Ultrasensitive and specific measurement of protease activity using functionalized photonic crystals.

Herein is presented a microsensor technology as a diagnostic tool for detecting specific matrix metalloproteinases (MMPs) at very low concentrations. MMP-2 and MMP-9 are detected using label free porous silicon (PSi) photonic crystals that have been made selective for a given MMP by filling the nanopores with synthetic polymeric substrates containing a peptide sequence for that MMP. Proteolytic cleavage of the peptide sequence results in a shift in wavelength of the main peak in the reflectivity spectrum of the PSi device, which is dependent on the amount of MMP present. The ability to detect picogram amounts of MMP-2 and MMP-9 released by primary retinal pigment epithelial (RPE) cells and iris pigment epithelial (IPE) cells stimulated with lipopolysaccharide (LPS) is demonstrated. It was found that both cell types secrete higher amounts of MMP-2 than MMP-9 in their stimulated state, with RPE cells producing higher amounts of MMPs than IPE cells. The microsensor performance was compared to conventional protease detection systems, including gelatin zymography and enzyme linked immunosorbent assay (ELISA). It was found that the PSi microsensors were more sensitive than gelatin zymography; PSi microsensors detected the presence of both MMP-2 and MMP-9 while zymography could only detect MMP-2. The MMP-2 and MMP-9 quantification correlated well with the ELISA. This new method of detecting protease activity shows superior performance to conventional protease assays and has the potential for translation to high-throughput multiplexed analysis.

Microbial diversity in dairy manure environment under liquid-solid separation systems.

In dairy manure, a wide array of microorganisms, including many pathogens, survive and grow under suitable conditions. This microbial community offers a tremendous opportunity for studying animal health, the transport of microbes into the soil, air, and water, and consequential impacts on public health. The aim of this study was to assess the impacts of manure management practices on the microbial community of manure. The key novelty of this work is to identify the impacts of various stages of manure management on microbes living in dairy manure. In general, the majority of dairy farms in California use a flush system to manage dairy manure, which involves liquid-solid separations. To separate liquid and solid in manure, Multi-stage Alternate Dairy Effluent Management Systems (ADEMS) that use mechanical separation systems (MSS) or weeping wall separation systems (WWSS) are used. Thus, this study was conducted to understand how these manure management systems affect the microbial community. We studied the microbial communities in the WWSS and MSS separation systems, as well as in the four stages of the ADEMS. The 16S rRNA gene from the extracted genomic DNA of dairy manure was amplified using the NovoSeq Illumina next-generation sequencing platform. The sequencing data were used to perform the analysis of similarity (ANOSIM) and multi-response permutation procedure (MRRP) statistical tests, and the results showed that microbial communities among WWSS and MSS were significantly different (p < 0.05). These findings have significant practical implications for the design and implementation of manure management practices in dairy farms.

Physico-chemical assessment of on-farm bioconversion of organic waste in dairy farms in context to sustainability and circular bioeconomy.

On a milk-producing dairy farm, milk production is correlated with manure production and the number of cattle, and manure is widely used as a soil fertilizer. However, excessive dairy manure production is linked with greenhouse gas emissions and water quality issues. On-farm planning of manure storage and application to enhance soil nutrients are essential in a circular economy to reduce environmental impact, where manure is not landfilled and incinerated. Instead, it creates a nutrient resource for crops and soil. Dairy manure, which is rich in nutrients, is a valuable fertilizer that contains many nutrients such as nitrogen (N), organic matter (OM), phosphorous (P), Potassium (K) and micronutrients. In this work, a pilot field research was conducted between 2016 and 2018 in various parts of California, USA (San Joaquin Valley, Sacramento Valley, Shasta Cascade, and the North Coast of California) to assess physio-chemical characteristics of solid fractions of dairy manure among various dairy farms. A total of 156 samples were collected from the gut (n = 107) and toe (n = 49) of the manure piles across California for determining total solid (TS), volatile solid (VS), temperature, moisture content and carbon-nitrogen ratio (C: N). Here, using the observations of field study and analysis, we show that C: N, OM and MC of solid fractions of dairy manure vary significantly among dairy farms. The average C: N ratio of manure (26-32) among various regions was close to an ideal C: N value of 24:1 for soil microbes to stimulate nutrient release to crops. Manure pH ranged between 7.0 and 8.0, which was close to an optimal pH range for common crops (6.0-8.0). Moreover, considering less cost and surplus availability, manure will likely continue providing a cost-effective organic fertilizer resource compared to commercial chemical fertilizers.

Oocyst wall formation and composition in coccidian parasites.

The oocyst wall of coccidian parasites is a robust structure that is resistant to a variety of environmental and chemical insults. This resilience allows oocysts to survive for long periods, facilitating transmission from host to host. The wall is bilayered and is formed by the sequential release of the contents of two specialized organelles - wall forming body 1 and wall forming body 2 - found in the macrogametocyte stage of Coccidia. The oocyst wall is over 90% protein but few of these proteins have been studied. One group is cysteine-rich and may be presumed to crosslink via disulphide bridges, though this is yet to be investigated. Another group of wall proteins is rich in tyrosine. These proteins, which range in size from 8-31 kDa, are derived from larger precursors of 56 and 82 kDa found in the wall forming bodies. Proteases may catalyze processing of the precursors into tyrosine-rich peptides, which are then oxidatively crosslinked in a reaction catalyzed by peroxidases. In support of this hypothesis, the oocyst wall has high levels of dityrosine bonds. These dityrosine crosslinked proteins may provide a structural matrix for assembly of the oocyst wall and contribute to its resilience.

Keratin 14 Expression in Epithelial Progenitor Cells of the Developing Human Cornea.

A healthy and transparent cornea is essential for exquisite vision. During adulthood, its epithelium is constantly replenished through the activity of its stem cells (SCs). Precisely when these cells develop and their distribution across the ocular surface remain incompletely characterized in man. We postulated that the human fetal cornea harbors SCs that can be identified with keratin (K) 14 and αv-integrin, two markers we and others previously used to identify their adult counterparts. Immunofluorescence, cell culture, quantitative real-time polymerase chain reaction (qRT-PCR), and colony-forming assays were performed on fetal and adult biomaterial to locate progenitors and establish their phenotypic and functional properties. K14 was used to map the spatiotemporal distribution of precursor cell activity across the developing cornea, divulging a dynamic pattern of vertical and horizontal consolidated expression with increasing gestational age. K14 was coexpressed with αv-integrin in fetal and adult corneas and cultured corneolimbal epithelium, and colony-forming efficiency (an indicator of SC activity) was similar in cells from both sources. Finally, fetal cells were adherent, grew well, and maintained a K14 phenotype on contact lenses, a substrate we previously used to deliver cells to patients with blinding corneal disease. This study provides valuable insights into the development of the cornea, including the formation of the SC repository, the distribution of these cells across the ocular surface, and a preliminary attempt at harnessing, phenotyping, and functionally characterizing these cells. Future studies will focus on isolating fetal SCs to determine their utility as an alternative cell therapy for patients suffering from corneal blindness.

Role of toll-like receptors in human iris pigment epithelial cells and their response to pathogen-associated molecular patterns.

BACKGROUND: Toll-like receptor (TLR) activation is hypothesized to contribute to inflammatory eye disease including uveitis, yet the distribution pattern of TLRs in human uveal tissues remains poorly described. The purpose of this study was to investigate the expression profile of TLRs in human iris pigment epithelial cells (IPE) at the gene and protein level and examine the effect of pathogen-associated molecular patterns (PAMPs), such as Pam3CSK4.3HCl, Poly(I:C), lipopolysaccharides (LPS from E. coli serotype O111:B4), Flagellin, MALP-2 (macrophage activating lipopeptide-2), Poly(U) and CpGODN2395 on the production of inflammatory mediators including interleukin-8 (IL-8) and monocyte chemotactic protein 1 (MCP-1) from human IPE and retinal pigment epithelial cells (RPE). METHODS: RT-PCR and Western blotting was employed to investigate the expression of TLRs 1-10 in primary IPE and RPE. Secretion of IL-8 or MCP-1 following treatment with PAMPs was measured by ELISA. The role of TLR2, TLR3 and TLR4 in mediating an inflammatory response was investigated using pharmacological TLR inhibitors. RESULTS: IPE and RPE expressed transcripts for TLR1-6 and 8-10; and proteins for TLR1-6 and 9. IPE secreted IL-8 or MCP-1 in response to Pam3CSK4.3HCl, Poly(I:C), LPS and MALP-2, whereas RPE produced IL-8 only after Poly(I:C), LPS or MALP-2 treatment. TLR inhibitors (OxPAPC, CI-095 and chloroquine) blocked IL-8 secretion in Poly(I:C), LPS or MALP-2-treated IPE and RPE. CONCLUSIONS: Ocular pigment epithelial cells respond to PAMPs through activation of TLRs, particularly TLR2, TLR3 and TLR4. Expression of TLRs in human IPE cells provides a basis for responses to many ocular pathogens and their activation may be involved in the pathogenesis of ocular inflammation.

The role of lumican in ocular disease.

Lumican is keratan sulfate proteoglycan of the small leucine rich proteoglycan family. Through studies in animal models lumican has been found to be critical in maintaining corneal clarity. It maintains ordered collagen fibrils which are vital in keeping the cornea transparent. It may also be important in primary open angle glaucoma influencing aqueous outflow. Lumican deficiency in mice results in increased axial length with fibromodulin deficiency and thinner sclerae. There is evidence suggesting that this characteristic may be pertinent in humans and lumican gene polymorphisms could be related to high myopia. Lumican plays a fundamental role in inflammation and wound healing. It localises macrophages to the site of corneal injury and recruits neutrophils in lipopolysaccharide-induced keratitis in mice. It has also been shown to bind lipopolysaccharide which may be critical in inflammatory diseases such as uveitis. Lumican is also important in wound healing revealing decreased synthesis in scar tissue and mediating Fas-Fas ligand interactions. It is present in human placenta and amniotic membrane suggesting that it may ensure viable amniotic membrane grafts. Lumican may also be involved in the formation of posterior capsular opacification following cataract surgery. Research into the pivotal role of lumican in the pathogenesis of ocular disease has resulted in greater understanding of the key role which proteoglycans play in human disease.

Peroxidase catalysed cross-linking of an intrinsically unstructured protein via dityrosine bonds in the oocyst wall of the apicomplexan parasite, Eimeria maxima.

Apicomplexan parasites such as Eimeria maxima possess a resilient oocyst wall that protects them upon excretion in host faeces and in the outside world, allowing them to survive between hosts. The wall is formed from the contents of specialised organelles - wall-forming bodies - found in macrogametes of the parasites. The presence of dityrosine in the oocyst wall suggests that peroxidase-catalysed dityrosine cross-linking of tyrosine-rich proteins from wall-forming bodies forms a matrix that is a crucial component of oocyst walls. Bioinformatic analyses showed that one of these tyrosine-rich proteins, EmGAM56, is an intrinsically unstructured protein, dominated by random coil (52-70%), with some α-helix (28-43%) but a relatively low percentage of ß-sheet (1-11%); this was confirmed by nuclear magnetic resonance and circular dichroism. Furthermore, the structural integrity of EmGAM56 under extreme temperatures and pH indicated its disordered nature. The intrinsic lack of structure in EmGAM56 could facilitate its incorporation into the oocyst wall in two ways: first, intrinsically unstructured proteins are highly susceptible to proteolysis, explaining the several differently-sized oocyst wall proteins derived from EmGAM56; and, second, its flexibility could facilitate cross-linking between these tyrosine-rich derivatives. An in vitro cross-linking assay was developed using a recombinant 42kDa truncation of EmGAM56. Peroxides, in combination with plant or fungal peroxidases, catalysed the rapid formation of dityrosine cross-linked polymers of the truncated EmGAM56, as determined by western blotting and HPLC, confirming this protein's propensity to form dityrosine bonds.

Conservation of proteins involved in oocyst wall formation in Eimeria maxima, Eimeria tenella and Eimeria acervulina.

Vaccination with proteins from gametocytes of Eimeria maxima protects chickens, via transfer of maternal antibodies, against infection with several species of Eimeria. Antibodies to E. maxima gametocyte proteins recognise proteins in the wall forming bodies of macrogametocytes and oocyst walls of E. maxima, Eimeria tenella and Eimeria acervulina. Homologous genes for two major gametocyte proteins - GAM56 and GAM82 - were found in E. maxima, E. tenella and E. acervulina. Alignment of the predicted protein sequences of these genes reveals that, as well as sharing regions of tyrosine richness, strong homology exists in their amino-terminal regions, where protective antibodies bind. This study confirms the conservation of the roles of GAM56 and GAM82 in oocyst wall formation and shows that antibodies to gametocyte antigens of E. maxima cross-react with homologous proteins in other species, helping to explain cross-species maternal immunity.

Characterisation of the antigenic and immunogenic properties of bacterially expressed, sexual stage antigens of the coccidian parasite, Eimeria maxima.

Coccidiosis in poultry is caused by the intestinal parasite Eimeria; it causes significant financial losses to the commercial poultry industry worldwide. CoxAbic is the first commercially available subunit vaccine against coccidiosis. The vaccine consists of affinity purified sexual stage (gametocyte) antigens (APGA) isolated from Eimeria maxima. Production of this vaccine is time-consuming and laborious and, therefore, a recombinant subunit vaccine substitute for CoxAbic is desirable. The genes encoding the two immunodominant components of CoxAbic, gam56 and gam82, were cloned into the bacterial expression vector, pTRCHisB, and the proteins expressed and purified. Both recombinant proteins were recognised by protective chicken antibodies that were raised to APGA, by immunoblotting. In a competitive ELISA, a combination of the recombinant proteins inhibited the binding of anti-APGA antibodies to APGA by 76%, which was comparable to the inhibition of 98% observed when APGA was used as the competing protein in the assay. In two breeds of chicken (Australorp and Cobb500), the recombinant proteins alone, or in combination, elicited a dose-dependent, antibody response that recognised APGA by ELISA, and gametocytes by immunoblotting. Together, the results suggested that the development of a recombinant subunit vaccine that maintains the antigenic and immunogenic properties of the native protein vaccine, CoxAbic, is feasible.

Oocyst wall formation and composition in coccidian parasites

The oocyst wall of coccidian parasites is a robust structure that is resistant to a variety of environmental and chemical insults. This resilience allows oocysts to survive for long periods, facilitating transmission from host to host. The wall is bilayered and is formed by the sequential release of the contents of two specialized organelles - wall forming body 1 and wall forming body 2 - found in the macrogametocyte stage of Coccidia. The oocyst wall is over 90 percent protein but few of these proteins have been studied. One group is cysteine-rich and may be presumed to crosslink via disulphide bridges, though this is yet to be investigated. Another group of wall proteins is rich in tyrosine. These proteins, which range in size from 8-31 kDa, are derived from larger precursors of 56 and 82 kDa found in the wall forming bodies. Proteases may catalyze processing of the precursors into tyrosine-rich peptides, which are then oxidatively crosslinked in a reaction catalyzed by peroxidases. In support of this hypothesis, the oocyst wall has high levels of dityrosine bonds. These dityrosine crosslinked proteins may provide a structural matrix for assembly of the oocyst wall and contribute to its resilience.