Depletion of calpain2 accelerates epithelial barrier establishment and reduces growth factor-induced cell scattering.

Calpain2 is a conventional member of the non-lysosomal calpain protease family that has been shown to affect the dynamics of focal and cell-cell adhesions by proteolyzing the components of adhesion complexes. Here, we inactivated calpain2 using CRISPR/Cas9 in epithelial MDCK cells. We show that depletion of calpain2 has multiple effects on cell morphology and function. Calpain2-depleted cells develop epithelial shape, however, they cover a smaller area, and cell clusters are more compact. Inactivation of calpain2 enhanced restoration of transepithelial electrical resistance after calcium switch, decreased cell migration, and delayed cell scattering induced by HGF/SF. In addition, calpain2 depletion prevented morphological changes induced by ERK2 overexpression. Interestingly, proteolysis of several calpain2 targets, including E-cadherin, ß-catenin, talin, FAK, and paxillin, was not discernibly affected by calpain2 depletion. Taken together, these data suggest that calpain2 regulates the stability of cell-cell and cell-substratum adhesions indirectly without affecting the proteolysis of these adhesion complexes.

The absorption of bovine milk small extracellular vesicles largely depends on galectin 3 and galactose ligands in human intestinal cells and C57BL/6J mice.

Small extracellular vesicles in milk (sMEVs) have attracted attention in drug delivery and as bioactive food compounds. Previous studies implicate galactose residues on the sMEV surface in sMEV transport across intestinal and endothelial barriers in humans, but details of glycoprotein-dependent transport are unknown. We used a combination of cell biology and genetics protocols to identify glycoproteins on the sMEV surface that facilitate sMEV absorption. We identified 256 proteins on the bovine sMEVs surface by using LC-MS/MS, and bioinformatics analysis suggested that 42, 13, and 13 surface proteins were N-, O-, and 13 C-glycosylated, respectively. Lectin blots confirmed the presence of mannose, galactose, N-acetyl galactose, fucose, and neuraminate. When surface proteins were removed by various treatment with various proteases, sMEV uptake decreased by up to 58% and 67% in FHs-74 Int and Caco-2 cells, respectively, compared with controls (P < 0.05). When glycans were removed by treatment with various glycosidases, sMEV uptake decreased by up to 54% and 74% in FHs-74 Int and Caco-2 cells, respectively (P < 0.05). When galactose and N-acetyl galactosamine residues were blocked with agglutinins, sMEV uptake decreased by more than 50% in FHs-74 Int cells (P < 0.05). When bovine sMEVs were administered to Galectin-3 knockout mice by oral gavage, hepatic sMEV accumulation decreased by 56% compared with wild-type mice (P < 0.05), consistent with a role of ß-galactoside glycan structures in the absorption of sMEVs. We conclude that sMEVs are decorated with glycoproteins, and Galectin-3 and its galactose ligands are particularly important for sMEV absorption.NEW & NOTEWORTHY This is the first paper to assess the role of unique glycans and their Galectin-3 receptor in the transport and distribution of small extracellular vesicles ("exosomes") from milk in mammals. The research assessed milk exosome transport and distribution by using multiple approaches and platforms including cell cultures, various exosome labels, knockout and mutant mice, enzymatic removal of surface proteins and glycans, and lectin blocking of glycans.

Comparative polar and lipid plasma metabolomics differentiate KSHV infection and disease states.

BACKGROUND: Kaposi sarcoma (KS) is a neoplastic disease etiologically associated with infection by the Kaposi sarcoma-associated herpesvirus (KSHV). KS manifests primarily as cutaneous lesions in individuals due to either age (classical KS), HIV infection (epidemic KS), or tissue rejection preventatives in transplantation (iatrogenic KS) but can also occur in individuals, predominantly in sub-Saharan Africa (SSA), lacking any obvious immune suppression (endemic KS). The high endemicity of KSHV and human immunodeficiency virus-1 (HIV) co-infection in Africa results in KS being one of the top 5 cancers there. As with most viral cancers, infection with KSHV alone is insufficient to induce tumorigenesis. Indeed, KSHV infection of primary human endothelial cell cultures, even at high levels, is rarely associated with long-term culture, transformation, or growth deregulation, yet infection in vivo is sustained for life. Investigations of immune mediators that distinguish KSHV infection, KSHV/HIV co-infection, and symptomatic KS disease have yet to reveal consistent correlates of protection against or progression to KS. In addition to viral infection, it is plausible that pathogenesis also requires an immunological and metabolic environment permissive to the abnormal endothelial cell growth evident in KS tumors. In this study, we explored whether plasma metabolomes could differentiate asymptomatic KSHV-infected individuals with or without HIV co-infection and symptomatic KS from each other. METHODS: To investigate how metabolic changes may correlate with co-infections and tumorigenesis, plasma samples derived from KSHV seropositive sub-Saharan African subjects in three groups, (A) asymptomatic (lacking neoplastic disease) with KSHV infection only, (B) asymptomatic co-infected with KSHV and HIV, and (C) symptomatic with clinically diagnosed KS, were subjected to analysis of lipid and polar metabolite profiles RESULTS: Polar and nonpolar plasma metabolic differentials were evident in both comparisons. Integration of the metabolic findings with our previously reported KS transcriptomics data suggests dysregulation of amino acid/urea cycle and purine metabolic pathways, in concert with viral infection in KS disease progression. CONCLUSIONS: This study is, to our knowledge, the first to report human plasma metabolic differentials between in vivo KSHV infection and co-infection with HIV, as well as differentials between co-infection and epidemic KS.

Collection of Plasma Samples in Areas with Limited Healthcare Access.

Regular monitoring of various biomarkers and molecular panels in plasma can significantly help to prevent disease onset and improve its management and final outcomes. Many groups can benefit from monitoring programs focusing on the prevention of cardiovascular diseases, evaluation of environmental exposure impacts, or the prevention/management of cancer. Improvement in therapeutic options in part due to targeted therapeutic agents and monoclonal antibody therapies has led to a significant sized population that can be described as "cancer survivors." These patients, although in remission from their original disease, are at significant risk for the recurring disease and must be monitored for adverse events. Monitoring is, however, not an easy task; requiring a high level of complexity in lab facilities and blood/plasma sampling, collection, and storage must occur under tightly controlled conditions. These demanding circumstances are especially difficult to attain in rural areas and in historically marginalized populations. The Telimmune Plasma Separation Card (TPS card or TPSC) has been developed to enable diagnostic plasma sampling, collection, and stabilization in locations that may be remote to laboratory or clinic. The TPSC requires a drop of blood applied to a top of a separation system consisting of a separation membrane and collection disk. In 3 min, the TPSC device separates plasma from erythrocytes and deposits a defined volume of plasma into a collection disc which is air-dried for 15 min to deliver a stabilized, volumetric plasma sample, which may be stored or shipped at ambient temperatures with minimal biological risk. Extraction of proteins and metabolites is then achieved in well-equipped laboratories using protocols discussed in this chapter.

The effects of mercury exposure on Amazonian fishes: An investigation of potential biomarkers.

Exposure to mercury can interfere with the expression of proteins and enzymes, compromise important pathways, such as apoptosis and glucose metabolism, and even induce the expression of metallothioneins. In this study, analytical techniques were used to determine the concentration of total mercury (THg) in muscle and liver tissue, protein pellets, and spots [using graphite furnace atomic absorption spectrometry (GFAAS)], and molecular techniques were used to identify metalloproteins present in mercury-associated protein spots. Thirty individuals from three different fish species, Cichla sp. (n = 10), Brachyplatystoma filamentosum (n = 10), and Semaprochilodus sp. (n = 10) from the Brazilian Amazon were used. Oxidative stress indicators [such as glutathione peroxidase (GSH-Px), catalase (CAT), superoxide dismutase (SOD), a marker of lipid peroxidation (LPO)] and the possible expression of metallothioneins in muscle and liver tissues were investigated. The two piscivorous species, Cichla sp. and B. filamentosum, presented the highest concentrations of mercury in their hepatic tissue, 1219 ± 15.00 and 1044 ± 13.6 µg kg-1, respectively, and in their muscle tissue, 101 ± 1.30 µg kg-1 and 87.4 ± 0.900 µg kg-1, respectively. The non-carnivorous species Semaprochilodus sp. had comparatively low concentrations of mercury in both its hepatic (852 ± 11.1 µg kg-1) and muscle (71.4 ± 0.930 µg kg-1) tissues. The presence of mercury was identified in 24 protein spots using GFAAS; concentrations ranged from 11.5 to 787 µg kg-1, and mass spectrometry identified 21 metal-binding proteins. The activities of GSH-Px, CAT, and SOD, related to oxidative stress, decreased proportionally as tissue Hg concentrations increased, while the levels of LPO markers increased, indicating the presence of stress. Our study results demonstrate possible mercury interference in oxidative stress markers (GSH-Px, CAT, SOD, and LPO), in addition to the identification of 21 metal-binding proteins as possible biomarkers of mercury exposure in fish.

Metalloproteomic approach to liver tissue of rats exposed to mercury.

The aims of this study were to identify mercury-associated protein spots in the liver tissue of rats exposed to low concentrations of mercury and to elucidate the physiological and functional aspects of the proteins identified in the protein spots. Therefore, proteomic analysis of the liver tissue of Wistar rats exposed to mercury chloride (4.60 µg kg-1 in Hg2+) was performed for thirty days (Hg-30 group) and sixty days (Hg-60 group). The proteomic profile of the liver tissue of the rats was obtained by two-dimensional electrophoresis (2D-PAGE), and the determinations of total mercury in the liver tissue, pellets and protein spots were performed by graphite furnace atomic absorption spectrometry (GFAAS). ImageMaster 2D Platinum 7.0 software was used to identify the differentially expressed mercury-associated protein spots, which were then characterized by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The determinations by GFAAS indicated a total mercury bioaccumulation of 2812% in the Hg-30 group and 3298% in the Hg-60 group and 10 mercury-associated protein spots with a concentration range of 51 ± 1.0 to 412 ± 6.00 mg kg-1 in the 2D PAGE gels from the liver tissue of the Hg-60 group. The LC-MS/MS analyses allowed the identification of 11 metal binding proteins in mercury-associated protein spots that presented fold change with upregulation >1.5, downregulation < -1.7 or that were expressed only in the Hg-60 group. Using the FASTA sequences of the proteins identified in the mercury-associated protein spots, bioinformatics analyses were performed to elucidate the physiological and functional aspects of the metal binding proteins, allowing us to infer that enzymes such as GSTM2 presented greater mercury concentrations and downregulation < -3; Acaa2 and Bhmt, which showed expression only in the Hg-60 group, among others, may act as potential mercury exposure biomarkers.

Metalloproteomic Investigation of Hg-Binding Proteins in Renal Tissue of Rats Exposed to Mercury Chloride.

Results obtained from rat studies indicate that, even at low concentrations, mercurial species cause harmful effects on the kidneys, by inducing the nephrotic oxidative stress response. In the present work, Hg-associated proteins were identified as possible mercury-exposure biomarkers in rat kidneys exposed to low mercury chloride concentrations for 30 days (Hg-30) and 60 days (Hg-60), using metalloproteomic strategies. The renal proteomic profile was fractioned by two-dimensional electrophoresis and the mercury determinations in kidney samples, protein pellets and protein spots were performed using graphite furnace atomic absorption spectrometry. The characterization of Hg-associated protein spots and the analysis of differentially expressed proteins were performed by liquid chromatography, coupled with tandem mass spectrometry. Eleven Hg-associated protein spots with a concentration range of 79 ± 1 to 750 ± 9 mg kg-1 in the Hg-60 group were identified. The characterization and expression analyses allowed the identification of 53 proteins that were expressed only in the Hg-60 group, 13 "upregulated" proteins (p > 0.95) and 47 "downregulated" proteins (p < 0.05). Actin isoforms and hemoglobin subunits were identified in protein spots of the Hg-60 group, with mercury concentrations in the range of 138 to 750 mg kg-1, which qualifies these proteins as potential mercury-exposure biomarkers.

Mercury metalloproteomic profile in muscle tissue of Arapaima gigas from the Brazilian Amazon.

Metalloproteomics is an innovative methodology for identifying of protein-associated mercury. Thus, we analyzed the muscle proteome of Arapaima gigas (pirarucu), collected in the Madeira River of the Brazilian Amazon, to identify protein-associated mercury, with the aim of identifying possible mercury biomarkers in fish muscle tissue. After obtaining the protein pellet, we conducted two-dimensional electrophoresis (2D PAGE) to fractionate the muscle proteome. Total mercury in muscle tissue and protein pellets and mapping of mercury content in protein spots of the 2D PAGE gels was determined using graphite furnace atomic absorption spectrometry (GFAAS). The protein-associated mercury identification was performed using liquid chromatography coupled with sequence mass spectrometry (LC‒MS/MS). Total mercury determinations by GFAAS indicated concentrations on the order of 153 ± 1.90 mg kg-1 and 142 ± 1.50 mg kg-1 (total precipitation of protein fraction) and 139 ± 1.45 mg kg-1 (fractional precipitation of protein fraction) in muscle tissue and protein pellets, respectively. Mercury concentrations in the range of 48 ± 0.90 to 165 ± 3.00 mg kg-1 were found in twelve protein spots. Among the 2D PAGE protein spots, eleven Hg-binding proteins were identified using LC‒MS/MS, which showed characteristics of mercury exposure biomarkers for important metabolic functions, such as five parvalbumin isoforms, triosephosphate isomerase, cofilin 2 (muscle), and fructose-bisphosphate aldolases.

Peptidomics analysis reveals changes in small urinary peptides in patients with interstitial cystitis/bladder pain syndrome.

Interstitial cystitis/bladder pain syndrome (IC/BPS) is a chronic and debilitating pain disorder of the bladder and urinary tract with poorly understood etiology. A definitive diagnosis of IC/BPS can be challenging because many symptoms are shared with other urological disorders. An analysis of urine presents an attractive and non-invasive resource for monitoring and diagnosing IC/BPS. The antiproliferative factor (APF) peptide has been previously identified in the urine of IC/BPS patients and is a proposed biomarker for the disorder. Nevertheless, other small urinary peptides have remained uninvestigated in IC/BPS primarily because protein biomarker discovery efforts employ protocols that remove small endogenous peptides. The purpose of this study is to investigate the profile of endogenous peptides in IC/BPS patient urine, with the goal of identifying putative peptide biomarkers. Here, a non-targeted peptidomics analysis of urine samples collected from IC/BPS patients were compared to urine samples from asymptomatic controls. Our results show a general increase in the abundance of urinary peptides in IC/BPS patients, which is consistent with an increase in inflammation and protease activity characteristic of this disorder. In total, 71 peptides generated from 39 different proteins were found to be significantly altered in IC/BPS. Five urinary peptides with high variable importance in projection (VIP) coefficients were found to reliably differentiate IC/BPS from healthy controls by receiver operating characteristic (ROC) analysis. In parallel, we also developed a targeted multiple reaction monitoring method to quantify the relative abundance of the APF peptide from patient urine samples. Although the APF peptide was found in moderately higher abundance in IC/BPS relative to control urine, our results show that the APF peptide was inconsistently present in urine, suggesting that its utility as a sole biomarker of IC/BPS may be limited. Overall, our results revealed new insights into the profile of urinary peptides in IC/BPS that will aid in future biomarker discovery and validation efforts.

Feedlot diets containing different starch levels and additives change the cecal proteome involved in cattle's energy metabolism and inflammatory response.

Diets for feedlot cattle must be a higher energy density, entailing high fermentable carbohydrate content. Feed additives are needed to reduce possible metabolic disorders. This study aimed to analyze the post-rumen effects of different levels of starch (25%, 35%, and 45%) and additives (monensin or a blend of essential oils and exogenous α-amylase) in diets for Nellore feedlot cattle. The cecum tissue proteome was analyzed via two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and then differentially expressed protein spots were identified with liquid chromatography-tandem mass spectrometry (LC-MS/MS). The use of blends of essential oils associated with α-amylase as a feed additive promoted the upregulation of enzymes such as triosephosphate isomerase, phosphoglycerate mutase, alpha-enolase, beta-enolase, fructose-bisphosphate aldolase, pyruvate kinase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), L-lactate dehydrogenase B, L-lactate dehydrogenase A chain, L-lactate dehydrogenase, and ATP synthase subunit beta, which promote the degradation of carbohydrates in the glycolysis and gluconeogenesis pathways and oxidative phosphorylation, support pyruvate metabolism through the synthesis of lactate from pyruvate, and participate in the electron transport chain, producing ATP from ADP in the presence of a proton gradient across the membrane. The absence of proteins related to inflammation processes (leukocyte elastase inhibitors) in the cecum tissues of animals fed essential oils and amylase may be because feed enzymes can remain active in the intestine and aid in the digestion of nutrients that escape rumen fermentation; conversely, the effect of monensin is more evident in the rumen and less than 10% results in post-ruminal action, corroborating the hypothesis that ionophore antibiotics have a limited effect on the microbiota and intestinal fermentation of ruminants. However, the increase in starch in these diets promoted a downregulation of enzymes linked to carbohydrate degradation, probably caused by damage to the cecum epithelium due to increased responses linked to inflammatory injuries.

Role of Specialized Pro-resolving Mediators in Reducing Neuroinflammation in Neurodegenerative Disorders.

Alzheimer's disease (AD) and Parkinson's disease (PD) are neurodegenerative disorders that affect millions of individuals worldwide. As incidence of these conditions increases with age, there will undoubtedly be an increased prevalence of cases in the near future. Neuroinflammation is a hallmark in the development and progression of neurodegenerative diseases and prevention or resolution of chronic neuroinflammation may represent a novel approach to treatment. The present review highlights the potential of the anti-inflammatory and pro-resolving effects of polyunsaturated fatty acid (PUFA)-derived mediators (Specialized Pro-resolving Mediators-SPM) in neurodegenerative disorders. PUFA-derived SPM are biosynthesized in response to chemicals produced from acute inflammatory responses. Preclinical studies from both AD and PD models suggest a dysregulation of SPM and their receptors in neurological disorders. Decreased SPM may be due to inadequate substrate, an imbalance between SPM and pro-inflammatory mediators or a disruption in SPM synthesis. SPMs hold great promise for neuroprotection in AD by altering expression of pro-inflammatory genes, modulating macrophage function, serving as a biomarker for AD status, and promoting resolution of neuroinflammation. In PD, data suggest SPM are able to cross the blood-brain barrier, inhibit microglial activation and decrease induced markers of inflammation, possibly as a result of their ability to downregulate NFκB signaling pathways. Several in vivo and in vitro studies suggest a benefit from administration of SPMs in both neurodegenerative disorders. However, extrapolation of these outcomes to humans is difficult as no models are able to replicate all features of AD or PD. Minimal data evaluating these PUFA-derived metabolites in humans with neurodegenerative disorders are available and a gap in knowledge exists regarding behavior of SPM and their receptors in patients with these conditions. There is also large gap in our knowledge regarding which lipid mediator would be most effective in which model of AD or PD and how dietary intake or supplementation can impact SPM levels. Future direction should include focused, translational efforts to investigate SPM as an add-on (in addition to standard treatment) or as standalone agents in patients with neurodegenerative disorders.

Ultrasonication of Milk Decreases the Content of Exosomes and MicroRNAs in an Exosome-Defined Rodent Diet.

BACKGROUND: Bovine milk exosomes (BMEs) harbor regulatory proteins, lipids, and microRNAs. Consumption of an exosome- and RNA-depleted (ERD) diet elicited phenotypes compared with controls fed an exosome- and RNA-sufficient (ERS) diet in mice. All other ingredients were identical in the diets. ERD and ERS diets were prepared by substituting ultrasonicated and nonultrasonicated milk, respectively, for casein in the AIN-93G formulation. OBJECTIVES: The objective of this study was to assess the effect of ultrasonication of milk on exosome content and bioavailability, and cargo content. METHODS: Bovine milk was ultrasonicated and exosomes were isolated by ultracentrifugation [ultrasonicated exosomes (USEs)]; controls were not ultrasonicated [nonultrasonicated exosomes (NSEs)]. Exosome count, size, and morphology were assessed using a nanoparticle tracker and electron microscopy. RNAs, lipids, and proteins were analyzed by RNA sequencing and MS. Intestinal transport, bioavailability, and distribution were measured by using fluorophore-labeled USEs and NSEs in Caco-2 cells, FHs 74 Int cells, and C57BL/6J mice (n = 3; age: 6-8 wk). RESULTS: The exosome count was 76% ± 22% lower in USEs than in NSEs (P < 0.05). Ultrasonication caused a degradation of ≤100% of microRNAs. USEs and NSEs contained 145 and 332 unique lipid signatures, respectively (P < 0.05). We detected a total of 525 and 484 proteins in USEs and NSEs, respectively. The uptake of USEs decreased by 46% ± 30% and 40% ± 27% compared with NSEs in Caco-2 and FHs 74 Int cells, respectively (P < 0.05). The hepatic accumulation of USEs was 48% ± 28% lower than the accumulation of NSEs in mice (P < 0.05). CONCLUSIONS: Ultrasonication of milk depletes bioavailable BMEs in studies of Caco-2 cells, FHs 74 Int cells, and C57BL/6J mice and causes a near-complete degradation of microRNA cargos.

Investigation of Protein Biomarkers and Oxidative Stress in Pinirampus pirinampu Exposed to Mercury Species from the Madeira River, Amazon-Brazil.

In recent decades, the scientific community has widely debated the contamination of fish in the Amazon region by mercury species. As the diet of riverside populations in the Amazon region is based mainly on fish, these populations are exposed to mercurial species that can cause serious and irreversible damage to their health. The risks of consuming fish exposed to mercurial species in the Amazon region have motivated toxicological investigations. However, the effect of mercurial species on protein and enzyme levels is still controversial. In this work, analytical and bioanalytical techniques Two-dimensional polyacrylamide gel electrophoresis [2D-PAGE] Graphite Furnace Atomic Absorption Spectrometry [GFAAS], and Mass Spectrometry in Sequence with Electrospray Ionization [ESI-MS/MS] were used to identify proteins associated with mercury (metal-binding protein) in muscle and liver tissues of the fish species Pinirampus pirinampu from the Madeira River, in the Brazilian Amazon. Enzymatic and lipid peroxidation analyses were also used to assess changes related to oxidative stress. Determinations of total mercury by GFAAS indicated higher concentrations in liver tissue (555 ± 19.0 µg kg-1) when compared to muscle tissue (60 ± 2.0 µg kg-1). The fractionation process of tissue proteomes by 2D-PAGE and subsequent mapping of mercury by GFAAS in the protein spots of the gels identified the presence of mercury in three spots of the liver tissue (concentrations in the range of 0.800 to 1.90 mg kg-1). The characterization of protein spots associated with mercury by ESI-MS/MS identified the enzymes triosephosphate isomerase A, adenylate kinase 2 mitochondrial, and glyceraldehyde-3-phosphate dehydrogenase as possible candidates for mercury exposure biomarkers. The muscle tissue did not show protein spots associated with mercury. Enzymatic activity decreased proportionally to the increase in mercury concentrations in the tissues.

Isolation of extracellular vesicles from byproducts of cheesemaking by tangential flow filtration yields heterogeneous fractions of nanoparticles.

Extracellular vesicles (EV) in milk, particularly exosomes, have attracted considerable attention as bioactive food compounds and for their use in drug delivery. The utility of small EV in milk (sMEV) as an animal feed additive and in drug delivery would be enhanced by cost-effective large-scale protocols for the enrichment of sMEV from byproducts in dairy plants. Here, we tested the hypothesis that sMEV may be enriched from byproducts of cheesemaking by tangential flow filtration (EV-FF) and that the sMEV have properties similar to sMEV prepared by ultracentrifugation (sMEV-UC). Three fractions of EV were purified from the whey fraction of cottage cheese making by using EV-FF that passed through a membrane with a 50-kDa cutoff (50 penetrate; 50P), and subfractions of 50P that were retained (100 retentate; 100R) or passed through (100 penetrate; 100P) a membrane with a 100-kDa cutoff; sMEV-UC controls were prepared by serial ultracentrifugation. The abundance of sMEV (<200 nm) was less than 0.3% in EV-FF compared with sMEV-UC (1012/mL of milk). Despite the low EV count, the protein content (mg/mL) of 100R (63 ± 0.02; ± standard deviation) was higher than that of 50P (0.75 ± 0.10), 100P (0.65 ± 0.40), and sMEV-UC (27 ± 0.02). There were 17, 14, 35, and 75 distinct proteins detected by nontargeted mass spectrometry analysis in 50P, 100R, 100P, and sMEV-UC, respectively. Exosome markers CD9, CD63, CD81, HSP-70, PDCD6IP, and TSG101 were detected in control sMEV-UC but not in EV-FF by using targeted mass spectrometry and immunoblot analyses. Negative exosome markers, APOB, ß-integrin, and histone H3 were below the limit of detection in EV-FF and control sMEV-UC analyzed by immunoblotting. The abundance of the major milk fat globule protein butyrophilin showed the following pattern: 100R ≫ 100P = 50P > sMEV-UC. More than 100 mature microRNA were detected in sMEV-UC by using sequencing analysis, compared with 36 to 60 microRNA in EV-FF. Only 100R and sMEV-UC yielded mRNA in quantities and qualities sufficient for sequencing analysis; an average of 276,000 and 838,000 reads were mapped to approximately 14,600 and 18,500 genes in 100R and sMEV-UC, respectively. In principal component analysis, microRNA, mRNA, and protein in EV-FF preparations clustered separately from control sMEV-UC. We conclude that under the conditions used here, flow filtration yields a heterogeneous population of milk EV.

An in silico, structural, and biological analysis of lactoferrin of different mammals.

Lactoferrin (LF) belongs to the family of transferrins having multifunctional roles associated with the immune system of animals. To follow the aims for this study was selected 20 sequences of LF from mammalian species to evaluate the chemical, biological, and structural properties. Bioinformatics approaches used programs such as MAFFT for sequence alignment; PartitionFinder and MrBayes for phylogenetic approaches; I-TASSER, PROCHECK, Molecular Operating Environment (MOE), SWISS Model server, Peptide DB and Expasy ProtParam to estimate the physicochemical properties, to model the protein and predicted secondary structures. A phylogenic analysis shows species with genetic similarities clustered by complexity and unique grouping between Capra hircus, Macaca mulatta, and Myotis lucifugus, since they presented more amino acids but not overall changes in the iron-binding sites or biological aspects. Structural deviations in these clusters obtained in LF from those species were found in residues 46 (position 406-450), that is part of alpha-helix, and 37 (position 295-331), that is part of the beta-sheets. Our predicted model can be used to investigate more about structural aspects of LF and be applied for medicinal research.

Biomechanical assessment of various punching techniques.

Punches without the use of instruments/objects are a common type of body violence and as such a frequent subject of medicolegal analyses. The assessment of the injuries occurred as well as of the potential of the assault to produce severe body harm is based on objective traces (especially the documented injuries of both parties involved) as well as the-often divergent-descriptions of the event. Quantitative data regarding the punching characteristics that could be used for the assessment are rare and originate mostly in sports science. The aim of this study was to provide physical data enabling/facilitating the assessment of various punching techniques. A total of 50 volunteers took part in our study (29 males and 21 females) and performed severe punches with the fist, with the small finger edge of the hand (karate chop), and with the open hand with both the dominant and the non-dominant hands in randomized order. The strikes were performed on a boxing pad attached to a KISTLER force plate (sampling frequency 10,000 Hz) mounted on a vertical wall. The punching velocity was defined as the hand velocity over the last 10 cm prior to the contact to the pad and ascertained by using a high-speed camera (2000 Hz). Apart from the strike velocity, the maximum force, the impulse (the integral of the force-time curve), the impact duration, and the effective mass of the punch (the ratio between the impulse and the strike velocity) were measured/calculated. The results show a various degree of dependence of the physical parameters of the strikes on the punching technique, gender, hand used, body weight, and other factors. On the other hand, a high degree of variability was observed that is likely attributable to individual punching capabilities. In a follow-up study, we plan to compare the "ordinary" persons with highly trained (boxers etc.) individuals. Even though the results must be interpreted with great caution and a direct transfer of the quantitative parameters to real-world situations is in general terms not possible, the study offers valuable insights and a solid basis for a qualified forensic medical/biomechanical assessment.

Changes in lipid profiles of epileptic mouse model.

INTRODUCTION: Approximately 1% of the world's population is impacted by epilepsy, a chronic neurological disorder characterized by seizures. One-third of epileptic patients are resistant to AEDs, or have medically refractory epilepsy (MRE). One non-invasive treatment that exists for MRE includes the ketogenic diet, a high-fat, low-carbohydrate diet. Despite the KD's success in seizure attenuation, it has a few risks and its mechanisms remain poorly understood. The KD has been shown to improve metabolism and mitochondrial function in epileptic phenotypes. Potassium channels have implications in epileptic conditions as they have dual roles as metabolic sensors and control neuronal excitation. OBJECTIVES: The goal of this study was to explore changes in the lipidome in hippocampal and cortical tissue from Kv1.1-KO model of epilepsy. METHODS: FT-ICR/MS analysis was utilized to examine nonpolar metabolome of cortical and hippocampal tissue isolated from a Kv1.1 channel knockout mouse model of epilepsy (n = 5) and wild-type mice (n = 5). RESULTS: Distinct metabolic profiles were observed, significant (p < 0.05) features in hippocampus often being upregulated (FC ≥ 2) and the cortex being downregulated (FC ≤ 0.5). Pathway enrichment analysis shows lipid biosynthesis was affected. Partition ratio analysis revealed that the ratio of most metabolites tended to be increased in Kv1.1-/-. Metabolites in hippocampal tissue were commonly upregulated, suggesting seizure initiation in the hippocampus. Aberrant mitochondrial function is implicated by the upregulation of cardiolipin, a common component in the mitochondrial membrane. CONCLUSION: Generally, our study finds that the lipidome is changed in the hippocampus and cortex in response to Kv1.1-KO indicating changes in membrane structural integrity and synaptic transmission.

Aberrant energy metabolism and redox balance in seizure onset zones of epileptic patients.

Epilepsy is a disorder that affects around 1% of the population. Approximately one third of patients do not respond to anti-convulsant drugs treatment. To understand the underlying biological processes involved in drug resistant epilepsy (DRE), a combination of proteomics strategies was used to compare molecular differences and enzymatic activities in tissue implicated in seizure onset to tissue with no abnormal activity within patients. Label free quantitation identified 17 proteins with altered abundance in the seizure onset zone as compared to tissue with normal activity. Assessment of oxidative protein damage by protein carbonylation identified additional 11 proteins with potentially altered function in the seizure onset zone. Pathway analysis revealed that most of the affected proteins are involved in energy metabolism and redox balance. Further, enzymatic assays showed significantly decreased activity of transketolase indicating a disruption of the Pentose Phosphate Pathway and diversion of intermediates into purine metabolic pathway, resulting in the generation of the potentially pro-convulsant metabolites. Altogether, these findings suggest that imbalance in energy metabolism and redox balance, pathways critical to proper neuronal function, play important roles in neuronal network hyperexcitability and can be used as a primary target for potential therapeutic strategies to combat DRE. SIGNIFICANCE: Epileptic seizures are some of the most difficult to treat neurological disorders. Up to 40% of patients with epilepsy are resistant to first- and second-line anticonvulsant therapy, a condition that has been classified as refractory epilepsy. One potential therapy for this patient population is the ketogenic diet (KD), which has been proven effective against multiple refractory seizure types However, compliance with the KD is extremely difficult, and carries severe risks, including ketoacidosis, renal failure, and dangerous electrolyte imbalances. Therefore, identification of pathways disruptions or shortages can potentially uncover cellular targets for anticonvulsants, leading to a personalized treatment approach depending on a patient's individual metabolic signature.

Validity of plasma collection cards for ferritin assessment-A proof-of-concept study.

OBJECTIVES: Iron depletion is common around the world and among certain risk groups in developed countries. The overall purpose was to test the suitability of a novel plasma collection card for minimally invasive iron status assessment. METHODS: Twenty participants (10 f/10 m) participated in this cross-sectional study. Ferritin and hemoglobin were measured from blood collected from a forearm vein, serving as reference method. Blood was also collected from the fingertip using the NoviplexTM Plasma Prep Card as well as capillary collection tubes. RESULTS: There was substantial concordance between ferritin measured from samples collected via NoviplexTM and venous ferritin (concordance correlation coefficient (CCC) = 0.96) with a mean bias of -0.8 ng/mL. Storing NoviplexTM cards at room temperature for 2 weeks resulted in slightly lower but good concordance when compared to venous ferritin (CCC = 0.95). Capillary hemoglobin (CCC = 0.42) and hematocrit (CCC = 0.25) were in poor agreement with venous data. CONCLUSIONS: NoviplexTM cards offer a suitable alternative for a minimally invasive ferritin screening in the field when compared to capillary collection tubes. Despite overall substantial concordance with the reference method, findings indicative of iron status abnormalities should be confirmed in venous samples.

Preparation, FPLC Purification and LC-FT-ICR-MS of Proteins.

High magnetic field Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometers provide extremely high mass resolution (resolving power of ~200,000 at 400 m/z) protein detection across a broad mass range, enabling analysis of fine structure of isotopic peak clusters that is missed in other types of mass spectrometers. The protocol detailed here describes preparation of cellular extracts for purification of DNA-binding proteins using multiple chromatographic chemistries via fast protein liquid chromatography (FPLC), and identification and quantitation of the protein isoforms and their post-translational modifications by liquid chromatography coupled to Fourier transform ion cyclotron resonance mass spectrometry (LC-FT-ICR-MS). This protocol benefits from selectively purifying proteins for identification and quantitation by high resolution FT-ICR, which has the resolution to definitively distinguish between acetylation and trimethylation post-translational modification (PTM) additions.