The Influence of Viral Infections on Iron Homeostasis and the Potential for Lactoferrin as a Therapeutic in the Age of the SARS-CoV-2 Pandemic.

The association of hyperinflammation and hyperferritinemia with adverse outcomes in SARS-CoV-2-infected patients suggests an integral role for iron homeostasis in pathogenesis, a commonly described symptom of respiratory viral infections. This dysregulated iron homeostasis results in viral-induced lung injury, often lasting long after the acute viral infection; however, much remains to be understood mechanistically. Lactoferrin is a multipurpose glycoprotein with key immunomodulatory, antimicrobial, and antiviral functions, which can be found in various secreted fluids, but is most abundantly characterized in milk from all mammalian species. Lactoferrin is found at its highest concentrations in primate colostrum; however, the abundant availability of bovine-dairy-derived lactoferrin (bLf) has led to the use of bLf as a functional food. The recent research has demonstrated the potential value of bovine lactoferrin as a therapeutic adjuvant against SARS-CoV-2, and herein this research is reviewed and the potential mechanisms of therapeutic targeting are considered.

Evaluation of protein expression in bovine bronchoalveolar fluid following challenge with Mannheimia haemolytica.

Proteomics analysis of bovine bronchoalveolar fluid (BAF) following induction of pneumonia with Mannheimia haemolytica using nanoflow liquid chromatography coupled with tandem mass spectrometry (nanoLC-MS/MS) resulted in the identification of 88 unique proteins. Proteins detected in BAF included antimicrobial peptides (AMPs), complement factors, acute-phase proteins, protease inhibitors, and proteins involved in oxidation-reduction. Notwithstanding biological variation, differences in relative protein abundance, determined using normalized peptide counts, were detected for select proteins in BAF from genuinely infected versus sham-infected animals. To demonstrate the applicability of using normalized peptide counts to assess protein expression trends, LC-MS/MS data for the acute-phase protein haptoglobin (HPT) were compared with ELISA data, and statistical evaluation of the relationship between the data revealed a strong measure of association. Differences were detected between sham- and genuinely infected animals for haptoglobin, as well as the AMPs cathelicidin-1 and cathelicidin-4, and inter-α-trypsin inhibitor heavy chain-4, a fairly novel protein involved in the acute phase response. Though the small sample size limited the scope of the inferences, the results indicate the likely importance of AMPs and acute-phase proteins during respiratory infection, and provide additional information regarding potential mechanisms involved in the bovine mucosal barrier defense.

A No Observable Adverse Effects Level (NOAEL) for pigs fed melamine and cyanuric acid.

Ingesting melamine adulterated milk products led to kidney stones in many infants in 2008. This differs from the renal failure caused by intratubular crystal formation after co-ingestion of melamine (MEL) and cyanuric acid (CYA) in adulterated pet foods in 2007. To better understand the potential risk of developing crystal nephropathy following co-ingestion of MEL and CYA, we fed 16 weanling pigs 0, 1, 3.3, 10, 33, or 100 mg/kg bw/day of each MEL and CYA, or 200 mg/kg bw/day of either compound individually for 7 days. Crystals were found in the renal medulla and cortex and urine sediments of all pigs fed both MEL and CYA each at 10 mg/kg bw/day (or greater). Crystals were also found in one of the two pigs fed 200 mg/kg bw/day MEL-only. In a 28 day study, 36 weanling pigs were fed 0, 1, or 3.3 mg/kg bw/day of MEL and CYA or 200 mg/kg bw/day MEL-only. Only one of the 3.3 mg/kg MEL and CYA pig kidneys contained crystals. The no-observed-adverse-effect level (NOAEL) for pigs fed MEL and CYA for 28 days was concluded to be 1.0 mg/kg bw/day corresponding to 25 mg/kg (ppm) MEL and 25 mg/kg (ppm) CYA in dry feed.

The proteomic advantage: label-free quantification of proteins expressed in bovine milk during experimentally induced coliform mastitis.

Coliform mastitis remains a primary focus of dairy cattle disease research due in part to the lack of efficacious treatment options for the deleterious side effects of exposure to LPS, including profound intra-mammary inflammation. To facilitate new veterinary drug approvals, reliable biomarkers are needed to evaluate the efficacy of adjunctive therapies for the treatment of inflammation associated with coliform mastitis. Most attempts to characterize the host response to LPS, however, have been accomplished using ELISAs. Because a relatively limited number of bovine-specific antibodies are commercially available, reliance on antibodies can be very limiting for biomarker discovery. Conversely, proteomic approaches boast the capability to analyze an unlimited number of protein targets in a single experiment, independent of antibody availability. Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), a widely used proteomic strategy for the identification of proteins in complex mixtures, has gained popularity as a means to characterize proteins in various bovine milk fractions, both under normal physiological conditions as well as during clinical mastitis. The biological complexity of bovine milk has, however, precluded the complete annotation of the bovine milk proteome. Conventional approaches to reducing sample complexity, including fractionation and the removal of high abundance proteins, has improved proteome coverage, but the dynamic range of proteins present, and abundance of a relatively small number of proteins, continues to hinder comparative proteomic analyses of bovine milk. Nonetheless, advances in both liquid chromatography and mass spectrometry instrumentation, including nano-flow liquid chromatography (nano-LC), nano-spray ionization, and faster scanning speeds and ionization efficiency of mass spectrometers, have improved analyses of complex samples. In the current paper, we review the proteomic approaches used to conduct comparative analyses of milk from healthy cows and cows with clinical mastitis, as well as proteins related to the host response that have been identified in mastitic milk. Additionally, we present data that suggests the potential utility of LC-MS/MS label-free quantification as an alternative to costly labeling strategies for the relative quantification of individual proteins in complex mixtures. Temporal expression patterns generated using spectral counts, an LC-MS/MS label-free quantification strategy, corresponded well with ELISA data for acute phase proteins with commercially available antibodies. Combined, the capability to identify low abundance proteins, and the potential to generate temporal expression profiles, indicate the advantages of using proteomics as a screening tool in biomarker discovery analyses to assess biologically relevant proteins modulated during disease, including previously uncharacterized targets.

Genomic organization and functional characterization of the human concentrative nucleoside transporter-3 isoform (hCNT3) expressed in mammalian cells.

Human CNT3 encodes the concentrative nucleoside transport N3 system. Previous expression studies in oocytes showed that the Km values for nucleosides of the cloned hCNT3 were 7- to 25-fold lower than the endogenous N3 transporter in HL60 cells. Therefore, in the present study we re-examined the kinetic properties of the cloned hCNT3 using mammalian cell expression systems by transient expression in Cos7L cells and stably expression in nucleoside transporter deficient PK15NTD cells. We demonstrated that hCNT3 is a Na-dependent, broadly-selective nucleoside transporter with affinities (<11 microM) for nucleosides closely resembling the endogenous N3 transporter. Pharmacological studies showed that phloridzin is a mixed-type inhibitor of hCNT3 (Ki=15 microM), and the dideoxyuridine analogs are poor substrates. By epitope-tagging, we further demonstrated that hCNT3 is N-glycosylated as PNGase F and Endo H deglycosylated hCNT3 from 67 kDa to 58 kDa. Searching the human genome database, we identified the genomic organization of hCNT3. This gene contains 19 exons and its exon-intron boundaries within the coding sequence exactly match with those of hCNT1 and hCNT2 with one additional exon in the N-terminus. Our data suggest that hCNT3 gene is evolutionarily conserved with hCNT1 and hCNT2. Physiologically, hCNT3 is a glycoprotein, which transports purine and pyrimidine nucleosides in a Na-dependent manner with high affinities.

Functional analysis of site-directed glycosylation mutants of the human equilibrative nucleoside transporter-2.

Protein glycosylation is important for nucleoside transport, and this has been demonstrated for the human equilibrative nucleoside transporter-1 (hENT1). It is not known whether glycosylation affects the functions of hENT2 or where hENT2 is glycosylated. We address these questions using N-glycosylation mutants (N48D, N57D, and N48/57D) and demonstrate that hENT2 is glycosylated at Asn(48) and Asn(57). Our results show that although the apparent affinities for [3H]uridine and [3H]cytidine of the mutants were indistinguishable from those of the wild-type protein, N-glycosylation was required for efficient targeting of hENT2 to the plasma membrane. All mutants had a two- to threefold increase in IC(50) for dipyridamole. N57D and N48/57D, but not N48D, also had a twofold increase in IC(50) for NBMPR. We conclude that the relative insensitivity of hENT2 to inhibitors is primarily due to its primary structure and not to glycosylation. Glycosylation modulates hENT1 function, but is not required for hENT2.