No abstract

Summary Phosphatidylinositol 3,4,5-trisphosphate (PIP3)-dependent Rac exchanger 1 (P-Rex1) is a Rho guanine nucleotide exchange factor synergistically activated by PIP3 and Gβγ that plays an important role in the metastasis of breast, prostate, and skin cancer, making it an attractive therapeutic target. However, the molecular mechanisms behind P-Rex1 regulation are poorly understood. We determined structures of the P-Rex1 pleckstrin homology (PH) domain bound to the head group of PIP3 and resolved that PIP3 binding to the PH domain is required for P-Rex1 activity in cells but not for membrane localization, which points to an allosteric activation mechanism by PIP3. We also determined structures of the P-Rex1 tandem Dbl homology/PH domains in complexes with two of its substrate GTPases, Rac1 and Cdc42. Collectively, this study provides important molecular insights into P-Rex1 regulation and tools for targeting the PIP3-binding pocket of P-Rex1 for a new generation of cancer chemotherapeutic agents.

Direct-fed microbials (DFM) supplemented in sow diets may confer health benefits to the host and their piglets by reducing pathogens in the sow and environment. In this study we evaluated the effect of a Bacillus-based DFM on the gastrointestinal microbiota of neonatal piglets. A total of 208 sows were divided into 2 treatments: a control diet and the control diet supplemented with a Bacillus subtilis-based DFM (3.75 × 10(5) cfu/g feed). Twenty-one piglets sampled from each sow treatment group were euthanized on d 3 of lactation followed by an additional 15 piglets per treatment on d 10 of lactation. Litters from DFM-supplemented sows had greater (P = 0.02) weaning weights and a tendency (P = 0.09) for improvement in litter ADG. Sows supplemented with the DFM weaned more pigs (P = 0.06) than control sows which was reflected in numerically lower but not statistically different (P = 0.12) decrease in piglet mortality in DFM litters. Terminal RFLP was used to characterize gastrointestinal (GI) microbial populations in the ileum and colon of the piglets. Terminal restriction fragments (T-RF) were compared between control and DFM treatments. There was a greater incidence and quantity of T-RF B423 and H330 (binary P = 0.01, 0.08; quantitative P = 0.01, 0.05, respectively), putatively identified as Lactobacillus gasseri/johnsonii, in the ileum of pigs nursing sows supplemented with DFM at d 3. Terminal restriction fragment peaks B423 and H330 were also greater (binary P = 0.01, 0.08; quantitative P = 0.01, 0.01, respectively) in the colon of pigs nursing sows supplemented with DFM at d 3. Peaks M495 and B394, putatively identified as E. coli, were greater (binary P = 0.01, 0.04; quantitative P = 0.01, 0.01, respectively) in the colon of the control pigs at d 3. At d 10, both the presence and quantity of Lactobacillus species were greater (P < 0.05) in the colon of pigs with the DFM treatment. Additionally, there was a tendency for T-RF B227 and H257 (binary P = 0.07, 0.07, respectively), putatively identified as Clostridium perfringens, to be present in the ileum of the control pigs at d 10 compared with treated pigs. Results of this study reveal that the developing gastrointestinal microbiota of a neonatal piglet can be affected by DFM supplementation to the sow.

The objective of this study was to investigate the effects of Bacillus subtilis-based probiotic supplementation in broiler chicken diets on growth performance, feed efficiency, intestinal cytokine, and tight junction (TJ) protein mRNA expression. Zero-day-old broiler chicks (n = 140) were randomly assigned to one of five dietary treatments: basal diet (CON); basal diet supplemented with either antibiotic bacitracin methylene disalicylate (BMD); or probiotics, namely, B. subtilis strain 1781 (PB1), a combination of B. subtilis strain 1104 + strain 747 (PB2), or B. subtilis strain 1781 + strain 747 (PB3). Body weight and feed intake were measured at 14 days of age, and the feed conversion ratio (FCR) was calculated. At 14 days of age, ileal samples were collected and used for intestinal cytokine, TJ protein, and mucin gene expression analysis using qRT-PCR. The chickens supplemented with antibiotic (BMD) and B. subtilis strain 1781 alone (PB1) had significantly higher body weights compared to controls of the same age. Dietary supplementation with antibiotic (BMD) or probiotics (PB1, PB2, PB3) significantly improved the feed efficiency as evidenced by decreased FCR compared to controls. No differences were observed in the expression of IL1β, IL17F, IFNγ, and MUC2 gene among the different treatment groups. However, elevated expression of IL6 (BMD, PB1, PB2), IL8 (PB2), and TNFSF15 (PB1, PB2, PB3) compared to controls was observed in the ileum. IL2 and IL10 expression was upregulated in chicks in the PB2 and PB3 groups, and IL4 was elevated in the PB1 group. IL13 was elevated in all probiotic-fed groups (PB1, PB2, PB3). Probiotic supplementation was also shown to significantly increase the expression of TJ proteins JAM2, ZO1 (PB2, PB3), and occludin (PB1, PB2). Taken together, B. subtilis supplementation altered intestinal immune activity and influenced gut barrier integrity through increased tight junction gene expression.

The effects of above-ambient pressure on ion mobility on resolving power, resolution, and ion current were investigated using a small, stand-alone ion mobility spectrometer (IMS). This work demonstrates the first example of ion mobility spectrometry at pressures above ambient. Ion mobility spectra of chemical warfare agent (CWA) stimulant dimethyl methylphosphonate (DMMP) and several other standard compounds are shown for superambient conditions. The IMS was operated at pressures from 700 to 4560 Torr. An optimal resolving power was obtained at a specific voltage as a function of pressure, with higher optimal resolving powers obtained at higher voltages, as predicted from standard IMS theory. At high pressures, however, resolving power did not increase as much as theory predicted, presumably due to ion clustering. Nevertheless, an increase in pressure was found to improve resolution in IMS. One example where high pressure improved resolution was the separation of cyclohexylamine (K0 = 1.83) and 2-hexanone (K0 = 1.86) (where K0 is the reduced mobility value). The product ions of these two compounds could not be separated at ambient pressure but could be nearly baseline separated when the pressure of the buffer gas was raised to 2280 Torr. Total ion current was also examined at pressures from ambient up to 4560 Torr. Total ion current, when investigated with pressure, was found to reach a maximum, initially rising with increased pressure.