👤 S Nichols

Platelet concentrate (PC) is an alternative therapy to treat mastitis in dairy cattle and is an alternative treatment for reproduction problems such as endometritis. Unfortunately, double-centrifugation processing methods described are time-consuming, require specialized laboratory equipment, and are usually done in a heterologous way, which risks herd health. To overcome this limitation, we evaluated single-step bovine PC processing methods readily applicable to a farm setting using an autologous conditioned plasma (ACP) production system. We investigated the hematologic findings, cytokines, and growth factors of the obtained PC samples. Autologous conditioned plasma was prepared using whole blood (WB) from 4 cows (group 1) using single-step centrifugation and 16 different processing methods. The 2 protocols that yielded the highest ratio of platelet to white blood cell (WBC) concentration were ACP-1 [720 × g (2,200 rpm), 5 min] and ACP-2 [929 × g (2,500 rpm), 3 min]. They were subsequently reproduced and compared using WB from 8 cows (group 2). Hematologic findings were quantified, IL-1β (cytokine) and growth factors [platelet-derived growth factor (PDGF), transforming growth factor (TGF)-β, bovine fibroblast growth factor (b-FGF)] were measured, and enrichment factors were compared between samples and processing methods. Hematological characteristics and platelet enrichment varied markedly among tested protocols and all were statistically different from WB. Protocol ACP-2 resulted in significantly greater platelet enrichment (mean 169% of WB) than ACP-1 (125% of WB). We found no significant difference between the 2 ACP preparation protocols with regard to leukocyte reduction (7.53-9.75% WBC compared with WB) or growth factor enrichment (124-125% PDGF, 95-100% TGF-β, 102-104% b-FGF, and 56-74% IL-1β compared with WB). In conclusion, both ACP protocols yielded a platelet concentration shown to promote healing for clinical applications in cattle, and the ACP-2 protocol resulted in a greater degree of platelet enrichment. Therefore, this protocol could be used for ACP production for clinical applications in cattle. Show less

Phosphatidylinositol-3-phosphate (PI(3)P), and Vps34, the type III phosphatidylinositol 3-kinase primarily responsible for its production, are important for function and survival of sensory neurons, where they have key roles in membrane processing events, such as autophagy, endosome processing, and fusion of membranes bearing ubiquitinated cargos with lysosomes. We examined their roles in the most abundant class of secondary neurons in the vertebrate retina, the ON-bipolar cells (ON-BCs). A conditional Vps34 knockout mouse line was generated by crossing Vps34 floxed mice with transgenic mice expressing Cre recombinase in ON-BCs. Structural changes in the retina were determined by immunofluorescence and electron microscopy, and bipolar cell function was determined by electroretinography. Vps34 deletion led to selective death of ON-BCs, a thinning of the inner nuclear layer, and a progressive decline of electroretinogram b-wave amplitudes. There was no evidence for loss of other retinal neurons, or disruption of rod-horizontal cell contacts in the outer plexiform layer. Loss of Vps34 led to aberrant accumulation of membranes positive for autophagy markers LC3, p62, and ubiquitin, accumulation of endosomal membranes positive for Rab7, and accumulation of lysosomes. Similar effects were observed in Purkinje cells of the cerebellum, leading to severe and progressive ataxia. These results support an essential role for PI(3)P in fusion of autophagosomes with lysosomes and in late endosome maturation. The cell death resulting from Vps34 knockout suggests that these processes are essential for the health of ON-BCs. Show less

How host and microbial factors combine to structure gut microbial communities remains incompletely understood. Redox potential is an important environmental feature affected by both host and microbial actions. We assessed how antibiotics, which can impact host and microbial function, change redox state and how this contributes to post-antibiotic succession. We showed gut redox potential increased within hours of an antibiotic dose in mice. Host and microbial functioning changed under treatment, but shifts in redox potentials could be attributed specifically to bacterial suppression in a host-free ex vivo human gut microbiota model. Redox dynamics were linked to blooms of the bacterial family Enterobacteriaceae. Ecological succession to pre-treatment composition was associated with recovery of gut redox, but also required dispersal from unaffected gut communities. As bacterial competition for electron acceptors can be a key ecological factor structuring gut communities, these results support the potential for manipulating gut microbiota through managing bacterial respiration. Show less

Mitogen-activated protein (MAP) kinases play a central role in controlling a wide range of cellular functions following their activation by a variety of extracellular stimuli. MAP kinase phosphatases (MKPs) represent a subfamily of dual specificity phosphatases, which negatively regulate MAP kinases. Although ERK2 activity is regulated by its phosphorylation state, MKP3 is regulated by physical interaction with ERK2, independent of its enzymatic activity (Camps, M., Nichols, A., Gillieron, C., Antonsson, B., Muda, M., Chabert, C., Boschert, U., and Arkinstall, S., (1998) Science 280, 1262-1265; Farooq, A., Chaturvedi, G., Mujtaba, S., Plotnikova, O., Zeng, L., Dhalluin, C., Ashton, R., and Zhou, M. M. (2001), Mol. Cell 7, 387-399; Zhou, B., and Zhang, Z. Y. (1999) J. Biol. Chem. 274, 35526-35534). The interaction of ERK2 and MKP3 allows the reciprocal cross-regulation of their catalytic activity. Indeed, MKP3 acts as a negative regulator on ERK2-MAP kinase signal transduction activity, representing thus a negative feedback for this MAPK pathway. To identify novel proteins able to complex MKP3, we used the yeast two-hybrid system. Here we report that MKP3 and protein kinase CK2 form a protein complex, which can include ERK2. The phosphatase activity of MKP3 is then slightly increased in vitro, whereas in transfected cells, ERK2 dephosphorylation is reduced. In addition, we demonstrated that CK2 selectively phosphorylates MKP3, suggesting cross-regulation between CK2alpha and MKP3, as well as a modulation of ERK2-MAPK signaling by CK2alpha via MKP3. Show less