An Rtn4/Nogo-A-interacting micropeptide modulates synaptic plasticity with age.

Micropeptides, encoded from small open reading frames of 300 nucleotides or less, are hidden throughout mammalian genomes, though few functional studies of micropeptides in the brain are published. Here, we describe a micropeptide known as the Plasticity-Associated Neural Transcript Short (Pants), located in the 22q11.2 region of the human genome, the microdeletion of which conveys a high risk for schizophrenia. Our data show that Pants is upregulated in early adulthood in the mossy fiber circuit of the hippocampus, where it exerts a powerful negative effect on long-term potentiation (LTP). Further, we find that Pants is secreted from neurons, where it associates with synapses but is rapidly degraded with stimulation. Pants dynamically interacts with Rtn4/Nogo-A, a well-studied regulator of adult plasticity. Pants interaction with Nogo-A augments its influence over postsynaptic AMPA receptor clustering, thus gating plasticity at adult synapses. This work shows that neural micropeptides can act as architectural modules that increase the functional diversity of the known proteome.

Pain in the Prehospital Setting in Rwanda: Results of a Mixed-Methods Quality Improvement Project.

Introduction: Pain is a universal human experience tied to an individual's health but difficult to understand. It is especially important in health emergencies. We performed a two-step quality improvement project to assess pain management by the SAMU ambulance service in Kigali, Rwanda, examining how pain is assessed and treated by ambulance staff to facilitate development of standardized guidelines of pain management in the prehospital setting, which did not exist at the time of the study. Materials and Methods: Deidentified ambulance service records from December 2012 to May 2016 were analyzed descriptively for patient demographics, emergency conditions, pain assessment, and medications given. Then, anonymized, semistructured interviews of ambulance staff were conducted until thematic saturation was achieved. Data were analyzed using a grounded theory approach. Results: SAMU managed 11,161 patients over the study period, of which 6,168 (55%) were documented as reporting pain and 5,010 (45%) received pain medications. Men had greater odds of receiving pain medications compared to women (OR = 3.8, 95% CI (3.5, 4.1), p < 0.01). Twenty interviews were conducted with SAMU staff. They indicated that patients communicate pain in different ways. They reported using informal ways to measure pain or a standardized granular numeric scale. The SAMU team reviewed these results and developed plans to modify practices. Conclusions: We reviewed the existing quality of pain management in the prehospital setting in Kigali, Rwanda, assessed the SAMU staff's perceptions of pain, and facilitated standardization of prehospital pain management through context-specific guidelines.

Detecting episomal or integrated human papillomavirus 16 DNA using an exonuclease V-qPCR-based assay.

Screening for human papillomavirus (HPV) integration into host cell chromosomes typically requires large amounts of time and reagents. We developed a rapid and sensitive assay based on exonuclease V (ExoV) and quantitative polymerase chain reaction (qPCR) to determine HPV genome configurations in cell lines and tissues. We established the assay using genomic DNA from cell lines known to harbor integrated or episomal HPV16. DNA was incubated with ExoV, which is specific for linear DNA, and the DNA fraction resistant to digestion was measured by qPCR. The percent of DNA resistant to ExoV digestion was calculated relative to undigested DNA for determination of episomal or integrated HPV16. The ExoV assay was accurate, capable of distinguishing episomal from integrated HPV16 in cell lines and tissues. Future applications of the ExoV assay may include screening of HPV genome configurations in the progression of HPV-associated cancers.

Epstein-Barr Virus Infection Promotes Epithelial Cell Growth by Attenuating Differentiation-Dependent Exit from the Cell Cycle.

Epstein-Barr virus (EBV) is a human herpesvirus that is associated with lymphomas as well as nasopharyngeal and gastric carcinomas. Although carcinomas account for almost 90% of EBV-associated cancers, progress in examining EBV's role in their pathogenesis has been limited by difficulty in establishing latent infection in nontransformed epithelial cells. Recently, EBV infection of human telomerase reverse transcriptase (hTERT)-immortalized normal oral keratinocytes (NOKs) has emerged as a model that recapitulates aspects of EBV infection in vivo, such as differentiation-associated viral replication. Using uninfected NOKs and NOKs infected with the Akata strain of EBV (NOKs-Akata), we examined changes in gene expression due to EBV infection and differentiation. Latent EBV infection produced very few significant gene expression changes in undifferentiated NOKs but significantly reduced the extent of differentiation-induced gene expression changes. Gene set enrichment analysis revealed that differentiation-induced downregulation of the cell cycle and metabolism pathways was markedly attenuated in NOKs-Akata relative to that in uninfected NOKs. We also observed that pathways induced by differentiation were less upregulated in NOKs-Akata. We observed decreased differentiation markers and increased suprabasal MCM7 expression in NOKs-Akata versus NOKs when both were grown in raft cultures, consistent with our transcriptome sequencing (RNA-seq) results. These effects were also observed in NOKs infected with a replication-defective EBV mutant (AkataΔRZ), implicating mechanisms other than lytic-gene-induced host shutoff. Our results help to define the mechanisms by which EBV infection alters keratinocyte differentiation and provide a basis for understanding the role of EBV in epithelial cancers.IMPORTANCE Latent infection by Epstein-Barr virus (EBV) is an early event in the development of EBV-associated carcinomas. In oral epithelial tissues, EBV establishes a lytic infection of differentiated epithelial cells to facilitate the spread of the virus to new hosts. Because of limitations in existing model systems, the effects of latent EBV infection on undifferentiated and differentiating epithelial cells are poorly understood. Here, we characterize latent infection of an hTERT-immortalized oral epithelial cell line (NOKs). We find that although EBV expresses a latency pattern similar to that seen in EBV-associated carcinomas, infection of undifferentiated NOKs results in differential expression of a small number of host genes. In differentiating NOKs, however, EBV has a more substantial effect, reducing the extent of differentiation and delaying the exit from the cell cycle. This effect may synergize with preexisting cellular abnormalities to prevent exit from the cell cycle, representing a critical step in the development of cancer.

Inhibition of Epstein-Barr Virus Replication in Human Papillomavirus-Immortalized Keratinocytes.

Epstein-Barr virus (EBV) is implicated in the pathogenesis of human papillomavirus (HPV)-associated oropharyngeal squamous cell carcinoma (OSCC). EBV-associated cancers harbor a latent EBV infection characterized by a lack of viral replication and the expression of viral oncogenes. Cellular changes promoted by HPV are comparable to those shown to facilitate EBV latency, though whether HPV-positive cells support a latent EBV infection has not been demonstrated. Using a model of direct EBV infection into HPV16-immortalized tonsillar cells grown in organotypic raft culture, we showed robust EBV replication in HPV-negative rafts but little to no replication in HPV-immortalized rafts. The reduced EBV replication was independent of immortalization, as human telomerase-immortalized normal oral keratinocytes supported robust EBV replication. Furthermore, we observed reduced EBV lytic gene expression and increased expression of EBER1, a noncoding RNA highly expressed in latently infected cells, in the presence of HPV. The use of human foreskin keratinocyte rafts expressing the HPV16 E6 and/or E7 oncogene(s) (HPV E6 and E7 rafts) showed that E7 was sufficient to reduce EBV replication. EBV replication is dependent upon epithelial differentiation and the differentiation-dependent expression of the transcription factors KLF4 and PRDM1. While KLF4 and PRDM1 levels were unaltered, the expression levels of KLF4 transcriptional targets, including late differentiation markers, were reduced in HPV E6 and E7 rafts compared to their levels in parental rafts. However, the HPV E7-mediated block in EBV replication correlated with delayed expression of early differentiation markers. Overall, this study reveals an HPV16-mediated block in EBV replication, through E7, that may facilitate EBV latency and long-term persistence in the tumor context.IMPORTANCE Using a model examining the establishment of EBV infection in HPV-immortalized tissues, we showed an HPV-induced interruption of the normal EBV life cycle reminiscent of a latent EBV infection. Our data support the notion that a persistent EBV epithelial infection depends upon preexisting cellular alterations and suggest the ability of HPV to promote such changes. More importantly, these findings introduce a model for how EBV coinfection may influence HPV-positive (HPV-pos) OSCC pathogenesis. Latently EBV-infected epithelial cells, as well as other EBV-associated head-and-neck carcinomas, exhibit oncogenic phenotypes commonly seen in HPV-pos OSCC. Therefore, an HPV-induced shift in the EBV life cycle toward latency would not only facilitate EBV persistence but also provide additional viral oncogene expression, which can contribute to the rapid progression of HPV-pos OSCC. These findings provide a step toward defining a role for EBV as a cofactor in HPV-positive oropharyngeal tumors.

Epstein-Barr virus in the pathogenesis of oral cancers.

Epstein-Barr virus (EBV) is a ubiquitous gamma-herpesvirus that establishes a lifelong persistent infection in the oral cavity and is intermittently shed in the saliva. EBV exhibits a biphasic life cycle, supported by its dual tropism for B lymphocytes and epithelial cells, which allows the virus to be transmitted within oral lymphoid tissues. While infection is often benign, EBV is associated with a number of lymphomas and carcinomas that arise in the oral cavity and at other anatomical sites. Incomplete association of EBV in cancer has questioned if EBV is merely a passenger or a driver of the tumorigenic process. However, the ability of EBV to immortalize B cells and its prevalence in a subset of cancers has implicated EBV as a carcinogenic cofactor in cellular contexts where the viral life cycle is altered. In many cases, EBV likely acts as an agent of tumor progression rather than tumor initiation, conferring malignant phenotypes observed in EBV-positive cancers. Given that the oral cavity serves as the main site of EBV residence and transmission, here we review the prevalence of EBV in oral malignancies and the mechanisms by which EBV acts as an agent of tumor progression.

Reasons of repeat dental treatment under general anaesthesia: A retrospective study.

AIM: The purpose of this chart review study was to investigate the common factors that exist in paediatric patients requiring a repeat dental treatment under general anaesthesia (GA2) within four years after the initial dental treatment under general anaesthesia (GA1). MATERIALS AND METHODS: The Electronic Health Records of one to 12 year-old children who received dental treatment under general anaesthesia (GA) between April 2004 and October 2009 were identified and analysed by a single examiner. Children who had GA2, within a four year period following GA1 were categorised as cases. Children who had only one dental treatment under GA were considered the control pool. Each case was matched to three controls based on sex and age range at GA1 of ± 6 months. Other recorded variables included: date of birth, date of GAs (GA1 and GA2 for cases; GA1 for controls), type of payment, dmfs before GA1, dental treatments provided under GA, return of 1-week post-GA1 follow-up, frequency of recare/recall visits following one-year post-GA1 visit and the type and frequency of post GA1 emergency visits. RESULTS: Out of 581 subjects, 29 (4.99%) cases were matched to 87 controls. Medically compromised patients had four times the risk of GA2. At GA1, cases received statistically significant less sealants (p=0.026), less extractions (p<0.0001), and more composite restorations (p=0.0002) compared to controls. CONCLUSION: Medically compromised children and children treated with more composites and fewer sealants and extractions at their initial dental treatment under general anaesthesia were more likely to have a repeat dental treatment under general anaesthesia within 4 years.

The interaction between human papillomavirus and other viruses.

The etiological role of human papillomavirus (HPV) in anogenital tract and head and neck cancers is well established. However, only a low percentage of HPV-positive women develop cancer, indicating that HPV is necessary but not sufficient in carcinogenesis. Several biological and environmental cofactors have been implicated in the development of HPV-associated carcinoma that include immune status, hormonal changes, parity, dietary habits, tobacco usage, and co-infection with other sexually transmissible agents. Such cofactors likely contribute to HPV persistent infection through diverse mechanisms related to immune control, efficiency of HPV infection, and influences on tumor initiation and progression. Conversely, HPV co-infection with other factors may also harbor anti-tumor effects. Here, we review epidemiological and experimental studies investigating human immunodeficiency virus (HIV), herpes simplex virus (HSV) 1 and 2, human cytomegalovirus (HCMV), Epstein-Barr virus (EBV), BK virus (BKV), JC virus (JCV), and adeno-associated virus (AAV) as viral cofactors in or therapeutic factors against the development of genital and oral HPV-associated carcinomas.

Characterization of surface antigen from Lyme disease spirochete Borrelia burgdorferi.

Borrelia burgdorferi, the Lyme disease spirochete, possesses a surface protein, VlsE (variable major protein-like sequence, expressed), that undergoes antigenic variation. Unlike conserved regions of other proteins involved in antigenic variation, the most conserved invariable region of VlsE is immunodominant in Lyme-disease patients. Physicochemical analyses of pure recombinant VlsE yielded the following results: The protein appeared oligomeric in solution, with a secondary structure dominated by alpha-helices. Thermal denaturation (pH 7) probed by calorimetry involved two transitions: oligomer-to-monomer conversion (around 40 degrees C) followed by protein unfolding (55 +/- 1 degrees C). Chemical denaturation monitored by far-UV circular dichroism (20 degrees C, pH 7) sensed only polypeptide unfolding and took place in a single transition (Delta G(U)(H(2)O) = 23 +/- 2 kJ/mol). VlsE did not adopt a native structure at pH 3; at pH 10 the stability was significantly reduced. Knowledge of biophysical properties of VlsE may aid in understanding the mechanism of VlsE antigenic variation in B. burgdorferi.

The Medical Education Commission report on trends in graduate medical education 2001.

The state of Louisiana Medical Education Commission offers the second publication in the Journal of Louisiana State Medical Society on Graduate Medical Education (GME) in Louisiana. The four previous annual reports of the Commission have provided data to focus on trends in size, status, proportions, and issues on GME. The Commission was established by Act 3 of the 1997 Louisiana Legislature. This summary report for 2001 provides the detailed and updated data on all GME residents and fellows in Louisiana for the last academic year. The presentation of data and trends poses three questions for analysis. First, what are the trends in Graduate Medical Education in Louisiana for total residents and fellows? Second, how do these trends relate to the size, status, and proportions of Graduate Medical Education in Louisiana and as compared to the United States? Third, where do these trends lead to the future in predictions and recommendations for Graduate Medical Education in Louisiana? The 4-year trend in the total number of Graduate Medical Education filled positions has been relatively consistent, with some recent increases in primary care residency positions, especially in Family Medicine and Medicine/Pediatrics. These increases will plateau as current trends on new program formation stabilize, requiring emphasis on recruitment and factors promoting recruitment for GME in the state of Louisiana. The state of Louisiana has participated proportionately relative to population in the growth of medical education in the last century and compares favorably with other states and the nation. Louisiana has exceeded national averages in the increases in primary care programs and residents and in the retention of trainees in practice sites in the state.

High stability of a ferredoxin from the hyperthermophilic archaeon A. ambivalens: involvement of electrostatic interactions and cofactors.

The ferredoxin from the thermophilic archaeon Acidianus ambivalens is a small monomeric seven-iron protein with a thermal midpoint (T(m)) of 122 degrees C (pH 7). To gain insight into the basis of its thermostability, we have characterized unfolding reactions induced chemically and thermally at various pHs. Thermal unfolding of this ferredoxin, in the presence of various guanidine hydrochloride (GuHCl) concentrations, yields a linear correlation between unfolding enthalpies (DeltaH[T(m)]) and T(m) from which an upper limit for the heat capacity of unfolding (DeltaC(P)) was determined to be 3.15 +/- 0.1 kJ/(mole * K). Only by the use of the stronger denaturant guanidine thiocyanate (GuSCN) is unfolding of A. ambivalens ferredoxin at pH 7 (20 degrees C) observed ([GuSCN](1/2) = 3.1 M; DeltaG(U)[H(2)O] = 79 +/- 8 kJ/mole). The protein is, however, less stable at low pH: At pH 2.5, T(m) is 64 +/- 1 degrees C, and GuHCl-induced unfolding shows a midpoint at 2.3 M (DeltaG(U)[H(2)O] = 20 +/- 1 kJ/mole). These results support that electrostatic interactions contribute significantly to the stability. Analysis of the three-dimensional molecular model of the protein shows that there are several possible ion pairs on the surface. In addition, ferredoxin incorporates two iron-sulfur clusters and a zinc ion that all coordinate deprotonated side chains. The zinc remains bound in the unfolded state whereas the iron-sulfur clusters transiently form linear three-iron species (in pH range 2.5 to 10), which are associated with the unfolded polypeptide, before their complete degradation.

Hybridization of 2'-ribose modified mixed-sequence oligonucleotides: thermodynamic and kinetic studies.

In this study, we characterize the thermodynamics of hybridization, binding kinetics and conformations of four ribose-modified (2'-fluoro, 2'-O-propyl, 2'-O-methoxyethyl and 2'-O-aminopropyl) decameric mixed-sequence oligonucleotides. Hybridization to the complementary non-modified DNA or RNA decamer was probed by fluorescence and circular-dichroism spectroscopy and compared to the same duplex formed between two non-modified strands. The thermal melting points of DNA-DNA duplexes were increased by 1.8, 2.2, 0.3 and 1.3 degrees C for each propyl, methoxyethyl, aminopropyl and fluoro modification, respectively. In the case of DNA-RNA duplexes, the melting points were increased by 3.1, 4.1 and 1.0 degrees C for each propyl, methoxyethyl and aminopropyl modification, respectively. The high stability of the duplexes formed with propyl-, methoxyethyl- and fluoro-modified oligonucleotides correlated with high preorganization in these single-strands. Despite higher thermodynamic duplex stability, hybridization kinetics to complementary DNA or RNA was slower for propyl- and methoxyethyl-modified oligonucleotides than for the non-modified control. In contrast, the positively-charged aminopropyl-modified oligonucleotide showed rapid binding to the complementary DNA or RNA.

Probing copper ligands in denatured Pseudomonas aeruginosa azurin: unfolding His117Gly and His46Gly mutants.

Azurin is a single-domain beta-barrel protein with a redox-active copper cofactor. Upon Pseudomonas aeruginosa azurin unfolding, the cofactor remains bound to the polypeptide, coordinating three ligands: cysteine-112, one histidine imidazole, and a third, unknown ligand. In order to identify which histidine (histidine-117 and histidine-46 both coordinate copper in native azurin) is involved in copper coordination in denatured azurin, two single-site (histidine to glycine) mutants, His117Gly and His46Gly azurin, are investigated here. Equilibrium denaturation experiments of His46Gly azurin loaded with copper demonstrate that copper remains bound to this mutant in high urea concentrations where the protein's secondary structure is lost. In contrast, for copper-loaded His117Gly azurin, copper does not stay coordinated upon polypeptide unfolding. The copper absorption at 370 nm in denatured His46Gly azurin agrees with that for copper in complex with a peptide corresponding to residues 111-123 in azurin, suggesting similar metal coordination. We conclude that histidine-117 (and not histidine-46) is the histidine copper ligand in denatured azurin. This is also in accord with the proximity of histidine-117 to cysteine-112 in the primary sequence.

Equilibrium unfolding of dimeric desulfoferrodoxin involves a monomeric intermediate: iron cofactors dissociate after polypeptide unfolding.

Here we report the conformational stability of homodimeric desulfoferrodoxin (dfx) from Desulfovibrio desulfuricans (ATCC 27774). The dimer is formed by two dfx monomers linked through beta-strand interactions in two domains; in addition, each monomer contains two different iron centers: one Fe-(S-Cys)(4) center and one Fe-[S-Cys+(N-His)(4)] center. The dissociation constant for dfx was determined to be 1 microM (DeltaG = 34 kJ/mol of dimer) from the concentration dependence of aromatic residue emission. Upon addition of the chemical denaturant guanidine hydrochloride (GuHCl) to dfx, a reversible fluorescence change occurred at 2-3 M GuHCl. This transition was dependent upon protein concentration, in accord with a dimer to monomer reaction [DeltaG(H(2)O) = 46 kJ/mol of dimer]. The secondary structure did not disappear, according to far-UV circular dichroism (CD), until 6 M GuHCl was added; this transition was reversible (for incubation times of < 1 h) and independent of dfx concentration [DeltaG(H(2)O) = 50 kJ/mol of monomer]. Thus, dfx equilibrium unfolding is at least three-state, involving a monomeric intermediate with native-like secondary structure. Only after complete polypeptide unfolding (and incubation times of > 1 h) did the iron centers dissociate, as monitored by disappearance of ligand-to-metal charge transfer absorption, fluorescence of an iron indicator, and reactivity of cysteines to Ellman's reagent. Iron dissociation took place over several hours and resulted in an irreversibly denatured dfx. It appears as if the presence of the iron centers, the amino acid composition, and, to a lesser extent, the dimeric structure are factors that aid in facilitating dfx's unusually high thermodynamic stability for a mesophilic protein.

Apparent heparin resistance from elevated factor VIII during pregnancy.

BACKGROUND: Heparin resistance is the need for more than 35,000 units of heparin per 24 hours to achieve therapeutic activated partial thromboplastin time (APTT) values. Elevated factor VIII can cause apparent heparin resistance by suppressing the APTT result without inhibiting the antithrombotic effect of heparin. CASE: A 41-year-old gravida 2 para 0 presented at 25 weeks of a twin gestation with a deep venous thrombosis that required unusually high doses of heparin, resulting in hematuria. Apparent heparin resistance caused by elevated factor VIII was diagnosed, and the heparin dose was appropriately decreased with anti-Xa heparin monitoring. The deep venous thrombosis and hematuria resolved. CONCLUSION: Factor VIII rises significantly during pregnancy, and can cause apparent heparin resistance. When this occurs, anti-Xa heparin levels are superior to APTT for monitoring heparin therapy.

Cytochrome c(553), a small heme protein that lacks misligation in its unfolded state, folds with rapid two-state kinetics.

Cytochrome c(553) (cyt c(553)) from Desulfovibrio vulgaris is a small helical heme protein that displays apparent two-state equilibrium-unfolding behavior. The covalently attached heme is low-spin, ligated by Met and His residues, in the native state but becomes high-spin upon unfolding at pH 7. Here, we show that in contrast to other c-type heme proteins, where misligations in the unfolded states are prominent, cyt c(553) refolding kinetics at pH 7 proceeds rapidly without detectable intermediates. The extrapolated folding rate constant in water for oxidized cyt c(553) matches exactly that predicted from the cyt c(553) native-state topology: 5300 s(-1 )(experimental) versus 5020 s(-1) (predicted). We therefore conclude that the presence of the oxidized cofactor does not affect the intrinsic formation speed of the cyt c(553 )structural motif.

No cofactor effect on equilibrium unfolding of Desulfovibrio desulfuricans flavodoxin.

Flavodoxins are proteins with an alpha/beta doubly wound topology that mediate electron transfer through a non-covalently bound flavin mononucleotide (FMN). The FMN moiety binds strongly to folded flavodoxin (K(D)=0.1 nM, oxidized FMN). To study the effect of this organic cofactor on the conformational stability, we have characterized apo and holo forms of Desulfovibrio desulfuricans flavodoxin by GuHCl-induced denaturation. The unfolding reactions for both holo- and apo-flavodoxin are reversible. However, the unfolding curves monitored by far-UV circular dichroism and fluorescence spectroscopy do not coincide. For both apo- and holo-flavodoxin, a native-like intermediate (with altered tryptophan fluorescence but secondary structure as the folded form) is present at low GuHCl concentrations. There is no effect on the flavodoxin stability imposed by the presence of the FMN cofactor (DeltaG=20(+/-2) and 19(+/-1) kJ/mol for holo- and apo-flavodoxin, respectively). A thermodynamic cycle, connecting FMN binding to folded and unfolded flavodoxin with the unfolding free energies for apo- and holo-flavodoxin, suggests that the binding strength of FMN to unfolded flavodoxin must be very high (K(D)=0.2 nM). In agreement, we discovered that the FMN remains coordinated to the polypeptide upon unfolding.