Biomarkers of Chronic Pancreatitis: A systematic literature review.

BACKGROUND: Chronic pancreatitis (CP) does not have diagnostic or prognostic biomarkers. CP is the end stage of a progressive inflammatory syndrome that is diagnosed at late stages by morphologic features. To diagnose earlier stages of the disease, a new mechanistic definition was established based on identifying underlying pathogenic processes and biomarker evidence of disease activity and stage. Although multiple risk factors are known, the corresponding biomarkers needed to make a highly accurate diagnosis of earlier disease stages have not been established. The goal of this study is to systematically analyze the literature to identify the most likely candidates for development into biomarkers of CP. METHODS: We conducted a systematic review of candidate analytes from easily accessible biological fluids and identified 67 studies that compared CP to nonpancreatic-disease controls. We then ranked candidate biomarkers for sensitivity and specificity by area under the receiver operator curves (AUROCs). RESULTS: Five biomarkers had a large effect size (an AUROC > 0.96), whereas 30 biomarkers had a moderate effect size (an AUROC between 0.96 and 0.83) for distinguishing CP cases from controls or other diseases. However, the studies reviewed had marked variability in design, enrollment criteria, and biospecimen sample handling and collection. CONCLUSIONS: Several biomarkers have the potential for evaluation in prospective cohort studies and should be correlated with risk factors, clinical features, imaging studies and outcomes. The Consortium for the Study of Chronic Pancreatitis, Diabetes and Pancreas Cancer provides recommendations for avoiding design biases and heterogeneity in sample collection and handling in future studies.

Delayed Processing of Secretin-Induced Pancreas Fluid Influences the Quality and Integrity of Proteins and Nucleic Acids.

OBJECTIVES: Endoscopic pancreatic function tests are used to diagnose pancreatic diseases and are a viable source for the discovery of biomarkers to better characterize pancreatic disorders. However, pancreatic fluid (PF) contains active enzymes that degrade biomolecules. Therefore, we tested how preservation methods and time to storage influence the integrity and quality of proteins and nucleic acids. METHODS: We obtained PF from 9 subjects who underwent an endoscopic pancreatic function test. Samples were snap frozen at the time of collection; after 1, 2, and 4 hours on ice; or after storage overnight at 4°C with or without RNase or protease inhibitors (PIs). Electrophoresis and mass spectrometry analysis determined protein abundance and quality, whereas nucleic acid integrity values determined DNA and RNA degradation. RESULTS: Protein degradation increased after 4 hours on ice and DNA degradation after 2 hours on ice. Adding PIs delayed degradation. RNA was significantly degraded under all conditions compared with the snap frozen samples. Isolated RNA from PF-derived exosomes exhibited similar poor quality as RNA isolated from matched PF samples. CONCLUSIONS: Adding PIs immediately after collecting PF and processing the fluid within 4 hours of collection maintains the protein and nucleic acid integrity for use in downstream molecular analyses.

MG53 suppresses interferon-ß and inflammation via regulation of ryanodine receptor-mediated intracellular calcium signaling.

TRIM family proteins play integral roles in the innate immune response to virus infection. MG53 (TRIM72) is essential for cell membrane repair and is believed to be a muscle-specific TRIM protein. Here we show human macrophages express MG53, and MG53 protein expression is reduced following virus infection. Knockdown of MG53 in macrophages leads to increases in type I interferon (IFN) upon infection. MG53 knockout mice infected with influenza virus show comparable influenza virus titres to wild type mice, but display increased morbidity accompanied by more accumulation of CD45+ cells and elevation of IFNß in the lung. We find that MG53 knockdown results in activation of NFκB signalling, which is linked to an increase in intracellular calcium oscillation mediated by ryanodine receptor (RyR). MG53 inhibits IFNß induction in an RyR-dependent manner. This study establishes MG53 as a new target for control of virus-induced morbidity and tissue injury.

Biological Functions and Therapeutic Potential of Lipocalin 2 in Cancer.

Lipocalin-2 (LCN2) is a secreted glycoprotein linked to several physiological roles, including transporting hydrophobic ligands across cell membranes, modulating immune responses, maintaining iron homeostasis, and promoting epithelial cell differentiation. Although LNC2 is expressed at low levels in most human tissues, it is abundant in aggressive subtypes of cancer, including breast, pancreas, thyroid, ovarian, colon, and bile duct cancers. High levels of LCN2 have been associated with increased cell proliferation, angiogenesis, cell invasion, and metastasis. Moreover, LCN2 modulates the degradation, allosteric events, and enzymatic activity of matrix metalloprotease-9, a metalloprotease that promotes tumor cell invasion and metastasis. Hence, LCN2 has emerged as a potential therapeutic target against many cancer types. This review summarizes the most relevant findings regarding the expression, biological roles, and regulation of LCN2, as well as the proteins LCN2 interacts with in cancer. We also discuss the approaches to targeting LCN2 for cancer treatment that are currently under investigation, including the use of interference RNAs, antibodies, and gene editing.

Correction to: A novel organ preservation solution with efficient clearance of red blood cells improves kidney transplantation in a canine model.

[This corrects the article DOI: 10.1186/s13578-018-0226-2.].

Calcium-dependent Protein Kinases in Malaria Parasite Development and Infection.

Apicomplexan parasites have challenged researchers for nearly a century. A major challenge to developing efficient treatments and vaccines is the parasite's ability to change its cellular and molecular makeup to develop intracellular and extracellular niches in its hosts. Ca2+ signaling is an important messenger for the egress of the malaria parasite from the infected erythrocyte, gametogenesis, ookinete motility in the mosquito, and sporozoite invasion of mammalian hepatocytes. Calcium-dependent protein kinases (CDPKs) have crucial functions in calcium signaling at various stages of the parasite's life cycle; this therefore makes them attractive drug targets against malaria. Here, we summarize the functions of the various CDPK isoforms in relation to the malaria life cycle by emphasizing the molecular mechanism of developmental progression within host tissues. We also discuss the current development of anti-malarial drugs, such as how specific bumped kinase inhibitors (BKIs) for parasite CDPKs have been shown to reduce infection in Toxoplasma gondii, Cryptosporidium parvum, and Plasmodium falciparum. Our suggested combinations of BKIs, artemisinin derivatives with peroxide bridge, and inhibitors on the Ca(2+)-ATPase PfATP6 as a potential target should be inspected further as a treatment against malaria.

Lipocalin-2 expression and function in pancreatic diseases.

Lipocalin-2 (LCN2) is a secreted molecule, expressed in various cell types, that is involved in the progression of numerous diseases and disorders. The biological functions and expression levels of LCN2 in diseases including pancreatic cancer, pancreatitis (acute and chronic), and diabetes mellitus, suggest the potential role of LCN2 as a biomarker and/or therapeutic target. However, findings on the role of LCN2 in pancreatic diseases have been contradictory. In pancreatic cancer and pancreatitis, LCN2 has been identified as a potential biomarker; increased expression levels in various biological specimens correlate with the presence of the disease and may be able to differentiate cancer and chronic pancreatitis from healthy subjects. LCN2 is also known to be an adipokine; it is upregulated in obesity and is a common co-factor in the development of pancreatic diseases. Emerging research suggests LCN2 is elevated in type 2 diabetes mellitus, but the exact role of LCN2 in this disease is not clear. In this review, we summarize research on LCN2 as it relates to pancreatic diseases, highlighting the discrepancies in the literature. By explaining and clarifying the role of LCN2 in these disorders, we aim to promote research in developing novel diagnostic and treatment strategies to reduce the burden of pancreatic diseases.

The TRIM protein Mitsugumin 53 enhances survival and therapeutic efficacy of stem cells in murine traumatic brain injury.

BACKGROUND: Traumatic brain injury (TBI) is a common neurotrauma leading to brain dysfunction and death. Human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) hold promise in the treatment of TBI. However, their efficacy is modest due to low survival and differentiation under the harsh microenvironment of the injured brain. MG53, a member of TRIM family protein, plays a vital role in cell and tissue damage repair. The present study aims to test whether MG53 preserves hUC-MSCs against oxidative stress and enhances stem cell survival and efficacy in TBI treatment. METHODS: In this study, we performed a series of in vitro and in vivo experiments in hUC-MSCs and mice to define the function of MG53 enhancing survival, neurogenesis, and therapeutic efficacy of stem cells in murine traumatic brain injury. RESULTS: We found that recombinant human MG53 (rhMG53) protein protected hUC-MSCs against H2O2-induced oxidative damage and stimulated hUC-MSC proliferation and migration. In a mouse model of contusion-induced TBI, intravenous administration of MG53 protein preserved the survival of transplanted hUC-MSCs, mitigated brain edema, reduced neurological deficits, and relieved anxiety and depressive-like behaviors. Co-treatment of MG53 and hUC-MSCs enhanced neurogenesis by reducing apoptosis and improving PI3K/Akt-GSK3ß signaling. CONCLUSION: MG53 enhances the efficacy of hUC-MSCs in the recovery of TBI, indicating that such adjunctive therapy may provide a novel strategy to lessen damage and optimize recovery for brain injury.

Sustained elevation of MG53 in the bloodstream increases tissue regenerative capacity without compromising metabolic function.

MG53 is a muscle-specific TRIM-family protein that presides over the cell membrane repair response. Here, we show that MG53 present in blood circulation acts as a myokine to facilitate tissue injury-repair and regeneration. Transgenic mice with sustained elevation of MG53 in the bloodstream (tPA-MG53) have a healthier and longer life-span when compared with littermate wild type mice. The tPA-MG53 mice show normal glucose handling and insulin signaling in skeletal muscle, and sustained elevation of MG53 in the bloodstream does not have a deleterious impact on db/db mice. More importantly, the tPA-MG53 mice display remarkable dermal wound healing capacity, enhanced muscle performance, and improved injury-repair and regeneration. Recombinant human MG53 protein protects against eccentric contraction-induced acute and chronic muscle injury in mice. Our findings highlight the myokine function of MG53 in tissue protection and present MG53 as an attractive biological reagent for regenerative medicine without interference with glucose handling in the body.

MG 53 Protein Protects Aortic Valve Interstitial Cells From Membrane Injury and Fibrocalcific Remodeling.

Background The aortic valve of the heart experiences constant mechanical stress under physiological conditions. Maladaptive valve injury responses contribute to the development of valvular heart disease. Here, we test the hypothesis that MG 53 (mitsugumin 53), an essential cell membrane repair protein, can protect valvular cells from injury and fibrocalcific remodeling processes associated with valvular heart disease. Methods and Results We found that MG 53 is expressed in pig and human patient aortic valves and observed aortic valve disease in aged Mg53-/- mice. Aortic valves of Mg53-/- mice showed compromised cell membrane integrity. In vitro studies demonstrated that recombinant human MG 53 protein protects primary valve interstitial cells from mechanical injury and that, in addition to mediating membrane repair, recombinant human MG 53 can enter valve interstitial cells and suppress transforming growth factor-ß-dependent activation of fibrocalcific signaling. Conclusions Together, our data characterize valve interstitial cell membrane repair as a novel mechanism of protection against valvular remodeling and assess potential in vivo roles of MG 53 in preventing valvular heart disease.

Skeletal Muscle Lysosomal Function via Cathepsin Activity Measurement.

Muscle wasting or cachexia is commonly associated with aging and many diseases such as cancer, infection, autoimmune disorders, and trauma. Decrease in muscle mass, or muscle atrophy, is often caused by dysfunction of protein proteolytic systems, such as lysosomes, which regulate protein turnover and homeostasis. Lysosomes contain many hydrolases and proteases and, thus, represent the major organelle that control protein turnover. Recently, lysosomes have emerged as a signaling hub to integrate cellular functions of nutrient sensing and metabolism, autophagy, phagocytosis, and endocytosis, which are all related to tissue homeostasis. In this chapter, we describe the protocol used to measure lysosomal proteinase (cathepsins) activity in the skeletal muscle. A better understanding of lysosomal function in muscle homeostasis is critical in developing new therapeutic approaches to prevent muscle wasting.

A novel organ preservation solution with efficient clearance of red blood cells improves kidney transplantation in a canine model.

Organ preservation solutions are designed to minimize organ damage during transplantation. A novel preservation solution, WMO-II, was developed to have a low viscosity and to improve microvasculature perfusion for kidneys. In an autologous canine transplantation model, kidney function and recovery were evaluated after organs were flushed and cold-stored with WMO-II or HTK solution, a perfusate currently approved for clinical use. The average number of red blood cells remaining in a single glomerulus after flushing with WMO-II was significantly reduced when compared with HTK solution. Additionally, WMO-II reduced the number of apoptotic bodies in stored kidneys compared to HTK treated tissue after 48 h of cold storage by reducing expression of Caspase-9, BiP, Chop, and Caspase-12. WMO-II solution reduced serum creatinine levels and serum potassium in kidneys stored for 48 h when compared to HTK perfusion. WMO-II preserves kidney function as evidenced by the reduction in serum creatinine and potassium during graft transplantation.

MicroRNA regulation of autophagy in cardiovascular disease.

Autophagy, a form of lysosomal degradation capable of eliminating dysfunctional proteins and organelles, is a cellular process associated with homeostasis. Autophagy functions in cell survival by breaking down proteins and organelles and recycling them to meet metabolic demands. However, aberrant up regulation of autophagy can function as an alternative to apoptosis. The duality of autophagy, and its regulation over cell survival/death, intimately links it with human disease. Non-coding RNAs regulate mRNA levels and elicit diverse effects on mammalian protein expression. The most studied non-coding RNAs to-date are microRNAs (miRNA). MicroRNAs function in post-transcriptional regulation, causing profound changes in protein levels, and affect many biological processes and diseases. The role and regulation of autophagy, whether it is beneficial or harmful, is a controversial topic in cardiovascular disease. A number of recent studies have identified miRNAs that target autophagy-related proteins and influence the development, progression, or treatment of cardiovascular disease. Understanding the mechanisms by which these miRNAs work can provide promising insight and potential progress towards the development of therapeutic treatments in cardiovascular disease.

Fast two-dimensional super-resolution image reconstruction algorithm for ultra-high emitter density.

Single-molecule localization microscopy achieves sub-diffraction-limit resolution by localizing a sparse subset of stochastically activated emitters in each frame. Its temporal resolution is limited by the maximal emitter density that can be handled by the image reconstruction algorithms. Multiple algorithms have been developed to accurately locate the emitters even when they have significant overlaps. Currently, compressive-sensing-based algorithm (CSSTORM) achieves the highest emitter density. However, CSSTORM is extremely computationally expensive, which limits its practical application. Here, we develop a new algorithm (MempSTORM) based on two-dimensional spectrum analysis. With the same localization accuracy and recall rate, MempSTORM is 100 times faster than CSSTORM with ℓ(1)-homotopy. In addition, MempSTORM can be implemented on a GPU for parallelism, which can further increase its computational speed and make it possible for online super-resolution reconstruction of high-density emitters.

3D multifocus astigmatism and compressed sensing (3D MACS) based superresolution reconstruction.

Single molecule based superresolution techniques (STORM/PALM) achieve nanometer spatial resolution by integrating the temporal information of the switching dynamics of fluorophores (emitters). When emitter density is low for each frame, they are located to the nanometer resolution. However, when the emitter density rises, causing significant overlapping, it becomes increasingly difficult to accurately locate individual emitters. This is particularly apparent in three dimensional (3D) localization because of the large effective volume of the 3D point spread function (PSF). The inability to precisely locate the emitters at a high density causes poor temporal resolution of localization-based superresolution technique and significantly limits its application in 3D live cell imaging. To address this problem, we developed a 3D high-density superresolution imaging platform that allows us to precisely locate the positions of emitters, even when they are significantly overlapped in three dimensional space. Our platform involves a multi-focus system in combination with astigmatic optics and an ℓ 1-Homotopy optimization procedure. To reduce the intrinsic bias introduced by the discrete formulation of compressed sensing, we introduced a debiasing step followed by a 3D weighted centroid procedure, which not only increases the localization accuracy, but also increases the computation speed of image reconstruction. We implemented our algorithms on a graphic processing unit (GPU), which speeds up processing 10 times compared with central processing unit (CPU) implementation. We tested our method with both simulated data and experimental data of fluorescently labeled microtubules and were able to reconstruct a 3D microtubule image with 1000 frames (512×512) acquired within 20 seconds.

MG53-mediated cell membrane repair protects against acute kidney injury.

Injury to the renal proximal tubular epithelium (PTE) represents the underlying consequence of acute kidney injury (AKI) after exposure to various stressors, including nephrotoxins and ischemia/reperfusion (I/R). Although the kidney has the ability to repair itself after mild injury, insufficient repair of PTE cells may trigger inflammatory and fibrotic responses, leading to chronic renal failure. We report that MG53, a member of the TRIM family of proteins, participates in repair of injured PTE cells and protects against the development of AKI. We show that MG53 translocates to acute injury sites on PTE cells and forms a repair patch. Ablation of MG53 leads to defective membrane repair. MG53-deficient mice develop pronounced tubulointerstitial injury and increased susceptibility to I/R-induced AKI compared to wild-type mice. Recombinant human MG53 (rhMG53) protein can target injury sites on PTE cells to facilitate repair after I/R injury or nephrotoxin exposure. Moreover, in animal studies, intravenous delivery of rhMG53 ameliorates cisplatin-induced AKI without affecting the tumor suppressor efficacy of cisplatin. These findings identify MG53 as a vital component of reno-protection, and targeting MG53-mediated repair of PTE cells represents a potential approach to prevention and treatment of AKI.

Lysosomal two-pore channel subtype 2 (TPC2) regulates skeletal muscle autophagic signaling.

Postnatal skeletal muscle mass is regulated by the balance between anabolic protein synthesis and catabolic protein degradation, and muscle atrophy occurs when protein homeostasis is disrupted. Autophagy has emerged as critical in clearing dysfunctional organelles and thus in regulating protein turnover. Here we show that endolysosomal two-pore channel subtype 2 (TPC2) contributes to autophagy signaling and protein homeostasis in skeletal muscle. Muscles derived from Tpcn2(-/-) mice exhibit an atrophic phenotype with exacerbated autophagy under starvation. Compared with wild types, animals lacking TPC2 demonstrated an enhanced autophagy flux characterized by increased accumulation of autophagosomes upon combined stress induction by starvation and colchicine treatment. In addition, deletion of TPC2 in muscle caused aberrant lysosomal pH homeostasis and reduced lysosomal protease activity. Association between mammalian target of rapamycin and TPC2 was detected in skeletal muscle, allowing for appropriate adjustments to cellular metabolic states and subsequent execution of autophagy. TPC2 therefore impacts mammalian target of rapamycin reactivation during the process of autophagy and contributes to maintenance of muscle homeostasis.

Superresolution microscope image reconstruction by spatiotemporal object decomposition and association: application in resolving t-tubule structure in skeletal muscle.

One key factor that limits resolution of single-molecule superresolution microscopy relates to the localization accuracy of the activated emitters, which is usually deteriorated by two factors. One originates from the background noise due to out-of-focus signals, sample auto-fluorescence, and camera acquisition noise; and the other is due to the low photon count of emitters at a single frame. With fast acquisition rate, the activated emitters can last multiple frames before they transiently switch off or permanently bleach. Effectively incorporating the temporal information of these emitters is critical to improve the spatial resolution. However, majority of the existing reconstruction algorithms locate the emitters frame by frame, discarding or underusing the temporal information. Here we present a new image reconstruction algorithm based on tracklets, short trajectories of the same objects. We improve the localization accuracy by associating the same emitters from multiple frames to form tracklets and by aggregating signals to enhance the signal to noise ratio. We also introduce a weighted mean-shift algorithm (WMS) to automatically detect the number of modes (emitters) in overlapping regions of tracklets so that not only well-separated single emitters but also individual emitters within multi-emitter groups can be identified and tracked. In combination with a maximum likelihood estimator method (MLE), we are able to resolve low to medium density of overlapping emitters with improved localization accuracy. We evaluate the performance of our method with both synthetic and experimental data, and show that the tracklet-based reconstruction is superior in localization accuracy, particularly for weak signals embedded in a strong background. Using this method, for the first time, we resolve the transverse tubule structure of the mammalian skeletal muscle.

Reconstituted human myosin light chain phosphatase reveals distinct roles of two inhibitory phosphorylation sites of the regulatory subunit, MYPT1.

The myosin light chain phosphatase (MLCP) is a cytoskeleton-associated protein phosphatase-1 (PP1) holoenzyme and a RhoA/ROCK effector, regulating cytoskeletal reorganization. ROCK-induced phosphorylation of the MLCP regulatory subunit (MYPT1) at two sites, Thr696 and Thr853, suppresses the activity, although little is known about the difference in the role. Here, we developed a new method for the preparation of the recombinant human MLCP complex and determined the molecular and cellular basis of inhibitory phosphorylation. The recombinant MLCP partially purified from mammalian cell lysates retained characteristics of the native enzyme, such that it was fully active without Mn(2+) and sensitive to PP1 inhibitor compounds. Selective thio-phosphorylation of MYPT1 at Thr696 with ROCK inhibited the MLCP activity 30%, whereas the Thr853 thio-phosphorylation did not alter the phosphatase activity. Interference with the docking of phospho-Thr696 at the active site weakened the inhibition, suggesting selective autoinhibition induced by phospho-Thr696. Both Thr696 and Thr853 sites underwent autodephosphorylation. Compared with that of Thr853, phosphorylation of Thr696 was more stable, and it facilitated Thr853 phosphorylation. Endogenous MYPT1 at Thr696 was spontaneously phosphorylated in quiescent human leiomyosarcoma cells. Serum stimulation of the cells resulted in dissociation of MYPT1 from myosin and PP1C in parallel with an increase in the level of Thr853 phosphorylation. The C-terminal domain of human MYPT1(495-1030) was responsible for the binding to the N-terminal portion of myosin light meromyosin. The spontaneous phosphorylation at Thr696 may adjust the basal activity of cellular MLCP and affect the temporal phosphorylation at Thr853 that is synchronized with myosin targeting.