Antisense oligonucleotides targeting the miR-29b binding site in the GRN mRNA increase progranulin translation.

Heterozygous GRN (progranulin) mutations cause frontotemporal dementia (FTD) due to haploinsufficiency, and increasing progranulin levels is a major therapeutic goal. Several microRNAs, including miR-29b, negatively regulate progranulin protein levels. Antisense oligonucleotides (ASOs) are emerging as a promising therapeutic modality for neurological diseases, but strategies for increasing target protein levels are limited. Here, we tested the efficacy of ASOs as enhancers of progranulin expression by sterically blocking the miR-29b binding site in the 3' UTR of the human GRN mRNA. We found 16 ASOs that increase progranulin protein in a dose-dependent manner in neuroglioma cells. A subset of these ASOs also increased progranulin protein in iPSC-derived neurons and in a humanized GRN mouse model. In FRET-based assays, the ASOs effectively competed for miR-29b from binding to the GRN 3' UTR RNA. The ASOs increased levels of newly synthesized progranulin protein by increasing its translation, as revealed by polysome profiling. Together, our results demonstrate that ASOs can be used to effectively increase target protein levels by partially blocking miR binding sites. This ASO strategy may be therapeutically feasible for progranulin-deficient FTD as well as other conditions of haploinsufficiency.

Discriminating aging and protein-to-fat ratio in Cheddar cheese using sensory analysis and a potentiometric electronic tongue.

The objectives of this study were to evaluate the flavor and taste attributes of full-fat Cheddar cheeses with different protein-to-fat ratios (PFR) over aging time using a descriptive sensory analysis panel and a consumer panel, and to correlate these attributes with instrumental parameters obtained by the potentiometric electronic tongue. Three Cheddar cheese formulations (PFR of 0.74, 0.85, and 1.01) were produced in triplicate and composition was verified. Cheese was aged at 7.2°C and evaluated at 2, 5, 8, 10, 11, and 12 mo by a trained panel (n = 10) for 8 flavor and 5 taste attributes and using an electronic tongue for 7 nonvolatile taste attributes. Cheese aged for 12 mo was also evaluated by a consumer sensory panel for liking and intensity attributes. Principal component analysis was performed to discriminate cheese based on aging time and PFR, whereas correlation between sensory and instrumental attributes was assessed using partial least squares regression. Descriptive sensory analysis of flavor and taste attributes differentiated Cheddar cheeses over aging time, but not among PFR formulations. The electronic tongue distinguished changes among cheese samples due to PFR formulation and aging time. The electronic tongue proved successful in characterizing the nonvolatile flavor components in Cheddar cheese and correlated with taste perceptions measured by descriptive sensory analysis. Consumer evaluations showed distinctive attribute profiles for the 3 PFR Cheddar cheese formulations. Overall, higher fat content was associated with increased flavor intensities in Cheddar cheese and drove consumer acceptability and purchase intent ratings. The electronic tongue detected smaller changes in tastes (bitter, metallic, salty, sour, spicy, sweet, and umami) of the 3 PFR formulations over time when compared with the trained panelists, who detected no differences, suggesting that the electronic tongue may be more sensitive to tastants than humans and may have the capability for early detection or identification of problems in a batch of cheese during aging. Results suggest taste quality of cheese may be monitored using the electronic tongue with greater sensitivity than a trained panel, and may be more objective, rapid, and cost effective than human panelists.

Biochemical, biomarker, and behavioral characterization of the GrnR493X mouse model of frontotemporal dementia.

Heterozygous loss-of-function mutations in the progranulin gene (GRN) are a major cause of frontotemporal dementia due to progranulin haploinsufficiency; complete deficiency of progranulin causes neuronal ceroid lipofuscinosis. Several progranulin-deficient mouse models have been generated, including both knockout mice and knockin mice harboring a common patient mutation (R493X). However, the GrnR493X mouse model has not been characterized completely. Additionally, while homozygous GrnR493X and Grn knockout mice have been extensively studied, data from heterozygous mice is still limited. Here, we performed more in-depth characterization of heterozygous and homozygous GrnR493X knockin mice, which includes biochemical assessments, behavioral studies, and analysis of fluid biomarkers. In the brains of homozygous GrnR493X mice, we found increased phosphorylated TDP-43 along with increased expression of lysosomal genes, markers of microgliosis and astrogliosis, pro-inflammatory cytokines, and complement factors. Heterozygous GrnR493X mice did not have increased TDP-43 phosphorylation but did exhibit limited increases in lysosomal and inflammatory gene expression. Behavioral studies found social and emotional deficits in GrnR493X mice that mirror those observed in Grn knockout mouse models, as well as impairment in memory and executive function. Overall, the GrnR493X knockin mouse model closely phenocopies Grn knockout models. Lastly, in contrast to homozygous knockin mice, heterozygous GrnR493X mice do not have elevated levels of fluid biomarkers previously identified in humans, including neurofilament light chain (NfL) and glial fibrillary acidic protein (GFAP) in both plasma and CSF. These results may help to inform pre-clinical studies that use this Grn knockin mouse model and other Grn knockout models.

Biochemical, Biomarker, and Behavioral Characterization of the GrnR493X Mouse Model of Frontotemporal Dementia.

Heterozygous loss-of-function mutations in the progranulin gene (GRN) are a major cause of frontotemporal dementia due to progranulin haploinsufficiency; complete deficiency of progranulin causes neuronal ceroid lipofuscinosis. Several progranulin-deficient mouse models have been generated, including both knockout mice and knockin mice harboring a common patient mutation (R493X). However, the GrnR493X mouse model has not been characterized completely. Additionally, while homozygous GrnR493X and Grn knockout mice have been extensively studied, data from heterozygous mice is still limited. Here, we performed more in-depth characterization of heterozygous and homozygous GrnR493X knockin mice, which includes biochemical assessments, behavioral studies, and analysis of fluid biomarkers. In the brains of homozygous GrnR493X mice, we found increased phosphorylated TDP-43 along with increased expression of lysosomal genes, markers of microgliosis and astrogliosis, pro-inflammatory cytokines, and complement factors. Heterozygous GrnR493X mice did not have increased TDP-43 phosphorylation but did exhibit limited increases in lysosomal and inflammatory gene expression. Behavioral studies found social and emotional deficits in GrnR493X mice that mirror those observed in Grn knockout mouse models, as well as impairment in memory and executive function. Overall, the GrnR493X knockin mouse model closely phenocopies Grn knockout models. Lastly, in contrast to homozygous knockin mice, heterozygous GrnR493X mice do not have elevated levels of fluid biomarkers previously identified in humans, including neurofilament light chain (NfL) and glial fibrillary acidic protein (GFAP) in both plasma and CSF. These results may help to inform pre-clinical studies that use this Grn knockin mouse model and other Grn knockout models.

Targeting nonsense-mediated RNA decay does not increase progranulin levels in the Grn R493X mouse model of frontotemporal dementia.

A common cause of frontotemporal dementia (FTD) are nonsense mutations in the progranulin (GRN) gene. Because nonsense mutations activate the nonsense-mediated RNA decay (NMD) pathway, we sought to inhibit this RNA turnover pathway as a means to increase progranulin levels. Using a knock-in mouse model harboring a common patient mutation, we tested whether either pharmacological or genetic inhibition of NMD upregulates progranulin in these GrnR493X mice. We first examined antisense oligonucleotides (ASOs) targeting an exonic region in GrnR493X mRNA predicted to block its degradation by NMD. As we previously reported, these ASOs effectively increased GrnR493X mRNA levels in fibroblasts in vitro. However, following CNS delivery, we found that none of the 8 ASOs we tested increased Grn mRNA levels in the brains of GrnR493X mice. This result was obtained despite broad ASO distribution in the brain. An ASO targeting a different mRNA was effective when administered in parallel to wild-type mice. As an independent approach to inhibit NMD, we examined the effect of loss of an NMD factor not required for embryonic viability: UPF3b. We found that while Upf3b deletion effectively perturbed NMD, it did not increase Grn mRNA levels in Grn+/R493X mouse brains. Together, our results suggest that the NMD-inhibition approaches that we used are likely not viable for increasing progranulin levels in individuals with FTD caused by nonsense GRN mutations. Thus, alternative approaches should be pursued.

Influence of storage time and elevated ripening temperature on the chemical and sensory properties of white Cheddar cheese.

Aged cheese is an increasingly popular dairy product. One approach to reduce Cheddar cheese maturation time is by utilizing elevated temperature, despite potential problems including development of imbalanced or off-flavors and negative changes in texture. Therefore, the objective of this study was to evaluate the influence of elevated ripening temperature on chemical and sensory properties of aged white Cheddar cheese. White Cheddar cheese was aged at 7.2, 10, or 12.8 °C for 12 months, with samples evaluated at 2, 5, 8, 10, 11, and 12 months by a trained sensory panel (n = 10). Two consumer sensory panels (n = 120) assessed 8- and 12-month aged cheese for comparison to a commercially available reference sample of the same cheese, aged for 12 months. An electronic tongue methodology was developed for analysis of nonvolatile compounds. Trained panel results showed that 2-month cheeses were described by milkfat flavor and sweet taste, 5-month cheeses were described by nutty aroma and white color, and 8-, 10-, 11-, and 12-month cheeses developed aged characteristics, such as umami and bitter tastes, brothy aroma, and aged flavor. Consumer panel results showed similar overall liking scores for the reference cheese and cheeses aged at 10 or 12.8 °C for both evaluations. The electronic tongue could classify samples according to aging month with a validity value of 92.59%. In conclusion, the electronic tongue served as a valid method of instrumental analysis for Cheddar cheese samples throughout maturation. This study demonstrated that aging white Cheddar cheese for 8 months at an elevated storage temperature of 10 °C produced cheese similar in consumer acceptance to that aged at 7 °C for 12 months. PRACTICAL APPLICATION: This study showed that aged white Cheddar storage at a higher temperature was perceived similarly by consumers as one stored for 1 year at a slightly lower temperature. This may be useful to those in the dairy industry exploring ways to accelerate aging, reducing devoted resources, while still producing an acceptable product. Also, the electronic tongue was effective at distinguishing among aged white Cheddar cheese samples showing another application for this technology.

Enhanced functionality of pea-rice protein isolate blends through direct steam injection processing.

Direct steam injection (DSI) processing with pH adjustment was investigated to enhance the functionality of pea-rice protein isolate blends (PR). Protein slurries at concentration of 5%(w/w) of commercial pea and rice protein isolates in the ratio of 2:1(w/w) across a range of steam temperatures (66-107°C) and pH values (2-11) were studied. After DSI treatment, the PR were freeze-dried to obtain the final dry protein powder. Based on protein solubility profiles, the optimal DSI processing conditions were 107°C and pH 11. Available lysine was not reduced (P>0.05) in the blend. Solubility (from 3 to 41%, at pH 7), emulsifying activity index (from 5.9 to 52.5m2/g), foam stability (from 68.2 to 82.8%), and oil holding capacity (from 1.8 to 4.9g/g) values increased (P<0.05) compared to the untreated PR. DSI can modify the functionality of PR without affecting the essential amino acid composition.

Influence of High-Pressure Processing at Low Temperature and Nisin on Listeria innocua Survival and Sensory Preference of Dry-Cured Cold-Smoked Salmon.

Cold-smoked salmon (CSS) production lacks a validated kill step for Listeria monocytogenes. Although Listeria spp. are reduced by nisin or high-pressure processing (HPP), CSS muscle discoloration is often observed after HPP. Effects of nisin and low-temperature HPP on L. innocua survival (nonpathogenic surrogate for L. monocytogenes), spoilage organism growth, color, and sensory preference and peelability of CSS were studied. Cold-smoked sockeye salmon (Oncorhynchus nerka) fillets ± nisin (10 µg/g) were inoculated with a 3-strain L. innocua cocktail, vacuum-packaged, frozen at - 30 °C, and high-pressure processed in an ice slurry within an insulated sleeve. Initial experiments indicated that nisin and HPP for 120 s at 450 MPa (N450) and 600 MPa (N600) were most effective against L. innocua, and thus were selected for further storage studies. L. innocua in N450 and N600-treated CSS was reduced 2.63 ± 0.15 and 3.99 ± 0.34 Log CFU/g, respectively, immediately after HPP. L. innocua and spoilage growth were not observed in HPP-treated CSS during 36 d storage at 4 °C. Low-temperature HPP showed a smaller increase in lightness of CSS compared to ambient-temperature HPP performed in previous studies. Sensory evaluation indicated that overall liking of CSS treated with N450 and N600 were preferred over the control by 61% and 62% of panelists, respectively (P < 0.05). Peelability of sliced CSS was reduced by HPP (P < 0.05). Nisin in combination with low-temperature HPP was effective in controlling L. innocua in CSS while maintaining consumer acceptability. PRACTICAL APPLICATION: Cold-smoked salmon is a high-risk ready-to-eat product that may be contaminated with L. monocytogenes. Results showed that nisin combined with high-pressure processing at low temperature, reduced the population of Listeria and controlled the spoilage organisms during storage. As an added benefit, high-pressure processing at low temperature may reduce lightening of the salmon flesh, leading to enhanced consumer preference.

Use of Volatiles as Indicators of Lipid Oxidation in Muscle Foods.

Lipid oxidation has long been recognized as a leading cause of quality deterioration in muscle foods and is often the decisive factor in determining food product storage life. Lipid oxidation generates a number of products, including volatile compounds, which are the major contributors to the development of rancid off-flavors and odors. Over the years, methodologies have been developed to quantify lipid oxidation products in muscle foods. This article reviews the analytical methods that have been used to quantify volatile compounds as indicators of lipid oxidation in muscle foods. The sampling methodologies of distillation/solvent extraction and headspace analysis, and isolation methods associated with gas chromatographic (GC) and high-performance liquid chromatography (HPLC) analyses are discussed. Within gas chromatographic methodologies, headspace (HS) sampling (static HS, dynamic purge-and-trap HS techniques, and solid-phase microextraction [SPME]) are addressed.

Gelling properties of heat-denatured beta-lactoglobulin aggregates in a high-salt buffer.

Thermal denaturation, rheological, and microstructural properties of gels prepared from native beta-lactoglobulin (beta-LG) and preheated or heat-denatured beta-LG (HDLG) aggregates were compared. The HDLG was prepared by heating solutions of 4% beta-LG in deionized water, pH 7.0, at 80 degrees C for 30 min and then diluted to the desired concentration in 0.6 M NaCl and 0.05 M phosphate buffer at pH 6.0, 6.5, and 7.0. When reheated to 71 degrees C, HDLG formed a gel at a concentration of 2% protein. At pH 7.0, 3% HDLG gelled at 52.5 degrees C and had a storage modulus (G') of 2200 Pa after cooling. beta-LG (3%) in 0.6 M NaCl and 0.05 M phosphate buffer, pH 7.0, did not gel when heated to 71 degrees C. The gel point of 3% HDLG decreased by 10.5 degrees C and the G' did not change when the pH was decreased to 6.0. The HDLG gel microstructure was composed of strands and clumps of small globular aggregates in contrast to beta-LG gels, which contained a particulate network of compacted globules. The HDLG formed a gel at a lower concentration and lower temperature than beta-LG in the high-salt buffer, suggesting an application in meat systems or other food products prepared with salt and processed at temperatures of < or =71 degrees C.

Gelation of chicken pectoralis major myosin and heat-denatured beta-lactoglobulin.

Thermal, rheological, and microstructural properties of myosin (1 and 2% protein) were compared to mixtures of 1% myosin and 1% heat-denatured beta-lactoglobulin aggregates (myosin/HDLG) and 1% myosin and 1% native beta-lactoglobulin (myosin/beta-LG) in 0.6 M NaCl and 0.05 M sodium phosphate buffer, pH 6.0, 6.5, and 7.0 during heating to 71 degrees C. Thermal denaturation patterns of myosin and myosin/HDLG were similar except for the appearance of an endothermic peak at 54-56 degrees C in the mixed system. At pH 7.0, 2% myosin began to gel at 48 degrees C and had a storage modulus (G') of 500 Pa upon cooling. Myosin/HDLG (2% total protein) had a gel point of 48 degrees C and a G' of 650 Pa, whereas myosin/beta-LG had a gel point of 49 degrees C but the G' was lower (180 Pa). As the pH was decreased, the gel points of myosin and myosin/HDLG decreased and the G' after cooling increased. The HDLG was incorporated within the myosin gel network, whereas beta-LG remained soluble.

Solid phase microextraction-gas chromatography for quantifying headspace hexanal above freeze-dried chicken myofibrils.

A method using solid phase microextraction (SPME) combined with gas chromatography/mass spectrometry (GC/MS) was developed and used to determine the oxidation of freeze-dried chicken myofibrils spiked with methyl linoleate. Freeze-dried chicken myofibrils were found to act as a significant reservoir for hexanal. Recovery of hexanal emissions from the headspace above spiked myofibrils was 95% using a 5 min sampling time, with a total analysis time of approximately 12 min/sample. The SPME-GC/MS working linear response was from 0.01 to 10 mg hexanal/L (r( 2) = 0.995). Freeze-dried chicken myofibrils with added methyl linoleate (0.6 mmol/g of protein) were stored at 50 degrees C at water activities of 0.30 and 0.75 for 0, 12, 27, and 50 h. Lipid oxidation was determined using SPME-GC/MS to measure headspace hexanal concentration, the thiobarbituric acid reactive substances assay (TBARS) to quantify malonaldehyde, and a conjugated diene assay. Lipid oxidation was influenced by storage time and water activity. A strong correlation (r = 0.938) existed between SPME-GC/MS and TBARS. The use of SPME-GC/MS was a sensitive and rapid method for detecting hexanal as an indicator of lipid oxidation in chicken myofibrils.

ELISA for monitoring lipid oxidation in chicken myofibrils through quantification of hexanal-protein adducts.

The objectives of this study were to optimize a monoclonal competitive indirect enzyme-linked immunosorbent assay (CI-ELISA) for hexanal detection, optimize solubilization and alkylation procedures for the formation of hexanal-protein adducts, and compare the ability the CI-ELISA, thiobarbituric acid reactive substances assay (TBARS), and a solid-phase microextraction-gas chromatography-mass spectrometry (GC/MS-SPME) method for monitoring lipid oxidation in freeze-dried chicken protein. Freeze-dried myofibrils with added methyl linoleate (0.6 mmol/g of protein) were stored at 50 degrees C at two water activities (a(w) = 0.30 and 0.75) for 5 days. Hexanal was measured by GC/MS-SPME and CI-ELISA, and malonaldehyde by TBARS. At an a(w) of 0.30, 34.7 and 39.7 microg of hexanal/g of myofibril were detected by GC/MS-SPME and CI-ELISA, respectively, after 4 days of storage. At an a(w) of 0.75, 39.8 and 61.1 microg of hexanal/g of myofibril were detected by GC/MS-SPME and CI-ELISA, respectively, after 4 days of storage. The CI-ELISA was well correlated with the GC/MS-SPME (r = 0.78) and TBARS (r = 0.87) methods. The correlation of the hexanal-specific CI-ELISA to both GC/MS-SPME and TBARS verified the ability of the CI-ELISA to be used as an index of lipid oxidation, offering the convenience for use in a kit to be utilized within a food-processing facility.

Lactate Dehydrogenase as Safe Endpoint Cooking Indicator in Poultry Breast Rolls: Development of Monoclonal Antibodies and Application to Sandwich Enzyme-Linked Immunosorbent Assay (ELISA).

A sandwich enzyme-linked immunosorbent assay (ELISA) was developed to detect lactate dehydrogenase (LDH) as a marker protein for verifying endpoint cooking of uncured poultry products. Monoclonal antibodies were prepared against chicken muscle LDH and used with rabbit polyclonal antibodies developed against turkey or chicken muscle LDH for capture and detection in the assay, respectively. Minimum assay detection limits for turkey and chicken muscle LDH were 1 ng/ml. Turkey and chicken muscle LDH, but not LDH from other species cross reacted in the ELISA. The ELISA was further verified using extracts of turkey breast rolls processed to internal temperatures between 68.3 and 72.1°C. The LDH content of extracts diluted 3- to 6-fold was below 15 ng/ml for turkey rolls processed to 70.9 and 72.1°C. At a 6-fold dilution, LDH content of extracts from rolls processed to 69.7°C was approximately 10 times greater than those processed to 70.9°C. A survey of market precooked poultry products indicated assay validity with precooked turkey roast, but not turkey hams with maximum internal temperature requirements of 68.3°C. Results suggested the sandwich ELISA should be applicable for determining whether turkey breast rolls are processed to the required U.S. Department of Agriculture endpoint temperature of 71.1°C.

Lactate Dehydrogenase Polyclonal Antibody Sandwich ELISA for Determination of Endpoint Heating Temperatures of Ground Beef.

An enzyme-linked immunosorbent assay (ELISA) for the protein marker lactate dehydrogenase (LDH) was developed to determine endpoint heating temperature (EPT) of ground beef. Ground beef was placed in 10 by 75 mm tubes and heated to temperatures between 62 and 74°C at 2°C intervals. Electrophoresis and immunoblotting of meat extracts indicated a decrease in the intensity of the LDH band as temperature was increased. Polyclonal antibodies (PAb) against bovine muscle LDH were produced and a sandwich ELISA devised using biotinylated PAb as the detecting antibody. The LDH content decreased (P < 0.05) in ground beef heated between 66 and 74°C and was less than 4 µg/g of meat at the recommended minimum endpoint of 69°C. The ELISA was used to estimate the EPT of commercially cooked beef patties.

Thermal Inactivation of Escherichia coli O157: H7, Salmonella senftenberg , and Enzymes with Potential as Time-Temperature Indicators in Ground Beef.

The USDA has established processing schedules for beef products based on the destruction of pathogens. Several enzymes have been suggested as potential indicators of heat processing. However, no relationship between the inactivation rates of these enzymes and those of pathogenic microorganisms has been determined. Our objective was to compare the thermal inactivation of Escherichia coli O157:H7 and Salmonella senftenberg to those of endogenous muscle proteins. Inoculated and noninoculated ground beef samples were heated at four temperatures for predetermined intervals of time in thermal-death-time studies. Bacterial counts were determined and enzymes were assayed for residual activity. The D values for E. coli O157:H7 were 46.10, 6.44, 0.43, and 0.12 min at 53, 58, 63, and 68°C, respectively, with a z value of 5.60°C. The D values for S. senftenberg were 53.00, 15.17, 2.08, and 0.22 min at 53, 58, 63, and 68°C, respectively, with a z value of 6.24°C. Apparent D values at 53, 58, 63, and 68°C were 352.93, 26.31, 5.56, and 3.33 min for acid phosphatase; 6968.64, 543.48, 19.61, and 1.40 min for lactate dehydrogenase; and 3870.97, 2678.59, 769.23, and 42.92 min for peroxidase; with z values of 7.41,3.99, and 7.80°C, respectively. Apparent D values at 53, 58, 63, and 66°C were 325.03, 60.07, 3.07, and 1.34 min for phosphoglycerate mutase; 606.72, 89.86, 4.40, and 1.28 min for glyceraldehyde-3-phosphate dehydrogenase; and 153.06, 20.13, 2.25, and 0.74 min for triose phosphate isomerase; with z values of 5.18, 4.71, and 5.56°C, respectively. The temperature dependence of triose phosphate isomerase was similar to those of both E. coli O157 :H7 and S. senftenberg , suggesting that this enzyme could be used as an endogenous time-temperature indicator in beef products.