When several models can describe a biological process, the equation that best fits the data is typically considered the best. However, models are most useful when they also possess biologically-meaningful parameters. In particular, model parameters should be stable, physically interpretable, and transferable to other contexts, e.g. for direct indication of system state, or usage in other model types. As an example of implementing these recommended requirements for model parameters, we evaluated twelve published empirical models for temperature-dependent tropical seagrass photosynthesis, based on two criteria: (1) goodness of fit, and (2) how easily biologically-meaningful parameters can be obtained. All models were formulated in terms of parameters characterising the thermal optimum (Topt) for maximum photosynthetic rate (Pmax). These parameters indicate the upper thermal limits of seagrass photosynthetic capacity, and hence can be used to assess the vulnerability of seagrass to temperature change. Our study exemplifies an approach to model selection which optimises the usefulness of empirical models for both modellers and ecologists alike.
Background. Artemisinin resistance in Plasmodium falciparum has emerged in Southeast Asia and poses a threat to malaria control and elimination. Mutations in a P. falciparum gene encoding a kelch protein on chromosome 13 have been associated with delayed parasite clearance following artemisinin treatment elsewhere in the region, but not yet in China.
Methods. Therapeutic efficacy studies of artesunate and dihydroartemisinin-piperaquine were conducted from 2009 to 2012 in the Yunnan Province of China near the border with Myanmar. K13 mutations were genotyped by capillary sequencing of DNA extracted from dried blood spots collected in these clinical trials and in routine surveillance. Associations between K13 mutations and delayed parasite clearance were tested using regression models.
Results. Parasite clearance half-lives were prolonged after artemisinin treatment, with 44% of infections having half-lives >5 hours (n = 109). Fourteen mutations in K13 were observed, with an overall prevalence of 47.7% (n = 329). A single mutation, F446I, predominated, with a prevalence of 36.5%. Infections with F446I were significantly associated with parasitemia on day 3 following artemisinin treatment and with longer clearance half-lives.
Conclusions. Plasmodium falciparum infections in southern China displayed markedly delayed clearance following artemisinin treatment. F446I was the predominant K13 mutation and was associated with delayed parasite clearance.
artemisinin resistance; China; kelch 13; malaria; Plasmodium falciparum
Malaria infections during pregnancy lead to sequestration of parasite infected red blood cells in the placenta. Placental infection can result in adverse outcomes for mothers and infants. Despite many studies, it remains unclear which peripheral blood infections during pregnancy lead to development of placental malaria. Understanding the timing of peripheral infections that lead to placental malaria and the ability of intermittent preventive treatment with sulfadoxine–pyrimethamine (SP-IPT) and artemisinin-based combination therapy to clear infections will enable the rational design of new interventions to decrease the burden of malaria in pregnancy.
Microsatellite markers were used to genotype peripheral and placental malaria infections in an observational cohort in Blantyre, Malawi. Genotypes were compared to determine the timing of infections that sequester in the placenta. The effects of SP-IPT and artemether–lumefantrine as curative treatment were also evaluated by assessing the occurrence of peripheral infections or matching genotypes between peripheral and placental parasites following treatment.
Genotypes from 92 peripheral samples prior to delivery, 26 peripheral samples at delivery, and 29 placental samples were compared. Thirty percent of women with genotyped parasites in their placentas that had peripheral infections detected during pregnancy had matching peripheral-placental genotypes. Matching genotypes were not associated with gestational age and occurred from 13 to 39 weeks. Among women with more than one genotyped peripheral infection during pregnancy, 80 % had persistent infection with the same genotype while the remaining were new infections. Among infections treated with SP or artemether–lumefantrine, 28/84 (33 %) and 9/56 (16 %) had infection detected after treatment, respectively. Recrudescent infections were detected after both treatments and occurred up to 76 days after treatment. Women treated with SP-IPT and artemether–lumefantrine had genotypes matching treated infections detected in the placenta.
Placental malaria can occur at any time during pregnancy. In the context of late enrollment in antenatal care, interventions that protect all women of childbearing age and throughout pregnancy are needed. Currently used medications do not always clear peripheral or placental infections. The ability of anti-malarial drugs to prevent or clear placental infections should be considered in the development of future interventions.
Malaria in pregnancy; Placental malaria; Molecular epidemiology; Genotyping; Microsatellite markers; Submicroscopic; Sulfadoxine–pyrimethamine; Intermittent preventive treatment; Artemether–lumefantrine
To investigate endoluminal ultrasound applicator configurations for volumetric thermal ablation and hyperthermia of pancreatic tumors using 3D acoustic and biothermal finite element models.
Materials and Methods
Parametric studies compared endoluminal heating performance for varying applicator transducer configurations (planar, curvilinear-focused, or radial-diverging), frequencies (1–5 MHz), and anatomical conditions. Patient-specific pancreatic head and body tumor models were used to evaluate feasibility of generating hyperthermia and thermal ablation using an applicator positioned in the duodenal or stomach lumen. Temperature and thermal dose were calculated to define ablation (>240 EM43°C) and moderate hyperthermia (40–45 °C) boundaries, and to assess sparing of sensitive tissues. Proportional-integral control was incorporated to regulate maximum temperature to 70–80 °C for ablation and 45 °C for hyperthermia in target regions.
Parametric studies indicated that 1–3 MHz planar transducers are most suitable for volumetric ablation, producing 5–8 cm3 lesion volumes for a stationary 5 minute sonication. Curvilinear-focused geometries produce more localized ablation to 20–45 mm depth from the GI tract and enhance thermal sparing (Tmax<42 °C) of the luminal wall. Patient anatomy simulations show feasibility in ablating 60.1–92.9% of head/body tumor volumes (4.3–37.2 cm3) with dose <15 EM43°C in the luminal wall for 18–48 min treatment durations, using 1–3 applicator placements in GI lumen. For hyperthermia, planar and radial-diverging transducers could maintain up to 8 cm3 and 15 cm3 of tissue, respectively, between 40–45 °C for a single applicator placement.
Modeling studies indicate the feasibility of endoluminal ultrasound for volumetric thermal ablation or hyperthermia treatment of pancreatic tumor tissue.
Ultrasound; thermal therapy; pancreatic cancer; catheter-based ultrasound; thermal ablation; hyperthermia
The natural history of clinical symptoms in the spinocerebellar ataxias (SCA)s has been well characterised. However there is little longitudinal data comparing cognitive changes in the most common SCA subtypes over time. The present study provides a preliminary longitudinal characterisation of the clinical and cognitive profiles in patients with SCA1, SCA2, SCA3, SCA6 and SCA7, with the aim of elucidating the role of the cerebellum in cognition.
13 patients with different SCAs all caused by CAG repeat expansion (SCA1, n = 2; SCA2, n = 2; SCA3, n = 2; SCA6, n = 4; and SCA7, n = 3) completed a comprehensive battery of cognitive and mood assessments at two time points, a mean of 7.35 years apart. All patients were evaluated clinically using the Scale for the Rating and Assessment of Ataxia (SARA) and the Inventory of Non-Ataxia Signs (INAS). Patients underwent structural MRI imaging at follow-up.
Clinical scale scores increased in all patients over time, most prominently in the SCA1 (SARA) and SCA3 (INAS) groups. New impairments on neuropsychological tests were most commonly observed with executive functions, speed, attention, visual memory and Theory of Mind. Results suggest possible differences in cognitive decline in SCA subtypes, with the most rapid cognitive decline observed in the SCA1 patients, and the least in the SCA6 patients, congruent with observed patterns of motor deterioration. Minimal changes in mood were observed, and MRI measures of atrophy did not correlate with cognitive decline.
As well as increasing physical impairment, cognitive decline over time appears to be a distinct aspect of the SCA phenotype, in keeping with the cerebellar cognitive-affective syndrome. Our data suggest a trend of cognitive decline that is different for each SCA subtype, and for the majority is related to the severity of cerebellar motor impairment.
Electronic supplementary material
The online version of this article (doi:10.1186/s13023-016-0447-6) contains supplementary material, which is available to authorized users.
Ataxia; Spinocerebellar ataxia; Cognition
Artemisinin-resistant Plasmodium falciparum malaria has been documented in southeast Asia and may already be spreading in that region. Molecular markers are important tools for monitoring the spread of antimalarial drug resistance. Recently, single-nucleotide polymorphisms (SNPs) in the PF3D7_1343700 kelch propeller (K13-propeller) domain were shown to be associated with artemisinin resistance in vivo and in vitro. The prevalence and role of K13-propeller mutations are poorly known in sub-Saharan Africa. K13-propeller mutations were genotyped by direct sequencing of nested polymerase chain reaction (PCR) amplicons from dried blood spots of pre-treatment falciparum malaria infections collected before and after the use of artemisinin-based combination therapy (ACT) as first-line therapy in Mali. Although K13-propeller mutations previously associated with delayed parasite clearance in Cambodia were not identified, 26 K13-propeller mutations were identified in both recent samples and pre-ACT infections. Parasite clearance time was comparable between infections with non-synonymous K13-propeller mutations and infections with the reference allele. These findings suggest that K13-propeller mutations are present in artemisinin-sensitive parasites and that they preceded the wide use of ACTs in Mali.
Background. The emergence of artemisinin-resistant Plasmodium falciparum in Southeast Asia threatens malaria treatment efficacy. Mutations in a kelch protein encoded on P. falciparum chromosome 13 (K13) have been associated with resistance in vitro and in field samples from Cambodia.
Methods. P. falciparum infections from artesunate efficacy trials in Bangladesh, Cambodia, Laos, Myanmar, and Vietnam were genotyped at 33 716 genome-wide single-nucleotide polymorphisms (SNPs). Linear mixed models were used to test associations between parasite genotypes and parasite clearance half-lives following artesunate treatment. K13 mutations were tested for association with artemisinin resistance, and extended haplotypes on chromosome 13 were examined to determine whether mutations arose focally and spread or whether they emerged independently.
Results. The presence of nonreference K13 alleles was associated with prolonged parasite clearance half-life (P = 1.97 × 10−12). Parasites with a mutation in any of the K13 kelch domains displayed longer parasite clearance half-lives than parasites with wild-type alleles. Haplotype analysis revealed both population-specific emergence of mutations and independent emergence of the same mutation in different geographic areas.
Conclusions. K13 appears to be a major determinant of artemisinin resistance throughout Southeast Asia. While we found some evidence of spreading resistance, there was no evidence of resistance moving westward from Cambodia into Myanmar.
malaria; artemisinin resistance; Southeast Asia; Plasmodium falciparum; kelch
Defects in primary cilium biogenesis underlie the ciliopathies, a growing group of genetic disorders. We describe a whole genome siRNA-based reverse genetics screen for defects in biogenesis and/or maintenance of the primary cilium, obtaining a global resource. We identify 112 candidate ciliogenesis and ciliopathy genes, including 44 components of the ubiquitin-proteasome system, 12 G-protein-coupled receptors, and three pre-mRNA processing factors (PRPF6, PRPF8 and PRPF31) mutated in autosomal dominant retinitis pigmentosa. The PRPFs localise to the connecting cilium, and PRPF8- and PRPF31-mutated cells have ciliary defects. Combining the screen with exome sequencing data identified recessive mutations in PIBF1/CEP90 and C21orf2/LRRC76 as causes of the ciliopathies Joubert and Jeune syndromes. Biochemical approaches place C21orf2 within key ciliopathy-associated protein modules, offering an explanation for the skeletal and retinal involvement observed in individuals with C21orf2-variants. Our global, unbiased approaches provide insights into ciliogenesis complexity and identify roles for unanticipated pathways in human genetic disease.
cilia; ciliopathies; reverse genetics; whole-genome siRNA screen; Jeune syndrome; Joubert syndrome
Highly sensitive, scalable diagnostic methods are needed to guide malaria elimination interventions. While traditional microscopy and rapid diagnostic tests (RDTs) are suitable for the diagnosis of symptomatic malaria infection, more sensitive tests are needed to screen for low-density, asymptomatic infections that are targeted by interventions aiming to eliminate the entire reservoir of malaria infection in humans.
A reverse transcription polymerase chain reaction (RT- PCR) was developed for multiplexed detection of the 18S ribosomal RNA gene and ribosomal RNA of Plasmodium falciparum and Plasmodium vivax. Simulated field samples stored for 14 days with sample preservation buffer were used to assess the analytical sensitivity and specificity. Additionally, 1750 field samples from Southeastern Myanmar were tested both by RDT and ultrasensitive RT-PCR.
Limits of detection (LoD) were determined under simulated field conditions. When 0.3 mL blood samples were stored for 14 days at 28 °C and 80 % humidity, the LoD was less than 16 parasites/mL for P. falciparum and 19.7 copies/µL for P. vivax (using a plasmid surrogate), about 10,000-fold lower than RDTs. Of the 1739 samples successfully evaluated by both ultrasensitive RT-PCR and RDT, only two were RDT positive while 24 were positive for P. falciparum, 108 were positive for P. vivax, and 127 were positive for either P. vivax and/or P. falciparum using ultrasensitive RT-PCR.
This ultrasensitive RT-PCR method is a robust, field-tested screening method that is vastly more sensitive than RDTs. Further optimization may result in a truly scalable tool suitable for widespread surveillance of low-level asymptomatic P. falciparum and P. vivax parasitaemia.
Electronic supplementary material
The online version of this article (doi:10.1186/s12936-015-1038-z) contains supplementary material, which is available to authorized users.
Malaria; Malaria elimination; Plasmodium falciparum; Plasmodium vivax; RT-PCR; Limits of detection; Nucleic acid
An ultrasound applicator for endoluminal thermal therapy of pancreatic tumors has been introduced and evaluated through acoustic/biothermal simulations and ex vivo experimental investigations. Endoluminal therapeutic ultrasound constitutes a minimally invinvasive conformal therapy and is compatible with ultrasound or MR-based image guidance. The applicator would be placed in the stomach or duodenal lumen, and sonication would be performed through the luminal wall into the tumor, with concurrent water cooling of the wall tissue to prevent its thermal injury. A finite-element (FEM) 3D acoustic and biothermal model was implemented for theoretical analysis of the approach. Parametric studies over transducer geometries and frequencies revealed that operating frequencies within 1-3 MHz maximize penetration depth and lesion volume while sparing damage to the luminal wall. Patient-specific FEM models of pancreatic head tumors were generated and used to assess the feasibility of performing endoluminal ultrasound thermal ablation and hyperthermia of pancreatic tumors. Results indicated over 80% of the volume of small tumors (~2 cm diameter) within 35 mm of the duodenum could be safely ablated in under 30 minutes or elevated to hyperthermic temperatures at steady-state. Approximately 60% of a large tumor (~5 cm diameter) model could be safely ablated by considering multiple positions of the applicator along the length of the duodenum to increase coverage. Prototype applicators containing two 3.2 MHz planar transducers were fabricated and evaluated in ex vivo porcine carcass heating experiments under MR temperature imaging (MRTI) guidance. The applicator was positioned in the stomach adjacent to the pancreas, and sonications were performed for 10 min at 5 W/cm2 applied intensity. MRTI indicated over 40°C temperature rise in pancreatic tissue with heating penetration extending 3 cm from the luminal wall.
ultrasound ablation; pancreatic cancer; endoluminal; thermal modeling; MR guided ultrasound; catheter-based ultrasound; thermal therapy
Controlled human malaria infection (CHMI) is a powerful tool to evaluate malaria vaccine and prophylactic drug efficacy. Until recently CHMI was only carried out by the bite of infected mosquitoes. A parenteral method of CHMI would standardize Plasmodium falciparum sporozoite (PfSPZ) administration, eliminate the need for expensive challenge facility infrastructure, and allow for use of many P. falciparum strains. Recently, intradermal (ID) injection of aseptic, purified, cryopreserved PfSPZ was shown to induce P. falciparum malaria; however, 100% infection rates were not achieved by ID injection. To optimize ID PfSPZ dosing so as to achieve 100% infection, 30 adults aged 18–45 years were randomized to one of six groups composed of five volunteers each. The parameters of dose (1 × 104 versus 5 × 104 PfSPZ total dose per volunteer), number of injections (two versus eight), and aliquot volume per ID injection (10 μL versus 50 μL) were studied. Three groups attained 100% infection: 1 × 104 PfSPZ in 50 μL/2 doses, 1 × 104 PfSPZ in 10 μL/2 doses, and 5 × 104 PfSPZ in 10 μL/8 doses. The group that received 5 × 104 PfSPZ total dose in eight 10 μL injections had a 100% infection rate and the shortest prepatent period (mean of 12.7 days), approaching the prepatent period for the current CHMI standard of five infected mosquitoes.
INTRODUCTION MicroRNAs, single-stranded non-coding RNAs that function by reducing translation or causing degradation of target mRNAs, have been shown to play roles in multiple hallmarks of GBM, suggesting they may be of therapeutic importance. METHODS: The miRIDIAN mimic library (Dharmacon) that encompasses all human microRNAs annotated in miRBase v16.0 was used with a high-throughput imaging platform (Operetta) to identify microRNAs with potent effects on GBM cell proliferation and survival. Screens were performed in duplicate on U251 (adult) and KNS42 (paediatric) GBM cell lines. Cell number (nuclei count) was assessed 72h post-transfection and expressed as a z-score for each microRNA. MicroRNAs were considered significant candidates if their mean z-score was below zero and if it differed from that of the negative control by at least two standard deviations. Validation included RTqPCR, imaging and flow cytometry based assays. RESULTS: The functional screens resulted in approximately 100 candidates per cell line with 70% overlap between them. Based on available knowledge and potential relevance for GBM therapy, seven initial microRNAs candidates were chosen for further validation in a panel of four adult and two paediatric GBM cell lines: miR-34a-5p, miR-34b-5p, miR-34c-5p, miR-449a-5p, miR-449b-5p which all share a common seed sequence; miR-340-5p and miR-X. Of these candidates miR-X was identified as having the most potent, robust effects. MiR-X consistently caused a G2M cell cycle arrest followed by apoptosis in all cell lines and has predicted targets involved in the induction of the cytoskeleton rearrangement and non-canonical Wnt pathways following the G2M phase transition. This phenotype was specific to mimic-miR-X transfected cells. CONCLUSION: We have identified a novel potent pro-apoptotic microRNA in GBM that may be relevant for therapy. Work is ongoing to characterise the mechanism responsible for this effect and test for efficacy in vivo.
Following various types of naturally-occurring traumatic injury to an articular joint, the lubricating ability of synovial fluid is impaired, with a correlated alteration in the concentration and/or structure of lubricant molecules, hyaluronan and proteoglycan-4. However, the effect of arthroscopic cartilage repair surgery on synovial fluid lubricant function and composition is unknown.
Arthroscopic treatment of full-thickness chondral defects in horses with (1) platelet-enriched fibrin or (2) platelet-enriched fibrin+mesenchymal stem cells leads to equine synovial fluid with impaired lubricant function and hyaluronan and proteoglycan-4 composition.
Controlled Laboratory Study.
Equine synovial fluid was aspirated from normal joints at a pre-injury state (0 days) and at 10 days and 3 months following fibrin or fibrin+mesenchymal stem cell repair of full thickness chondral defects. Equine synovial fluid samples were analyzed for friction-lowering boundary lubrication of normal articular cartilage (static and kinetic friction coefficients) and concentrations of hyaluronan and proteoglycan-4, as well as molecular weight distribution of hyaluronan. Experimental groups deficient in lubrication function were also tested for the ability of exogenous high-molecular weight hyaluronan to restore lubrication function.
Lubrication and biochemical data varied with time after surgery but generally not between repair groups. Relative to pre-injury, kinetic friction was higher (+94%) at 10 days but returned to baseline levels at 3 months while static friction was not altered. Correspondingly, hyaluronan concentration was transiently lower (-64%) and shifted towards lower molecular weight forms, while proteoglycan-4 concentration was increased (+210%) in 10-day samples relative to pre-injury levels. Regression analysis revealed that kinetic friction decreased with increasing total and high molecular weight hyaluronan. Addition of high molecular weight hyaluronan to bring 10-day hyaluronan levels to 2.0mg/ml restored kinetic friction to pre-injury levels.
Following arthroscopic surgery for cartilage defect repair, synovial fluid lubrication function is transiently impaired, in association with decreased hyaluronan concentration. This functional deficiency in synovial fluid lubrication can be counteracted in vitro by addition of high molecular weight hyaluronan.
Synovial fluid lubrication is deficient shortly following arthroscopic cartilage repair surgery, and supplementation with high molecular weight hyaluronan may be beneficial.
The Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family mediates parasite sequestration in small capillaries through tissue-specific cytoadherence. The best characterized of these proteins is VAR2CSA, which is expressed on the surface of infected erythrocytes that bind to chondroitin sulfate in the placental matrix. Antibodies to VAR2CSA prevent placental cytoadherence and protect against placental malaria. The size and complexity of the VAR2CSA protein pose challenges for vaccine development, but smaller constitutive domains may be suitable for subunit vaccine development. A protein microarray was printed to include five overlapping fragments of the 3D7 VAR2CSA extracellular region. Malian women with a history of at least one pregnancy had antibody recognition of four of these fragments and had stronger reactivity against the two distal fragments than did nulliparous women, children, and men from Mali, suggesting that the C-terminal extracellular VAR2CSA domains are a potential focus of protective immunity. With carefully chosen sera from longitudinal studies of pregnant women, this approach has the potential to identify seroreactive VAR2CSA domains associated with protective immunity against pregnancy-associated malaria.
Background. Hemoglobin C trait, like hemoglobin S trait, protects against severe malaria in children, but it is unclear whether hemoglobin C trait also protects against uncomplicated malaria. We hypothesized that Malian children with hemoglobin C trait would have a lower risk of clinical malaria than children with hemoglobin AA.
Methods. Three hundred children aged 0–6 years were enrolled in a cohort study of malaria incidence in Bandiagara, Mali, with continuous passive and monthly active follow-up from June 2009 to June 2010.
Results. Compared to hemoglobin AA children (n = 242), hemoglobin AC children (n = 39) had a longer time to first clinical malaria episode (hazard ratio [HR], 0.19; P = .001; 364 median malaria-free days vs 181 days), fewer episodes of clinical malaria, and a lower cumulative parasite burden. Similarly, hemoglobin AS children (n = 14) had a longer time to first clinical malaria episode than hemoglobin AA children (HR, 0.15; P = .015; 364 median malaria-free days vs 181 days), but experienced the most asymptomatic malaria infections of any group.
Conclusions. Both hemoglobin C and S traits exerted a protective effect against clinical malaria episodes, but appeared to do so by mechanisms that differentially affect the response to infecting malaria parasites.
cohort study; hemoglobin C; hemoglobin S; malaria; Mali; Plasmodium falciparum
We propose and mathematically examine a theory of calcium profile formation in unwounded mammalian epidermis based on: changes in keratinocyte proliferation, fluid and calcium exchange with the extracellular fluid during these cells’ passage through the epidermal sublayers, and the barrier functions of both the stratum corneum and tight junctions localised in the stratum granulosum. Using this theory, we develop a mathematical model that predicts epidermal sublayer transit times, partitioning of the epidermal calcium gradient between intracellular and extracellular domains, and the permeability of the tight junction barrier to calcium ions. Comparison of our model’s predictions of epidermal transit times with experimental data indicates that keratinocytes lose at least 87% of their volume during their disintegration to become corneocytes. Intracellular calcium is suggested as the main contributor to the epidermal calcium gradient, with its distribution actively regulated by a phenotypic switch in calcium exchange between keratinocytes and extracellular fluid present at the boundary between the stratum spinosum and the stratum granulosum. Formation of the extracellular calcium distribution, which rises in concentration through the stratum granulosum towards the skin surface, is attributed to a tight junction barrier in this sublayer possessing permeability to calcium ions that is less than 15 nm s−1 in human epidermis and less than 37 nm s−1 in murine epidermis. Future experimental work may refine the presented theory and reduce the mathematical uncertainty present in the model predictions.
Following various types of naturally occurring traumatic injury to an articular joint, the lubricating ability of synovial fluid is impaired, with a correlated alteration in the concentration and/or structure of lubricant molecules, hyaluronan, and proteoglycan-4 (PRG4). However, the effect of arthroscopic cartilage repair surgery on synovial fluid lubricant function and composition is unknown.
Arthroscopic treatment of full-thickness chondral defects in horses with (1) platelet-enriched fibrin or (2) platelet-enriched fibrin + mesenchymal stem cells leads to equine synovial fluid with impaired lubricant function and hyaluronan and PRG4 composition.
Controlled laboratory study.
Equine synovial fluid was aspirated from normal joints at a preinjury state (0 days) and at 10 days and 3 months following fibrin or fibrin + mesenchymal stem cell repair of full-thickness chondral defects. Equine synovial fluid samples were analyzed for friction-lowering boundary lubrication of normal articular cartilage (static and kinetic friction coefficients) and concentrations of hyaluronan and PRG4, as well as molecular weight distribution of hyaluronan. Experimental groups deficient in lubrication function were also tested for the ability of exogenous high–molecular weight hyaluronan to restore lubrication function.
Lubrication and biochemical data varied with time after surgery but generally not between repair groups. Relative to preinjury, kinetic friction was higher (+94%) at 10 days but returned to baseline levels at 3 months, while static friction was not altered. Correspondingly, hyaluronan concentration was transiently lower (−64%) and shifted toward lower molecular weight forms, while PRG4 concentration was increased (+210%) in 10-day samples relative to preinjury levels. Regression analysis revealed that kinetic friction decreased with increasing total and high–molecular weight hyaluronan. Addition of high–molecular weight hyaluronan to bring 10-day hyaluronan levels to 2.0 mg/mL restored kinetic friction to preinjury levels.
Following arthroscopic surgery for cartilage defect repair, synovial fluid lubrication function is transiently impaired, in association with decreased hyaluronan concentration. This functional deficiency in synovial fluid lubrication can be counteracted in vitro by addition of high–molecular weight hyaluronan.
Synovial fluid lubrication is deficient shortly after arthroscopic cartilage repair surgery, and supplementation with high–molecular weight hyaluronan may be beneficial.
cartilage repair; synovial fluid; lubrication; hyaluronan; proteoglycan-4
The role of serotonin-1B receptors (5-HT1BRs) in modulating cocaine abuse-related behaviors has been controversial due to discrepancies between pharmacological and gene knockout approaches, and opposite influences on cocaine selfadministration versus cocaine-seeking behavior. We hypothesized that modulation of these behaviors via 5-HT1BRs in the mesolimbic pathway may vary depending on the stage of the addiction cycle.
To test this hypothesis, we examined the effects of increasing 5-HT1BR production by microinfusing a viral vector expressing either green fluorescent protein (GFP) and 5-HT1BR or GFP alone into the medial nucleus accumbens shell of rats either during maintenance of cocaine self-administration (i.e. active drug use) or during protracted withdrawal.
5-HT1BR-gene transfer during maintenance shifted the dose–response curve for cocaine self-administration upward and to the left and increased break points and cocaine intake on a progressive ratio (PR) schedule, consistent with enhanced reinforcing effects of cocaine. In contrast, following 21 days of forced abstinence 5-HT1BR-gene transfer attenuated break points and cocaine intake on a PR schedule of reinforcement, as well as cue- and cocaine-primed reinstatement of cocaineseeking behavior.
This unique pattern of effects suggests that mesolimbic 5-HT1BRs differentially modulate cocaine abuse-related behaviors, with a facilitative influence during periods of active drug use in striking contrast to an inhibitory influence during protracted withdrawal. These findings suggest that targeting 5-HT1BRs may lead to a novel treatment for cocaine dependence and that the therapeutic efficacy of these treatments may vary depending on the stage of the addiction cycle.
reward; addiction; reinforcement; relapse; craving; reinstatement
Nucleic acid testing (NAT) for malaria parasites is an increasingly recommended diagnostic endpoint in clinical trials of vaccine and drug candidates and is also important in surveillance of malaria control and elimination efforts. A variety of reported NAT assays have been described, yet no formal external quality assurance (EQA) program provides validation for the assays in use. Here, we report results of an EQA exercise for malaria NAT assays. Among five centers conducting controlled human malaria infection trials, all centers achieved 100% specificity and demonstrated limits of detection consistent with each laboratory's pre-stated expectations. Quantitative bias of reported results compared to expected results was generally <0.5 log10 parasites/mL except for one laboratory where the EQA effort identified likely reasons for a general quantitative shift. The within-laboratory variation for all assays was low at <10% coefficient of variation across a range of parasite densities. Based on this study, we propose to create a Molecular Malaria Quality Assessment program that fulfills the need for EQA of malaria NAT assays worldwide.
Toxicity is a major cause of failure in drug discovery and development, and whilst robust toxicological testing occurs, efficiency could be improved if compounds with cytotoxic characteristics were identified during primary compound screening. The use of high-content imaging in primary screening is becoming more widespread, and by utilising phenotypic approaches it should be possible to incorporate cytotoxicity counter-screens into primary screens. Here we present a novel phenotypic assay that can be used as a counter-screen to identify compounds with adverse cellular effects. This assay has been developed using U2OS cells, the PerkinElmer Operetta high-content/high-throughput imaging system and Columbus image analysis software. In Columbus, algorithms were devised to identify changes in nuclear morphology, cell shape and proliferation using DAPI, TOTO-3 and phosphohistone H3 staining, respectively. The algorithms were developed and tested on cells treated with doxorubicin, taxol and nocodazole. The assay was then used to screen a novel, chemical library, rich in natural product-like molecules of over 300 compounds, 13.6% of which were identified as having adverse cellular effects. This assay provides a relatively cheap and rapid approach for identifying compounds with adverse cellular effects during screening assays, potentially reducing compound rejection due to toxicity in subsequent in vitro and in vivo assays.
•Acute application of apo(a) to smooth muscle cells induced chemorepulsion.•Chronic application (>24 h) induced stress fibre formation and cell spreading.•Effects of apo(a) were mediated by integrin αVβ3, tyrosine kinases and RhoA/ROCK.•Apo(a) impaired SMC motility which potentially contributes to vascular dysfunction.
Lipoprotein(a) (Lp(a)) is an independent risk factor for the development of cardiovascular disease. Vascular smooth muscle cell (SMC) motility and plasticity, functions that are influenced by environmental cues, are vital to adaptation and remodelling in vascular physiology and pathophysiology. Lp(a) is reportedly damaging to SMC function via unknown molecular mechanisms. Apolipoprotein(a) (apo(a)), a unique glycoprotein moiety of Lp(a), has been demonstrated as its active component. The aims of this study were to determine functional effects of recombinant apo(a) on human vascular SMC motility and explore the underlying mechanism(s). Exposure of SMC to apo(a) in migration assays induced a potent, concentration-dependent chemorepulsion that was RhoA and integrin αVβ3-dependent, but transforming growth factor β-independent. SMC manipulation through RhoA gene silencing, Rho kinase inhibition, statin pre-treatment, αVβ3 neutralising antibody and tyrosine kinase inhibition all markedly inhibited apo(a)-mediated SMC migration. Our data reveal unique and potent activities of apo(a) that may negatively influence SMC remodelling in cardiovascular disease. Circulating levels of Lp(a) are resistant to lipid-lowering strategies and hence a greater understanding of the mechanisms underlying its functional effects on SMC may provide alternative therapeutic targets.
Ao, aortic; α-SMA, alpha smooth muscle actin; apo(a), apolipoprotein(a); DMEM, Dulbecco's modified eagle medium; ERK, extracellular signal-regulated kinase; FCS, foetal calf serum; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HP, high power; Lp(a), lipoprotein(a); MAPK, mitogen activated protein kinase; NFκB, nuclear factor kappa B; PDGF, platelet-derived growth factor-BB; ROCK, Rho kinase; SV, saphenous vein; SMC, smooth muscle cell; TGFβ, transforming growth factor beta; Lipoprotein(a); Vascular smooth muscle cells; Migration; RhoA; Remodelling