Role of FBXW2 in explant cultures of bovine periosteum-derived cells

Objective Bone regeneration is a potential technique for treating osteoporosis. A previous study reported that F-box and WD-40 domain-containing protein 2 (FBXW2) localized with osteocalcin in bovine periosteum after 5 weeks of explant culture. However, the osteoblastic functions of FBXW2 remain unclear. In this study, double-fluorescent immunostaining was used to investigate the potential role of FBXW2 and its relationship with osteocalcin. Results At day 0, FBXW2 was expressed in the cambium layer between the bone and periosteum, while osteocalcin was expressed in bone. After explant culture, changes in the periosteum were observed from weeks 1 to 7. At week 1, partial FBXW2 expression was seen with a small amount of osteocalcin. At week 2, a layer of FBXW2 was observed. From weeks 3 to 7, tube-like structures of FBXW and osteocalcin were observed, and periosteum-derived cells were released from the periosteum in areas where no FBXW2 was observed. Bovine periosteum-derived cells can form a three-dimensional cell pellet, because multilayered cell sheets are formed inside of the periosteum in vitro. It is shown that in results FBXW2 is produced in periosteal explants near sites where initial osteogenic activity is observed, suggesting that it may be involved in periosteal osteogenesis.

Abstract P328: Regulation Of Monocyte Physiology By Il-4 Stimulated Memory Cd8+ T-cells

Following a myocardial infarction (MI) monocytes and T-cells begin to infiltrate into the ischemic area in effort to remove necrotic debris and initiate formation of scar tissue. Interleukin (IL)-4 has been linked to improved cardiac wound healing via alterations in both the macrophage and T-cell populations. The goal of this study was determine if proteins secreted by IL-4 stimulated CD8+ T-cells would regulate monocyte physiology that would ultimately improve cardiac healing after an MI. Isolated splenic naïve CD8+ T-cells from day 0 (no MI) mice (n=5/sex/stimulation)were cultured in RPMI with either 0.1% FBS (unstimulated) or 0.1% FBS+ IL-4. After 24 hours of stimulation, the cells and media were collected and separated by centrifugation. The cell pellet was stained and analyzed for markers of activation (CD44) and memory (CD27) by flowcytometry. Conditioned media (secretome) was collected for stimulation of bone marrow monocytes (n=4; females only). After stimulation with the secretome, monocytes were analyzed for viability, phagocytosis, and macrophage phenotype by flow cytometry. Migration of the monocytes after stimulation was also measured using electric cell-substrate impedance sensing (ECIS). After IL-4 stimulation, there was a shift from effector (CD44+ CD27-) to the memory phenotype (CD44+ CD27+; p<0.05 vs unstimulated cells). Interestingly, bone marrow monocyte viability was decreased by 15% when stimulated with the secretome of IL-4 treated CD8+ T-cells compared to unstimulated CD8+ T-cells (0.1%). Phagocytosis was slightly elevated though not significant (p=0.07) in monocytes that were stimulated with the secretome from the IL-4 group compared to the unstimulated CD8+ T-cells. No differences were found in expression of macrophage markers F4/80 (p=0.532) or M1 marker CD86 (p=0.471). The secretome of IL-4 stimulated T-cells increased monocyte migration after wounding similar to levels of the positive control (monocytes in 10% FBS only). The data collected showed that IL-4 stimulated CD8+ T-cells were able to upregulate memory marker CD27. These memory-like CD8+ T-cells initiated monocyte phagocytosis and migration but decreased monocyte viability suggesting that they may play a role in regulating macrophage biology post-MI.

Extracellular Proteome Analysis Shows the Abundance of Histidine Kinase Sensor Protein, DNA Helicase, Putative Lipoprotein Containing Peptidase M75 Domain and Peptidase C39 Domain Protein in Leptospira interrogans Grown in EMJH Medium

Leptospirosis is a re-emerging form of zoonosis that is caused by the spirochete pathogen Leptospira. Extracellular proteins play critical roles in the pathogenicity and survival of this pathogen in the host and environment. Extraction and analysis of extracellular proteins is a difficult task due to the abundance of enrichments like serum and bovine serum albumin in the culture medium, as is distinguishing them from the cellular proteins that may reach the analyte during extraction. In this study, extracellular proteins were separated as secretory proteins from the culture supernatant and surface proteins were separated during the washing of the cell pellet. The proteins identified were sorted based on the proportion of the cellular fractions and the extracellular fractions. The results showed the identification of 56 extracellular proteins, out of which 19 were exclusively extracellular. For those proteins, the difference in quantity with respect to their presence within the cell was found to be up to 1770-fold. Further, bioinformatics analysis elucidated characteristics and functions of the identified proteins. Orthologs of extracellular proteins in various Leptospira species were found to be closely related among different pathogenic forms. In addition to the identification of extracellular proteins, this study put forward a method for the extraction and identification of extracellular proteins.

Identification of effector metabolites using exometabolite profiling of diverse microalgae

Dissolved metabolites mediate algal interactions in aquatic ecosystems, but microalgal exometabolomes remain understudied. We conducted an untargeted metabolomic analysis of non-polar exometabolites exuded from four phylogenetically and ecologically diverse eukaryotic microalgal strains grown in the laboratory: freshwater Chlamydomonas reinhardtii, brackish Desmodesmus sp., marine Phaeodactylum tricornutum, and marine Microchloropsis salina, to identify released metabolites based on relative enrichment in the exometabolomes compared to cell pellet metabolomes. Exudates from the different taxa were distinct, but we did not observe clear phylogenetic patterns. We used feature based molecular networking to explore the identities of these metabolites, revealing several distinct di- and tripeptides secreted by each of the algae, lumichrome, a compound that is known to be involved in plant growth and bacterial quorum sensing, and novel prostaglandin-like compounds. We further investigated the impacts of exogenous additions of eight compounds selected based on exometabolome enrichment on algal growth. Of the these, five (lumichrome, 5’-S-methyl-5'-thioadenosine, 17-phenyl trinor prostaglandin A2, dodecanedioic acid, and aleuritic acid) impacted growth in at least one of the algal cultures. Two of these (dodecanedioic acid and aleuritic acid) produced contrasting results, increasing growth in some algae and decreasing growth in others. Together, our results reveal new groups of microalgal exometabolites, some of which could alter algal growth when provided exogenously, suggesting potential roles in allelopathy and algal interactions.

Analysis of Sirtuin 1 and Sirtuin 3 at Enzyme and Protein Levels in Human Breast Milk during the Neonatal Period

Breast feeding is regarded as the preferred nutrition modality for children during the first few months of life. It not only furthers growth and development but also is supposed to impact later life. The first 1000 days are regarded as a critical window for development, even beyond infancy. The physiological basis underlying this beneficial effect is not clear. Sirtuins are important regulatory proteins of metabolism and are supposed to play a critical role in ageing and longevity as well as in diseases. In the present study, we developed novel methods to assay sirtuin 1 and sirtuin 3 at enzyme activity (via fluorometry) and protein levels (by Western blot) in the aqueous phase and in the cell pellet of human breast milk and assessed the impact of ongoing lactation during the neonatal period. Sirtuin activities in the aqueous phase were negatively correlated with the duration of lactation in the neonatal period. There was no correlation of sirtuin activities in the cell pellet with the duration of lactation. The amounts of sirtuin 1 and sirtuin 3 measured by Western blot were negatively correlated with the lactation period.

Rift Valley fever virus detection in susceptible hosts with special emphasis in insects

Rift Valley fever phlebovirus (RVFV, Phenuiviridae) is an emerging arbovirus that can cause potentially fatal disease in many host species including ruminants and humans. Thus, tools to detect this pathogen within tissue samples from routine diagnostic investigations or for research purposes are of major interest. This study compares the immunohistological usefulness of several mono- and polyclonal antibodies against RVFV epitopes in tissue samples derived from natural hosts of epidemiologic importance (sheep), potentially virus transmitting insect species (Culex quinquefasciatus, Aedes aegypti) as well as scientific infection models (mouse, Drosophila melanogaster, C6/36 cell pellet). While the nucleoprotein was the epitope most prominently detected in mammal and mosquito tissue samples, fruit fly tissues showed expression of glycoproteins only. Antibodies against non-structural proteins exhibited single cell reactions in salivary glands of mosquitoes and the C6/36 cell pellet. However, as single antibodies exhibited a cross reactivity of varying degree in non-infected specimens, a careful interpretation of positive reactions and consideration of adequate controls remains of critical importance. The results suggest that primary antibodies directed against viral nucleoproteins and glycoproteins can facilitate RVFV detection in mammals and insects, respectively, and therefore will allow RVFV detection for diagnostic and research purposes.

Application of Bio-Friendly Formulations of Chitinase-Producing Streptomyces cellulosae Actino 48 for Controlling Peanut Soil-Borne Diseases Caused by Sclerotium rolfsii

Of ten actinobacterial isolates, Streptomyces cellulosae Actino 48 exhibited the strongest suppression of Sclerotium rolfsii mycelium growth and the highest chitinase enzyme production (49.2 U L−1 min−1). The interaction between Actino 48 and S. rolfsii was studied by scanning electron microscope (SEM), which revealed many abnormalities, malformations, and injuries of the hypha, with large loss of S. rolfsii mycelia density and mass. Three talc-based formulations with culture broth, cell-free supernatant, and cell pellet suspension of chitinase-producing Actino 48 were characterized using SEM, Fourier transform infrared spectroscopy (FTIR), and a particle size analyzer. All formulations were evaluated as biocontrol agents for reducing damping-off, root rot, and pods rot diseases of peanut caused by S. rolfsii under greenhouse and open-field conditions. The talc-based culture broth formulation was the most effective soil treatment, which decreased the percentage of peanut diseases under greenhouse and open-field conditions during two successive seasons. The culture broth formulation showed the highest increase in the dry weight of peanut shoots, root systems, and yielded pods. The transcriptional levels of three defense-related genes (PR-1, PR-3, and POD) were elevated in the culture broth formulation treatment compared with other formulations. Subsequently, the bio-friendly talc-based culture broth formulation of chitinase-producing Actino 48 could potentially be used as a biocontrol agent for controlling peanut soil-borne diseases caused by S. rolfsii.

Centrifugation does not remove bacteria from the fat fraction of human milk

Analysis of the human milk microbiome is complicated by the presence of a variable quantity of fat. The fat fraction of human milk is typically discarded prior to analysis. It is assumed that all cells are pelleted out of human milk by high speed centrifugation; however, studies of bovine milk have reported that bacteria may remain trapped within the fat fraction. Here, the bacterial DNA profiles of the fat fraction and cell pellet of human milk (n = 10) were analysed. Human and bacterial DNA was consistently recovered from the fat fraction of human milk (average of 12.4% and 32.7%, respectively). Staphylococcus epidermidis was significantly more abundant in the cell pellet compared to the fat fraction (P = 0.038), and three low-abundance species (< 5% relative abundance) were recovered from one fraction only. However, inclusion of fat reduced the efficiency of DNA extraction by 39%. Culture-based methods were used to quantify the distribution of an exogenously added strain of Staphylococcus aureus in human milk fractions. S. aureus was consistently recovered from the fat fraction (average 28.9%). Bacterial DNA profiles generated from skim milk or cell pellets are not representative of the entire human milk microbiome. These data have critical implications for the design of future work in this field.