Redox proteomics of PANC-1 cells reveals the significance of HIF-1 signaling protein oxidation in pancreatic ductal adenocarcinoma pathogenesis.

BACKGROUND: Protein cysteine oxidation is substantially involved in various biological and pathogenic processes, but its implications in pancreatic cancer development remains poorly understood. METHODS AND RESULTS: In this study, we performed a global characterization of protein oxidation targets in PDAC cells through iodoTMT-based quantitative proteomics, which identified over 4300 oxidized cysteine sites in more than 2100 proteins in HPDE6c7 and PANC-1 cells. Among them, 1715 cysteine residues were shown to be differentially oxidized between HPDE6c7 and PANC-1 cells. Also, charged amino acids including aspartate, glutamate and lysine were significantly overrepresented in flanking sequences of oxidized cysteines. Differentially oxidized proteins in PANC-1 cells were enriched in multiple cancer-related biological processes and signaling pathways. Specifically, the HIF-1 signaling proteins exhibited significant oxidation alterations in PANC-1 cells, and the reduced PHD2 oxidation in human PDAC tissues was correlated with lower survival time in pancreatic cancer patients. CONCLUSION: These investigations provided new insights into protein oxidation-regulated signaling and biological processes during PDAC pathogenesis, which might be further explored for pancreatic cancer diagnosis and treatment.

Neutrophil CD64 index as a new early predictive biomarker for infected pancreatic necrosis in acute pancreatitis.

OBJECTIVE: Infectious pancreatic necrosis (IPN) is a serious complication of acute pancreatitis, and early recognition and timely intervention are the keys to improving clinical outcomes. The purpose of this study was to investigate the predictive capacity of the neutrophil CD64 index (nCD64 index) on IPN in patients with acute pancreatitis METHODS: This study comprises two independent cohorts: the training cohort consisted of 202 patients from Hunan Provincial People's Hospital, and the validation cohort consisted of 100 patients from Changsha Central Hospital. Peripheral blood samples were collected on the day of admission and on the 3rd, 5th, 7th, and 10th days of hospitalization, and the nCD64 index was detected by flow cytometry. Additionally, relevant clinical characteristics and laboratory biomarkers were collected and analyzed. RESULTS: We observed that nCD64 index on admission was significantly higher in the IPN group than Non-IPN group (p < 0.001). In the training cohort, a higher occurrence rate of IPN was observed in the high nCD64 index group compared to the moderate and low nCD64 index group (p < 0.001). Further analysis showed that nCD64 index was significant positive correlated with the incidence rate of IPN (p < 0.001, correlation coefficient = 0.972). Furthermore, logistic regression analysis showed that high expression of the nCD64 index on admission was a risk factor for the occurrence of IPN (OR = 2.971, p = 0.038). We further found that the nCD64 index of IPN patients was significantly higher than the Non-IPN patients on the days 1, 3, and 5 after admission, and the nCD64 index of IPN patients before and after the onset (p < 0.05). At the same time, this study revealed that the nCD64 index on admission showed good predictive efficacy for IPN (AUC = 0.859, sensitivity = 80.8%, specificity = 87.5%), which was comparable to APACHE II score. And this finding was further validated in an independent cohort of 100 participants (AUC = 0.919, Sensitivity = 100.0%, Specificity = 76.6%). CONCLUSION: This study demonstrated the clinical value of nCD64 index in patients with IPN patients for the first time through two independent cohort studies. The nCD64 index can be used as an early prediction and risk assessment tool for the occurrence of IPN, contributing to the improvement of patient outcomes and efficiency of medical resource allocation.

Serum metabolomics study for acute attack of chronic pancreatitis.

BACKGROUND & AIMS: Chronic pancreatitis (CP) is an inflammatory disease characterized by irreversible changes. However, acute CP attacks can lead to various complications and affect patient prognosis. Therefore, this study aimed to identify reliable candidate metabolic biomarkers for diagnosing acute CP attacks and complement candidate diagnostic markers for CP. METHODS: A total of 139 serum specimens were prospectively included in three consecutive exploratory, identification, and validation studies. All samples were analyzed for candidate diagnostic biomarkers and metabolic pathways using a liquid chromatography-mass spectrometer. RESULTS: Serum metabolic profiles differed between patients with CP and non-pancreatic disease controls, and 239 potential metabolic biomarkers for diagnosing CP were identified. Based on identification and validation studies, Diacylglycerol(16:0/18:4), 16-F1-PhytoP, N-(hexacosanoyl)-tetradecasphing-4-enine, carnosic acid, and Auxin b were identified as biomarkers for distinguishing acute attacks from non-acute attacks in patients with CP. The area under the curve of the Diacylglycerol(16:0/18:4) was 0.969 (95% confidence interval, 0.869-1) in the validation study. CONCLUSIONS: To the best of our knowledge, this is the first prospective cohort study to identify and validate a metabolomic signature in serum for diagnosing acute attacks of CP. In addition, our study identified 239 potential biomarkers for CP diagnosis.

Enhanced sucrose-mediated cryoprotection of siRNA-loaded poly (lactic-co-glycolic acid) nanoparticles.

The present study aimed to determine the effects of sucrose on the physical stability, cellular entry pathways and functional efficacy of poly(lactic-co-glycolic acid) nanoparticles (PLGA-NPs). PLGA-NPs were synthesized in the absence or presence of 10 % sucrose, using HEI-101, an unmodified small interfering RNA (siRNA), as a drug model. The newly synthesized HEI-101-loaded PLGA-NPs (HEI-101-NPs) were exposed to repeated freeze-thaw cycles and iteratively tested over a six-month evaluation period. The effect of sucrose stabilization on HEI-101-NPs was independently tested in vitro for biocompatibility and cellular uptake in IMO-2B1 cells. Data analyses suggest that, without sucrose, freeze-thaw cycles of HEI-101-NPs resulted in increased particle diameter, increased polydispersity index, and reduced zeta potential. In contrast, a substantial improvement in the physical stability of HEI-101-NPs was observed in the presence of 10 % sucrose. The data revealed that the release of HEI-101 from the PLGA-NPs was governed by polymer erosion and drug diffusion. Data from cellular uptake study in IMO-2B1 cells demonstrated that, 10 % sucrose significantly reduced the inhibitory effect of nocodazole on the microtubule-dependent uptake of PLGA-NPs. In addition, the presence of 10 % sucrose seemed to lessen the inhibitory effect of sodium azide on the energy-dependent uptake of PLGA-NPs. Overall, the current data suggest that the cellular internalization of PLGA-NPs occurred through the polymerization of actin filaments under the control of the microtubules. Our findings reveal cryoprotective effect of 10 % sucrose on HEI-101-NPs that confers marked improvements in the stability, cellular uptake and efficiency for the delivery of biomolecules to inner ear cells.

Electrophysiological assessment and pharmacological treatment of blast-induced tinnitus.

Tinnitus, the phantom perception of sound, often occurs as a clinical sequela of auditory traumas. In an effort to develop an objective test and therapeutic approach for tinnitus, the present study was performed in blast-exposed rats and focused on measurements of auditory brainstem responses (ABRs), prepulse inhibition of the acoustic startle response, and presynaptic ribbon densities on cochlear inner hair cells (IHCs). Although the exact mechanism is unknown, the "central gain theory" posits that tinnitus is a perceptual indicator of abnormal increases in the gain (or neural amplification) of the central auditory system to compensate for peripheral loss of sensory input from the cochlea. Our data from vehicle-treated rats supports this rationale; namely, blast-induced cochlear synaptopathy correlated with imbalanced elevations in the ratio of centrally-derived ABR wave V amplitudes to peripherally-derived wave I amplitudes, resulting in behavioral evidence of tinnitus. Logistic regression modeling demonstrated that the ABR wave V/I amplitude ratio served as a reliable metric for objectively identifying tinnitus. Furthermore, histopathological examinations in blast-exposed rats revealed tinnitus-related changes in the expression patterns of key plasticity factors in the central auditory pathway, including chronic loss of Arc/Arg3.1 mobilization. Using a formulation of N-acetylcysteine (NAC) and disodium 2,4-disulfophenyl-N-tert-butylnitrone (HPN-07) as a therapeutic for addressing blast-induced neurodegeneration, we measured a significant treatment effect on preservation or restoration of IHC ribbon synapses, normalization of ABR wave V/I amplitude ratios, and reduced behavioral evidence of tinnitus in blast-exposed rats, all of which accorded with mitigated histopathological evidence of tinnitus-related neuropathy and maladaptive neuroplasticity.

Alterations of Asymmetric Dimethylarginine (ADMA)-Containing Protein Profiles Associated with Chronic Pancreatitis Pathogenesis.

OBJECTIVE: The pathophysiological mechanisms of chronic pancreatitis (CP) still remain poorly understood. In this study, we aimed to characterize asymmetric dimethylarginine (ADMA)-containing proteins in pancreatic tissues and its relationship with CP pathogenesis. METHODS: Totally 36 patients with CP were enrolled in this study. Seven other cholangiocarcinoma patients without pancreas involvements or patients with benign pancreatic tumors were included as the control group. Total proteins in human pancreatic tissues were digested by trypsin, and ADMA-containing peptides were enriched via immunoaffinity purification. The LC-MS/MS was performed to characterize ADMA-containing peptides and their modification sites in CP tissues. Relative asymmetric arginine dimethylation levels of HNRNPA3 proteins in human pancreatic tissues were detected by the immunoprecipitation combined with Western blot. The serum inflammatory factors were determined via the ELISA method. RESULTS: A total of 134 ADMA sites in the control group and 137 ADMA sites in CP tissues were characterized by mass spectrometry, which belong to 93 and 94 ADMA-containing proteins in the control group and CP tissues, respectively. Glycine and proline residues were significantly overrepresented in the flanking sequences of ADMA sites. ADMA-containing proteins in the CP tissues were associated with various biological processes, especially the RNA metabolism and splicing pathways. Multiple protein members of the spliceosome pathway such as HNRNPA3 possess ADMA sites in the CP tissues. HNRNPA3 dimethylation levels were greatly increased in CP tissues, which were positively correlated with inflammatory factors. CONCLUSION: The pathogenesis of CP is associated with alterations of asymmetric arginine dimethylation in pancreatic tissues.

Comprehensive Design of the High-Sulfur-Loading Li-S Battery Based on MXene Nanosheets.

The lithium-sulfur battery is the subject of much recent attention due to the high theoretical energy density, but practical applications are challenged by fast decay owing to polysulfide shuttle and electrode architecture degradation. A comprehensive study of the sulfur host microstructure design and the cell architecture construction based on the MXene phase (Ti3C2Tx nanosheets) is performed, aiming at realize stable cycling performance of Li-S battery with high sulfur areal loading. The interwoven KB@Ti3C2Tx composite formed by self-assembly of MXene and Ktejen black, not only provides superior conductivity and maintains the electrode integrality bearing the volume expansion/shrinkage when used as the sulfur host, but also functions as an interlayer on separator to further retard the polysulfide cross-diffusion that possibly escaped from the cathode. The KB@Ti3C2Tx interlayer is only 0.28 mg cm-2 in areal loading and 3 µm in thickness, which accounts a little contribution to the thick sulfur electrode; thus, the impacts on the energy density is minimal. By coupling the robust KB@Ti3C2Tx cathode and the effective KB@Ti3C2Tx modified separator, a stable Li-S battery with high sulfur areal loading (5.6 mg cm-2) and high areal capacity (6.4 mAh cm-2) at relatively lean electrolyte is achieved.

Highly Selective and Potent Human ß-Secretase†2 (BACE2) Inhibitors against Type†2 Diabetes: Design, Synthesis, X-ray Structure and Structure-Activity Relationship Studies.

Herein we present the design, synthesis, and biological evaluation of potent and highly selective ß-secretase 2 (memapsin 1, beta-site amyloid precursor protein cleaving enzyme 2, or BACE 2) inhibitors. BACE2 has been recognized as an exciting new target for type 2 diabetes. The X-ray structure of BACE1 bound to inhibitor 2 a {N3 -[(1S,2R)-1-benzyl-2-hydroxy-3-[[(1S,2S)-2-hydroxy-1-(isobutylcarbamoyl)propyl]amino]propyl]-5-[methyl(methylsulfonyl)amino]-N1 -[(1R)-1-phenylpropyl]benzene-1,3-dicarboxamide} containing a hydroxyethylamine isostere was determined. Based on this structure, a computational docking study was performed which led to inhibitor 2 a-bound BACE2 models. These were used to optimize the potency and selectivity of inhibitors. A systematic structure-activity relationship study led to the identification of determinants of the inhibitors' potency and selectivity toward the BACE2 enzyme. Inhibitors 2 d [N3 -[(1S,2R)-1-benzyl-2-hydroxy-3-[[(1S,2S)-2-hydroxy-1-(isobutylcarbamoyl)pentyl]amino]propyl]-N1 -methyl-N1 -[(1R)-1-phenylpropyl]benzene-1,3-dicarboxamide; Ki =0.031 nm, selectivity over BACE1: ≈174 000-fold] and 3 l [N1 -((2S,3R)-3-hydroxy-1-phenyl-4-((3-(trifluoromethyl)benzyl)amino)butan-2-yl)-N3 ,5-dimethyl-N3 -((R)-1-phenylethyl)isophthalamide; Ki =1.6 nm, selectivity over BACE1: >500-fold] displayed outstanding potency and selectivity. Inhibitor 3 l is nonpeptide in nature and may pave the way to the development of a new class of potent and selective BACE2 inhibitors with clinical potential.

Design, synthesis, X-ray studies, and biological evaluation of novel BACE1 inhibitors with bicyclic isoxazoline carboxamides as the P3 ligand.

We describe the design, synthesis, X-ray studies, and biological evaluation of novel BACE1 inhibitors containing bicyclic isoxazoline carboxamides as the P3 ligand in combination with methyl cysteine, methylsulfonylalanine and Boc-amino alanine as P2 ligands. Inhibitor 3a displayed a BACE1 Ki value of 10.9 nM and EC50 of 343 nM. The X-ray structure of 3a bound to the active site of BACE1 was determined at 2.85 Šresolution. The structure revealed that the major molecular interactions between BACE1 and the bicyclic tetrahydrofuranyl isoxazoline heterocycle are van der Waals in nature.

Regeneration of Cochlear Hair Cells and Hearing Recovery through Hes1 Modulation with siRNA Nanoparticles in Adult Guinea Pigs.

Deafness is commonly caused by the irreversible loss of mammalian cochlear hair cells (HCs) due to noise trauma, toxins, or infections. We previously demonstrated that small interfering RNAs (siRNAs) directed against the Notch pathway gene, hairy and enhancer of split 1 (Hes1), encapsulated within biocompatible poly(lactic-co-glycolic acid) nanoparticles (PLGA NPs) could regenerate HCs within ototoxin-ablated murine organotypic cultures. In the present study, we delivered this sustained-release formulation of Hes1 siRNA (siHes1) into the cochleae of noise-injured adult guinea pigs. Auditory functional recovery was measured by serial auditory brainstem responses over a nine-week follow-up period, and HC regeneration was evaluated by immunohistological evaluations and scanning electron microscopy. Significant HC restoration and hearing recovery were observed across a broad tonotopic range in ears treated with siHes1 NPs, beginning at three weeks and extending out to nine weeks post-treatment. Moreover, both ectopic and immature HCs were uniquely observed in noise-injured cochleae treated with siHes1 NPs, consistent with de novo HC production. Our results indicate that durable cochlear HCs were regenerated and promoted significant hearing recovery in adult guinea pigs through reversible modulation of Hes1 expression. Therefore, PLGA-NP-mediated delivery of siHes1 to the cochlea represents a promising pharmacologic approach to regenerate functional and sustainable mammalian HCs in vivo.

Design, synthesis, and X-ray structural studies of BACE-1 inhibitors containing substituted 2-oxopiperazines as P1'-P2' ligands.

We report the design and synthesis of a series of BACE1 inhibitors incorporating mono- and bicyclic 6-substituted 2-oxopiperazines as novel P1' and P2' ligands and isophthalamide derivative as P2-P3 ligands. Among mono-substituted 2-oxopiperazines, inhibitor 5a with N-benzyl-2-oxopiperazine and isophthalamide showed potent BACE1 inhibitory activity (Ki=2nM). Inhibitor 5g, with N-benzyl-2-oxopiperazine and substituted indole-derived P2-ligand showed a reduction in potency. The X-ray crystal structure of 5g-bound BACE1 was determined and used to design a set of disubstituted 2-oxopiperazines and bicyclic derivatives that were subsequently investigated. Inhibitor 6j with an oxazolidinone derivative showed a BACE1 inhibitory activity of 23nM and cellular EC50 of 80nM.

Design of Potent and Highly Selective Inhibitors for Human ß-Secretase 2 (Memapsin 1), a Target for Type 2 Diabetes.

Design, synthesis and evaluation of very potent and selective ß-Secretase 2 (memapsin 1, BACE 2) inhibitors are described. The inhibitors were designed specifically to interact with the S2'-site of ß-secretase 2 to provide >170,000-fold selectivity over ß-secretase (BACE 1) and >15,000-fold selectivity over cathepsin D. BACE 2 is implicated in Type 2 diabetes. The studies serve as an important guide to selective BACE 2 inhibitors.

Structure-based design, synthesis and biological evaluation of novel ß-secretase inhibitors containing a pyrazole or thiazole moiety as the P3 ligand.

We describe structure-based design, synthesis, and biological evaluation of a series of novel inhibitors bearing a pyrazole (compounds 3a-h) or a thiazole moiety (compounds 4a-e) as the P3 ligand. We have also explored Boc-ß-amino-l-alanine as a novel P2 ligand. A number of inhibitors have displayed ß-secretase inhibitory potency. Inhibitor 4c has shown potent BACE1 inhibitory activity, Ki=0.25nM, cellular EC50 of 194nM, and displayed good selectivity over BACE2. A model of 4c was created based upon the X-ray structure of 2-bound ß-secretase which revealed critical interactions in the active site.

Expression and processing of fluorescent fusion proteins of amyloid precursor protein (APP).

Processing of ß-amyloid precursor protein (APP) by ß- and γ-secretases in neurons produces amyloid-ß (Aß), whose excess accumulation leads to Alzheimer's disease (AD). Knowledge on subcellular trafficking pathways of APP and its fragments is important for the understanding of AD pathogenesis. We designed fusion proteins comprising a C-terminal fragment of APP (app) and fluorescent proteins GFP (G) and DsRed (D) to permit the tracking of the fusion proteins and fragments in cells. CAD cells expressing these proteins emitted colocalized green and red fluorescence and produce ectodomains, sGapp and sRapp, and Aß, whose level was reduced by inhibitors of ß- and γ-secretases. The presence of GappR in endosomes was observed via colocalization with Rab5. These observations indicated that the fusion proteins were membrane inserted, transported in vesicles and proteolytically processed by the same mechanism for APP. By attenuating fusion protein synthesis with cycloheximide, individual fluorescent colors from the C-terminus of the fusion proteins appeared in the cytosol which was strongly suppressed by ß-secretase inhibitor, suggesting that the ectodomains exit the cell rapidly (t1/2 about 20min) while the C-terminal fragments were retained longer in cells. In live cells, we observed the fluorescence of the ectodomains located between parental fusion proteins and plasma membrane, suggesting that these ectodomain positions are part of their secretion pathway. Our results indicate that the native ectodomain does not play a decisive role for the key features of APP trafficking and processing and the new fusion proteins may lead to novel insights in intracellular activities of APP.

Structure-based design of highly selective ß-secretase inhibitors: synthesis, biological evaluation, and protein-ligand X-ray crystal structure.

The structure-based design, synthesis, and X-ray structure of protein-ligand complexes of exceptionally potent and selective ß-secretase inhibitors are described. The inhibitors are designed specifically to interact with S(1)' active site residues to provide selectivity over memapsin 1 and cathepsin D. Inhibitor 5 has exhibited exceedingly potent inhibitory activity (K(i) = 17 pM) and high selectivity over BACE 2 (>7000-fold) and cathepsin D (>250000-fold). A protein-ligand crystal structure revealed important molecular insight into these selectivities. These interactions may serve as an important guide to design selectivity over the physiologically important aspartic acid proteases.

Structure-based design, synthesis, and biological evaluation of dihydroquinazoline-derived potent ß-secretase inhibitors.

Structure-based design, synthesis, and biological evaluation of a series of dihydroquinazoline-derived ß-secretase inhibitors incorporating thiazole and pyrazole-derived P2-ligands are described. We have identified inhibitor 4f which has shown potent enzyme inhibitory (K(i)=13 nM) and cellular (IC(50)=21 nM in neuroblastoma cells) assays. A model of 4f was created based upon the X-ray structure of 3a-bound ß-secretase. The model suggested possible interactions in the active site.

Beta-secretase inhibitor GRL-8234 rescues age-related cognitive decline in APP transgenic mice.

Alzheimer disease is intimately linked to an excess amount of amyloid-ß (Aß) in the brain. Thus, therapeutic inhibition of Aß production is an attractive clinical approach to treat this disease. Here we provide the first direct experimental evidence that the treatment of Tg2576 transgenic mice with an inhibitor of ß-secretase, GRL-8234, rescues the age-related cognitive decline. We demonstrated that the injected GRL-8234 effectively enters the brain and rapidly decreases soluble Aß in the brain of Tg2576 mice. The rescue of cognition, which was observed only after long-term inhibitor treatment ranging from 5 to 7.5 mo, was associated with a decrease of brain amyloid-ß plaque load. We also found no accumulation of amyloid-ß precursor protein after several months of inhibitor treatment. These observations substantiate the idea that Aß accumulation plays a major role in the cognitive decline of Tg2576 mice and support the concept of Aß reduction therapy as a treatment of AD.

Amyloid-beta reduction by memapsin 2 (beta-secretase) immunization.

Memapsin 2 (beta-secretase, BACE1) is the protease that initiates cleavage of beta-amyloid precursor protein leading to the production of amyloid-beta (Abeta) and the onset of Alzheimer's disease (AD). Reducing Abeta by targeting memapsin 2 is a major strategy in developing new AD therapy. Here, in a proof-of-concept study, we show that immunization of transgenic AD mice (Tg2576) with memapsin 2 resulted in Abeta reduction and cognitive improvement. To study the basis of this therapy, we demonstrated that anti-memapsin 2 (anti-M2) antibodies were rapidly internalized and reduced Abeta production in cultured cells. These antibodies also effectively crossed the blood-brain barrier to reach the brain. Two- and 10-month Tg2576 mice were immunized and monitored over 10 and 6 months, respectively. We observed a significant decrease of plasma and brain Abeta40 and Abeta42 (approximately 35%) in the immunized mice as compared to controls. Immunized mice also showed better cognitive performance than controls in both cohorts. Brain histological analyses found no evidence of T cell/microglia/astrocyte activation in the immunized mice, suggesting the absence of inflammatory responses. These results suggest that memapsin 2 immunization in Tg2576 was effective in reducing Abeta production and improving cognitive function and that the current approach warrants further investigation as a therapy for AD.

Activation profiles of the zymogen of aspergilloglutamic peptidase.

Aspergilloglutamic peptidase produced by Aspergillus niger var. macrosporus belongs to the novel glutamic peptidase family. Its zymogen is autocatalytically activated under acidic conditions to the mature enzyme with a two-chain structure. Analyses by SDS-PAGE and mass spectrometry of the activation products of the recombinant zymogen showed that the major pathway of activation includes initial fast cleavage at Glu12-Ala13, followed by stepwise cleavages in the N-terminal and intervening propeptide regions. Essentially the same activation profile was obtained with the recombinant zymogen lacking the N-terminal 12-aa sequence. The missing region includes the most prominent cluster of basic residues of the propeptide, indicating low importance of this cluster for activation and refolding of the zymogen.

Structural features of human memapsin 2 (beta-secretase) and their biological and pathological implications.

Memapsin 2 (beta-secretase) is the membrane-anchored aspartic protease that initiates the cleavage of beta-amyloid precursor protein (APP) leading to the production of amyloid-beta (Abeta), a major factor in the pathogenesis of Alzheimer's disease (AD). Since memapsin 2 is a major target for the development of inhibitor drugs for AD, it has been intensively studied during the past five years. Here we discuss the structural features of the catalytic/specificity apparatus, transmembrane domain, cytosolic domain and the implications of these features in the physiological and pathological roles of this protease.