Mechanisms of mono- and poly-ubiquitination : ubiquitination specificity depends on compatibility between the E2 catalytic core and amino acid residues proximal to the lysine

Ubiquitination involves the attachment of ubiquitin to lysine residues on substrate proteins or itself, which can result in protein monoubiquitination or polyubiquitination. Ubiquitin attachment to different lysine residues can generate diverse substrate-ubiquitin structures, targeting proteins to different fates. The mechanisms of lysine selection are not well understood. Ubiquitination by the largest group of E3 ligases, the RING-family E3 s, is catalyzed through co-operation between the non-catalytic ubiquitin-ligase (E3) and the ubiquitin-conjugating enzyme (E2), where the RING E3 binds the substrate and the E2 catalyzes ubiquitin transfer. Previous studies suggest that ubiquitination sites are selected by E3-mediated positioning of the lysine toward the E2 active site. Ultimately, at a catalytic level, ubiquitination of lysine residues within the substrate or ubiquitin occurs by nucleophilic attack of the lysine residue on the thioester bond linking the E2 catalytic cysteine to ubiquitin. One of the best studied RING E3/ E2 complexes is the Skp1/Cul1/F box protein complex, SCFCdc4, and its cognate E2, Cdc34, which target the CDK inhibitor Sic1 for K48-linked polyubiquitination, leading to its proteasomal degradation. Our recent studies of this model system demonstrated that residues surrounding Sic1 lysines or lysine 48 in ubiquitin are critical for ubiquitination. This sequence-dependence is linked to evolutionarily conserved key residues in the catalytic region of Cdc34 and can determine if Sic1 is mono- or poly-ubiquitinated. Our studies indicate that amino acid determinants in the Cdc34 catalytic region and their compatibility to those surrounding acceptor lysine residues play important roles in lysine selection. This may represent a general mechanism in directing the mode of ubiquitination in E2 s.

Plasmodium falciparum parasites lacking histidine-rich protein 2 and 3 : a review and recommendations for accurate reporting

Malaria rapid diagnostic tests (RDTs) play a critical role in malaria case management, surveillance and case investigations. Test performance is largely determined by design and quality characteristics, such as detection sensitivity, specificity, and thermal stability. However, parasite characteristics such as variable or absent expression of antigens targeted by RDTs can also affect RDT performance. Plasmodium falciparum parasites lacking the PfHRP2 protein, the most common target antigen for detection of P. falciparum, have been reported in some regions. Therefore, accurately mapping the presence and prevalence of P. falciparum parasites lacking pfhrp2 would be an important step so that RDTs targeting alternative antigens, or microscopy, can be preferentially selected for use in such regions. Herein the available evidence and molecular basis for identifying malaria parasites lacking PfHRP2 is reviewed, and a set of recommended procedures to apply for future investigations for parasites lacking PfHRP2, is proposed.

Modelling the dynamics of Plasmodium falciparum histidine-rich protein 2 in human malaria to better understand malaria rapid diagnostic test performance

BACKGROUND: Effective diagnosis of malaria is a major component of case management. Rapid diagnostic tests (RDTs) based on Plasmodium falciparumhistidine-rich protein 2 (PfHRP2) are popular for diagnosis of this most virulent malaria infection. However, concerns have been raised about the longevity of the PfHRP2 antigenaemia following curative treatment in endemic regions. METHODS: A model of PfHRP2 production and decay was developed to mimic the kinetics of PfHRP2 antigenaemia during infections. Data from two human infection studies was used to fit the model, and to investigate PfHRP2 kinetics. Four malaria RDTs were assessed in the laboratory to determine the minimum detectable concentration of PfHRP2. RESULTS: Fitting of the PfHRP2 dynamics model indicated that in malaria naive hosts, P. falciparum parasites of the 3D7 strain produce 1.4 x 10(-)(1)(3) g of PfHRP2 per parasite per replication cycle. The four RDTs had minimum detection thresholds between 6.9 and 27.8 ng/mL. Combining these detection thresholds with the kinetics of PfHRP2, it is predicted that as few as 8 parasites/muL may be required to maintain a positive RDT in a chronic infection. CONCLUSIONS: The results of the model indicate that good quality PfHRP2-based RDTs should be able to detect parasites on the first day of symptoms, and that the persistence of the antigen will cause the tests to remain positive for at least seven days after treatment. The duration of a positive test result following curative treatment is dependent on the duration and density of parasitaemia prior to treatment and the presence and affinity of anti-PfHRP2 antibodies.

Blood transfer devices for malaria rapid diagnostic tests : evaluation of accuracy, safety and ease of use

Background: Malaria rapid diagnostic tests (RDTs) are increasingly used by remote health personnel with minimal training in laboratory techniques. RDTs must, therefore, be as simple, safe and reliable as possible. Transfer of blood from the patient to the RDT is critical to safety and accuracy, and poses a significant challenge to many users. Blood transfer devices were evaluated for accuracy and precision of volume transferred, safety and ease of use, to identify the most appropriate devices for use with RDTs in routine clinical care. Methods: Five devices, a loop, straw-pipette, calibrated pipette, glass capillary tube, and a new inverted cup device, were evaluated in Nigeria, the Philippines and Uganda. The 227 participating health workers used each device to transfer blood from a simulated finger-prick site to filter paper. For each transfer, the number of attempts required to collect and deposit blood and any spilling of blood during transfer were recorded. Perceptions of ease of use and safety of each device were recorded for each participant. Blood volume transferred was calculated from the area of blood spots deposited on filter paper. Results: The overall mean volumes transferred by devices differed significantly from the target volume of 5 microliters (p < 0.001). The inverted cup (4.6 microliters) most closely approximated the target volume. The glass capillary was excluded from volume analysis as the estimation method used is not compatible with this device. The calibrated pipette accounted for the largest proportion of blood exposures (23/225, 10%); exposures ranged from 2% to 6% for the other four devices. The inverted cup was considered easiest to use in blood collection (206/ 226, 91%); the straw-pipette and calibrated pipette were rated lowest (143/225 [64%] and 135/225 [60%] respectively). Overall, the inverted cup was the most preferred device (72%, 163/227), followed by the loop (61%, 138/227). Conclusions: The performance of blood transfer devices varied in this evaluation of accuracy, blood safety, ease of use, and user preference. The inverted cup design achieved the highest overall performance, while the loop also performed well. These findings have relevance for any point-of-care diagnostics that require blood sampling.

Artemisinin-induced parasite dormancy : a plausible mechanism for treatment failure

Background Artemisinin-combination therapy is a highly effective treatment for uncomplicated falciparum malaria but parasite recrudescence has been commonly reported following artemisinin (ART) monotherapy. The dormancy recovery hypothesis has been proposed to explain this phenomenon, which is different from the slower parasite clearance times reported as the first evidence of the development of ART resistance. Methods In this study, an existing P. falciparum infection model is modified to incorporate the hypothesis of dormancy. Published in vitro data describing the characteristics of dormant parasites is used to explore whether dormancy alone could be responsible for the high recrudescence rates observed in field studies using monotherapy. Several treatment regimens and dormancy rates were simulated to investigate the rate of clinical and parasitological failure following treatment. Results The model output indicates that following a single treatment with ART parasitological and clinical failures occur in up to 77% and 67% of simulations, respectively. These rates rapidly decline with repeated treatment and are sensitive to the assumed dormancy rate. The simulated parasitological and clinical treatment failure rates after 3 and 7 days of treatment are comparable to those reported from several field trials. Conclusions Although further studies are required to confirm dormancy in vivo, this theoretical study adds support for the hypothesis, highlighting the potential role of this parasite sub-population in treatment failure following monotherapy and reinforcing the importance of using ART in combination with other anti-malarials.

An improved method for undertaking limiting dilution assays for in vitro cloning of Plasmodium falciparum parasites

Background Obtaining single parasite clones is required for many techniques in malaria research. Cloning by limiting dilution using microscopy-based assessment for parasite growth is an arduous and labor-intensive process. An alternative method for the detection of parasite growth in limiting dilution assays is using a commercial ELISA histidine-rich protein II (HRP2) detection kit. Methods Detection of parasite growth was undertaken using HRP2 ELISA and compared to thick film microscopy. An HRP2 protein standard was used to determine the detection threshold of the HRP2 ELISA assay, and a HRP2 release model was used to extrapolate the amount of parasite growth required for a positive result. Results The HRP2 ELISA was more sensitive than microscopy for detecting parasite growth. The minimum level of HRP2 protein detection of the ELISA was 0.11ng/ml. Modeling of HRP2 release determined that 2,116 parasites are required to complete a full erythrocytic cycle to produce sufficient HRP2 to be detected by the ELISA. Under standard culture conditions this number of parasites is likely to be reached between 8 to 14 days of culture. Conclusions This method provides an accurate and simple way for the detection of parasite growth in limiting dilution assays, reducing time and resources required in traditional methods. Furthermore the method uses spent culture media instead of the parasite-infected red blood cells, enabling culture to continue.

Outbreak detection algorithms for seasonal disease data : a case study using Ross River virus disease

Background Detection of outbreaks is an important part of disease surveillance. Although many algorithms have been designed for detecting outbreaks, few have been specifically assessed against diseases that have distinct seasonal incidence patterns, such as those caused by vector-borne pathogens. Methods We applied five previously reported outbreak detection algorithms to Ross River virus (RRV) disease data (1991-2007) for the four local government areas (LGAs) of Brisbane, Emerald, Redland and Townsville in Queensland, Australia. The methods used were the Early Aberration Reporting System (EARS) C1, C2 and C3 methods, negative binomial cusum (NBC), historical limits method (HLM), Poisson outbreak detection (POD) method and the purely temporal SaTScan analysis. Seasonally-adjusted variants of the NBC and SaTScan methods were developed. Some of the algorithms were applied using a range of parameter values, resulting in 17 variants of the five algorithms. Results The 9,188 RRV disease notifications that occurred in the four selected regions over the study period showed marked seasonality, which adversely affected the performance of some of the outbreak detection algorithms. Most of the methods examined were able to detect the same major events. The exception was the seasonally-adjusted NBC methods that detected an excess of short signals. The NBC, POD and temporal SaTScan algorithms were the only methods that consistently had high true positive rates and low false positive and false negative rates across the four study areas. The timeliness of outbreak signals generated by each method was also compared but there was no consistency across outbreaks and LGAs. Conclusions This study has highlighted several issues associated with applying outbreak detection algorithms to seasonal disease data. In lieu of a true gold standard, a quantitative comparison is difficult and caution should be taken when interpreting the true positives, false positives, sensitivity and specificity.

A large proportion of asymptomatic Plasmodium infections with low and sub-microscopic parasite densities in the low transmission setting of Temotu Province, Solomon Islands : challenges for malaria diagnostics in an elimination setting

Background Many countries are scaling up malaria interventions towards elimination. This transition changes demands on malaria diagnostics from diagnosing ill patients to detecting parasites in all carriers including asymptomatic infections and infections with low parasite densities. Detection methods suitable to local malaria epidemiology must be selected prior to transitioning a malaria control programme to elimination. A baseline malaria survey conducted in Temotu Province, Solomon Islands in late 2008, as the first step in a provincial malaria elimination programme, provided malaria epidemiology data and an opportunity to assess how well different diagnostic methods performed in this setting. Methods During the survey, 9,491 blood samples were collected and examined by microscopy for Plasmodium species and density, with a subset also examined by polymerase chain reaction (PCR) and rapid diagnostic tests (RDTs). The performances of these diagnostic methods were compared. Results A total of 256 samples were positive by microscopy, giving a point prevalence of 2.7%. The species distribution was 17.5% Plasmodium falciparum and 82.4% Plasmodium vivax. In this low transmission setting, only 17.8% of the P. falciparum and 2.9% of P. vivax infected subjects were febrile (≥38°C) at the time of the survey. A significant proportion of infections detected by microscopy, 40% and 65.6% for P. falciparum and P. vivax respectively, had parasite density below 100/μL. There was an age correlation for the proportion of parasite density below 100/μL for P. vivax infections, but not for P. falciparum infections. PCR detected substantially more infections than microscopy (point prevalence of 8.71%), indicating a large number of subjects had sub-microscopic parasitemia. The concordance between PCR and microscopy in detecting single species was greater for P. vivax (135/162) compared to P. falciparum (36/118). The malaria RDT detected the 12 microscopy and PCR positive P. falciparum, but failed to detect 12/13 microscopy and PCR positive P. vivax infections. Conclusion Asymptomatic malaria infections and infections with low and sub-microscopic parasite densities are highly prevalent in Temotu province where malaria transmission is low. This presents a challenge for elimination since the large proportion of the parasite reservoir will not be detected by standard active and passive case detection. Therefore effective mass screening and treatment campaigns will most likely need more sensitive assays such as a field deployable molecular based assay.

Global sequence variation in the histidine-rich proteins 2 and 3 of Plasmodium falciparum: implications for the performance of malaria rapid diagnostic tests

Background Accurate diagnosis is essential for prompt and appropriate treatment of malaria. While rapid diagnostic tests (RDTs) offer great potential to improve malaria diagnosis, the sensitivity of RDTs has been reported to be highly variable. One possible factor contributing to variable test performance is the diversity of parasite antigens. This is of particular concern for Plasmodium falciparum histidine-rich protein 2 (PfHRP2)-detecting RDTs since PfHRP2 has been reported to be highly variable in isolates of the Asia-Pacific region. Methods The pfhrp2 exon 2 fragment from 458 isolates of P. falciparum collected from 38 countries was amplified and sequenced. For a subset of 80 isolates, the exon 2 fragment of histidine-rich protein 3 (pfhrp3) was also amplified and sequenced. DNA sequence and statistical analysis of the variation observed in these genes was conducted. The potential impact of the pfhrp2 variation on RDT detection rates was examined by analysing the relationship between sequence characteristics of this gene and the results of the WHO product testing of malaria RDTs: Round 1 (2008), for 34 PfHRP2-detecting RDTs. Results Sequence analysis revealed extensive variations in the number and arrangement of various repeats encoded by the genes in parasite populations world-wide. However, no statistically robust correlation between gene structure and RDT detection rate for P. falciparum parasites at 200 parasites per microlitre was identified. Conclusions The results suggest that despite extreme sequence variation, diversity of PfHRP2 does not appear to be a major cause of RDT sensitivity variation.

Building the youth sector from the bottom up : case studies from the Queensland CPLAN Initiative 2012-2013

As part of YANQ's decentralisation across the state, YANQ have set up 10 Networks across Queensland, with Facilitators based in each of the regions. We encourage you to get in contact with your local Facilitator if you would like to have input on Workforce Development or youth policy issues. CPLANs aim to create an ongoing and sustainable structure across ten regions in Queensland to support a consistent focus on: ⋅ Policy issues relevant to young people; and ⋅ Workforce development strategies for the youth sector from a local, regional and state perspective. The ten CPLANs fall under the existing structure of YANQ and utlise and lever off the comprehensive network of youth inter-­‐agencies and networks across the state. The ten CPLANs are made up of representatives from the youth sector in each region who have an interest in contributing to policy development and workforce issues.

Herpes simplex 2 risk among women in a polygynous setting in rural West Africa

OBJECTIVES: To determine risk factors for herpes simplex 2 (HSV2) infection in women in a polygynous rural Gambian population. METHODS: Data from women who participated in a cross-sectional survey of reproductive health were matched to their own and, for women who had been or were married (ever-married), their spouses' data collected in a cross-sectional survey of fertility interests, including information on marital histories. RESULTS: Data were available on 150 never-married and 525 ever-married women. HSV2 prevalence was 16% amongst never-married women and 36% amongst ever-married women. For ever-married women, their own personal characteristics (age, ethnicity and genital cutting status) and events from their husbands' marriage history were important determinants of HSV2 infection. Women whose husbands married for the first time over age 35 were at greater risk than women whose husbands married by age 24 [odds ratio (OR) 2.72, 95% confidence interval (CI) 1.20-6.10]. Women whose husband reported interest in a new marriage were more likely to be HSV2 positive (OR 1.91, 95% CI 1.18-3.09). Women whose husbands were currently monogamous but had had previous marriages (OR 2.76, 95% CI 1.30-5.88) and women in currently polygynous marriages (OR 2.88, 95% CI 1.66-5.01) were three times as likely to be HSV2 positive as women who were their husband's only wife ever. CONCLUSION: Much transmission of HSV2 in this setting occurs within marriage where opportunity for personal protection is limited. High levels of transmission within marriage may undermine the impact of sexual behaviour change programmes aiming to reduce HSV2 and HIV incidence and complicate their evaluation.

Slaving and release in co-infection control

Background Animal and human infection with multiple parasite species is the norm rather than the exception, and empirical studies and animal models have provided evidence for a diverse range of interactions among parasites. We demonstrate how an optimal control strategy should be tailored to the pathogen community and tempered by species-level knowledge of drug sensitivity with use of a simple epidemiological model of gastro-intestinal nematodes. Methods We construct a fully mechanistic model of macroparasite co-infection and use it to explore a range of control scenarios involving chemotherapy as well as improvements to sanitation. Results Scenarios are presented whereby control not only releases a more resistant parasite from antagonistic interactions, but risks increasing co-infection rates, exacerbating the burden of disease. In contrast, synergisms between species result in their becoming epidemiologically slaved within hosts, presenting a novel opportunity for controlling drug resistant parasites by targeting co-circulating species. Conclusions Understanding the effects on control of multi-parasite species interactions, and vice versa, is of increasing urgency in the advent of integrated mass intervention programmes.

Health education and the control of intestinal worm infections in China : a new vision

Background: The transmission of soil-transmitted helminths (STHs) is associated with poverty, poor hygiene behaviour, lack of clean water and inadequate waste disposal and sanitation. Periodic administration of benzimidazole drugs is the mainstay for global STH control but it does not prevent re-infection, and is unlikely to interrupt transmission as a stand-alone intervention. Findings: We reported recently on the development and successful testing in Hunan province, PR China, of a health education package to prevent STH infections in Han Chinese primary school students. We have recently commenced a new trial of the package in the ethnically diverse Xishuangbanna autonomous prefecture in Yunnan province and the approach is also being tested in West Africa, with further expansion into the Philippines in 2015. Conclusions: The work in China illustrates well the direct impact that health education can have in improving knowledge and awareness, and in changing hygiene behaviour. Further, it can provide insight into the public health outcomes of a multi-component integrated control program, where health education prevents re-infection and periodic drug treatment reduces prevalence and morbidity.

Nonvolatile multilevel data storage memory device from controlled ambipolar charge trapping mechanism

The capability of storing multi-bit information is one of the most important challenges in memory technologies. An ambipolar polymer which intrinsically has the ability to transport electrons and holes as a semiconducting layer provides an opportunity for the charge trapping layer to trap both electrons and holes efficiently. Here, we achieved large memory window and distinct multilevel data storage by utilizing the phenomena of ambipolar charge trapping mechanism. As fabricated flexible memory devices display five well-defined data levels with good endurance and retention properties showing potential application in printed electronics.

Mandatory reporting laws

As part of the effort to protect children from significant abuse and neglect, each state and territory in Australia has enacted legislation commonly known as "mandatory reporting laws". There is much confusion about the nature and effects of these laws, both generally and within each jurisdiction. Accordingly, the main aim of this chapter is to review and explain the legislative principles across Australia. In doing so, the chapter will identify differences between the state and territory laws and will situate the laws as part of a system of responses to the whole spectrum of child abuse and neglect. We will also highlight the need for effective reporter training and public awareness, especially given the tension between the widely perceived need for a community response to child abuse and neglect and the simultaneous concern to avoid unnecessary reporting of innocuous events and situations.