No abstract

YchF is one of two universally conserved GTPases with unknown cellular function. As a first step toward elucidating YchF’s cellular role, we performed a detailed biochemical characterization of the protein from Escherichia coli. Our data from fluorescence titrations not only confirmed the surprising finding that YchFE.coli binds adenine nucleotides more efficiently than guanine nucleotides, but also provides the first evidence suggesting that YchF assumes two distinct conformational states (ATP- and ADP-bound) consistent with the functional cycle of a typical GTPase. Based on an in vivo pull-down experiment using a His-tagged variant of YchF from E. coli (YchFE.coli), we were able to isolate a megadalton complex containing the 70S ribosome. Based on this finding, we report the successful reconstitution of a YchF•70S complex in vitro, revealing an affinity (KD) of the YchFE.coli•ADPNP complex for 70S ribosomes of 3 μM. The in vitro reconstitution data also suggests that the identity of the nucleotide-bound state of YchF (ADP or ATP) modulates its affinity for 70S ribosomes. A detailed Michaelis-Menten analysis of YchF’s catalytic activity in the presence and the absence of the 70S ribosome and its subunits revealed for the first time that the 70S ribosome is able to stimulate YchF’s ATPase activity (~10-fold), confirming the ribosome as part of the functional cycle of YchF. Our findings taken together with previously reported data for the human homolog of YchF (hOLA1) indicate a high level of evolutionary conservation in the enzymatic properties of YchF and suggest that the ribosome is the main functional partner of YchF not only in bacteria.

Piglets are abruptly separated from their sow at weaning and are quickly transitioned from sow’s milk to a plant-based diet. This is the most important period in commercial swine production, yet the effect of weaning age on the long-term development of the pig gut microbiome is largely unknown.

The present study compared the growth-inhibitory effects of four common branched chain fatty acids (BCFAs) found in beef and dairy fats including iso 15:0, anteiso 15:0, iso 17:0, and anteiso 17:0. MCF-7 human breast cancer cells were exposed for 72 h to media containing increasing doses (50 to −400 μM) of the four BCFA. Cell viability was not affected by any of the BCFA treatments at doses less than 200 μM. Culturing cells with 200 μM of iso-15:0 or iso-17:0 reduced cell viability by 27 ± 2.8 and 43 ± 8.3% at 24 h, 35 ± 4.6 and 49 ± 9.1% at 48 h, and 44 ± 6.8 and 57 ± 8.8% at 72 h posttreatment. In contrast, culturing cells with 200 μM of anteiso-15:0 or anteiso-17:0 did not affect cell viability for any durations tested. The incorporation of iso 15:0 and iso 17:0 into cells (19.1 ± 1.3 and 21.2 ± 1.4 μmol/mg protein, respectively) was greater (P < 0.01) than that of anteiso 15:0 and anteiso 17:0 (11.8 ± 0.7 and 13.8 ± 0.8 μmol/mg protein, respectively). Iso-15:0 and iso-17:0 downregulated (P < 0.01) the expression of antiapoptotic Bcl-2 (0.71 ± 0.6-fold and 0.64 ± 0.09-fold, respectively) and upregulated (P < 0.01) the expression of proapoptotic Bax (1.72 ± 0.14-fold and 2.15 ± 0.24-fold, respectively) compared to the control, whereas their corresponding anteiso isomers did not affect the expression of any apoptosis-related genes. Our findings suggest that the branching structure influences anticarcinogenic effects of BCFAs, with iso being more potent than anteiso.

Human liver cells (HepG2) were cultured with individual trans (t) 18:1 including t6-, t12-, t13-, t14-, t15- and t16-18:1, and retention times of their Δ-9 desaturation products were determined using 100-m biscyanopropyl-polysiloxane and SLB-IL111 columns. Corresponding peaks were found in beef adipose tissues known to have different delta-9 desaturase activities. Further lines of evidence indicating the presence of Δ-9 desaturation products of t-18:1 isomers in beef fat were developed by analysis of fatty acid methyl esters (FAME) fractionated using Ag-TLC, and by GC/MS. Some of the Δ-9 desaturation products of t-18:1 have been previously identified in ruminant fat (c9, t12- and c9, t13-18:2). Some of the Δ-9 desaturation products of t-18:1 (c9, t14- and c9, t15-18:2) have been previously tentatively identified as different fatty acids, and for the first time we provide evidence of the presence of c9, t16-18:2, and where t6, c9-18:2 may elute during analysis of FAME from beef fat.

Piglets are often weaned between 19 and 22 d of age in North America although in some swine operations this may occur at 14 d or less. Piglets are abruptly separated from their sow at weaning and are quickly transitioned from sow's milk to a plant-based diet. The effect of weaning age on the long-term development of the pig gut microbiome is largely unknown. In this study, pigs were weaned at either 14, 21, or 28 d of age and fecal samples collected 21 times from d 4 (neonatal) through to marketing at d 140. The fecal microbiome was characterized using 16S rRNA gene and shotgun metagenomic sequencing. The fecal microbiome of all piglets shifted significantly three to seven days post-weaning with an increase in microbial diversity. Several Prevotella spp. increased in relative abundance immediately after weaning as did butyrate-producing species such as Butyricicoccus porcorum, Faecalibacterium prausnitzii, and Megasphaera elsdenii. Within 7 days of weaning, the gut microbiome of pigs weaned at 21 and 28 days of age resembled that of pigs weaned at 14 d. Resistance genes to most antimicrobial classes decreased in relative abundance post-weaning with the exception of those conferring resistance to tetracyclines and macrolides-lincosamides-streptogramin B. The relative abundance of microbial carbohydrate-active enzymes (CAZymes) changed significantly in the post-weaning period with an enrichment of CAZymes involved in degradation of plant-derived polysaccharides. These results demonstrate that pigs tend to have a more similar microbiome as they age and that weaning age has only a temporary effect on the gut microbiome.

Antimicrobial resistance (AMR) is a serious challenge facing the agricultural industry. Understanding the flow of antimicrobial-resistant bacteria through the beef fabrication process and into ground beef is an important step in identifying intervention points for reducing AMR.

Early life microbial colonization and factors affecting colonization patterns are gaining interest due to recent developments suggesting that early life microbiome may play a role in Developmental Origins of Health and Disease. In cattle, limited information exists on the early microbial colonization of anatomical sites involved in bovine health beyond the gastrointestinal tract. Here, we investigated 1) the initial microbial colonization of seven different anatomical locations in newborn calves and 2) whether these early life microbial communities and 3) serum cytokine profiles are influenced by prenatal vitamin and mineral (VTM) supplementation. Samples were collected from the hoof, liver, lung, nasal cavity, eye, rumen (tissue and fluid), and vagina of beef calves that were born from dams that either received or did not receive VTM supplementation throughout gestation (n = 7/group). Calves were separated from dams immediately after birth and fed commercial colostrum and milk replacer until euthanasia at 30 h post-initial colostrum feeding. The microbiota of all samples was assessed using 16S rRNA gene sequencing and qPCR. Calf serum was subjected to multiplex quantification of 15 bovine cytokines and chemokines. Our results indicated that the hoof, eye, liver, lung, nasal cavity, and vagina of newborn calves were colonized by site-specific microbiota, whose community structure differed from the ruminal-associated communities (0.64 ≥ R2 ≥ 0.12, p ≤ 0.003). The ruminal fluid microbial community was the only one that differed by treatment (p &lt; 0.01). However, differences (p &lt; 0.05) by treatment were detected in microbial richness (vagina); diversity (ruminal tissue, fluid, and eye); composition at the phylum and genus level (ruminal tissue, fluid, and vagina); and in total bacterial abundance (eye and vagina). From serum cytokines evaluated, concentration of chemokine IP-10 was greater (p = 0.02) in VTM calves compared to control calves. Overall, our results suggest that upon birth, the whole-body of newborn calves are colonized by relatively rich, diverse, and site-specific bacterial communities. Noticeable differences were observed in ruminal, vaginal, and ocular microbiota of newborn calves in response to prenatal VTM supplementation. These findings can derive future hypotheses regarding the initial microbial colonization of different body sites, and on maternal micronutrient consumption as a factor that may influence early life microbial colonization.