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Tau aggregates are present in a large number of neurodegenerative diseases known as “tauopathies”, including Alzheimer’s disease (AD). As there are six human tau isoforms in brain tissues and both 3R and 4R isoforms have been observed in the neuronal inclusions, we tested whether tau isoforms behave differently in aggregation. We discovered that all six tau isoforms are capable of forming PHF-tau like filaments and the 3R tau isoforms aggregate significantly faster than their 4R counterparts. We further mapped key segments of tau isoforms that contribute to their aggregation kinetics, where it was determined that microtubule binding domains R2 and R3 were the major contributors to tau aggregation. To evaluate the feasibility of using the six recombinant tau isoforms as substrates to amplify misfolded tau, we demonstrated that full-length human tau isoforms can seed and detect misfolded tau from the post-mortem AD brain tissues with high specificity by an ultrasensitive technology termed real-time quaking-induced conversion (RT-QuIC). Mass spectrometric analysis of PHF-tau samples extracted from AD brains identified peptides corresponding to all major forms of human brain tau isoforms along with a consensus hyperphosphorylated peptide near the C-terminus. Together, our findings not only reveal new aggregation kinetic properties of human tau isoforms, support the development of methods to quantitatively measure misfolded human tau isoforms in AD brains, but also uncover the capability of full-length human tau isoforms as substrates for “prion-like” tau seeding by RT-QuIC assays that may be used for new biomarker development for AD and other tauopathy diagnosis.

In Methanocaldococcus jannaschii, methylglyoxal (MG) is required for aromatic amino acid biosynthesis. Previously, the reduction of MG to lactaldehyde in Methanocaldococcus jannaschii cell extracts using either NADPH or F420 H2 was demonstrated; however, the enzyme responsible was not identified. Using NADPH as the reductant, the unknown enzyme was purified from cell extracts of Methanocaldococcus jannaschii and determined to be the F420 -dependent N5 ,N10 -methylenetetrahydromethanopterin reductase (Mer). Here, we report that the recombinantly overexpressed Mer is able to use NADPH and MG (KM of 1.6 and 1.0 mm, respectively) to produce lactaldehyde. Additionally, Mer does not catalyze the reduction of MG to lactaldehyde in the presence of reduced Fo, the precursor of F420 .

Cancer patients receiving chemotherapy often experience taste and smell abnormalities (TSA). To date, the underlying molecular mechanisms of this frequent side-effect have not been determined and effective treatments are not available. This study assessed the feasibility of lactoferrin (LF) supplementation as a treatment for TSA and investigate the related mechanisms through salivary proteome analysis. Nineteen cancer patients with established TSA following chemotherapy administration were enrolled in this study. Cancer patients and additional 12 healthy subjects took LF supplements, 3 tablets per day (250 mg per tablet), for 30 days. Saliva was collected at three timepoints: baseline, 30-day LF supplementation, and 30-day post-LF supplementation. Patient's TSA level, salivary proteome, and salivary minerals at each LF treatment stage were analyzed. High TSA level was associated with high concentration of salivary Fe and loss of critical salivary immune proteins. LF supplementation significantly decreased the concentration of salivary Fe (P = 0.025), increased the abundance (P < 0.05) of salivary α-amylase and Zn-α-2-GP, and led to an overall increase of expression (≥2-fold changes) of immune proteins including immunoglobulin heavy chain, annexin A1, and proteinase inhibitor. Abundance of α-amylase and SPLUNC2 were further increased (P < 0.05) at 30-day post-LF supplementation in cancer patients. At the same time, total TSA score was significantly reduced (P < 0.001) in chemotherapy patients. This study demonstrated the feasibility of developing lactoferrin supplementation as a treatment to reduce TSA caused by chemotherapy and improve cancer patient's oral immunity.

A commercially reared domesticated turkey (Meleagris gallopavo) was purchased from a local market and sections of tissue representing leg, thigh, and breast were harvested and processed for analysis of the lipids and proteins present. Leg and thigh tissue was enriched in mitochondrial proteins whereas the breast tissue was enriched in glycolytic enzymes as well as the cytosolic and mitochondrial forms of glycerol-3-phosphate dehydrogenase. A potential marker for breast tissue muscle formation and/or function was also identified. The tissues could clearly be separated based upon their lipid profiles with little differences in cardiolipin levels suggesting that mitochondrial surface areas may be similar across the tissues. The most significant differences in the lipids were found to be higher levels of oxidized lipids in thigh meat. This work provides the first untargeted proteome and lipidome datasets for the domesticated turkey. The proteome dataset is accessible from ProteomeXchange Consortium via the PRIDE partner repository with the identifier PXD008207.

The eukaryotic protein kinase (ePK) paradigm provides integral components for signal transduction cascades throughout nature. However, while so-called typical ePKs permeate the Eucarya and Bacteria, atypical ePKs dominate the kinomes of the Archaea. Intriguingly, the catalytic domains of the handful of deduced typical ePKs from the archaeon Sulfolobus solfataricus P2 exhibit significant resemblance to the protein kinases that phosphorylate translation initiation factor 2α (eIF2α) in response to cellular stresses. We cloned and expressed one of these archaeal eIF2α protein kinases, SsoPK4. SsoPK4 exhibited protein-serine/threonine kinase activity toward several proteins, including the S. solfataricus homolog of eIF2α, aIF2α. The activity of SsoPK4 was inhibited in vitro by 3ʹ,5ʹ-cyclic AMP (Ki of ~23 µM) and was activated by oxidized Coenzyme A, an indicator of oxidative stress in the Archaea. Activation enhanced the apparent affinity for protein substrates, Km, but had little effect on Vmax. Autophosphorylation activated SsoPK4 and rendered it insensitive to oxidized Coenzyme A. Published version

Specific AA affect rates of milk protein synthesis in the mammary glands of lactating cows. The objective of this study was to quantify the rate of αS1-casein synthesis in response to Ile, Leu, Met, and Thr supplementation, and to test the single-limiting AA theory for milk protein synthesis by exploring interactions among these AA. Effects of Ile, Leu, Met, and Thr were studied in vitro with a composite design containing a central point repeated 4 times, with 2 axial points per AA and a complete 2(4) factorial. Other AA were at the concentration in Dulbecco's modified Eagle medium/F12 medium (DMEM). The experiment was replicated with mammary tissue from 5 lactating cows. Mammary tissue slices (0.12 ± 0.02 g) were incubated for 4h at 37°C in 5 mL of treatment medium containing (2)H5-Phe. Caseins were precipitated from cell homogenate supernatants. Enrichment with (2)H5-Phe of the N[34]LLRFFVAPFPE αS1 peptide was determined by matrix-assisted laser desorption/ionization-tandem time-of-flight (MALDI-TOF-TOF), which was used to determine enrichment of Phe in the transfer (t)RNA pool and αS1-casein fractional synthesis rates (CFSR). Data were analyzed with a polynomial mixed model containing linear, quadratic, and 2-factor interactions for Ile, Leu, Met, and Thr, and cow and residual as random factors. Interactions were not significant at P

Cranberry products have long been used to treat urinary tract infections. It is believed that the A-type proanthocyanidins in cranberries contribute to this function. Peanut is one of the other, few food sources that primarily contain A-type proanthocyanidins. The skin on the outside of the peanut kernels (testa), which is treated as an agriculture waste product, contains high levels of A-type proanthocyanidins. In this study, an HPLC diol column separation method and MALDI-TOF MS were used to characterize and compare the proanthocyanidin compositions of peanut skins and cranberries. MALDI-TOF MS in linear mode was able to detect a group of proanthocyanidins with DP (degree of polymerization) 10 in peanut skin extract, but was only able to detect DP 8 in cranberry extract. The reflectron mode showed clusters of clear narrow peaks at DP 7 in peanut skin extract, while the highest DP resolved for cranberry extract was only 3 in reflectron mode. This might be due to the low response intensity of the cranberry samples with the current cleanup method and the matrix. Based on the resolved peaks in reflectron mode, pPeanut skins and cranberries have similar proanthocyanidins composition; they contain both A-type and B-type proanthocyanidins, with the A-type being predominant. This result may inspire future studies on the comparison of biological functions between peanut skins and cranberries and further comparison of their polymeric proanthocyanidin composition.

Water makes up more than 80% of the total weight of milk. However, the influence of water chemistry on the milk proteome has not been extensively studied. The objective was to evaluate interaction of water-sourced iron (low, medium, and high levels) on milk proteome and implications on milk oxidative state and mineral content. Protein composition, oxidative stability, and mineral composition of milk were investigated under conditions of iron ingestion through bovine drinking water (infused) as well as direct iron addition to commercial milk in 2 studies. Four ruminally cannulated cows each received aqueous infusions (based on water consumption of 100L) of 0, 2, 5, and 12.5mg/L Fe(2+) as ferrous lactate, resulting in doses of 0, 200, 500 or 1,250mg of Fe/d, in a 4×4Latin square design for a 14-d period. For comparison, ferrous sulfate solution was directly added into commercial retail milk at the same concentrations: control (0mg of Fe/L), low (2mg of Fe/L), medium (5mg of Fe/L), and high (12.5mg of Fe/L). Two-dimensional electrophoresis coupled with matrix-assisted laser desorption/ionization-tandem time-of-flight (MALDI-TOF/TOF) high-resolution tandem mass spectrometry analysis was applied to characterize milk protein composition. Oxidative stability of milk was evaluated by the thiobarbituric acid reactive substances (TBARS) assay for malondialdehyde, and mineral content was measured by inductively coupled plasma mass spectrometry. For milk from both abomasal infusion of ferrous lactate and direct addition of ferrous sulfate, an iron concentration as low as 2mg of Fe/L was able to cause oxidative stress in dairy cattle and infused milk, respectively. Abomasal infusion affected both caseins and whey proteins in the milk, whereas direct addition mainly influenced caseins. Although abomasal iron infusion did not significantly affect oxidation state and mineral balance (except iron), it induced oxidized off-flavor and partial degradation of whey proteins. Direct iron addition to milk led to lipid oxidation during storage at 4°C. Oxidation level was positively associated with the concentration of added iron. Minerals (Mg, P, Na, K, Ca, Zn) in milk were not affected by the added iron in milk. This study indicated that a small amount of iron contamination in bovine drinking water at the farm or incidental iron addition from potable water sources causes oxidation, affects milk protein composition and stability, and affects final milk quality.

Dairy cattle only capture about 25% of dietary N in milk protein, with the remainder being excreted. By coordinating AA supply with demand, mammary cells reduce recycling of AA to the splanchnic tissues, thereby increasing efficiency. Several essential AA regulate the rate of synthesis of casein by altering the phosphorylation state of intracellular signaling proteins that affect the activity of initiation (eIF) and elongation (eEF) factors (Appuhamy et al., 2012). Therefore, supplementing the specific AA that exert the most influence on pathways controlling eIF and eEF should allow a reduction in CP supply to the animal, which will increase post-absorptive efficiency and reduce N excretion. The objective of this study was to determine the quantitative responses in milk protein synthesis to individual essential AA in mammary tissue.