Subchondral trabecular bone integrity changes following ACL injury and reconstruction: a cohort study with a nested, matched case-control analysis.

OBJECTIVE: There is limited information regarding changes in bone architecture following anterior cruciate ligament (ACL) injury. The objective of this study was to evaluate differences in tibial fractal signature in the medial and lateral compartments following ACL injury and describe how these values change following ACL-reconstruction and return to activity. DESIGN: This was a prospective cohort study with a nested case-control analysis. ACL-injured subjects and matched controls were evaluated at pre-surgical baseline and post ACL reconstruction follow-up at a mean of 46 months. Serial Fractal Dimensions (FD) of tibial subchondral bone architecture were calculated in medial and lateral regions of interest in the horizontal and vertical dimensions. RESULTS: In the medial and lateral compartments, there were significant differences in the vertical FD signature for ACL-injured subjects at final follow-up, when compared to the contralateral healthy tibia (medial P < 0.0001; lateral P < 0.0001) and the control group (medial P = 0.01; lateral P < 0.0001). Similarly, in the lateral compartment, there were significant differences in the horizontal FD profile for ACL-injured subjects at final follow-up, when compared to the contralateral healthy tibia (P = 0.003) and the controls (P < 0.0001). There were no significant side-to-side differences in FDs among healthy control subjects in the medial or lateral compartments at baseline or final follow-up. CONCLUSION: At 46-month follow-up, FD profiles are significantly different, and show an overall lower FD signature, for ACL-injured knees when compared to the contralateral healthy knee and uninjured controls. Additionally, this study provided the first side-to-side symmetry data of medial and lateral FD values in healthy controls.

Evaluating vaccination strategies to control foot-and-mouth disease: a country comparison study.

Vaccination is increasingly being recognised as a potential tool to supplement 'stamping out' for controlling foot-and-mouth disease (FMD) outbreaks in non-endemic countries. Infectious disease simulation models provide the opportunity to determine how vaccination might be used in the face of an FMD outbreak. Previously, consistent relative benefits of specific vaccination strategies across different FMD simulation modelling platforms have been demonstrated, using a UK FMD outbreak scenario. We extended this work to assess the relative effectiveness of selected vaccination strategies in five countries: Australia, New Zealand, the USA, the UK and Canada. A comparable, but not identical, FMD outbreak scenario was developed for each country with initial seeding of Pan Asia type O FMD virus into an area with a relatively high density of livestock farms. A series of vaccination strategies (in addition to stamping out (SO)) were selected to evaluate key areas of interest from a disease response perspective, including timing of vaccination, species considerations (e.g. vaccination of only those farms with cattle), risk area vaccination and resources available for vaccination. The study found that vaccination used with SO was effective in reducing epidemic size and duration in a severe outbreak situation. Early vaccination and unconstrained resources for vaccination consistently outperformed other strategies. Vaccination of only those farms with cattle produced comparable results, with some countries demonstrating that this could be as effective as all species vaccination. Restriction of vaccination to higher risk areas was less effective than other strategies. This study demonstrates consistency in the relative effectiveness of selected vaccination strategies under different outbreak start up conditions conditional on the assumption that each of the simulation models provide a realistic estimation of FMD virus spread. Preferred outbreak management approaches must however balance the principles identified in this study, working to clearly defined outbreak management objectives, while having a good understanding of logistic requirements and the socio-economic implications of different control measures.

Application of Next Generation Sequencing for Diagnostic Testing of Tree Fruit Viruses and Viroids.

Conventional detection of viruses and virus-like diseases of plants is accomplished using a combination of molecular, serological, and biological indexing. These are the primary tools used by plant virologists to monitor and ensure trees are free of known viral pathogens. The biological indexing assay, or bioassay, is considered to be the "gold standard" as it is the only method of the three that can detect new, uncharacterized, or poorly characterized viral disease agents. Unfortunately, this method is also the most labor intensive and can take up to three years to complete. Next generation sequencing (NGS) is a technology with rapidly expanding possibilities including potential applications for the detection of plant viruses. In this study, comparisons are made between tree fruit testing by conventional and NGS methods, to demonstrate the efficacy of NGS. A comparison of 178 infected trees, many infected with several viral pathogens, demonstrated that conventional and NGS were equally capable of detecting known viruses and viroids. Comparable results were obtained for 170 of 178 of the specimens. Of the remaining eight specimens, some discrepancies were observed between viruses detected by the two methods, representing less than 5% of the specimens. NGS was further demonstrated to be equal or superior for the detection of new or poorly characterized viruses when compared with a conventional bioassay. These results validated both the effectiveness of conventional virus testing methods and the use of NGS as an additional or alternative method for plant virus detection.

Observations on the distribution and control of Salmonella in commercial duck hatcheries in the UK.

Salmonella infection causes a significant number of cases of gastroenteritis and more serious illnesses in people in the UK and EU. The serovars Salmonella Enteritidis and Salmonella Typhimurium are most frequently associated with foodborne illness in Europe. Whilst control programmes exist to monitor these serovars in the chicken and turkey sectors, no regulatory programme is currently in place for the duck sector. A voluntary industry scheme (Duck Assurance Scheme) was launched in the UK in 2010. Hatcheries act as focal points of Salmonella contamination, in particular if Salmonella-contaminated eggs from positive breeding farms enter the hatchery. Five duck hatcheries were visited in this study and four were positive for Salmonella. S. Typhimurium DT8 and S. Indiana were isolated from hatchery 1 and S. Typhimurium DT41 and S. Senftenberg were isolated from hatchery 3. S. Kottbus, S. Bovismorbificans and S. Senftenberg were isolated from hatchery 2 and S. Kedougou was isolated from hatchery 4. Advice on the control/elimination of Salmonella was provided at each visit and a longitudinal study was undertaken to monitor its effectiveness. Extensive sampling was carried out in the hatcheries visited and the tray wash area and waste/external areas had the highest probability of being contaminated. The hatcher area was also found to be a primary focus of contamination. Improvements of farm and hatchery biosecurity standards have resulted in a reduction of hatchery contamination in this study and in previous investigations. Hatcheries 1 and 5 were cleared of Salmonella, demonstrating that elimination of Salmonella contamination from duck hatcheries is achievable.

An in-vitro investigation into the efficacy of disinfectants used in the duck industry against Salmonella.

In 2010, 81 confirmed cases of Salmonella Typhimurium DT8 were reported across England and Northern Ireland - an increase of 26% from 2009 and 41% since 2008. Five cases were hospitalized and one death reported, with a strong association found between cases and the consumption of duck eggs. Once present on farms, Salmonella may become persistent and can survive for long periods of time in residual organic matter, increasing risk of infection for follow-on flocks if cleaning and disinfection is not carried out effectively. The aim of this study was to investigate the efficacy of a range of disinfectants used by the duck industry against Salmonella using laboratory models. Sixteen products were selected from seven chemical groups and the Minimum Inhibitory Concentration and Minimum Bactericidal Concentrations determined. Each product was also tested at the recommended general orders (GO) concentration using a faecal suspension model to mimic boot dips and a surface contamination model to simulate contaminated building fabric and equipment. In the faecal suspension model, all products were effective at 2 × GO concentration, and activity was more inconsistent at GO concentration. At 0.5 × GO concentration, iodine-based and quaternary-ammonium-compound-based products were significantly less effective than products within other chemical groups (P < 0.001). Glutaraldehyde-based products were significantly more effective than the other products in the surface contamination tests (P < 0.001). Chlorocresol-based products were found to be most effective for use in boot dips and aldehyde-based products for surface disinfection, although there was variability between products within a chemical group.

Evaluating vaccination strategies to control foot-and-mouth disease: a model comparison study.

Simulation models can offer valuable insights into the effectiveness of different control strategies and act as important decision support tools when comparing and evaluating outbreak scenarios and control strategies. An international modelling study was performed to compare a range of vaccination strategies in the control of foot-and-mouth disease (FMD). Modelling groups from five countries (Australia, New Zealand, USA, UK, The Netherlands) participated in the study. Vaccination is increasingly being recognized as a potentially important tool in the control of FMD, although there is considerable uncertainty as to how and when it should be used. We sought to compare model outputs and assess the effectiveness of different vaccination strategies in the control of FMD. Using a standardized outbreak scenario based on data from an FMD exercise in the UK in 2010, the study showed general agreement between respective models in terms of the effectiveness of vaccination. Under the scenario assumptions, all models demonstrated that vaccination with 'stamping-out' of infected premises led to a significant reduction in predicted epidemic size and duration compared to the 'stamping-out' strategy alone. For all models there were advantages in vaccinating cattle-only rather than all species, using 3-km vaccination rings immediately around infected premises, and starting vaccination earlier in the control programme. This study has shown that certain vaccination strategies are robust even to substantial differences in model configurations. This result should increase end-user confidence in conclusions drawn from model outputs. These results can be used to support and develop effective policies for FMD control.

Twelve-month outcomes following midurethral sling procedures for stress incontinence: impact of obesity.

OBJECTIVE: To evaluate impact of body mass index (BMI) ≥30 on objective and subjective cure rates 12 months after midurethral sling surgery. DESIGN: Secondary analysis. SETTING: Three hospitals in Calgary, Canada, 2005-07. POPULATION: A total of 182 women enrolled in a randomised control trial of tension-free vaginal tape versus transobturator tape. METHODS: Women were classified as obese or nonobese from height and weight on day of surgery. Women underwent postoperative standardised pad tests, self-reporting of urinary incontinence, and quality of life scores. Categorical data compared with chi-square or Fisher's exact, continuous data compared with Mann-Whitney U test. MAIN OUTCOME MEASURES: Primary outcome was objective cure, defined as <1 g urine loss on postoperative 1-hour pad test. Secondary outcomes were subjective cure of incontinence (no stress incontinence in previous 7 days), presence of urinary urgency in previous 7 days, Urogenital Distress Inventory (UDI-6) scores, Incontinence Impact Questionnaire (IIQ-7) scores, and surgical complication rates. RESULTS: Objective cure differed, with 85.6% of nonobese women leaking <1 g on 1-hour pad test, versus 67.8% of obese women (P = 0.006, risk difference [RD] 17.8%, 95% confidence interval [95% CI] 4.2-31.4%). Subjective cure was 85.8% for nonobese women versus 70.7% for obese women (P = 0.016, RD 15.1%, 95% CI 1.9-28.4%). For both groups, improvement was seen for postoperative UDI-6 (median -33.3 [-44.4 to -22.2] and -27.2 [-44.4 to -16.7]) and IIQ-7 scores (median -26.2 [-45.2 to -14.3] and -23.8 [-42.9 to -14.3]). No differences existed in rates of operative complications between the two groups. CONCLUSION: Twelve months after midurethral sling surgery, obese women experience lower rates of cure than those who are nonobese.

Anchor placement and subsequent movement in a mesh kit with self-fixating tips: 6-month follow-up of a prospective cohort.

OBJECTIVE: To describe the initial placement of Elevate single-incision mesh kit device tips relative to the sacrospinous ligament, and to measure tip movement over a 6-month period from initial placement. DESIGN: Prospective cohort. SETTING: Tertiary care urogynaecology centre in Calgary, Canada. POPULATION: Women electing for surgical management of anterior vaginal wall prolapse. METHODS: Ten women underwent anterior prolapse repair using the Elevate single-incision mesh kit with a metallic fiducial marker attached to the tips of the surgical device. Women were imaged by magnetic resonance imaging (MRI) within 48 hours of surgery, and again 6 months later to investigate the position of the device tips and change ≥4 mm over the 6-month postoperative period. MAIN OUTCOME MEASURE(S): Position of self-fixating tips within 48 hours of surgery, and at six months post-operative. RESULTS: Anchor insertion was directly into the sacrospinous ligament in 10 of 20 insertion points (50%, 95% CI 27-73%). Movement was most often noted in the cranial-caudal direction: a change in location of ≥4 mm was observed for 8/20 anchors (40%, 95% CI 19-64%). Cranial-caudal movement was observed less frequently among sacrospinous anchors than among anchors inserted into other pelvic structures (1/10 versus 7/10, P = 0.020, difference in proportion -60%, 95% CI -94 to -26%). PFDI-20 scores improved statistically significantly by 6 months (P = 0.008, mean change -62.9%, 95% CI -105.1 to -20.7%), but PFIQ-7 scores did not change statistically significantly over the same time period (P = 0.523, mean change -12.4%, 95% CI -54.5 to 29.8%). CONCLUSIONS: The novel self-fixating anchoring tips of this single-incision mesh kit do not reliably anchor into the sacrospinous ligament. The tips have been shown to move with time, although not all cases of anchor movement were associated with recurrent prolapse.

Randomized clinical trial of enhanced recovery versus standard care after laparoscopic sleeve gastrectomy.

BACKGROUND: Optimized perioperative care within an enhanced recovery after surgery (ERAS) protocol is designed to reduce morbidity after surgery, resulting in a shorter hospital stay. The present study evaluated this approach in the context of sleeve gastrectomy for patients with morbid obesity. METHODS: Patients were allocated to perioperative care according to a bariatric ERAS protocol or a control group that received standard care. These groups were also compared with a historical group of patients who underwent laparoscopic sleeve gastrectomy at the same institution between 2006 and 2010, selected using matched propensity scores. The primary outcome was median length of hospital stay. Secondary outcomes included readmission rates, postoperative morbidity, postoperative fatigue and mean cost per patient. RESULTS: Of 116 patients included in the analysis, 78 were allocated to the ERAS (40) or control (38) group and there were 38 in the historical group. There were no differences in baseline characteristics between groups. Median hospital stay was significantly shorter in the ERAS group (1 day) than in the control (2 days; P < 0·001) and historical (3 days; P < 0·001) groups. It was also shorter in the control group than in the historical group (P = 0·010). There was no difference in readmission rates, postoperative complications or postoperative fatigue. The mean cost per patient was significantly higher in the historical group than in the ERAS (P = 0·010) and control (P = 0·018) groups. CONCLUSION: The ERAS protocol in the setting of bariatric surgery shortened hospital stay and was cost-effective. There was no increase in perioperative morbidity. REGISTRATION NUMBER: NCT01303809 (http://www.clinicaltrials.gov).

"Mothers get really exhausted!" The lived experience of pregnancy in extreme heat: Qualitative findings from Kilifi, Kenya.

Heat exposure in pregnancy is associated with a range of adverse health and wellbeing outcomes, yet research on the lived experience of pregnancy in high temperatures is lacking. We conducted qualitative research in 2021 in two communities in rural Kilifi County, Kenya, a tropical savannah area currently experiencing severe drought. Pregnant and postpartum women, their male spouses and mothers-in-law, community health volunteers, and local health and environment stakeholders were interviewed or participated in focus group discussions. Pregnant women described symptoms that are classically regarded as heat exhaustion, including dizziness, fatigue, dehydration, insomnia, and irritability. They interpreted heat-related tachycardia as signalling hypertension and reported observing more miscarriages and preterm births in the heat. Pregnancy is conceptualised locally as a 'normal' state of being, and women continue to perform physically demanding household chores in the heat, even when pregnant. Women reported little support from family members to reduce their workload at this time, reflecting their relative lack of autonomy within the household, but also potentially the 'normalisation' of heat in these communities. Climate change risk reduction strategies for pregnant women in low-resource settings need to be cognisant of local household gender dynamics that constrain women's capacity to avoid heat exposures.

Accuracy of surface landmark identification for cannula cricothyroidotomy.

Cannula cricothyroidotomy is recommended for emergency transtracheal ventilation by all current airway guidelines. Success with this technique depends on the accurate and rapid identification of percutaneous anatomical landmarks. Six healthy subjects underwent neck ultrasound to delineate the borders of the cricothyroid membrane. The midline and bisecting transverse planes through the membrane were marked with an invisible ink pen which could be revealed with an ultraviolet light. Eighteen anaesthetists were then invited to mark an entry point for cricothyroid membrane puncture. Only 32 (30%) attempts by anaesthetists accurately marked the skin area over the cricothyroid membrane. Of these only 11 (10%) marked over the centre point of the membrane. Entry point accuracy was not significantly affected by subjects' weight, height, body mass index, neck circumference or cricothyroid dimensions. Consultant and registrar anaesthetists were significantly more accurate than senior house officers at correctly identifying the cricothyroid membrane. Accuracy of percutaneously identifying the cricothyroid membrane was poor. Ultrasound may assist in identifying anatomical landmarks for cricothyroidotomy.

A quantitative assessment of the efficacy of surgical and N95 masks to filter influenza virus in patients with acute influenza infection.

We assessed the in vivo efficacy of surgical and N95 (respirator) masks to filter reverse transcription-polymerase chain reaction (RT-PCR)-detectable virus when worn correctly by patients with laboratory-confirmed acute influenza. Of 26 patients with a clinical diagnosis of influenza, 19 had the diagnosis confirmed by RT-PCR, and 9 went on to complete the study. Surgical and N95 masks were equally effective in preventing the spread of PCR-detectable influenza.

First Report of Plum pox virus Recombinant Strain on Prunus spp. in Canada.

In July of 2008, during the Canadian Plum pox virus (PPV) eradication survey, three Prunus spp. trees (B-5, B-6, and C-1) in a home owner's yard in Grimsby, ON, Canada were found to be infected with PPV by triple antibody sandwich (TAS)-ELISA using the 5B generic monoclonal detecting antibody (1) and reverse transcription (RT)-PCR using the well-validated universal primer set P1/P2. All three trees were grafted on an unknown plum rootstock cultivar. Tree B-5 contained grafts of unknown peach (P. persica) and plum (P. domestica) cultivars, tree B-6 contained the same peach graft as tree B-5 and an unknown apricot (P. armeniaca) cultivar, and tree C-1 was grafted with the same plum cultivar as tree B-5. Strain typing was done by TAS-ELISA using strain-specific monoclonal antibodies for D, M, C, and EA strains. Positive results were obtained with the M-specific test. Strain typing by RT-PCR also was done using primers specific for D, M, W, and recombinant (Rec) strains. Positive results were obtained with the M and Rec primers (4). The 605-bp fragment generated by the PPV Rec primers, which spans the recombination site, was cloned and sequenced. The nucleotide sequences obtained from B-5, B-6, and C-1 are 99% identical to each other and approximately 98 and 99% identical to the PPV Rec isolates BOR-3 and J4c, respectively. Correspondingly, percentage identities are approximately 90% for PPV M, 84% for PPV isolate D-Fan (a typical Canadian D isolate), and 69% for PPV W. The deduced amino acid sequence of B-5, B-6, and C-1 are 98 to 99% identical to each other, 99% identical to the PPV Rec isolates BOR-3 and J4c, 92% identical to isolates of PPV M, and only 84% identical to the typical Canadian D isolate PPV Fan. The P3-6K1 genomic region was amplified using primers that generate a 836-bp fragment (2). This region was 97 to 98% identical to the PPV Rec isolates BOR-3 and J4c, 96 to 97% identical to isolates of PPV D, but only 86% identical to isolates of PPV M. The data above confirm that the PPV isolates B-5, B-6, and C-1 belong to the strain PPV Rec (3). To our knowledge, this is the first report of PPV Rec in North America, and together with PPV D and W, it represents the third PPV strain found on this continent. An intensive survey of all Prunus spp. within a 1.5-km radius area surrounding the home owner's property failed to reveal any additional PPV-positive plants. The three positive plants were removed. References: (1) M. Cambra et al. EPPO Bull. 24:569, 1994. (2) M. Glasa et al. Arch. Virol. 147:563, 2002. (3) M. Glasa et al. J. Gen. Virol. 85:2671, 2004. (4) A. Subr et al. Acta Virol. 48:173, 2004.

Molecular characterization of a novel fastidious mycobacterium causing lepromatous lesions of the skin, subcutis, cornea, and conjunctiva of cats living in Victoria, Australia.

Between 1999 and 2006, 15 cats were diagnosed with disease attributable to a novel mycobacterial species. The infections consisted of granulomatous lesions in the skin, subcutis, and ocular or periocular tissues with an indolent but progressive clinical course. Lesions typically were found in facial regions or on the distal limbs. Cats of all ages and both sexes were affected. Infections often were challenging to treat, although they could be cured using surgery in concert with combination antimicrobial therapy. Microscopically, lesions were granulomatous to pyogranulomatous and contained numerous acid-fast bacilli. Scanty cultures of the causal microorganisms occasionally could be obtained in mycobacterial broth, but subculture to solid media failed. When cultures were not available, DNA was extracted from fresh tissue, lyophilized material, and formalin-fixed, paraffin-embedded tissues from lesions. PCR amplification of the 5' end of the 16S rRNA gene and regions within four additional loci (ITS1, hsp65, rpoB, and sodA) was performed with various efficiencies using mycobacterial primers. Nucleotide sequences were unique for each locus tested. Nucleotide sequences obtained from individual cases were identical for each locus for which the amplification was successful. Phylogenetic analysis performed using concatenated partial 16S rRNA and hsp65 gene sequences indicated that this novel mycobacterial species from Victoria is a member of the Mycobacterium simiae-related group, taxonomically related to the mycobacterium causing leproid granulomas in dogs throughout the world. Based on the clustering of cases, we refer to this novel species as Mycobacterium sp. strain Tarwin.

Evidence for late stage compartmentalization of HIV-1 resistance mutations between lymph node and peripheral blood mononuclear cells.

OBJECTIVE: To determine the overall distribution of drug-resistance mutations to nucleoside reverse transcriptase inhibitors of HIV strains recovered from the lymph nodes (LN) and peripheral blood mononuclear cell (PBMC) compartments of four HIV-infected patients receiving zidovudine and didanosine and to compare them with antiretroviral-naive patients. DESIGN: Molecular comparison of major and minor HIV-1 env and pol region variants residing in LN and PBMC compartments. MATERIALS AND METHODS: Proviral DNA sequences were amplified by PCR from both PBMC and LN compartments, cloned into PGEM-T II Easy vector and sequenced. The clones were subjected to molecular and phylogenetic analysis. RESULTS: Comparison of PBMC and LN-derived HIV-1 variants in the env V3 region showed that nucleotide and amino acid variability was a characteristic feature of LN-derived variants. In contrast, a majority of resistance mutations to reverse transcriptase inhibitors were localized in the PBMC compartment rather than in LN, which is thought to be a reservoir of HIV. CONCLUSIONS: Distinct compartmentalization or independent evolution of pol and env gene variants between LN and PBMC could be due to the differential selection pressure imposed by the combination drug regimen, hence the bimodal distribution of resistance variants between LN and PBMC compartments.

Syncytium-inducing phenotype and zidovudine susceptibility of HIV-1 isolated from post-mortem tissue.

OBJECTIVE: To establish the syncytium-inducing (SI) phenotype and zidovudine (ZDV) susceptibility of HIV-1 isolates obtained from autopsy specimens. METHODS: Isolation of HIV was attempted from autopsy specimens obtained from 76 AIDS patients. Specimens of lymph node, spleen, spinal cord, brain and cerebrospinal fluid (CSF) were processed and cultured with peripheral blood mononuclear cells (PBMC) from seronegative donors. Biological phenotype was determined in a T-lymphocyte line (MT-2). ZDV susceptibility was evaluated in a PBMC-based assay. Sequencing of amino-acid codons in the reverse transcriptase gene previously shown to be associated with ZDV resistance was carried out on a subgroup of isolates. RESULTS: HIV was recovered from tissue specimens and CSF up to 5 days post-mortem, but recovery rate of infectious virus decreased as the time between autopsy and specimen processing increased. There was a lack of concordance between PBMC isolates and isolates from different tissue sites with respect to SI phenotype. ZDV-resistant virus was isolated from post-mortem specimens of patients who had received long-term ZDV therapy up until or shortly before their death. ZDV-sensitive virus re-emerged in the lymph node of patients who ceased treatment several months prior to death. Phenotypically sensitive virus obtained from lymph node tissue of three patients after a relatively short time off ZDV (4-6 months) retained some of the amino-acid substitutions known to be associated with resistance. CONCLUSION: The data suggests that ZDV resistance and re-emergence of sensitive virus does not originate in peripheral cells, and that these cells and tissues are seeded with virus present elsewhere, possibly in the germinal centres of the lymph node.

Monocytes harbour replication-competent, non-latent HIV-1 in patients on highly active antiretroviral therapy.

OBJECTIVE: To determine whether HIV-1 can be recovered from blood monocytes as well as resting, memory CD4 T lymphocytes of patients on highly active antiretroviral therapy (HAART) with undetectable plasma viraemia and whether infection is active or latent. DESIGN: Five patients with plasma HIV-1-RNA levels of less than 500 copies/ml for at least 3 months and less than 50 copies/ml at the time of sampling were initially selected, followed by an additional five patients with viral loads of less than 50 copies/ml for 3 months or more. METHODS: Monocytes were isolated from blood by plastic adherence, then further purified by a second adherence step or CD3 depletion before co-culture with CD8-depleted donor peripheral blood mononuclear cells. Virus isolates were examined for mutations conferring resistance to reverse transcriptase or protease inhibitors and for genotype. The highly purified monocytes were also analysed for the presence of proviral and unintegrated viral DNA and multiply spliced (MS) viral mRNA by polymerase chain reaction. RESULTS: Virus was recovered from monocytes of five patients. Sequencing of the recovered viruses did not reveal multiple drug resistance, and was consistent with a non-syncytium-inducing/CCR5 phenotype. Proviral DNA was detectable in monocytes from all subjects, and unintegrated HIV-1 DNA and MS RNA was found in four out of five populations examined. CONCLUSION: Recovery of replication-competent virus from some HAART patients indicates that monocytes can also harbour HIV-1. Detection of circular, viral DNA and spliced RNA, albeit at very low levels, in these cells suggests that their infection is recent and transcriptionally active rather than latent.

Synthesis, stability, antiviral activity, and protease-bound structures of substrate-mimicking constrained macrocyclic inhibitors of HIV-1 protease.

Three new peptidomimetics (1-3) have been developed with highly stable and conformationally constrained macrocyclic components that replace tripeptide segments of protease substrates. Each compound inhibits both HIV-1 protease and viral replication (HIV-1, HIV-2) at nanomolar concentrations without cytotoxicity to uninfected cells below 10 microM. Their activities against HIV-1 protease (K(i) 1.7 nM (1), 0.6 nM (2), 0.3 nM (3)) are 1-2 orders of magnitude greater than their antiviral potencies against HIV-1-infected primary peripheral blood mononuclear cells (IC(50) 45 nM (1), 56 nM (2), 95 nM (3)) or HIV-1-infected MT2 cells (IC(50) 90 nM (1), 60 nM (2)), suggesting suboptimal cellular uptake. However their antiviral potencies are similar to those of indinavir and amprenavir under identical conditions. There were significant differences in their capacities to inhibit the replication of HIV-1 and HIV-2 in infected MT2 cells, 1 being ineffective against HIV-2 while 2 was equally effective against both virus types. Evidence is presented that 1 and 2 inhibit cleavage of the HIV-1 structural protein precursor Pr55(gag) to p24 in virions derived from chronically infected cells, consistent with inhibition of the viral protease in cells. Crystal structures refined to 1.75 A (1) and 1.85 A (2) for two of the macrocyclic inhibitors bound to HIV-1 protease establish structural mimicry of the tripeptides that the cycles were designed to imitate. Structural comparisons between protease-bound macrocyclic inhibitors, VX478 (amprenavir), and L-735,524 (indinavir) show that their common acyclic components share the same space in the active site of the enzyme and make identical interactions with enzyme residues. This substrate-mimicking minimalist approach to drug design could have benefits in the context of viral resistance, since mutations which induce inhibitor resistance may also be those which prevent substrate processing.

Cellular topoisomerase I activity associated with HIV-1.

Topoisomerase I activity was detected in detergent-disrupted human immunodeficiency virus type 1 (HIV-1) particles. The enzyme did not require ATP for its conversion of SC DNA to an RC form, had divalent cation requirements similar to those of eukaryotic topoisomerase I, and was significantly inhibited by the specific topoisomerase I inhibitor camptothecin. However, camptothecin failed to inhibit replication of HIV in infected cells at nontoxic concentrations, and an active site motif for topoisomerase I could not be detected on the HIV genome. These results suggests that HIV does not encode a novel topoisomerase I, but rather packages the cellular enzyme.

Stolon growth and branching in Glechoma hederacea L.: an application of a plastochron index.

Stolon growth and branching in Glechoma hederacea L. was analyzed, using a plastochron index based on the number of ramets on each stolon and internode growth. New methods, employing this index, were used to detect fluctuations in the rate of stolon extension, and to analyse the development of secondary stolons. The plastochron index had a strong linear correlation with time, with a new primary ramet appearing every 3.5 d. The overall stolon extension rate fluctuated between 1.9 and 2.9 cm d-1 through each plastochron cycle. The age at which each node produced secondary stolons was related to its position on the plant, but the initial rates of secondary stolon development differed little between nodes. Rate of development of secondary stolons was strongly correlated with the time at which they started to grow. Thus, later developing secondary stolons grew more slowly than those developing earlier from nodes in equivalent positions.