Investigations, actions and learning from an outbreak of SARS-CoV-2 infection among healthcare workers in the United Kingdom.

BACKGROUND: We report an outbreak of SARS coronavirus-2 (SARS-CoV-2) infection among healthcare workers (HCW) in an NHS elective healthcare facility. METHODOLOGY: A narrative chronological account of events after declaring an outbreak of SARS-CoV-2 among HCWs. As part of the investigations, HCWs were offered testing during the outbreak. These were: (1) screening by real-time reverse transcriptase polymerase chain reaction (RT- PCR) to detect a current infection; and (2) serum samples to determine seroprevalence. RESULTS: Over 180 HCWs were tested by real-time RT-PCR for SARS-CoV-2 infection. The rate of infection was 15.2% (23.7% for clinical or directly patient-facing HCWs vs. 4.8% in non-clinical non-patient-facing HCWs). Of the infected HCWs, 57% were asymptomatic. Seroprevalence (SARS-CoV-2 IgG) among HCWs was 13%. It was challenging to establish an exact source for the outbreak. The importance of education, training, social distancing and infection prevention practices were emphasised. Additionally, avoidance of unnecessary transfer of patients and minimising cross-site working for staff and early escalation were highlighted. Establishing mass and regular screening for HCWs are also crucial to enabling the best care for patients while maintaining the wellbeing of staff. CONCLUSION: To our knowledge, this is the first UK outbreak report among HCWs and we hope to have highlighted some key issues and learnings that can be considered by other NHS staff and HCWs globally when dealing with such a task in future.

Integrative roles of nitric oxide and hydrogen sulfide in melatonin-induced tolerance of pepper (Capsicum annuum L.) plants to iron deficiency and salt stress alone or in combination.

There seems to be no report in the literature on the effect of melatonin (MT) in relieving the detrimental effects of combined application of salt stress (SS) and iron deficiency (ID). Therefore, the effect of MT on the accumulation/synthesis of endogenous nitric oxide (NO) and hydrogen sulphide (H2 S) and how far these molecules are involved in MT-improved tolerance to the combined application of ID and SS in pepper (Capsicum annuum L) were tested. Hence, two individual trials were set up. The treatments in the first experiment comprised: Control, ID (0.1 mM FeSO4 ), SS (100 mM NaCl) and ID + SS. The detrimental effects of combined stresses were more prominent than those by either of the single stress, with respect to growth, oxidative stress and antioxidant defense attributes. Single stress or both in combination improved the endogenous H2 S and NO, and foliar-applied MT (100 µM) led to a further increase in NO and H2 S levels. In the second experiment, 0.1 mM scavenger of NO, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potassium salt (cPTIO) and that of H2 S, hypotuarine (HT) were applied along with MT to get further evidence whether NO and H2 S are involved in MT-induced tolerance to ID and SS. MT combined with cPTIO and HT under a single or combined stress showed that NO effect was reversed by the NO scavenger, cPTIO, alone but the H2 S effect was inhibited by both scavengers. These findings suggested that tolerance to ID and SS induced by MT may be involved in downstream signal crosstalk between NO and H2 S.

Comparative analysis of silage fermentation and in vitro digestibility of tropical grass prepared with Acremonium and Tricoderma species producing cellulases.

OBJECTIVE: To find out ways of improving fermentation quality of silage, the comparative analysis of fermentation characteristics and in vitro digestibility of tropical grasses silage applied with cellulases produced from Acremonium or Tricoderma species were studied in Thailand. METHODS: Fresh and wilted Guinea grass and Napier grass silages were prepared with cellulases from Acremonium (AC) or Trichoderma (TC) at 0.0025%, 0.005%, and 0.01% on a fresh matter (FM), and their fermentation quality, chemical composition and in vitro digestibility were analyzed. RESULTS: All silages of fresh Napier grass were good quality with lower pH, butyric acid, and ammonia nitrogen, but higher lactic acid content than wilted Napier grass and Guinea grass silage. Silages treated with AC 0.01% had the best result in terms of fermentation quality. They also had higher in vitro dry matter digestibility and in vitro organic matter digestibility at 6 and 48 h after incubation than other silages. Silages treated with lower levels at 0.005% or 0.0025% of AC and all levels of TC did not improve silage fermentation. CONCLUSION: The AC could improve silage fermentation and in vitro degradation of Guinea grass and Napier grass silages, and the suitable addition ration is 0.01% (73.5 U) of FM for tropical silage preparation.

Fermentation quality and in vitro methane production of sorghum silage prepared with cellulase and lactic acid bacteria.

OBJECTIVE: The effects of lactic acid bacteria (LAB) and cellulase enzyme on fermentation quality, microorganism population, chemical composition and in vitro gas production of sorghum silages were studied. METHODS: Commercial inoculant Lactobacillus plantarum Chikuso 1 (CH), local selected strain Lactobacillus casei (L. casei) TH 14 and Acremonium cellulase (AC) were used as additives in sorghum silage preparation. RESULTS: Prior to ensiling Sorghum contained 104 LAB and 106 cfu/g fresh matter coliform bacteria. The chemical compositions of sorghum was 26.6% dry matter (DM), 5.2% crude protein (CP), and 69.7% DM for neutral detergent fiber. At 30 days of fermentation after ensiling, the LAB counts increased to a dominant population; the coliform bacteria and molds decreased to below detectable level. All sorghum silages were good quality with a low pH (<3.5) and high lactic acid content (>66.9 g/kg DM). When silage was inoculated with TH14, the pH value was significantly (p<0.05) lower and the CP content significantly (p<0.05) higher compared to control, CH and AC-treatments. The ratio of in vitro methane production to total gas production and DM in TH 14 and TH 14+AC treatments were significantly (p<0.05) reduced compared with other treatments while in vitro dry matter digestibility and gas production did not differ among treatments. CONCLUSION: The results confirmed that L. casei TH14 could improve sorghum silage fermentation, inhibit protein degradation and decrease methane production.

Natural lactic acid bacteria population of tropical grasses and their fermentation factor analysis of silage prepared with cellulase and inoculant.

Natural lactic acid bacteria (LAB) populations in tropical grasses and their fermentation characteristics on silage prepared with cellulase enzyme and LAB inoculants were studied. A commercial inoculant Lactobacillus plantarum Chikuso 1 (CH), a local selected strain Lactobacillus casei TH14 (TH14), and 2 cellulases, Acremonium cellulase (AC) and Maicelase (MC; Meiji Seika Pharma Co. Ltd., Tokyo, Japan), were used as additives to silage preparation with fresh and wilted (6 h) Guinea grass and Napier grass. Silage was prepared using a laboratory-scale fermentation system. Treatments were CH, TH14, AC at 0.01% fresh matter, AC 0.1%, MC 0.01%, MC 0.1%, CH+AC 0.01%, CH+AC 0.1%, CH+MC 0.01%, CH+MC 0.1%, TH14+AC 0.1%, TH14+AC 0.01%, TH14+MC 0.1%, and TH14+MC 0.01%. Microorganism counts of Guinea grass and Napier grass before ensiling were 102 LAB and 106 aerobic bacteria; these increased during wilting. Based on morphological and biochemical characteristics, and 16S rRNA gene sequence analysis, natural strains from both grasses were identified as L. plantarum, L. casei, Lactobacillus acidipiscis, Leuconostoc pseudomesenteroides, Leuconostoc garlicum, Weissella confusa, and Lactococcus lactis. Lactobacillus plantarum and L. casei are the dominant species and could grow at lower pH and produce more lactic acid than the other isolates. Crude protein and neutral detergent fiber were 5.8 and 83.7% of dry matter (DM) for Guinea grass, and 7.5 and 77.1% of DM for Napier grass. Guinea grass had a low level of water-soluble carbohydrates (0.39% of DM). Guinea grass silage treated with cellulase had a lower pH and higher lactic acid content than control and LAB treatments. The 0.1% AC and MC treatments had the best result for fermentation quality. All high water-soluble carbohydrate (2.38% DM) Napier grass silages showed good fermentation quality. Compared with control and LAB-inoculated silage, the cellulase-treated silages had significantly higher crude protein content and lower neutral detergent fiber and acid detergent fiber contents. The results confirmed that cellulase could improve tropical silage quality, inhibiting protein degradation and promoting fiber degradation.

Characterization and application of lactic acid bacteria for tropical silage preparation.

Strains TH 14, TH 21 and TH 64 were isolated from tropical silages, namely corn stover, sugar cane top and rice straw, respectively, prepared in Thailand. These strains were selected by low pH growth range and high lactic acid-producing ability, similar to some commercial inoculants. Based on the analysis of 16S ribosomal RNA gene sequence and DNA-DNA relatedness, strain TH 14 was identified as Lactobacillus casei, and strains TH 21 and TH 64 were identified as L. plantarum. Strains TH 14, TH 21, TH 64 and two commercial inoculants, CH (L. plantarum) and SN (L. rhamnosus), were used as additives to fresh and wilted purple Guinea and sorghum silages prepared using a small-scale fermentation method. The number of epiphytic lactic acid bacteria (LAB) in the forages before ensilage was relatively low but the numbers of coliform and aerobic bacteria were higher. Sorghum silages at 30 days of fermentation were all well preserved with low pH (3.56) and high lactic acid production (72.86 g/kg dry matter). Purple Guinea silage inoculated with LAB exhibited reduced count levels of aerobic and coliform bacteria, lower pH, butyric acid and ammonia nitrogen and increased lactic acid concentration, compared with the control. Strain TH 14 more effectively improved lactic acid production compared with inoculants and other strains. © 2016 Japanese Society of Animal Science.

Food Shortage Causes Differential Effects on Body Composition and Tissue-Specific Gene Expression in Salmon Modified for Increased Growth Hormone Production.

Growth hormone (GH) transgenic salmon possesses markedly increased metabolic rate, appetite, and feed conversion efficiency, as well as an increased ability to compete for food resources. Thus, the ability of GH-transgenic fish to withstand periods of food deprivation as occurs in nature is potentially different than that of nontransgenic fish. However, the physiological and genetic effects of transgenic GH production over long periods of food deprivation remain largely unknown. Here, GH-transgenic coho salmon (Oncorhynchus kisutch) and nontransgenic, wild-type coho salmon were subjected to a 3-month food deprivation trial, during which time performance characteristics related to growth were measured along with proximate compositions. To examine potential genetic effects of GH-transgenesis on long-term food deprivation, a group of genes related to muscle development and liver metabolism was selected for quantitative PCR analysis. Results showed that GH-transgenic fish lose weight at an increased rate compared to wild-type even though proximate compositions remained relatively similar between the groups. A total of nine genes related to muscle physiology (cathepsin, cee, insulin-like growth factor, myostatin, murf-1, myosin, myogenin, proteasome delta, tumor necrosis factor) and five genes related to liver metabolism (carnitine palmitoyltransferase, fatty acid synthase, glucose-6-phosphatase, glucose-6-phosphate dehydrogenase, glucokinase) were shown to be differentially regulated between GH-transgenic and wild-type coho salmon over time. These genetic and physiological responses assist in identifying differences between GH-transgenic and wild-type salmon in relation to fitness effects arising from elevated growth hormone during periods of long-term food shortage.

Flesh residue concentrations of organochlorine pesticides in farmed and wild salmon from British Columbia, Canada.

The present study reports measured levels of organochlorine pesticides (OCPs) in commercial salmon feed (n = 8) and farmed Atlantic, coho, and chinook salmon (n = 110), as well as wild coho, chinook, chum, sockeye, and pink salmon (n = 91). Flesh residue concentrations (ng/g wet weight) of dichlorodiphenyltrichloroethanes (DDTs), hexachlorocyclohexanes (HCHs), chlordanes, chlorobenzenes (CBz) and cyclodiene pesticides (e.g., dieldrin, mirex) were 2 to 11 times higher (p < 0.05) in farmed salmon compared with wild salmon. Concentrations were positively correlated with flesh lipid levels. Farmed Atlantic salmon (12-15% lipid) typically exhibited the greatest OCP burdens compared with other salmon species. However, when expressed on a lipid weight basis, concentrations of OCPs (ng/g lipid weight) in wild salmon, in many cases, exceeded those levels in farmed salmon. Observed interspecies and site-specific variations of OCP concentrations in farmed and wild salmon may be attributed to divergent life history, prey/feed characteristics and composition, bioenergetics, or ambient environmental concentrations. Calculated biomagnification factors (BMF = C(F)/C(D), lipid wt) of OCPs in farmed salmon typically ranged between two and five. Biomagnification of chemicals such as DDTs, chlordanes, and mirex was anticipated, because those compounds tend to exhibit high dietary uptake and slow depuration rates in fish because of relatively high octanol-water partition coefficients (K(OW)s > 105). Surprisingly, less hydrophobic pesticides such as hexachlorocyclohexanes and endosulfans (K(OW) s < 105) consistently exhibited a high degree of biomagnification in farmed salmon species (BMFs > 5). This is contrary to previous laboratory and field observations demonstrating fish BMFs less than 1 for low K(OW) chemicals, because of efficient respiratory elimination of those compounds via gills. The results suggest that ambient seawater concentrations and bioconcentration-driven accumulation may play a key role in the bioaccumulation of these relatively more water-soluble contaminants in farmed salmon. Finally, OCP exposure through consumption of British Columbian salmon is found to be low relative to United States national average per capita total exposure levels and provisional tolerable daily intakes.

Dislocation of large diameter metal-on-metal bearings an indicator of metal reaction?

We report 3 patients who underwent total hip arthroplasty (THA) using large diameter metal-on-metal bearing. These patients initially presented with pain but went on to develop dislocation of the THA while awaiting investigations. Any pain following metal-on-metal bearing THA should be taken seriously and should trigger investigations to identify a metal reaction. If left untreated, these reactions can cause progressive soft tissue necrosis leading to instability. These patients should be considered for early revision of the bearing surface to prevent further soft tissue damage.

Postprandial gastrointestinal blood flow, oxygen consumption and heart rate in rainbow trout (Oncorhynchus mykiss).

The present study is the first to simultaneously and continuously measure oxygen consumption (MO(2)) and gastrointestinal blood flow (q(gi)) in fish. In addition, while it is the first to compare the effects of three isoenergetic diets on q(gi) in fish, no significant differences among diets were found for postprandial MO(2), q(gi) or heart rate (f(H)) in rainbow trout, Oncorhynchus mykiss. Postprandial q(gi), f(H) and MO(2) were significantly elevated above baseline levels by 4 h. Postprandial q(gi) peaked at 136% above baseline after 11 h, f(H) peaked at 110% above baseline after 14 h and MO(2) peaked at 96% above baseline after 27 h. Moreover, postprandial MO(2) remained significantly elevated above baseline longer than q(gi) (for 41 h and 30 h, respectively), perhaps because most of the increase in MO(2) associated with feeding is due to protein handling, a process that continues following the absorption of nutrients which is thought to be the primary reason for the elevation of q(gi). In addition to the positive relationships found between postprandial MO(2) and q(gi) and between postprandial MO(2) and f(H), we discovered a novel relationship between postprandial q(gi) and f(H).

Mercury and other trace elements in farmed and wild salmon from British Columbia, Canada.

The present study reports measured levels of Hg and other trace elements in commercial salmon feed; farmed Atlantic, coho, and chinook salmon (n = 110); and wild coho, chinook, chum, sockeye, and pink salmon (n = 91). Metal concentrations in farmed and wild salmon from British Columbia, Canada, were relatively low and below human health consumption guidelines. Methylmercury in all salmon samples (range, 0.03-0.1 microg/g wet wt) were below the 0.5 microg/g guideline set by Health Canada. Negligible differences in metal concentrations were observed between the various species of farmed and wild salmon. Metal concentrations generally were higher in commercial salmon feed compared to farmed salmon. Mercury showed slight bioaccumulation potential in farmed salmon, with biomagnification factors (BMFs) ranging between 0.8 and 1.9. Other metals, such as Cd, Pb, and Ni, exhibited biodilution, with BMFs of much less than one. The relatively low degree of biomagnification of metals observed in farmed salmon likely resulted from the combination of low gastrointestinal absorption efficiency, negligible transfer to muscle tissue relative to other compartments, and a high degree of growth dilution in these fish. Human dietary exposure calculations indicate intakes of Hg, Cd, Pb, Cu, As, and Ni via farmed and wild British Columbia salmon are a relatively small percentage of total intakes (0.05-32%) compared to other Canadian foodstuffs, such as fruits, vegetables, chicken, and beef (68-99%). Although total dietary exposure of Cd, Pb, and Cu approached provisional tolerable daily intake levels, the contribution from British Columbia salmon was less than 2%. Our findings indicate farmed and wild British Columbia salmon remain a safe source of omega-3 highly unsaturated fatty acid intake for cardioprotective and, possibly, other health benefits.

Implementation and assessment of a molecular biology and bioinformatics undergraduate degree program.

The Department of Biological Sciences at the University of Wisconsin-Parkside has developed and implemented an innovative, multidisciplinary undergraduate curriculum in Molecular Biology and Bioinformatics (MBB). The objective of the MBB program is to give students a hands-on facility with molecular biology theories and laboratory techniques, an understanding of mathematical and physical concepts, an ability to apply these concepts to MBB, and a proficiency with the computational tools and skills related to bioinformatics. We hypothesized that a greater exposure to bioinformatics methods, more rigorous requirements in math and computer science, and a constant demand for integrating information in hands-on laboratory courses would help students develop better analytical skills. Indeed, the assessment data support these predictions. Interestingly, 80% of MBB majors apply and are accepted into graduate schools.

Nuclear suppressors define three factors that participate in both 5' and 3' end processing of mRNAs in Chlamydomonas chloroplasts.

Chloroplast RNA processing and degradation are orchestrated by nucleus-encoded factors. Although several transcript-specific factors have been identified, those involved in global RNA metabolism have mostly remained elusive. Using Chlamydomonas reinhardtii, we have identified three pleiotropic nuclear mutations, mcd3, mcd4 and mcd5, which cause quantitative variation between polycistronic transcripts and accumulation of transcripts with novel 3' ends. The mcd3, mcd4 and mcd5 mutants were initially isolated as photoautotrophic suppressors of the petD 5' mutants LS2 and LS6, which harbour four nucleotide linker-scanning mutations near the 5' end of the mature transcript. The LS mutants accumulate 1-3% of the wild-type (WT) petD mRNA level and no cytochrome b6/f complex subunit IV, which is the petD gene product and required for photosynthesis. Each suppressor restores approximately 15% of the WT petD mRNA and subunit IV levels. Genetic analysis showed mcd4 to be recessive, and suggested that MCD4 interacts with the petD mRNA stability factor MCD1. To assess the specificity of mcd3, mcd4 and mcd5, transcripts from 32 chloroplast genes were analysed by RNA filter hybridizations. mcd3 and mcd4 displayed aberrant transcript patterns for 17 genes, whereas only three were altered in mcd5. Since the mutations affect multiple RNAs in a variety of ways, our data suggest that MCD3, MCD4 and MCD5 may participate in a series of multiprotein complexes responsible for RNA maturation and degradation in Chlamydomonas chloroplasts.

Regulatory sequences of orthologous petD chloroplast mRNAs are highly specific among Chlamydomonas species.

The 5' untranslated regions (UTR) of chloroplast mRNAs often contain regulatory sequences that control RNA stability and/or translation. The petD chloroplast mRNA in Chlamydomonas reinhardtii has three such essential regulatory elements in its 362-nt long 5' UTR. To further analyze these elements, we compared 5' UTR sequences from four Chlamydomonas species (C. reinhardtii, C. incerta, C. moewusii and C. eugametos) and five independent strains of C. reinhardtii. Overall, these petD 5' UTRs have relatively low sequence conservation across these species. In contrast, sequences of the three regulatory elements and their relative positions appear partially conserved. Functionality of the 5' UTRs was tested in C. reinhardtii chloroplasts using beta-glucuronidase reporter genes, and the nearly identical C. incerta petD functioned for mRNA stability and translation in C. reinhardtii chloroplasts while the more divergent C. eugametos petD did not. This identified what may be key features in these elements. We conclude that these petD regulatory elements, and possibly the corresponding trans-acting factors, function via mechanisms highly specific and surprisingly sensitive to minor sequence changes. This provides a new and broader perspective of these important regulatory sequences that affect photosynthesis in these algae.

Effects of dietary exposure of 4-nonylphenol on growth and smoltification of juvenile coho salmon (Oncorhynchus kisutch).

There is considerable concern that endocrine disrupting substances such as 4-nonylphenol (4-NP) in the freshwater environment may have adverse effects on the growth, survival, and osmoregulatory ability of salmonids during and after their transfer to sea water. This study was conducted to examine the effects of dietary exposure of coho salmon (Oncorhynchus kisutch) to 4-NP during the parr-smolt transformation phase of their life cycle. Under laboratory conditions, juvenile fish were fed by hand twice daily to satiation diets dosed with one of several concentrations of 4-NP (doses varied between 0 (control) and 2000 mg/kg) for 4 weeks, then immediately transferred to sea water. Growth was observed for two successive 6-week periods following sea water transfer when all groups were fed the control diet (no supplemental 4-NP) only. In addition to 4-NP measurement in fish tissues, thyroid hormone concentrations in blood plasma were followed and related to diet treatment and sampling time. Dietary treatment of 4-NP did not influence the growth and smoltification of coho salmon, a result that conflicts to some extent with other reports in which deleterious effects of water-borne 4-NP on the smoltification process of salmonids were linked to disruption of the endocrine system. Appreciable concentrations of 4-NP were present in the livers, gall bladders and tissues after the 4-week exposure of coho salmon to the highest dietary dose of 4-NP, but 4-NP appeared to be effectively eliminated from the fish by the biliary-fecal pathway after sea water transfer.

Microarray analysis confirms the specificity of a Chlamydomonas reinhardtii chloroplast RNA stability mutant.

The expression of chloroplast and mitochondrial genes depends on nucleus-encoded proteins, some of which control processing, stability, and/or translation of organellar RNAs. To test the specificity of one such RNA stability factor, we used two known Chlamydomonas reinhardtii nonphotosynthetic mutants carrying mutations in the Mcd1 nuclear gene (mcd1-1 and mcd1-2). We previously reported that these mutants fail to accumulate the chloroplast petD mRNA and its product, subunit IV of the cytochrome b6/f complex, which is essential for photosynthesis. Such mutants are generally presumed to be gene specific but are not tested rigorously. Here, we have used microarray analysis to assess changes in chloroplast, mitochondrial, and nuclear RNAs, and since few other RNAs were significantly altered in these mutants, conclude that Mcd1 is indeed specifically required for petD mRNA accumulation. In addition, a new unlinked nuclear mutation was discovered in mcd1-2, which greatly reduced chloroplast atpA mRNA accumulation. Genetic analyses showed failure to complement mda1-ncc1, where atpA-containing transcripts are similarly affected (D. Drapier, J. Girard-Bascou, D.B. Stern, F.-A. Wollman [2002] Plant J 31: 687-697), and we have named this putative new allele mda1-2. We conclude that DNA microarrays are efficient and useful for characterizing the specificity of organellar RNA accumulation mutants.

Effect of dietary vitamin E level on growth, tissue lipid peroxidation, and erythrocyte fragility of transgenic coho salmon, Oncorhynchus kisutch.

This study was conducted to investigate the effect of dietary vitamin E concentration on growth performance, iron-catalyzed lipid peroxidation in liver and muscle tissue, and erythrocyte fragility of transgenic growth hormone coho salmon (Oncorhynchus kisutch). Fish were fed one of four isoenergetic and isonitrogenous experimental diets that contained either 11, 29, 50, or 105 IU of vitamin E/kg. Following the 10-week feeding trial, no significant (P>0.05) diet-related differences were detected in growth, whole body proximate composition or erythrocyte fragility. The vitamin E contents of liver and muscle, however, were affected by the dietary treatment. Fish fed diets containing > or =50 IU of vitamin E/kg had significantly increased vitamin E concentrations in their tissues. Iron-catalyzed lipid peroxidation of liver and muscle tissue of fish fed elevated dietary vitamin E (> or =50 IU vitamin E/kg diet) was significantly lower (P<0.05) than that noted for fish fed the diet containing no supplemental vitamin E. The results indicated that changes in tissue lipid peroxidation measurements precede clinical signs of sub-optimal vitamin E intake.

Graduated compression stockings reduce the venous velocity augmentation of foot pumps.

The use of the A-V Impulse System of foot pumps and graduated compression stockings to reduce the incidence of thrombo-embolic disease after total hip arthroplasty is well recognised. It is not known if they have a synergistic effect as all clinical trials have used them in combination. We examined the effect compression stockings had on the ability of the A-V Impulse System to accelerate peak venous velocities in the common femoral vein in ten healthy volunteers using a duplex scanner. The use of foot pumps without stockings produced the greatest increase in peak venous velocity and this was 34% greater than using a foot pump with stockings. It is not known if this difference could account for a reduction in thrombo-embolic episodes and so we propose a randomised clinical trial.

An mRNA 3' processing site targets downstream sequences for rapid degradation in Chlamydomonas chloroplasts.

In Chlamydomonas chloroplasts, atpB pre-mRNA matures through a two-step process. Initially, endonuclease cleavage occurs 8-10 nt downstream of the mature 3' end, which itself lies at the end of a stem-loop-forming inverted repeat (IR) sequence. This intermediate product is then trimmed by a 3' -->5' exonuclease activity. Although the initial endonucleolytic cleavage by definition generates two products, the downstream product of atpB pre-mRNA endonucleolytic processing cannot be detected, even transiently. This product thus appears to be highly unstable, and it can be hypothesized that specific mechanisms exist to prevent its accumulation. In experiments described here, the atpB 3' maturation site was placed upstream of reporter genes in vivo. Constructs containing both the IR and endonuclease cleavage site (ECS) did not accumulate the reporter gene mRNA, whereas constructs containing only the IR did accumulate the reporter mRNA. The ECS alone gave an intermediate result, suggesting that the IR and ECS act synergistically. Additional secondary structures were used to test whether 5' -->3' and/or 3' -->5' exonuclease activities mediated degradation. Because these structures did not prevent degradation, rapid endonucleolytic cleavages most likely trigger RNA destruction after ECS cleavage. On the other hand, fragments resulting from cleavage within the endogenous atpB mRNA could occasionally be detected as antisense transcripts of the adjacent reporter genes. Because endonuclease cleavages are also involved in the 5' maturation of chloroplast mRNAs, where only the downstream cleavage product accumulates, it appears that chloroplast endoribonuclease activities have evolved mechanisms to selectively stabilize different ECS products.