. These findings suggest that mobile keystroke dynamics have the potential to be used for the early detection of loneliness and the development of targeted interventions.

Systemic Treatment with Fas-Blocking Peptide Attenuates Apoptosis in Brain Ischemia.

Apoptosis plays a crucial role in neuronal injury, with substantial evidence implicating Fas-mediated cell death as a key factor in ischemic strokes. To address this, inhibition of Fas-signaling has emerged as a promising strategy in preventing neuronal cell death and alleviating brain ischemia. However, the challenge of overcoming the blood-brain barrier (BBB) hampers the effective delivery of therapeutic drugs to the central nervous system (CNS). In this study, we employed a 30 amino acid-long leptin peptide to facilitate BBB penetration. By conjugating the leptin peptide with a Fas-blocking peptide (FBP) using polyethylene glycol (PEG), we achieved specific accumulation in the Fas-expressing infarction region of the brain following systemic administration. Notably, administration in leptin receptor-deficient db/db mice demonstrated that leptin facilitated the delivery of FBP peptide. We found that the systemic administration of leptin-PEG-FBP effectively inhibited Fas-mediated apoptosis in the ischemic region, resulting in a significant reduction of neuronal cell death, decreased infarct volumes, and accelerated recovery. Importantly, neither leptin nor PEG-FBP influenced apoptotic signaling in brain ischemia. Here, we demonstrate that the systemic delivery of leptin-PEG-FBP presents a promising and viable strategy for treating cerebral ischemic stroke. Our approach not only highlights the therapeutic potential but also emphasizes the importance of overcoming BBB challenges to advance treatments for neurological disorders.

Small interfering RNA (siRNA)-based therapeutic applications against viruses: principles, potential, and challenges.

RNA has emerged as a revolutionary and important tool in the battle against emerging infectious diseases, with roles extending beyond its applications in vaccines, in which it is used in the response to the COVID-19 pandemic. Since their development in the 1990s, RNA interference (RNAi) therapeutics have demonstrated potential in reducing the expression of disease-associated genes. Nucleic acid-based therapeutics, including RNAi therapies, that degrade viral genomes and rapidly adapt to viral mutations, have emerged as alternative treatments. RNAi is a robust technique frequently employed to selectively suppress gene expression in a sequence-specific manner. The swift adaptability of nucleic acid-based therapeutics such as RNAi therapies endows them with a significant advantage over other antiviral medications. For example, small interfering RNAs (siRNAs) are produced on the basis of sequence complementarity to target and degrade viral RNA, a novel approach to combat viral infections. The precision of siRNAs in targeting and degrading viral RNA has led to the development of siRNA-based treatments for diverse diseases. However, despite the promising therapeutic benefits of siRNAs, several problems, including impaired long-term protein expression, siRNA instability, off-target effects, immunological responses, and drug resistance, have been considerable obstacles to the use of siRNA-based antiviral therapies. This review provides an encompassing summary of the siRNA-based therapeutic approaches against viruses while also addressing the obstacles that need to be overcome for their effective application. Furthermore, we present potential solutions to mitigate major challenges.

Radiological Assessment of Morphological Changes of Lumbar Spine: From First to 9th Decade of Life.

STUDY DESIGN: Analysis of lumbar spine radiograms of 1,496 Jeju islanders of Korea. PURPOSE: To look into the age- and gender-matched incidences of morphological changes and their severities. OVERVIEW OF LITERATURE: There have been several prior research on the prevalence and severity of age-related diseases, both related and unrelated. Those offer some fundamental clinical data for clinicians. METHODS: Radiograms of 1,496 patients (555 males and 941 females) from the first to 9th decade were examined for this study. Sagittal and coronal alignment, disc space narrowing, spur formation including diffuse idiopathic spinal hyperostosis (DISH), spondylolisthesis, and ballooned discs associated with biconcave bodies due to osteoporosis were among the parameters of lumbar spine morphologies examined on high-quality radiographs by both human observers and computers. RESULTS: The alignment of the lumbar spine altered after birth and set at growth maturity, and then the curve was maintained till the end of the 5th decade afterward and the curve gradually hypolordotic. There were three types of coronal alignment abnormalities can be seen: idiopathic, osteopathic, and discogenic (degenerative lumbar scoliosis [DLS]). DLS developed after 6th decade. There was no scoliosis associated with spondylolysis or the post-laminofacetectomy period. Disc space narrowing and corporal spur formation were not seen till the end of 3rd decade comparatively speaking, the corporal spurs generated in the non-scoliotic spine were smaller than those in the scoliotic spine. DISH began to appear in the 5th-decade patients and its incidences increased gradually afterward. Porosis-related vertebral body collapse started to happen after 6th decade. There are three different types of spondylolisthesis: anterior, posterior, and lateral. The lateral slip occurred only in the scoliotic spine. All types were related to degenerative discs. CONCLUSIONS: It has been shown that the morphology of the lumbar spine changes throughout time.

Detection of Periodontal Disease Marker with Geometrical Transformation of Ag Nanoplates.

Alkaline phosphatase (ALP) and interleukin-1beta (IL-1ß) are crucial salivary biomarkers for the diagnosis of periodontal disease that harms the periodontal tissue along with tooth loss. However, there has been no way of sensitive and portable detection of both biomarkers in saliva with multivariate signal readout. In this work, we design the multicolorimetric ALP and IL-1ß sensing platform based on geometrical transformation of silver nanoplate transducer. By utilizing enzymatic activity of ALP that dephosphorylates p-aminophenol phosphate (p-APP) to p-aminophenol (p-AP), localized surface plasmon resonance properties of silver nanoplate vary with ALP and show a distinct color change from blue to yellow based on a controlled seed transformation from triangular to hexagonal, rounded pentagonal, and spherical shape. The multicolor sensor shows an ALP detection range of 0-25 U/L with a limit of detection (LOD) of 0.0011 U/L, which is the lowest range of LOD demonstrated to date for state-of-the-art ALP sensor. Furthermore, we integrate the sensor with the conventional ELISA to detect IL-1ß for multicolor signaling and it exhibits a linear detection range of 0-250 pg/mL and an LOD of 0.066 pg/mL, which is 2 orders of magnitude lower than the monochromic conventional ELISA (LOD of 3.8 pg/mL). The ALP multicolor sensor shows high selectivity with a recovery of 100.9% in real human saliva proving its reliability and suitability for the readily accessible periodontal diagnosis with multivariate signal readout.

Lower Serum Calcium Levels Associated with Disrupted Sleep and Rest-Activity Rhythm in Shift Workers.

Vitamin D deficiency is prevalent in many developed countries, and several studies suggest that vitamin D plays an essential role in brain function. A recent study showed that vitamin D deficiency was closely associated with daytime sleepiness and shorter sleep time. The relationshipbetween vitamin D levels and calcium levels is well established, and calcium level regulates slow-wave sleep generation. It is conceivable that the sleep disturbance in vitamin D deficiency may be due to an altered calcium level. Nonetheless, calcium levels, sleep disturbances, and activity rhythms have not been investigated directly. Therefore, we hypothesized that calcium and vitamin D levels might be important in regulating sleep and activity rhythm, and we analyzed the correlation with calcium levels by actigraphy analysis. Interestingly, a negative correlation was found between calcium level and sleep latency, total sleep time, use of sleep medicine, and daytime dysfunction among shift workers. In contrast, non-shift workers showed a negative correlation between the calcium level and the circadian phase. These findings suggest that low serum calcium levels may disrupt sleep-wake control and rest-activity rhythm, even if they are within the normal range.

Deficiency in RCAT-1 Function Causes Dopamine Metabolism Related Behavioral Disorders in Caenorhabditis elegans.

When animals are faced with food depletion, food search-associated locomotion is crucial for their survival. Although food search-associated locomotion is known to be regulated by dopamine, it has yet to investigate the potential molecular mechanisms governing the regulation of genes involved in dopamine metabolism (e.g., cat-1, cat-2) and related behavioral disorders. During the studies of the pheromone ascaroside, a signal of starvation stress in C. elegans, we identified R02D3.7, renamed rcat-1 (regulator of cat genes-1), which had previously been shown to bind to regulatory sequences of both cat-1 and cat-2 genes. It was found that RCAT-1 (R02D3.7) is expressed in dopaminergic neurons and functions as a novel negative transcriptional regulator for cat-1 and cat-2 genes. When a food source becomes depleted, the null mutant, rcat-1(ok1745), exhibited an increased frequency of high-angled turns and intensified area restricted search behavior compared to the wild-type animals. Moreover, rcat-1(ok1745) also showed defects in state-dependent olfactory adaptation and basal slowing response, suggesting that the mutants are deficient in either sensing food or locomotion toward food. However, rcat-1(ok1745) has normal cuticular structures and locomotion genes. The discovery of rcat-1 not only identifies a new subtype of dopamine-related behaviors but also provides a potential therapeutic target in Parkinson's disease.

Development and Validation of an Insulin Resistance Predicting Model Using a Machine-Learning Approach in a Population-Based Cohort in Korea.

BACKGROUND: Insulin resistance is a common etiology of metabolic syndrome, but receiver operating characteristic (ROC) curve analysis shows a weak association in Koreans. Using a machine learning (ML) approach, we aimed to generate the best model for predicting insulin resistance in Korean adults aged > 40 of the Ansan/Ansung cohort using a machine learning (ML) approach. METHODS: The demographic, anthropometric, biochemical, genetic, nutrient, and lifestyle variables of 8842 participants were included. The polygenetic risk scores (PRS) generated by a genome-wide association study were added to represent the genetic impact of insulin resistance. They were divided randomly into the training (n = 7037) and test (n = 1769) sets. Potentially important features were selected in the highest area under the curve (AUC) of the ROC curve from 99 features using seven different ML algorithms. The AUC target was ≥0.85 for the best prediction of insulin resistance with the lowest number of features. RESULTS: The cutoff of insulin resistance defined with HOMA-IR was 2.31 using logistic regression before conducting ML. XGBoost and logistic regression algorithms generated the highest AUC (0.86) of the prediction models using 99 features, while the random forest algorithm generated a model with 0.82 AUC. These models showed high accuracy and k-fold values (>0.85). The prediction model containing 15 features had the highest AUC of the ROC curve in XGBoost and random forest algorithms. PRS was one of 15 features. The final prediction models for insulin resistance were generated with the same nine features in the XGBoost (AUC = 0.86), random forest (AUC = 0.84), and artificial neural network (AUC = 0.86) algorithms. The model included the fasting serum glucose, ALT, total bilirubin, HDL concentrations, waist circumference, body fat, pulse, season to enroll in the study, and gender. CONCLUSION: The liver function, regular pulse checking, and seasonal variation in addition to metabolic syndrome components should be considered to predict insulin resistance in Koreans aged over 40 years.

Early Diagnostic Ability of Human Complement Factor B in Pancreatic Cancer Is Partly Linked to Its Potential Tumor-Promoting Role.

Although plasma complement factor B (CFB, NX_P00751), both alone and in combination with CA19-9 (i.e., the ComB-CAN), previously exhibited a reliable diagnostic ability for pancreatic cancer (PC), its detectability of the early stages and the cancer detection mechanism remained elusive. We first evaluated the diagnostic accuracy of ComB-CAN using plasma samples from healthy donors (HDs), patients with chronic pancreatitis (CP), and patients with different PC stages (I/II vs III/IV). An analysis of the area under the curve (AUC) by PanelComposer using logistic regression revealed that ComB-CAN has a superior diagnostic ability for early-stage PC (97.1.% [95% confidence interval (CI): (97.1-97.2)]) compared with CFB (94.3% [95% CI: 94.2-94.4]) or CA19-9 alone (34.3% [95% CI: 34.1-34.4]). In the comparisons of all stages of patients with PC vs CP and HDs, the AUC values of ComB-CAN, CFB, and CA19-9 were 0.983 (95% CI: 0.983-0.983), 0.950 (95% CI: 0.950-0.951), and 0.873 (95% CI: 0.873-0.874), respectively. We then investigated the molecular mechanism underlying the detection of early-stage PC by using stable cell lines of CFB knockdown and CFB overexpression. A global transcriptomic analysis coupled to cell invasion assays of both CFB-modulated cell lines suggested that CFB plays a tumor-promoting role in PC, which likely initiates the PI3K-AKT cancer signaling pathway. Thus our study establishes ComB-CAN as a reliable early diagnostic marker for PC that can be clinically applied for early PC screening in the general public.

Performance Evaluation and Fouling Propensity of Forward Osmosis (FO) Membrane for Reuse of Spent Dialysate.

The number of chronic renal disease patients has shown a significant increase in recent decades over the globe. Hemodialysis is the most commonly used treatment for renal replacement therapy (RRT) and dominates the global dialysis market. As one of the most water-consuming treatments in medical procedures, hemodialysis has room for improvement in reducing wastewater effluent. In this study, we investigated the technological feasibility of introducing the forward osmosis (FO) process for spent dialysate reuse. A 30 LMH of average water flux has been achieved using a commercial TFC membrane with high water permeability and salt removal. The water flux increased up to 23% with increasing flowrate from 100 mL/min to 500 mL/min. During 1 h spent dialysate treatment, the active layer facing feed solution (AL-FS) mode showed relatively higher flux stability with a 4-6 LMH of water flux reduction while the water flux decreased significantly at the active layer facing draw solution (AL-DS) mode with a 10-12 LMH reduction. In the pressure-assisted forward osmosis (PAFO) condition, high reverse salt flux was observed due to membrane deformation. During the membrane filtration process, scaling occurred due to the influence of polyvalent ions remaining on the membrane surface. Membrane fouling exacerbated the flux and was mainly caused by organic substances such as urea and creatinine. The results of this experiment provide an important basis for future research as a preliminary experiment for the introduction of the FO technique to hemodialysis.

Potential Regulatory Role of Human-Carboxylesterase-1 Glycosylation in Liver Cancer Cell Growth.

We previously reported that human carboxylesterase 1 (CES1), a serine esterase containing a unique N-linked glycosyl group at Asn79 (N79 CES1), is a candidate serological marker of hepatocellular carcinoma (HCC). CES1 is normally present at low-to-undetectable levels in normal human plasma, HCC tumors, and major liver cancer cell lines. To investigate the potential mechanism underlying the suppression of CES1 expression in liver cancer cells, we took advantage of the low detectability of this marker in tumors by overexpressing CES1 in multiple HCC cell lines, including stable Hep3B cells. We found that the population of CES1-overexpressing (OE) cells decreased and that their doubling time was longer compared with mock control liver cancer cells. Using interactive transcriptome, proteome, and subsequent Gene Ontology enrichment analysis of CES1-OE cells, we found substantial decreases in the expression levels of genes involved in cell cycle regulation and proliferation. This antiproliferative function of the N79 glycan of CES1 was further supported by quantitative real-time polymerase chain reaction, flow cytometry, and an apoptosis protein array assay. An analysis of the levels of key signaling target proteins via Western blotting suggested that CES1 overexpression exerted an antiproliferative effect via the PKD1/PKCµ signaling pathway. Similar results were also seen in another HCC cell line (PLC/RFP/5) after transient transfection with CES1 but not in similarly treated non-HCC cell lines (e.g., HeLa and Tera-1 cells), suggesting that CES1 likely exerts a liver cell-type-specific suppressive effect. Given that the N-linked glycosyl group at Asn79 (N79 glycan) of CES1 is known to influence CES1 enzyme activity, we hypothesized that the post-translational modification of CES1 at N79 may be linked to its antiproliferative activity. To investigate the regulatory effect of the N79 glycan on cellular growth, we mutated the single N-glycosylation site in CES1 from Asn to Gln (CES1-N79Q) via site-directed mutagenesis. Fluorescence 2-D difference gel electrophoresis protein expression analysis of cell lysates revealed an increase in cell growth and a decrease in doubling time in cells carrying the N79Q mutation. Thus our results suggest that CES1 exerts an antiproliferative effect in liver cancer cells and that the single N-linked glycosylation at Asn79 plays a potential regulatory role. These functions may underlie the undetectability of CES1 in human HCC tumors and liver cancer cell lines. Mass spectrometry data are available via ProteomeXchange under the identifier PXD021573.

Identification of ALDH6A1 as a Potential Molecular Signature in Hepatocellular Carcinoma via Quantitative Profiling of the Mitochondrial Proteome.

Various liver diseases, including hepatocellular carcinoma (HCC), have been linked to mitochondrial dysfunction, reduction of reactive oxygen species (ROS), and elevation of nitric oxide (NO). In this study, we subjected the human liver mitochondrial proteome to extensive quantitative proteomic profiling analysis and molecular characterization to identify potential signatures indicative of cancer cell growth and progression. Sequential proteomic analysis identified 2452 mitochondrial proteins, of which 1464 and 2010 were classified as nontumor and tumor (HCC) mitochondrial proteins, respectively, with 1022 overlaps. Further metabolic mapping of the HCC mitochondrial proteins narrowed our biological characterization to four proteins, namely, ALDH4A1, LRPPRC, ATP5C1, and ALDH6A1. The latter protein, a mitochondrial methylmalonate semialdehyde dehydrogenase (ALDH6A1), was most strongly suppressed in HCC tumor regions (∼10-fold decrease) in contrast to LRPPRC (∼6-fold increase) and was predicted to be present in plasma. Accordingly, we selected ALDH6A1 for functional analysis and engineered Hep3B cells to overexpress this protein, called ALDH6A1-O/E cells. Since ALDH6A1 is predicted to be involved in mitochondrial respiration, we assessed changes in the levels of NO and ROS in the overexpressed cell lines. Surprisingly, in ALDH6A1-O/E cells, NO was decreased nearly 50% but ROS was increased at a similar level, while the former was restored by treatment with S-nitroso-N-acetyl-penicillamine. The lactate levels were also decreased relative to control cells. Propidium iodide and Rhodamine-123 staining suggested that the decrease in NO and increase in ROS in ALDH6A1-O/E cells could be caused by depolarization of the mitochondrial membrane potential (ΔΨ). Taken together, our results suggest that hepatic neoplastic transformation appears to suppress the expression of ALDH6A1, which is accompanied by a respective increase and decrease in NO and ROS in cancer cells. Given the close link between ALDH6A1 suppression and abnormal cancer cell growth, this protein may serve as a potential molecular signature or biomarker of hepatocarcinogenesis and treatment responses.

A bio-inspired highly selective enzymatic glucose sensor using a red blood cell membrane.

In the development of enzymatic glucose sensors, accurate glucose sensing has been a challenging task because of the existence of numerous interfering molecules in the blood. Meanwhile, red blood cells (RBCs) selectively uptake glucose via a membrane protein called glucose transporter-1. In this study, we developed the RBC membrane (RBCM)-coated enzymatic glucose sensors that mimic the glucose uptake. The RBCM-coated sensors were examined via scanning electron microscopy, atomic force microscopy, and ATR-FTIR. We optimized the glucose permeability of the RBCM filter by controlling the thickness of the filter. The sensing range of the optimized sensor was 1-15 mM, the detection limit was 0.66 mM, and the sensitivity was 2.978 µA mM-1. Intriguingly, the RBCM-coated sensor was highly accurate and precise, despite the coexistence of glucose and interfering molecules (e.g., mannose, galactose, ascorbic acid, uric acid, and cysteine). For each interfering molecule, the errors of our sensor were 0.8 to 2.3%, which was 4.8-14.2 times more accurate than the uncoated one. A similar result was verified for a human serum sample containing countless interfering molecules. Also, the sensing performance of the sensor was consistent after 4 weeks of storage. The results suggest that applying RBCM may improve the selectivity of various types of glucose sensors including the continuous monitoring system.

FusionPro, a Versatile Proteogenomic Tool for Identification of Novel Fusion Transcripts and Their Potential Translation Products in Cancer Cells.

Fusion proteoforms are translation products derived from gene fusion. Although very rare, the fusion proteoforms play important roles in biomedical science. For example, fusion proteoforms influence the development of tumors by serving as cancer markers or cell cycle regulators. Although numerous studies have reported bioinformatics tools that can predict fusion transcripts, few proteogenomic tools are available that can predict and identify proteoforms. In this study, we develop a versatile proteogenomic tool "FusionPro," which facilitates the identification of fusion transcripts and their potential translatable peptides. FusionPro provides an independent gene fusion prediction module and can build sequence databases for annotated fusion proteoforms. FusionPro shows greater sensitivity than the available fusion finders when analyzing simulated or real RNA sequencing data sets. We use FusionPro to identify 18 fusion junction peptides and three potential fusion-derived peptides by MS/MS-based analysis of leukemia cell lines (Jurkat and K562) and ovarian cancer tissues from the Clinical Proteomic Tumor Analysis Consortium. Among the identified fusion proteins, we molecularly validate two fusion junction isoforms and a translation product of FAM133B:CDK6. Moreover, sequence analysis suggests that the fusion protein participates in the cell cycle progression. In addition, our prediction results indicate that fusion transcripts often have multiple fusion junctions and that these fusion junctions tend to be distributed in a nonrandom pattern at both the chromosome and gene levels. Thus, FusionPro allows users to detect various types of fusion translation products using a transcriptome-informed approach and to gain a comprehensive understanding of the formation and biological roles of fusion proteoforms.

Graphene oxide substrates with N-cadherin stimulates neuronal growth and intracellular transport.

Intracellular transport is fundamental for neuronal function and development and is dependent on the formation of stable actin filaments. N-cadherin, a cell-cell adhesion protein, is actively involved in neuronal growth and actin cytoskeleton organization. Various groups have explored how neurons behaved on substrates engineered to present N-cadherin; however, few efforts have been made to examine how these surfaces modulate neuronal intracellular transport. To address this issue, we assembled a substrate to which recombinant N-cadherin molecules are physiosorbed using graphene oxide (GO) or reduced graphene oxide (rGO). N-cadherin physisorbed on GO and rGO led to a substantial enhancement of intracellular mass transport along neurites relative to N-cadherin on glass, due to increased neuronal adhesion, neurite extensions, dendritic arborization and glial cell adhesion. This study will be broadly useful for recreating active neural tissues in vitro and for improving our understanding of the development, homeostasis, and physiology of neurons. STATEMENT OF SIGNIFICANCE: Intracellular transport of proteins and chemical cues is extremely important for culturing neurons in vitro, as they replenish materials within and facilitate communication between neurons. Various studies have shown that intracellular transport is dependent on the formation of stable actin filaments. However, the extent to which cadherin-mediated cell-cell adhesion modulates intracellular transport is not heavily explored. In this study, N-cadherin was adsorbed onto graphene oxide-based substrates to understand the role of cadherin at a molecular level and the intracellular transport within cells was examined using spatial light interference microscopy. As such, the results of this study will serve to better understand and harness the role of cell-cell adhesion in neuron development and regeneration.

ASV-ID, a Proteogenomic Workflow To Predict Candidate Protein Isoforms on the Basis of Transcript Evidence.

One of the goals of the Chromosome-Centric Human Proteome Project (C-HPP) is to map and characterize the functions of protein isoforms produced by alternative splicing of genes. However, identifying alternative splice variants (ASVs) via mass spectrometry remains a major challenge, because ASVs usually contain highly homologous peptide sequences. A routine protein sequence analysis suggests that more than half of the investigated proteins do not generate two or more uniquely mapping peptides that would enable their isoforms to be distinguished. Here, we develop a new proteogenomics method, named "ASV-ID" (alternative splicing variants identification), which enables identification of ASVs by using a cell type-specific protein sequence database that is supported by RNA-Seq data. Using this workflow, we identify 1935 distinct proteins under highly stringent conditions. In fact, transcript evidence on these 841 proteins helps us distinguish them from other isoforms, despite the fact that these proteins are not predicted to make 2 or more uniquely mapping peptides. We also demonstrate that ASV-ID enables detection of 19 differently expressed isoforms present in several cell lines. Thus, a new workflow using ASV-ID has the potential to map yet-to-be-identified difficult protein isoforms in a simple and robust way.

Launching the C-HPP neXt-CP50 Pilot Project for Functional Characterization of Identified Proteins with No Known Function.

An important goal of the Human Proteome Organization (HUPO) Chromosome-centric Human Proteome Project (C-HPP) is to correctly define the number of canonical proteins encoded by their cognate open reading frames on each chromosome in the human genome. When identified with high confidence of protein evidence (PE), such proteins are termed PE1 proteins in the online database resource, neXtProt. However, proteins that have not been identified unequivocally at the protein level but that have other evidence suggestive of their existence (PE2-4) are termed missing proteins (MPs). The number of MPs has been reduced from 5511 in 2012 to 2186 in 2018 (neXtProt 2018-01-17 release). Although the annotation of the human proteome has made significant progress, the "parts list" alone does not inform function. Indeed, 1937 proteins representing ∼10% of the human proteome have no function either annotated from experimental characterization or predicted by homology to other proteins. Specifically, these 1937 "dark proteins" of the so-called dark proteome are composed of 1260 functionally uncharacterized but identified PE1 proteins, designated as uPE1, plus 677 MPs from categories PE2-PE4, which also have no known or predicted function and are termed uMPs. At the HUPO-2017 Annual Meeting, the C-HPP officially adopted the uPE1 pilot initiative, with 14 participating international teams later committing to demonstrate the feasibility of the functional characterization of large numbers of dark proteins (CP), starting first with 50 uPE1 proteins, in a stepwise chromosome-centric organizational manner. The second aim of the feasibility phase to characterize protein (CP) functions of 50 uPE1 proteins, termed the neXt-CP50 initiative, is to utilize a variety of approaches and workflows according to individual team expertise, interest, and resources so as to enable the C-HPP to recommend experimentally proven workflows to the proteome community within 3 years. The results from this pilot will not only be the cornerstone of a larger characterization initiative but also enhance understanding of the human proteome and integrated cellular networks for the discovery of new mechanisms of pathology, mechanistically informative biomarkers, and rational drug targets.

O-GlcNAcylation of the Tumor Suppressor FOXO3 Triggers Aberrant Cancer Cell Growth.

Posttranslational modifications of tumor suppressors can induce abnormal cell growth. Here, we identify site-specific O-GlcNAcylation as a critical block of FOXO3 that may abrogate a part of the p53 pathway, resulting in aberrant cancer cell growth. Of seven O-GlcNAcylation sites identified within the FOXO3 transactivation domain, we found that changes in O-GlcNAcylation at Ser284 modulated p21-mediated cancer cell growth. Overexpression of either O-GlcNAcylated FOXO3 (FOX-OV) or a Ser-to-Ala mutant (S284A) in PANC-1 cells indicated that S284 O-GlcNAc acts as a critical block of the FOXO tumor suppressor and induces proliferation in PANC-1 cancer cells by stimulating the MDM2-p53-p21 axis. Furthermore, S284A mutant cells lacking S284 O-GlcNAc and FOX-OV cells exhibited opposing MDM2-p53-p21 axis expression patterns at both the mRNA and protein levels. Thus, our study provides evidence to support a role for S284 O-GlcNAc as a critical block of FOXO3 to induce subsequent cancer cell growth via abrogation of the p53 regulatory circuit.Significance: These findings highlight a posttranslational mechanism for indirect abrogation of the p53 pathway, one that may occur with some frequency in human cancer cells. Cancer Res; 78(5); 1214-24. ©2018 AACR.