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.

Recent improvements in tilt measurement techniques have greatly enhanced the resolution of hydraulic fracture-induced tilts, resulting in both greater mapping precision and an increase in the maximum mapping depth achievable with a surface tiltmeter array. With a previous depth limitation of around 6,000 ft., surface tiltmeter mapping was limited to areas with relatively shallow production. Application is greatly broadened now with a depth range down to 10,000 ft. In addition to the expanded depth range, there has been a marked improvement in the fracture mapping resolution. This paper begins with an overview of the tiltmeter fracture mapping concept, highlighting both the strengths of this technique and its limitations. Following that is a description of the technical advancements made over the last three years to allow fracture mapping at far greater depths. Finally, two brief case studies are presented to demonstrate fracture mapping at great depth, and also to provide insight on hydraulic fracture growth behavior in two different environments. As the case studies make clear, fracture growth is far more complex than is generally assumed. Better understanding of these complexities can lead to significantly enhanced fracture stimulation practices. P. 135

Clostridium perfringens and Clostridium difficile are associated with scours in the neonatal piglet and are an economic concern in swine production. The objective of this study was to characterize the prevalence and diversity of C. perfringens and C. difficile isolates obtained from scouring neonatal piglets in a large integrated production system, as well as in smaller independently owned regional farms. Rectal swabs were collected from 333 pigs at 11 sites in an integrated swine production system and from an additional 180 pigs at 16 regional farms located throughout the Midwest. C. perfringens was isolated from 89.8% of the pigs swabbed at the integrated sites, and C. difficile was isolated from 57.7% of these pigs. Of the pigs from the regional farms sampled, 95.6% were positive for isolation of C. perfringens and 27.2% were positive for C. difficile. Toxigenic isolates were typed using random amplified polymorphic DNA (RAPD) PCR, and were placed in four dendrograms for C. perfringens and C. difficile populations isolated from the integrated sites and regional farms. Diversity indices showed that there was greater diversity in C. difficile populations and in populations isolated from the regional farms. A subset of isolates from the C. difficile dendrograms were further toxinotyped by amplification of the pathogenicity locus and subsequent digestion by HincII, AccI, and EcoRI. Of the 45 isolates typed, 44 were determined to be toxinotype V. The results of this study illustrate the diversity of C. perfringens and C. difficile isolates and the prevalence of these pathogens in swine production sites.

Additive manufacturing (3D printing) is a technology with near-unlimited potential for the chemical educator. However, its adoption into higher education has been limited by the dual requirements of expertise in 3D printing and 3D computer-aided design (CAD). Thus, its reported utilization in the chemistry curriculum has been within the creation of 3D models for the macroscopic visualization of molecular models and processes. With the commercialization of inexpensive 3D printers, we seek to provide a series of optical mounts and tools for use in chemical research or education by designing systems which may be mounted on a breadboard and used to construct chemical instrumentation. These designs include mounts for cylindrical lenses or mirrors with 0.5, 1, and 2 in. diameters, 0.5 in. diffraction gratings, an adjustable optical slit, cuvette holders, stepper motor (NEMA 17) adapters, and a modular, 3D printed breadboard. All designs were created using Solidworks CAD, and have been provided in Supporting Information and uploaded to in both Solidworks native format (*.SLDPRT) and a standard file exchange format for 3D objects (*.STL) format for use, modification, or collaboration. All files are released under the MIT Open-Source license (modified for Design). In comparison with commercially available products, the prototypes presented herein provide significant cost-savings, with many designs 1–2 orders of magnitude cheaper than commercial equivalents. In addition, these designs have been utilized in an upper division instrumental analysis course as students designed and constructed a visible spectrophotometer using these optical devices. Data from the resultant instrument is presented and compared with a commercially available spectrophotometer.