Pan-Inhibition of Protein Disulfide Isomerase Caused Cell Death through Disrupting Cellular Proteostasis in Pancreatic Ductal Adenocarcinoma Cells.

The protein disulfide isomerase (PDI) family is a group of thioredoxin endoplasmic reticulum (ER)-resident enzymes and molecular chaperones that play crucial roles in the correct folding of proteins. PDIs are upregulated in multiple cancer types and are considered a novel target for cancer therapy. In this study, we found that a potent pan-PDI inhibitor, E64FC26, significantly decreased the proliferation of pancreatic ductal adenocarcinoma (PDAC) cells. As expected, E64FC26 treatment increased ER stress and the unfolded protein response (UPR), as evidenced by upregulation of glucose-regulated protein, 78-kDa (GRP78), phosphorylated (p)-PKR-like ER kinase (PERK), and p-eukaryotic initiation factor 2α (eIF2α). Persistent ER stress was found to lead to apoptosis, ferroptosis, and autophagy, all of which are dependent on lysosomal functions. First, there was little cleaved caspase-3 in E64FC26-treated cells according to Western blotting, but a higher dose of E64FC26 was needed to induce caspase activity. Then, E64FC26-induced cell death could be reversed by adding the iron chelator, deferoxamine, and the reactive oxygen species scavengers, ferrostatin-1 and N-acetylcysteine. Furthermore, the autophagosome-specific marker, light chain 3B (LC3B)-II, increased, but the autolysosome marker, sequestosome 1 (SQSTM1)/p62, was not degraded in E64FC26-treated cells. Using the FUW mCherry-LC3 plasmid and acridine orange staining, we also discovered a lower number of acidic vesicles, such as autolysosomes and mature lysosomes, in E64FC26-treated cells. Finally, E64FC26 treatment increased the cathepsin L precursor (pre-CTSL) but decreased mature CTSL expression according to Western blotting, indicating a defective lysosome. These results suggested that the PDI inhibitor, E64FC26, might initially impede proper folding of proteins, and then induce ER stress and disrupt proteostasis, subsequently leading to lysosomal defects. Due to defective lysosomes, the extents of apoptosis and ferroptosis were limited, and fusion with autophagosomes was blocked in E64FC26-treated cells. Blockade of autolysosomal formation further led to the autophagic cell death of PDAC cells.

Liquid Viscosity Sensor Using a Surface Acoustic Wave Device for Medical Applications Including Blood and Plasma.

Blood viscosity is the defining health indicator for hyperviscosity syndrome patients. This paper introduces an alternative approach for the real-time monitoring of blood viscosity by employing a surface-horizontal surface acoustic wave (SH-SAW) device at room temperature. A novel bi-layer waveguide is constructed on top of the SAW device. This device enables the SAW sensing of liquid droplets utilizing a bi-layer waveguide, consisting of a zinc oxide (ZnO) enhancement layer and Parlyene C, that facilitates the promotion of the surface horizontal mode. The ZnO piezoelectric thin-film layer enhanced the local particle displacement and dielectric coupling while the Parylene C layer constrained the wave mode at the interface of the piezoelectric material and polymer material. The device was tested with a liquid drop on the SAW delay-line path. Both experimental and finite element analysis results demonstrated the benefits of the bi-layer waveguide. The simulation results confirmed that the displacement field of local particles increased 9 times from 1.261 nm to 11.353 nm with the Parylene C/ZnO bi-layer waveguide structure. The device demonstrated a sensitivity of 3.57 ± 0.3125 kHz shift per centipoise enabling the potential for high precision blood viscosity monitoring.

Histone Deacetylase Inhibitors Downregulate Calcium Pyrophosphate Crystal Formation in Human Articular Chondrocytes.

Calcium pyrophosphate (CPP) deposition disease (CPPD) is a form of CPP crystal-induced arthritis. A high concentration of extracellular pyrophosphate (ePPi) in synovial fluid is positively correlated with the formation of CPP crystals, and ePPi can be upregulated by ankylosis human (ANKH) and ectonucleotide pyrophosphatase 1 (ENPP1) and downregulated by tissue non-specific alkaline phosphatase (TNAP). However, there is currently no drug that eliminates CPP crystals. We explored the effects of the histone deacetylase (HDAC) inhibitors (HDACis) trichostatin A (TSA) and vorinostat (SAHA) on CPP formation. Transforming growth factor (TGF)-ß1-treated human primary cultured articular chondrocytes (HC-a cells) were used to increase ePPi and CPP formation, which were determined by pyrophosphate assay and CPP crystal staining assay, respectively. Artificial substrates thymidine 5'-monophosphate p-nitrophenyl ester (p-NpTMP) and p-nitrophenyl phosphate (p-NPP) were used to estimate ENPP1 and TNAP activities, respectively. The HDACis TSA and SAHA significantly reduced mRNA and protein expressions of ANKH and ENPP1 but increased TNAP expression in a dose-dependent manner in HC-a cells. Further results demonstrated that TSA and SAHA decreased ENPP1 activity, increased TNAP activity, and limited levels of ePPi and CPP. As expected, both TSA and SAHA significantly increased the acetylation of histones 3 and 4 but failed to block Smad-2 phosphorylation induced by TGF-ß1. These results suggest that HDACis prevented the formation of CPP by regulating ANKH, ENPP1, and TNAP expressions and can possibly be developed as a potential drug to treat or prevent CPPD.

MCPIP1 Enhances TNF-α-Mediated Apoptosis through Downregulation of the NF-κB/cFLIP Axis.

Monocyte chemoattractant protein-1-induced protein 1 (MCPIP1) is rapidly produced under proinflammatory stimuli, thereby feeding back to downregulate excessive inflammation. In this study, we used the stable, inducible expressions of wild-type (WT) MCPIP1 and an MCPIP1-D141N mutant in T-REx-293 cells by means of a tetracycline on (Tet-on) system. We found that WT MCPIP1 but not MCPIP1-D141N mutant expression dramatically increased apoptosis, caspase-3, -7, -8, and -9 activation, and c-Jun N-terminal kinase (JNK) phosphorylation in TNF-α-treated cells. The pan-caspase inhibitor, z-VAD-fmk, and the caspase-1 inhibitor, z-YVAD-fmk, but not the JNK inhibitor, SP600125, significantly reversed apoptosis and caspase activation in TNF-α/MCPIP1-treated cells. Surprisingly, MCPIP1 itself was also cleaved, and the cleavage was suppressed by treatment with the pan-caspase inhibitor and caspase-1 inhibitor. Moreover, MCPIP1 was found to contain a caspase-1/-4 consensus recognition sequence located in residues 234~238. As expected, the WT MCPIP1 but not the MCPIP1-D141N mutant suppressed NF-κB activation, as evidenced by inhibition of IκB kinase (IKK) phosphorylation and IκB degradation using Western blotting, IKK activity using in vitro kinase activity, and NF-κB translocation to nuclei using an immunofluorescence assay. Interestingly, MCPIP1 also significantly inhibited importin α3 and importin α4 expressions, which are major nuclear transporter receptors for NF-κB. Inhibition of NF-κB activation further downregulated expression of the caspase-8 inhibitor, cFLIP. In summary, the results suggest that MCPIP1 could enhance the TNF-α-induced apoptotic pathway through decreasing NF-κB activation and cFLIP expression.

MEMS biosensor for monitoring water toxicity based on quartz crystal microbalance.

This paper presents the use of a commercial quartz crystal microbalance (QCM) to investigate live-cell activity in water-based toxic solutions. The QCM used in this research has a resonant frequency of 10 MHz and consists of an AT-cut quartz crystal with gold electrodes on both sides. This QCM was transformed into a functional biosensor by integrating with polydimethylsiloxane culturing chambers. Rainbow trout gill epithelial cells were cultured on the resonators as a sensorial layer. The fluctuation of the resonant frequency, due to the change of cell morphology and adhesion, is an indicator of water toxicity. The shift in the resonant frequency provides information about the viability of the cells after exposure to toxicants. The toxicity result shows distinct responses after exposing cells to 0.526 µM of pentachlorophenol (PCP) solution, which is the Military Exposure Guidelines concentration. This research demonstrated that the QCM is sensitive to a low concentration of PCP and no further modification of the QCM surface was required.

Stretchable Piezoelectric Power Generators Based on ZnO Thin Films on Elastic Substrates.

The paper describes a stretchable, microfabricated power generator that will be attached on the skin and will produce energy based on the movements of the human body. The device was fabricated on a polymeric, elastomeric, poly(dimethylsiloxane) (PDMS) sheet. It consists of a piezoelectric thin film of ZnO sandwiched between two stretchable gold electrodes. An innovative technique was used for the deposition of ZnO thin film on the gold electrode-coated polymeric substrate at low temperatures below 150 °C. This is the first attempt to use a uniform film of ZnO, for energy harvesting. The ZnO film had the thickness at the submicron scale and the surface at the centimeter scale. We demonstrated that under a strain of 8% the voltage output from this power generator was equal to 2 V, the power output was equal to 160 µW and the corresponding power density was 1.27 mW/cm2. This device has great potential for application in power sensors attached on the human body, such as temperature sensors or wearable electrocardiography systems.

Seroepidemiology of helminths and the association with severe malaria among infants and young children in Tanzania.

The disease burden of Wuchereria bancrofti and Plasmodium falciparum malaria is high, particularly in Africa, and co-infection is common. However, the effects of filarial infection on the risk of severe malaria are unknown. We used the remaining serum samples from a large cohort study in Muheza, Tanzania to describe vector-borne filarial sero-reactivity among young children and to identify associations between exposure to filarial parasites and subsequent severe malaria infections. We identified positive filarial antibody responses (as well as positive antibody responses to Strongyloides stercoralis) among infants as young as six months. In addition, we found a significant association between filarial seropositivity at six months of age and subsequent severe malaria. Specifically, infants who developed severe malaria by one year of age were 3.9 times more likely (OR = 3.9, 95% CI: 1.2, 13.0) to have been seropositive for filarial antigen at six months of age compared with infants who did not develop severe malaria.

A Malaria-Resistant Phenotype with Immunological Correlates in a Tanzanian Birth Cohort Exposed to Intense Malaria Transmission.

AbstractMalaria incidence is highly heterogeneous even in areas of high transmission, although no conclusive evidence exists that innate or naturally acquired resistance can prevent infection over an extended period of time. This longitudinal study examined immunoparasitological evidence for a malaria-resistant phenotype in which children do not develop malaria despite an extended period of exposure to parasites. Within a birth cohort followed from 2002 to 2006 in Muheza, Tanzania, an area of intense transmission, children (N = 687) provided blood smears biweekly during infancy and monthly thereafter. Maternal and childhood characteristics were obtained, cord-blood cytokines were measured, and antibody responses were assayed as measures of stage-specific exposure. Sixty-three (9.2%) children had no blood smear-positive slides over 2 years of follow-up (range: 1-3.5 years) and were identified as malaria resistant. Malaria-resistant children were similar to other children with respect to completeness of follow-up and all maternal and childhood characteristics except residence area. Antibody seroprevalence was similar for two sporozoite antigens, but malaria-resistant children had a lower antibody seroprevalence to merozoite antigens merozoite surface protein 1 (5.4% versus 30.2%; P < 0.0001) and apical membrane antigen 1 (7.2% versus 33.3%; P < 0.0001). Malaria-resistant children had higher cytokine levels in cord blood, particularly interleukin-1ß. In summary, a subset of children living in an area of intense transmission was exposed to malaria parasites, but never developed patent parasitemia; this phenotype was associated with a distinct cytokine profile at birth and antibody profile during infancy. Further research with malaria-resistant children may identify mechanisms for naturally acquired immunity.

The effects of loading conditions and specimen environment on the nanomechanical response of canine cortical bone.

Bone is a viscoelastic connective tissue composed primarily of mineral and type I collagen, which interacts with water, affecting its mechanical properties. Therefore, both the level of hydration and the loading rate are expected to influence the measured nanomechanical response of bone. In this study, we investigated the influence of three distinct hydration conditions, peak loads and loading/unloading rates on the elastic modulus and hardness of canine femoral cortical bone via nanoindentation. Sections from three canine femurs from multiple regions of the diaphysis were tested for a total of 670 indentations. All three hydration conditions (dry, moist and fully hydrated tissue) were tested at three different loading profiles (a triangular loading profile with peak loads of 600, 800 and 1000 µN at loading/unloading rate of 60, 80 and 100 µN/s, respectively; each test was 20s in duration). Significant differences were found for both the elastic modulus and hardness between the dry, moist and fully hydrated conditions (p≤0.02). For dry bone, elastic modulus and hardness values were not found to be significantly different between the different loading profiles (p>0.05). However, in both the moist and fully hydrated conditions, the elastic modulus and hardness were significantly different under all loading profiles (with the exception of the moist condition at the 600- and 800-µN peak load). Given these findings, it is critical to perform nanoindentation of bone under fully hydrated conditions to ensure physiologically relevant results. Furthermore, this work found that a 20-s triangular loading/unloading profile was sufficient to capture the viscoelastic behavior of bone in the 600- to 1000-µN peak load range. Lastly, specific peak load values and loading rates need to be selected based on the structural region for which the mechanical properties are to be measured.

Nanomechanical Characterization of Canine Femur Bone for Strain Rate Sensitivity in the Quasistatic Range under Dry versus Wet Conditions.

As a strain rate-dependent material, bone has a different mechanical response to various loads. Our aim was to evaluate the effect of water and different loading/unloading rates on the nanomechanical properties of canine femur cortical bone. Six cross-sections were cut from the diaphysis of six dog femurs and were nanoindented in their cortical area. Both dry and wet conditions were taken into account for three quasistatic trapezoid profiles with a maximum force of 2000 µN (holding time = 30 s) at loading/unloading rates of 10, 100, and 1000 µN/s, respectively. For each specimen, 254 ± 9 (mean ± SD) indentations were performed under different loading conditions. Significant differences were found for the elastic modulus and hardness between wet and dry conditions (P < 0.001). No influence of the loading/unloading rates was observed between groups except for the elastic modulus measured at 1000 µN/s rate under dry conditions (P < 0.001) and for the hardness measured at a rate of 10 µN/s under wet conditions (P < 0.001). Therefore, for a quasistatic test with peak load of 2000 µN held for 30 s, it is recommended to nanoindent under wet conditions at a loading/unloading rate of 100-1000 µN/s, so the reduced creep effect allows for a more accurate computation of mechanical properties.