Maternal Group B Streptococcus Prophylaxis Improvement using an Electronic Medical Record Dynamic Order Set.

OBJECTIVE: To develop and implement a Group B Streptococcal (GBS) dynamic order set to improve adherence to the American College of Obstetricians and Gynecologists/Centers for Disease Control and Prevention (ACOG/CDC) guidelines. STUDY DESIGN: A team of information technology and clinical experts developed a dynamic order block. The content was patterned after the CDC "Prevent GBS" mobile app. It was then embedded in the labor and delivery/induction order set and piloted at a single high-volume obstetric unit. Following the pilot and incorporation of the 2019 ACOG update of the CDC guidelines, the order set was rolled out in five additional hospitals within a region of a large health system. Information on GBS prophylaxis performance before and after implementation was available for the pilot site and four of the additional hospitals. Information before implementation was obtained electronically from electronic medical record (EMR) laboratory and pharmacy data and supplemented by manual chart review. Postimplementation data were obtained from discrete order set EMR data elements. Adherence to the guidelines before and after were compared using chi-squared test. RESULTS: There were 7,114 deliveries before implementation and 4,502 after implementation. Preterm delivery occurred in 6.8 and 6.9%, respectively. There was an increase in appropriate treatment of preterm patients (positive and unknown GBS) delivering after implementation (88.7-99.1%, p < 0.001). More patients were reported to have a penicillin allergy before implementation than after implementation (14.7 vs. 11.1%, respectively, p = 0.01). Associated changes in therapy noted after implementation included a nonsignificant decrease in the proportion reporting a high-risk allergy (50.3 vs. 41.9%, p = 0.18), an increase in the appropriate use of clindamycin and vancomycin (64.4 vs. 92.3%, p < 0.001) and a decrease in clindamycin use in those without sensitivity testing. CONCLUSION: Routine universal use of a dynamic admission labor/induction order set was associated with high and improved adherence to GBS prophylaxis guidelines. KEY POINTS: · Lapses in GBS prophylaxis are associated with early-onset GBS disease.. · Preterm delivery and penicillin allergic patients are commonly associated with lapses in prophylaxis.. · Dynamic EMR order set use can improve adherence to clinical guidelines..

Protons as second messenger regulators of G protein signaling.

In response to environmental stress, cells often generate pH signals that serve to protect vital cellular components and reprogram gene expression for survival. A major barrier to our understanding of this process has been the identification of signaling proteins that detect changes in intracellular pH. To identify candidate pH sensors, we developed a computer algorithm that searches proteins for networks of proton-binding sidechains. This analysis indicates that Gα subunits, the principal transducers of G protein-coupled receptor (GPCR) signals, are pH sensors. Our structure-based calculations and biophysical investigations reveal that Gα subunits contain networks of pH-sensing sidechains buried between their Ras and helical domains. Further, we show that proton binding induces changes in conformation that promote Gα phosphorylation and suppress receptor-initiated signaling. Together, our computational, biophysical, and cellular analyses reveal an unexpected function for G proteins as mediators of stress-response signaling.

Clinical spectrum of BCS1L Mitopathies and their underlying structural relationships.

The most frequent cause of isolated complex III deficits is mutations to the nuclear-encoded ATPase BCS1L. Disease phenotypes are varied and can be as mild as Björnstad syndrome, characterized by pili torti and sensorineural hearing loss, or as severe as GRACILE syndrome, characterized by growth restriction, aminoaciduria, cholestasis, iron overload, lactic acidosis, and early death. BCS1L mutations are also linked to an undefined complex III deficiency, a heterogeneous condition generally involving low birth weight, renal and hepatic pathologies, hypotonia, and developmental delays. We analyzed all published patient cases of mutations to BCS1L and modeled the tertiary and quaternary structure of the BCS1L protein to map the location of disease-causing BCS1L mutations. We show that higher order structural analysis can be used to understand the phenotype observed in a patient with the novel compound heterozygous c.550C>T(p.Arg184Cys) and c.838C>T(p.Leu280Phe) mutations. More broadly, higher order structural analysis reveals genotype-phenotype relationships within the intermediate complex III deficiency category that help to make sense of the spectrum of observed phenotypes. We propose a change in nomenclature that unifies the intermediate phenotype under "BCS1L Mitopathies". Patterns in genotype-phenotype correlations within these BCS1L Mitopathies are evident in the context of the tertiary and quaternary structure of BCS1L.

Systematic analysis of essential genes reveals important regulators of G protein signaling.

The yeast pheromone pathway consists of a canonical heterotrimeric G protein and MAP kinase cascade. To identify additional signaling components, we systematically evaluated 870 essential genes using a library of repressible-promoter strains. Quantitative transcription-reporter and MAPK activity assays were used to identify strains that exhibit altered pheromone sensitivity. Of the 92 newly identified essential genes required for proper G protein signaling, those involved with protein degradation were most highly represented. Included in this group are members of the Skp, Cullin, F box (SCF) ubiquitin ligase complex. Further genetic and biochemical analysis reveals that SCF(Cdc4) acts together with the Cdc34 ubiquitin-conjugating enzyme at the level of the G protein; promotes degradation of the G protein alpha subunit, Gpa1, in vivo; and catalyzes Gpa1 ubiquitination in vitro. These insights to the G protein signaling network reveal the essential genome as an untapped resource for identifying new components and regulators of signal transduction pathways.

Guanine nucleotide-binding protein (Gα) endocytosis by a cascade of ubiquitin binding domain proteins is required for sustained morphogenesis and proper mating in yeast.

Heterotrimeric G proteins are well known to transmit signals from cell surface receptors to intracellular effector proteins. There is growing appreciation that G proteins are also present at endomembrane compartments, where they can potentially interact with a distinct set of signaling proteins. Here, we examine the cellular trafficking function of the G protein α subunit in yeast, Gpa1. Gpa1 contains a unique 109-amino acid insert within the α-helical domain that undergoes a variety of posttranslational modifications. Among these is monoubiquitination, catalyzed by the NEDD4 family ubiquitin ligase Rsp5. Using a newly optimized method for G protein purification together with biophysical measures of structure and function, we show that the ubiquitination domain does not influence enzyme activity. By screening a panel of 39 gene deletion mutants, each lacking a different ubiquitin binding domain protein, we identify seven that are necessary to deliver Gpa1 to the vacuole compartment including four proteins (Ede1, Bul1, Ddi1, and Rup1) previously not known to be involved in this process. Finally, we show that proper endocytosis of the G protein is needed for sustained cellular morphogenesis and mating in response to pheromone stimulation. We conclude that a cascade of ubiquitin-binding proteins serves to deliver the G protein to its final destination within the cell. In this instance and in contrast to the previously characterized visual system, endocytosis from the plasma membrane is needed for proper signal transduction rather than for signal desensitization.

Differences in the regulation of K-Ras and H-Ras isoforms by monoubiquitination.

Ras GTPases are signaling switches that control critical cellular processes including gene expression, differentiation, and apoptosis. The major Ras isoforms (K, H, and N) contain a conserved core GTPase domain, but have distinct biological functions. Among the three Ras isoforms there are clear differences in post-translational regulation, which contribute to differences in localization and signaling output. Modification by ubiquitination was recently reported to activate Ras signaling in cells, but the mechanisms of activation are not well understood. Here, we show that H-Ras is activated by monoubiquitination and that ubiquitination at Lys-117 accelerates intrinsic nucleotide exchange, thereby promoting GTP loading. This mechanism of Ras activation is distinct from K-Ras monoubiquitination at Lys-147, which leads to impaired regulator-mediated GTP hydrolysis. These findings reveal that different Ras isoforms are monoubiquitinated at distinct sites, with distinct mechanisms of action, but with a common ability to chronically activate the protein in the absence of a receptor signal or oncogenic mutation.

GTP-cyclohydrolase and development in Teladorsagia circumcincta and Dictyocaulus viviparus (Nematoda: Strongylida).

GTP-Cyclohydrolase (GTP-CH) is necessary for the production of tetrahydrobiopterin, a required cofactor for the three aromatic amino acid hydroxylases and nitric oxide synthases. The gene encoding GTP-CH is transcribed at high levels in infective third larval stages of a number of parasitic trichostrongylid nematodes. We explore the potential role of GTP-CH within the processes of nematode development and environmentally-induced hypobiosis. For two species of parasitic nematode that are of major economic and welfare importance to livestock in temperate regions, Teladorsagia circumcincta and Dictyocaulus viviparus, we have demonstrated that each of the pre-parasitic larval stages transcribe high mean levels of cat-4 (the gene encoding GTP-CH). Using quantitative real-time polymerase chain reaction analysis and two different isolates of D. viviparus, only one of which is capable of entering hypobiosis, we have shown that there were only minor differences between these isolates in mean cat-4 transcript levels, both during the parasitic stages and during the earlier environmental life cycle stages (L(1)-L(3)). Taken together, these data indicate that, although both species of nematode produce high levels of cat-4 transcript in pre-parasitic larval stages, GTP-CH levels are unlikely to be involved in the induction of parasite hypobiosis. Alternative roles for GTP-CH in larval development are discussed.

Effects of recombinant protein expression on green fluorescent protein diffusion in Escherichia coli.

Fluorescence recovery after photobleaching was used to measure the diffusion coefficient of green fluorescent protein (GFP, 27 kDa) in Escherichia coli in the presence or absence of four coexpressed proteins: cytoplasmic maltose binding protein (42 kDa), tau-40 (45 kDa), alpha-synuclein (14 kDa), or calmodulin (17 kDa). The GFP diffusion coefficient remains constant regardless of the type of coexpresseed protein and whether or not the coexpressed protein was induced. We conclude that expression of these soluble proteins has little to no effect on the diffusion of GFP. These results have implications for the utility of in-cell nuclear magnetic resonance spectroscopy.

A sychnological cell penetrating peptide mimic of p21(WAF1/CIP1) is pro-apoptogenic.

Targeting chemotherapeutic agents directly to sites of DNA replication and repair within cancerous cells is problematic. This study attempts to address the issue of nuclear delivery of biologically active peptides with the potential to disrupt cancer cell growth. Herein, the protein transduction domain of the HIV-1 transactivator of transcription, Tat (Tat(48-60)), is used to deliver a cytotoxic peptide mimic of the cyclin-dependent kinase inhibitor, p21(WAF1/CIP1) into the nucleus. This construct, which we designate as Tat(48-60)-P10, contains the PCNA interacting protein (PIP) box. We demonstrate the utility of Tat(48-60) for peptide delivery to the nucleus and show that Tat(48-60)-P10 induces apoptosis specific to the inclusion of the wild type PIP box containing sequence. Colocalization of Tat(48-60)-P10 with nuclear PCNA was observed by immunofluorescence analysis, supporting the hypothesis that cytotoxicity is potentially related to disruption of nuclear PCNA function. The U251 and U373 glioma cell lines exhibited particular sensitivity to the construct.

The identification of a novel alternatively spliced form of the MBD4 DNA glycosylase.

Methyl-CpG binding protein 4 (MBD4) is a mismatch-specific G:T and G:U DNA glycosylase. During an analysis of MBD4 expression in HeLa cells we noted the presence of an unexpectedly short reverse transcribed product. This cDNA lacked the region encoding the methyl-binding domain and exon 3 of MBD4 but retained the glycosylase domain. Sequence comparison indicates the existence of a previously unreported cryptic splice site in the MBD4 genomic sequence thus illuminating a mechanism whereby a glycosylase acquired a methyl-binding capacity, thus targeting potential mutagenic CpG sites. In vitro assays of this highly purified species, refolded in arginine rich conditions, confirmed that this unique, short version of MBD4 possessed uracil DNA glycosylase but not thymine DNA glycosylase activity. We conclude that the identification of a transcript encoding a short version of MBD4 indicates that MBD4 expression may be more complex than previously reported, and is worthy of further investigation.

Site-specific monoubiquitination activates Ras by impeding GTPase-activating protein function.

KRas has recently been shown to be activated by monoubiquitination (mUb). Similar to oncogenic mutations, mUb of Ras at position 147 activates Ras by causing a defect in GTPase activating protein (GAP) function. To characterize the mechanism by which mUb impairs GAP-mediated downregulation of Ras, we made various modifications at position 147 of Ras and examined the impact on Ras sensitivity to GAP function. Whereas small modifications (iodoacetamide and glutathione) at position 147 of Ras do not affect GAP-mediated hydrolysis, ligation of Ras to Ub(G76C) (native linker), Ub(X77C) (one residue longer), and PDZ2 (with a native ubiquitin linker) was defective in GAP-mediated GTP hydrolysis. However, restoration of GAP activity was observed for Ras modified with the PDZ2 domain containing a shorter and stiffer linker region than ubiquitin. Therefore, the properties of the linker region dictate whether modification affects GAP-mediated hydrolysis, and our data indicate that the GAP defect requires a minimum linker length of 7 to 8 residues.

Site-specific monoubiquitination activates Ras by impeding GTPase-activating protein function.

Cell growth and differentiation are controlled by growth factor receptors coupled to the GTPase Ras. Oncogenic mutations disrupt GTPase activity, leading to persistent Ras signaling and cancer progression. Recent evidence indicates that monoubiquitination of Ras leads to Ras activation. Mutation of the primary site of monoubiquitination impairs the ability of activated K-Ras (one of the three mammalian isoforms of Ras) to promote tumor growth. To determine the mechanism of human Ras activation, we chemically ubiquitinated the protein and analyzed its function by NMR, computational modeling and biochemical activity measurements. We established that monoubiquitination has little effect on the binding of Ras to guanine nucleotide, GTP hydrolysis or exchange-factor activation but severely abrogates the response to GTPase-activating proteins in a site-specific manner. These findings reveal a new mechanism by which Ras can trigger persistent signaling in the absence of receptor activation or an oncogenic mutation.

Buprenorphine/naloxone and methadone maintenance treatment outcomes for opioid analgesic, heroin, and combined users: findings from starting treatment with agonist replacement therapies (START).

OBJECTIVE: The objective of this secondary analysis was to explore differences in baseline clinical characteristics and opioid replacement therapy treatment outcomes by type (heroin, opioid analgesic [OA], or combined [heroin and OA]) and route (injector or non-injector) of opioid use. METHOD: A total of 1,269 participants (32.2% female) were randomized to receive one of two study medications (methadone or buprenorphine/naloxone [BUP]). Of these, 731 participants completed the 24-week active medication phase. Treatment outcomes were opioid use during the final 30 days of treatment (among treatment completers) and treatment attrition. RESULTS: Non-opioid substance dependence diagnoses and injecting differentiated heroin and combined users from OA users. Non-opioid substance dependence diagnoses and greater heroin use differentiated injectors from non-injectors. Further, injectors were more likely to be using at end of treatment compared with non-injectors. OA users were more likely to complete treatment compared with heroin users and combined users. Non-injectors were more likely than injectors to complete treatment. There were no interactions between type of opioid used or injection status and treatment assignment (methadone or BUP) on either opioid use or treatment attrition. CONCLUSIONS: Findings indicate that substance use severity differentiates heroin users from OA users and injectors from non-injectors. Irrespective of medication, heroin use and injecting are associated with treatment attrition and opioid misuse during treatment. These results have particular clinical interest, as there is no evidence of superiority of BUP over methadone for treating OA users versus heroin users.

Melanisation of Teladorsagia circumcincta larvae exposed to sunlight: a role for GTP-cyclohydrolase in nematode survival.

Trichostrongylid nematode parasites of livestock inhabit two very different niches during their life-cycle; within the host and free-living in the environment. UV radiation plays a significant role in the survival of free-living, pre-parasitic nematode larvae, with different species exhibiting differing levels of sensitivity. In many eukaryotes, melanisation is a key protective mechanism against UV damage, however there is little information about this process in parasitic nematodes. Caenorhabditis elegans cat-4 mutants, which are deficient in the enzyme guanosine triphosphate-cyclohydrolase I (GTP-CH), have both depleted levels of melanin in their cuticles and an increased sensitivity to anthelmintic drugs. Some parasitic nematodes have very high levels of GTP-CH transcript in their pre-parasitic stages, suggesting an important role for this biopterin synthetic enzyme. Here, we show that the Tci-cat-4 gene, which encodes GTP-CH in Teladorsagia circumcincta, has a role in melanisation and is also capable of rescuing C. elegans cat-4 mutants. In addition, following exposure of T. circumcincta L3s to sunlight, there is a 32% increase in GTP-CH enzyme activity (P=0.019), and a 21% increase in levels of melanin (P=0.031) compared with unexposed larvae. These data suggest that one explanation for the high level of GTP-CH present in pre-parasitic stages of trichostrongylid nematodes is to facilitate melanisation in response to UV exposure.

Interactions of cell penetrating peptide Tat with model membranes: a biophysical study.

The protein transduction domain of the HIV-1 transactivator of transcription, Tat (Tat((48-60))), has been shown to transport P10, a cytotoxic peptide mimic of the cyclin dependent kinase inhibitor p21WAF1/CIP1, into the nucleus of cancerous cells and induce apoptosis. Here, monolayer studies were used to investigate the membrane interactions of Tat((48-60)), P10 and the construct Tat((48-60))P10. It was found that Tat((48-60)) showed no significant surface activity but that both P10 and Tat((48-60))P10, were highly surface active, inducing surface pressure changes of 9.7 and 8.9mNm(-1), respectively, with DMPS monolayers. The comparison of Tat((48-60))P10 and P10 surface interactions would be consistent with a hypothesis that the cargo attachment influences the capacity of the Tat-protein transduction domain to mediate transport across membranes either directly or via localisation of the construct at the membrane interface.