Prokaryotic Super Program Advisory Committee DOE Joint Genome Institute,Walnut Creek,CA, March 27, 2013

The Prokaryotic Super Program Advisory Committee met on March 27, 2013 for their annual review the Prokaryotic Super Program at the DOE Joint Genome Institute. As is the case with any site visit or program review, the objective is to evaluate progress in meeting organizational objectives, provide feedback to from the user-community and to assist the JGI in formulating plans for the coming year. The advisors want to commend the JGI for its central role in developing new technologies and capabilities, and for catalyzing the formation of new collaborative user communities. Highlights of the post-meeting exchanges among the advisors focused on the importance of programmatic initiatives including:• GEBA, which serves as a phylogenetic “base-map” on which our knowledge of functional diversity can be layered.• FEBA, which promises to provide new insights into the physiological capabilities of prokaryotes under highly standardized conditions.• Single-cell genomics technology, which is seen to significantly enhance our ability to interpret genomic and metagenomic data and broaden the scope of the GEBA program to encompass at least a part of the microbial “dark-matter”.• IMG, which is seen to play a central role in JGI programs and is viewed as a strategically important asset in the JGI portfolio.On this latter point, the committee encourages the formation of a strategic relationship between IMG and the Kbase to ensure that the intelligence, deep knowledge and experience captured in the former is not lost. The committee strongly urges the DOE to continue its support for maintaining this critical resource.     

Reactogenicity of fluid compared with adsorbed diphtheria-pertussis-tetanus vaccine.

Shortly after the introduction of adsorbed diphtheria-pertussis-tetanus (DPT) vaccine in British Columbia the frequency of reports of reactions to the vaccine increased. As the reasons for the increase were not clear a study was carried out in five health units to compare the reactions to adsorbed DPT vaccine manufactured by Wyeth Ltd. and Connaught Laboratories Ltd. and fluid DPT vaccines manufactured by Connaught, all the vaccines being injected in the anterolateral thigh. From the responses on 1619 questionnaires that the parents of vaccinated children had completed it was found that the relative risk of a reaction was higher with the fluid than with the adsorbed Connaught vaccine (1.7 for redness and 1.8 for swelling on the day of vaccination but 1.0 for drowsiness and 1.3 for persistent crying). The size and duration of local redness and swelling were also greater with the fluid than with the adsorbed Connaught vaccines. The results with the Wyeth and Connaught vaccine were very similar. Only 10% of the parents said that there had been no reaction; 9% said that the reaction was severe, and 6% said that it was completely unacceptable. The overall frequency of local reactions was 86.1%.     

ON THE NATURE OF FORCES OPERATING IN BLOOD CLOTTING : I. THE PARTICIPATION OF ELECTROSTATIC ATTRACTION

The results of the study of the inhibiting effect of neutral salts upon the clotting tendency of fibrinogen by thrombin may be summarised as follows: Salts like NaCl and KCl inhibit only weakly. Salts of the same cation (K^•) with monovalent anions of different ionic radius are the more active the larger the anion (Cl'',Br'',I''). Salts of the same cation with anions of different valency are the more active the higher the charge of the anion (1–1 <1–2 <1–3 <1–4). Salts with the same anion with cations of different valency show stronger inhibition in the case of cations of higher charge (K^•,Na^• < Mg^••, Ca^••, Sr^••, Ba^••). Salts with the same anion and cations of the same charge, but of different radius, are the more active the larger the cation (but with an inversion between Mg^•• and Ca^•• in the series of the alkali earths, which is not infrequent in biocolloids). These results show that the clotting of fibrinogen with thrombin is, at least partly, caused by a coacervation process, due to electrostatic attraction between positive and negative groups. Its nature and localisation will be dealt with in the next paper of this series.     

GRADED ABRASION—A TREATMENT FOR ACNE

Long term continuous superficial abrasion with abrasive compounds of various degrees of roughness was used over an 11-year period in over 1,500 patients with acne of all types. The following observations were made:• That when a material with a specific abrasive quality is used for a period, desquamation stops despite continued use of the material, and then use of material of the next higher degree of roughness is indicated until the patient''s skin is blackhead-free and minutely flaky.• When long term continuous superficial graded abrasion is used alone or with other treatment, it permits the young patients to outgrow the disease with little or no scarring.• The routine of abrasive washing seems in a large measure to replace the constant fingering of lesions by the patient.• No untoward effects were observed clinically. There was no hyperpigmentation of Negro or Caucasian skins.     

New Concepts of Digitalis

The data presented in this review have substantiated the following ideas:• There is a direct relationship between digitalis dosage and myocardial contractile force. A small dose of digitalis produces a small increase of the contractile force of the heart and a large dose increases the force of the heart''s contraction by a much greater amount, whether the heart is normal, has a poor reserve, or is in frank failure.• Digitalis is of benefit to the patient with cardiovascular disease whether his heart is in failure or not.     

Fn14?Trail Effectively Inhibits Hepatocellular Carcinoma Growth

Background

New strategies for the treatment of hepatocellular carcinoma (HCC) are needed, given that currently available chemotherapeutics are inefficient. Since tumor growth reflects the net balance between pro-proliferative and death signaling, agents shifting the equilibrium toward the latter are of considerable interest. The TWEAK:Fn14 signaling axis promotes tumor cell proliferation and tumor angiogenesis, while TRAIL:TRAIL-receptor (TRAIL-R) interactions selectively induce apoptosis in malignant cells. Fn14•TRAIL, a fusion protein bridging these two pathways, has the potential to inhibit tumor growth, by interfering with TWEAK:Fn14 signaling, while at the same time enforcing TRAIL:TRAIL-R-mediated apoptosis. Consequently, Fn14•TRAIL''s capacity to inhibit HCC growth was tested.

Results

Fn14•TRAIL induced robust apoptosis of multiple HCC cell lines, while sparing non-malignant hepatocyte cell lines. Differential susceptibility to this agent did not correlate with expression levels of TRAIL, TRAIL-R, TWEAK and Fn14 by these lines. Fn14•TRAIL was more potent than soluble TRAIL, soluble Fn14, or a combination of the two. The requirement of both of Fn14•TRAIL''s molecular domains for function was established using blocking antibodies directed against each of them. Subcutaneous injection of Fn14•TRAIL abrogated HCC growth in a xenograft model, and was well tolerated by the mice.

Conclusions

In this study, Fn14•TRAIL, a multifunctional fusion protein originally designed to treat autoimmunity, was shown to inhibit the growth of HCC, both in vitro and in vivo. The demonstration of this fusion protein’s potent anti-tumor activity suggests that simultaneous targeting of two signaling axes by a single fusion can serve as a basis for highly effective anti-cancer therapies.     

Clinical Use of a New Mitral Disc Valve

A disc valve of new design was used successfully for the replacement of the mitral valve in patients with rheumatic mitral valve disease. This valve would appear to have the following advantages over the mitral ball valve prosthesis:• Lower left atrial pressure after replacement.• Elimination of the hazard of left ventricular outflow tract obstruction with mitral valve replacement.• Decreased incidence of thromboembolization.• Abolition of possibility of ventricular septal irritation.Despite the better outlook for this valve compared with the ball valve for mitral valve substitution, the mitral valve should always be repaired whenever feasible. Repair is possible in the majority of patients.     

THE MECHANISM OF CHANGE IN RESISTANCE OF ERYTHROCYTES TO HYPOTONIC SALT SOLUTIONS

Ashby''s work on the effects of KCl and NaCl on the resistance to hypotonic hemolysis of K^•-rich and K^•-poor erythrocytes has been repeated with great attention to purity of materials and refinement of technique. The results fail to agree with those of Ashby. 1. KCl produces greater loss in resistance to hypotonic hemolysis than does NaCl, irrespective of the species of the animal from which the cells are taken. 2. While cases of an increase in resistance have been encountered in my experiments, they are either very slight, or else the particular determination is subject to very great uncertainty. The great increases in resistance found by Ashby are not even approached in any of the present series of experiments. 3. Ashby''s generalization that KCl and NaCl have opposite effects on red blood cells, and that the sense of these effects depends on whether the cell is K^•-rich or K^•-poor is not substantiated.     

Interplay Among Nitric Oxide and Reactive Oxygen Species: A Complex Network Determining Cell Survival or Death

Programmed cell death (PCD) is an integrated cellular process occurring in plant growth, development, and defense responses to facilitate normal growth and development and better survival against various stresses as a whole. As universal toxic chemicals in plant and animal cells, reactive oxygen or nitrogen species (ROS or RNS), mainly superoxide anion (O₂^−•), hydrogen peroxide (H₂O₂) or nitric oxide (^•NO), have been studied extensively for their roles in PCD induction. Physiological and genetic studies have convincingly shown their essential roles. However, the details and mechanisms by which ROS and ^•NO interplay and induce PCD are not well understood. Our recent study on Cupressus lusitanica culture cell death revealed the elicitor-induced co-accumulation of ROS and ^•NO and interactions between ^•NO and H₂O₂ or O₂-^• in different ways to regulate cell death. ^•NO and H₂O₂ reciprocally enhanced the production of each other whereas ^•NO and O₂^−• showed reciprocal suppression on each other''s production. It was the interaction between ^•NO and O₂-^• but not between ^•NO and H₂O₂ that induced PCD, probably through peroxynitrite (ONOO⁻). In this addendum, some unsolved issues in the study were discussed based on recent studies on the complex network of ROS and ^•NO leading to PCD in animals and plants.Key Words: cell death, nitric oxide, reactive oxygen species, interaction, posttranslational modification     

The Nitric Oxide Production in the Moss Physcomitrella patens Is Mediated by Nitrate Reductase

During the last 20 years multiple roles of the nitric oxide gas (•NO) have been uncovered in plant growth, development and many physiological processes. In seed plants the enzymatic synthesis of •NO is mediated by a nitric oxide synthase (NOS)-like activity performed by a still unknown enzyme(s) and nitrate reductase (NR). In green algae the •NO production has been linked only to NR activity, although a NOS gene was reported for Ostreococcus tauri and O. lucimarinus, no other Viridiplantae species has such gene. As there is no information about •NO synthesis neither for non-vascular plants nor for non-seed vascular plants, the interesting question regarding the evolution of the enzymatic •NO production systems during land plant natural history remains open. To address this issue the endogenous •NO production by protonema was demonstrated using Electron Paramagnetic Resonance (EPR). The •NO signal was almost eliminated in plants treated with sodium tungstate, which also reduced the NR activity, demonstrating that in P. patens NR activity is the main source for •NO production. The analysis with confocal laser scanning microscopy (CLSM) confirmed endogenous NO production and showed that •NO signal is accumulated in the cytoplasm of protonema cells. The results presented here show for the first time the •NO production in a non-vascular plant and demonstrate that the NR-dependent enzymatic synthesis of •NO is common for embryophytes and green algae.     

An active site rearrangement within the Tetrahymena group I ribozyme releases nonproductive interactions and allows formation of catalytic interactions

Biological catalysis hinges on the precise structural integrity of an active site that binds and transforms its substrates and meeting this requirement presents a unique challenge for RNA enzymes. Functional RNAs, including ribozymes, fold into their active conformations within rugged energy landscapes that often contain misfolded conformers. Here we uncover and characterize one such “off-pathway” species within an active site after overall folding of the ribozyme is complete. The Tetrahymena group I ribozyme (E) catalyzes cleavage of an oligonucleotide substrate (S) by an exogenous guanosine (G) cofactor. We tested whether specific catalytic interactions with G are present in the preceding E•S•G and E•G ground-state complexes. We monitored interactions with G via the effects of 2′- and 3′-deoxy (–H) and −amino (–NH₂) substitutions on G binding. These and prior results reveal that G is bound in an inactive configuration within E•G, with the nucleophilic 3′-OH making a nonproductive interaction with an active site metal ion termed M_A and with the adjacent 2′-OH making no interaction. Upon S binding, a rearrangement occurs that allows both –OH groups to contact a different active site metal ion, termed M_C, to make what are likely to be their catalytic interactions. The reactive phosphoryl group on S promotes this change, presumably by repositioning the metal ions with respect to G. This conformational transition demonstrates local rearrangements within an otherwise folded RNA, underscoring RNA''s difficulty in specifying a unique conformation and highlighting Nature''s potential to use local transitions of RNA in complex function.     

C5'- and C3'-sugar radicals produced via photo-excitation of one-electron oxidized adenine in 2'-deoxyadenosine and its derivatives

We report that photo-excitation of one-electron-oxidized adenine [A(-H)•] in dAdo and its 2′-deoxyribonucleotides leads to formation of deoxyribose sugar radicals in remarkably high yields. Illumination of A(-H)• in dAdo, 3′-dAMP and 5′-dAMP in aqueous glasses at 143 K leads to 80-100% conversion to sugar radicals at C5′ and C3′. The position of the phosphate in 5′- and 3′-dAMP is observed to deactivate radical formation at the site of substitution. In addition, the pH has a crucial influence on the site of sugar radical formation; e.g. at pH ~5, photo-excitation of A(-H)• in dAdo at 143 K produces mainly C5′• whereas only C3′• is observed at high pH ~12. ¹³C substitution at C5′ in dAdo yields ¹³C anisotropic couplings of (28, 28, 84) G whose isotropic component 46.7 G identifies formation of the near planar C5′•. A β-¹³C 16 G isotropic coupling from C3′• is also found. These results are found to be in accord with theoretically calculated ¹³C couplings at C5′ [DFT, B3LYP, 6-31(G) level] for C5′• and C3′•. Calculations using time-dependent density functional theory [TD-DFT B3LYP, 6-31G(d)] confirm that transitions in the near UV and visible induce hole transfer from the base radical to the sugar group leading to sugar radical formation.     

Nitric Oxide Modifies Global Histone Methylation by Inhibiting Jumonji C Domain-containing Demethylases

Methylation of lysine residues on histone tails is an important epigenetic modification that is dynamically regulated through the combined effects of methyltransferases and demethylases. The Jumonji C domain Fe(II) α-ketoglutarate family of proteins performs the majority of histone demethylation. We demonstrate that nitric oxide (^•NO) directly inhibits the activity of the demethylase KDM3A by forming a nitrosyliron complex in the catalytic pocket. Exposing cells to either chemical or cellular sources of ^•NO resulted in a significant increase in dimethyl Lys-9 on histone 3 (H3K9me2), the preferred substrate for KDM3A. G9a, the primary methyltransferase acting on H3K9me2, was down-regulated in response to ^•NO, and changes in methylation state could not be accounted for by methylation in general. Furthermore, cellular iron sequestration via dinitrosyliron complex formation correlated with increased methylation. The mRNA of several histone demethylases and methyltransferases was also differentially regulated in response to ^•NO. Taken together, these data reveal three novel and distinct mechanisms whereby ^•NO can affect histone methylation as follows: direct inhibition of Jumonji C demethylase activity, reduction in iron cofactor availability, and regulation of expression of methyl-modifying enzymes. This model of ^•NO as an epigenetic modulator provides a novel explanation for nonclassical gene regulation by ^•NO.     

Studies of translational misreading in vivo show that the ribosome very efficiently discriminates against most potential errors

Protein synthesis must rapidly and repeatedly discriminate between a single correct and many incorrect aminoacyl-tRNAs. We have attempted to measure the frequencies of all possible missense errors by tRNA, tRNA and tRNA. The most frequent errors involve three types of mismatched nucleotide pairs, U•U, U•C, or U•G, all of which can form a noncanonical base pair with geometry similar to that of the canonical U•A or C•G Watson–Crick pairs. Our system is sensitive enough to measure errors at other potential mismatches that occur at frequencies as low as 1 in 500,000 codons. The ribosome appears to discriminate this efficiently against any pair with non-Watson–Crick geometry. This extreme accuracy may be necessary to allow discrimination against the errors involving near Watson–Crick pairing.     

Formation of 8-oxo-7,8-dihydroguanine-radicals in gamma-irradiated DNA by multiple one-electron oxidations

Electron spin resonance (ESR) studies of radicals formed by radiation-induced multiple one-electron oxidations of guanine moieties in DNA are reported in this work. Annealing of gamma-irradiated DNA from 77 to 235 K results in the hydration of one electron oxidized guanine (G^•+) to form the 8-hydroxy-7,8-dihydroguanin-7-yl-radical (•GOH) having one β-proton coupling of 17–28 G and an anisotropic nitrogen coupling, A_‖, of ~20 G, A = 0 with g_‖ = 2.0026 and g = 2.0037. Further annealing to 258 K results in the formation of a sharp singlet at g = 2.0048 with line-width of 5.3 G that is identified as the 8-oxo-7,8-dihydroguanine one-electron-oxidized radical (8-oxo-G^•+). This species is formed via two one-electron oxidations of •GOH. These two one-electron oxidation steps leading to the formation of 8-oxo-G^•+ from •GOH in DNA, are in accordance with the expected ease of oxidation of •GOH and 8-oxo-G. The incorporation of oxygen from water in G^•+ leading to •GOH and to 8-oxo-G^•+ is verified by ESR studies employing ¹⁷O isotopically enriched water, which provide unambiguous evidence for the formation of both radicals. ESR analysis of irradiated-DNA in the presence of the electron scavenger, Tl³⁺, demonstrates that the cationic pathway leads to the formation of the 8-oxo-G^•+. In irradiated DNA–Tl³⁺ samples, Tl³⁺ captures electrons. Tl²⁺ thus produced is a strong oxidant (2.2 V), which is metastable at 77 K and is observed to increase the formation of G^•+ and subsequently of 8-oxo-G^•+ upon annealing. We find that in the absence of the electron scavenger the yield of 8-oxo-G^•+ is substantially reduced as a result of electron recombinations with G^•+ and possible reaction with •GOH.     

The Mentally Retarded Infant — Is Placement Feasible?

A 1968 questionnaire about placement resources for mentally retarded infants that was sent to public agencies in all 58 California counties showed:• Resources for counseling, placement, and financial aid for out-of-home placement are not uniformly available to all groups within the population.• Financial help with out-of-home placement of a member of a family that is not medically indigent may be available in less than one-fourth of the counties of the state.• Knowledge of resources is often inadequate. A family could be referred inappropriately from one agency to another.     

Impact of Community Based Peer Support in Type 2 Diabetes: A Cluster Randomised Controlled Trial of Individual and/or Group Approaches

Background

Diabetes peer support, where one person with diabetes helps guide and support others, has been proposed as a way to improve diabetes management. We have tested whether different diabetes peer support strategies can improve metabolic and/or psychological outcomes.

Methods

People with type 2 diabetes (n = 1,299) were invited to participate as either ‘peer’ or ‘peer support facilitator’ (PSF) in a 2x2 factorial randomised cluster controlled trial across rural communities (130 clusters) in England. Peer support was delivered over 8–12 months by trained PSFs, supported by monthly meetings with a diabetes educator. Primary end point was HbA1c. Secondary outcomes included quality of life, diabetes distress, blood pressure, waist, total cholesterol and weight. Outcome assessors and investigators were masked to arm allocation. Main factors were 1:1 or group intervention. Analysis was by intention-to-treat adjusting for baseline.

Results

The 4 arms were well matched (Group n = 330, 1:1(individual) n = 325, combined n = 322, control n = 322); 1035 (79•7%) completed the mid-point postal questionnaire and 1064 (81•9%) had a final HbA1c. A limitation was that although 92.6% PSFs and peers were in telephone contact, only 61.4% of intervention participants attended a face to face session. Mean baseline HbA1c was 57 mmol/mol (7•4%), with no significant change across arms. Follow up systolic blood pressure was 2•3mm Hg (0.6 to 4.0) lower among those allocated group peer-support and 3•0mm Hg (1.1 to 5.0) lower if the group support was attended at least once. There was no impact on other outcomes by intention to treat or significant differences between arms in self-reported adherence or medication.

Conclusions

Group diabetes peer support over 8–12 months was associated with a small improvement in blood pressure but no other significant outcomes. Long term benefits should be investigated.

Trial Registration

ISRCTN.com ISRCTN6696362166963621     

Reducing Deaths from Severe Pneumonia in Children in Malawi by Improving Delivery of Pneumonia Case Management

Objective

To evaluate the pneumonia specific case fatality rate over time following the implementation of a Child Lung Health Programme (CLHP) within the existing government health services in Malawi to improve delivery of pneumonia case management.

Methods

A prospective, nationwide public health intervention was studied to evaluate the impact on pneumonia specific case fatality rate (CFR) in infants and young children (0 to 59 months of age) following the implementation of the CLHP. The implementation was step-wise from October 1^st 2000 until 31^st December 2005 within paediatric inpatient wards in 24 of 25 district hospitals in Malawi. Data analysis compared recorded outcomes in the first three months of the intervention (the control period) to the period after that, looking at trend over time and variation by calendar month, age group, severity of disease and region of the country. The analysis was repeated standardizing the follow-up period by using only the first 15 months after implementation at each district hospital.

Findings

Following implementation, 47,228 children were admitted to hospital for severe/very severe pneumonia with an overall CFR of 9•8%. In both analyses, the highest CFR was in the children 2 to 11 months, and those with very severe pneumonia. The majority (64%) of cases, 2–59 months, had severe pneumonia. In this group there was a significant effect of the intervention Odds Ratio (OR) 0•70 (95%CI: 0•50–0•98); p = 0•036), while in the same age group children treated for very severe pneumonia there was no interventional benefit (OR 0•97 (95%CI: 0•72–1•30); p = 0•8). No benefit was observed for neonates (OR 0•83 (95%CI: 0•56–1•22); p = 0•335).

Conclusions

The nationwide implementation of the CLHP significantly reduced CFR in Malawian infants and children (2–59 months) treated for severe pneumonia. Reasons for the lack of benefit for neonates, infants and children with very severe pneumonia requires further research.     

Integrated Analysis of COX-2 and iNOS Derived Inflammatory Mediators in LPS-Stimulated RAW Macrophages Pre-Exposed to Echium plantagineum L. Bee Pollen Extract

Oxidative stress and inflammation play important roles in disease development. This study intended to evaluate the anti-inflammatory and antioxidant potential of Echium plantagineum L. bee pollen to support its claimed health beneficial effects. The hydromethanol extract efficiently scavenged nitric oxide (^•NO) although against superoxide (O₂ ^•−) it behaved as antioxidant at lower concentrations and as pro-oxidant at higher concentrations. The anti-inflammatory potential was evaluated in LPS-stimulated macrophages. The levels of ^•NO and L-citrulline decreased for all extract concentrations tested, while the levels of prostaglandins, their metabolites and isoprostanes, evaluated by UPLC-MS, decreased with low extract concentrations. So, E. plantagineum bee pollen extract can exert anti-inflammatory activity by reducing ^•NO and prostaglandins. The extract is able to scavenge the reactive species ^•NO and O₂ ^•− and reduce markers of oxidative stress in cells at low concentrations.     

Problems Related to the Use of Food Additives in the United States

Major problems encountered by enforcement agencies and by regulated industries, respectively, in implementing and conforming to recent food additive laws are reviewed. Decisions as to which substances fall within the broad terms of the legal definition, and which escape by virtue of “generally recognized as safe” (GRAS) status, are often difficult and complex. Distinctions cannot be made solely on the basis of whether substances are old or new, natural or synthetic. Registration of pesticides on a “no residue” basis and establishment of “zero tolerances” for food additives have created an anomalous situation as a result of improvements in sensitivity of analytical techniques which revealed the presence of minute amounts of substances where none were believed to exist. A solution has been recommended by a specially appointed committee of the National Academy of Sciences-National Research Council (U.S.A.). Enforcement of the new food additive laws warrants revision of present labelling requirements to provide for designating chemical ingredients by functional categories rather than by confusing chemical terminology.