Evaluating the Sensitivity of a Media-Fill Challenge Test Under Various Situations as a Reliable Method for Recommended Aseptic Technique Competency Assessment.

Background: Microbial contamination of compounded medications is a serious concern within hospital pharmacies as it can lead to severe patient injury. The United States Pharmacopeia <797> mandates that pharmacy personnel responsible for preparing compounded sterile preparations must annually demonstrate competency in aseptic technique by performing a media-fill challenge test. Objective: The purpose of this study is to evaluate the sensitivity of a commonly used media-fill test through proper and improper compounding techniques. Methods: Two aseptically trained pharmacy technicians performed media-fill challenge testing by carrying out 5 separate manipulations 5 times each for a total of 25 trials. Sterile vials, syringes, and intravenous bags were prepared. The first manipulation followed best-practice aseptic technique and sterile compounding procedures. Each of the following 4 manipulations removed one aspect of best-practice aseptic technique. The prepared products were incubated at 20°C to 25°C. A positive result for microbial contamination is indicated by visible turbidity within the vials, syringes, and intravenous bags at the following check points: 24 hours, 72 hours, 7 days, 14 days, 21 days, and >30 days. Results: Twenty-five trials, each containing 10 distinct admixtures, resulted in a total of 250 compounded preparations. No single preparation showed signs of turbidity, sedimentation, or visible microbial growth at any of the 6 checkpoints yielding a 0% contamination rate. However, the positive controls inoculated with bacteria did have positive microbial growth results. Conclusion: A more sensitive test needs to be developed to provide assurances that all poor aseptic practices are detected in compounding personnel.

Allosteric modulation of Ras and the PI3K/AKT/mTOR pathway: emerging therapeutic opportunities.

GTPases and kinases are two predominant signaling modules that regulate cell fate. Dysregulation of Ras, a GTPase, and the three eponymous kinases that form key nodes of the associated phosphatidylinositol 4,5-bisphosphate 3-kinase (PI3K)/AKT/mTOR pathway have been implicated in many cancers, including pancreatic cancer, a disease noted for its current lack of effective therapeutics. The K-Ras isoform of Ras is mutated in over 90% of pancreatic ductal adenocarcinomas (PDAC) and there is growing evidence linking aberrant PI3K/AKT/mTOR pathway activity to PDAC. Although these observations suggest that targeting one of these nodes might lead to more effective treatment options for patients with pancreatic and other cancers, the complex regulatory mechanisms and the number of sequence-conserved isoforms of these proteins have been viewed as significant barriers in drug development. Emerging insights into the allosteric regulatory mechanisms of these proteins suggest novel opportunities for development of selective allosteric inhibitors with fragment-based drug discovery (FBDD) helping make significant inroads. The fact that allosteric inhibitors of Ras and AKT are currently in pre-clinical development lends support to this approach. In this article, we will focus on the recent advances and merits of developing allosteric drugs targeting these two inter-related signaling pathways.

Structural and biochemical characterization of ZhuI aromatase/cyclase from the R1128 polyketide pathway.

Aromatic polyketides comprise an important class of natural products that possess a wide range of biological activities. The cyclization of the polyketide chain is a critical control point in the biosynthesis of aromatic polyketides. The aromatase/cyclases (ARO/CYCs) are an important component of the type II polyketide synthase (PKS) and help fold the polyketide for regiospecific cyclizations of the first ring and/or aromatization, promoting two commonly observed first-ring cyclization patterns for the bacterial type II PKSs: C7-C12 and C9-C14. We had previously reported the crystal structure and enzymological analyses of the TcmN ARO/CYC, which promotes C9-C14 first-ring cyclization. However, how C7-C12 first-ring cyclization is controlled remains unresolved. In this work, we present the 2.4 Å crystal structure of ZhuI, a C7-C12-specific first-ring ARO/CYC from the type II PKS pathway responsible for the production of the R1128 polyketides. Though ZhuI possesses a helix-grip fold shared by TcmN ARO/CYC, there are substantial differences in overall structure and pocket residue composition that may be important for directing C7-C12 (rather than C9-C14) cyclization. Docking studies and site-directed mutagenesis coupled to an in vitro activity assay demonstrate that ZhuI pocket residues R66, H109, and D146 are important for enzyme function. The ZhuI crystal structure helps visualize the structure and putative dehydratase function of the didomain ARO/CYCs from KR-containing type II PKSs. The sequence-structure-function analysis described for ZhuI elucidates the molecular mechanisms that control C7-C12 first-ring polyketide cyclization and builds a foundation for future endeavors into directing cyclization patterns for engineered biosynthesis of aromatic polyketides.

Multiple conformations of the cytidine repressor DNA-binding domain coalesce to one upon recognition of a specific DNA surface.

The cytidine repressor (CytR) is a member of the LacR family of bacterial repressors with distinct functional features. The Escherichia coli CytR regulon comprises nine operons whose palindromic operators vary in both sequence and, most significantly, spacing between the recognition half-sites. This suggests a strong likelihood that protein folding would be coupled to DNA binding as a mechanism to accommodate the variety of different operator architectures to which CytR is targeted. Such coupling is a common feature of sequence-specific DNA-binding proteins, including the LacR family repressors; however, there are no significant structural rearrangements upon DNA binding within the three-helix DNA-binding domains (DBDs) studied to date. We used nuclear magnetic resonance (NMR) spectroscopy to characterize the CytR DBD free in solution and to determine the high-resolution structure of a CytR DBD monomer bound specifically to one DNA half-site of the uridine phosphorylase (udp) operator. We find that the free DBD populates multiple distinct conformations distinguished by up to four sets of NMR peaks per residue. This structural heterogeneity is previously unknown in the LacR family. These stable structures coalesce into a single, more stable udp-bound form that features a three-helix bundle containing a canonical helix-turn-helix motif. However, this structure differs from all other LacR family members whose structures are known with regard to the packing of the helices and consequently their relative orientations. Aspects of CytR activity are unique among repressors; we identify here structural properties that are also distinct and that might underlie the different functional properties.