微流控芯片及其在病原体检测中的应用

微流控芯片又称芯片实验室,是一种以在微米尺度空间对流体进行操控为主要特征的技术平台。微型化、集成化的微流控芯片具有高效、快速、样品和试剂用量少、节约药品等优点,促进了其在病原体检测中的应用。本文对微流控芯片在病原体检测中的优势及其应用研究进展进行综述。     

Viewpoint: evaluating the impact of malaria control efforts on mortality in sub-Saharan Africa

Objective To describe an approach for evaluating the impact of malaria control efforts on malaria‐associated mortality in sub‐Saharan Africa, where disease‐specific mortality trends usually cannot be measured directly and most malaria deaths occur among young children. Methods Methods for evaluating changes in malaria‐associated mortality are examined; advantages and disadvantages are presented. Results All methods require a plausibility argument—i.e., an assumption that mortality reductions can be attributed to programmatic efforts if improvements are found in steps of the causal pathway between intervention scale‐up and mortality trends. As different methods provide complementary information, they can be used together. We recommend following trends in the coverage of malaria control interventions, other factors influencing childhood mortality, malaria‐associated morbidity (especially anaemia), and all‐cause childhood mortality. This approach reflects decreases in malaria’s direct and indirect mortality burden and can be examined in nearly all countries. Adding other information can strengthen the plausibility argument: trends in indicators of malaria transmission, information from demographic surveillance systems and sentinel sites where malaria diagnostics are systematically used, and verbal autopsies linked to representative household surveys. Health facility data on malaria deaths have well‐recognized limitations; however, in specific circumstances, they could produce reliable trends. Model‐based predictions can help describe changes in malaria‐specific burden and assist with program management and advocacy. Conclusions Despite challenges, efforts to reduce malaria‐associated mortality in Africa can be evaluated with trends in malaria intervention coverage and all‐cause childhood mortality. Where there are resources and interest, complementary data on malaria morbidity and malaria‐specific mortality could be added.     

A systems framework for remedying dysfunction in US democracy

Democracy often fails to meet its ideals, and these failures may be made worse by electoral institutions. Unwanted outcomes include elite polarization, unresponsive representatives, and the ability of a faction of voters to gain power at the expense of the majority. Various reforms have been proposed to address these problems, but their effectiveness is difficult to predict against a backdrop of complex interactions. Here we outline a path for systems-level modeling to help understand and optimize repairs to US democracy. Following the tradition of engineering and biology, models of systems include mechanisms with dynamical properties that include nonlinearities and amplification (voting rules), positive feedback mechanisms (single-party control, gerrymandering), negative feedback (checks and balances), integration over time (lifetime judicial appointments), and low dimensionality (polarization). To illustrate a systems-level approach, we analyze three emergent phenomena: low dimensionality, elite polarization, and antimajoritarianism in legislatures. In each case, long-standing rules now contribute to undesirable outcomes as a consequence of changes in the political environment. Theoretical understanding at a general level will also help evaluate whether a proposed reform’s benefits will materialize and be lasting, especially as conditions change again. In this way, rigorous modeling may not only shape new lines of research but aid in the design of effective and lasting reform.     

Bayesian design of synthetic biological systems

Here we introduce a new design framework for synthetic biology that exploits the advantages of Bayesian model selection. We will argue that the difference between inference and design is that in the former we try to reconstruct the system that has given rise to the data that we observe, whereas in the latter, we seek to construct the system that produces the data that we would like to observe, i.e., the desired behavior. Our approach allows us to exploit methods from Bayesian statistics, including efficient exploration of models spaces and high-dimensional parameter spaces, and the ability to rank models with respect to their ability to generate certain types of data. Bayesian model selection furthermore automatically strikes a balance between complexity and (predictive or explanatory) performance of mathematical models. To deal with the complexities of molecular systems we employ an approximate Bayesian computation scheme which only requires us to simulate from different competing models to arrive at rational criteria for choosing between them. We illustrate the advantages resulting from combining the design and modeling (or in silico prototyping) stages currently seen as separate in synthetic biology by reference to deterministic and stochastic model systems exhibiting adaptive and switch-like behavior, as well as bacterial two-component signaling systems.     

New Approaches to Multi-Parametric HIV-1 Genetics Using Multiple Displacement Amplification: Determining the What,How, and Where of the HIV-1 Reservoir

Development of potential HIV-1 curative interventions requires accurate characterization of the proviral reservoir, defined as host-integrated viral DNA genomes that drive rebound of viremia upon halting ART (antiretroviral therapy). Evaluation of such interventions necessitates methods capable of pinpointing the rare, genetically intact, replication-competent proviruses within a background of defective proviruses. This evaluation can be achieved by identifying the distinct integration sites of intact proviruses within host genomes and monitoring the dynamics of these proviruses and host cell lineages over longitudinal sampling. Until recently, molecular genetic approaches at the single proviral level have been generally limited to one of a few metrics, such as proviral genome sequence/intactness, host-proviral integration site, or replication competency. New approaches, taking advantage of MDA (multiple displacement amplification) for WGA (whole genome amplification), have enabled multiparametric proviral characterization at the single-genome level, including proviral genome sequence, host-proviral integration site, and phenotypic characterization of the host cell lineage, such as CD4 memory subset and antigen specificity. In this review, we will examine the workflow of MDA-augmented molecular genetic approaches to study the HIV-1 reservoir, highlighting technical advantages and flexibility. We focus on a collection of recent studies in which investigators have used these approaches to comprehensively characterize intact and defective proviruses from donors on ART, investigate mechanisms of elite control, and define cell lineage identity and antigen specificity of infected CD4+ T cell clones. The highlighted studies exemplify how these approaches and their future iterations will be key in defining the targets and evaluating the impacts of HIV curative interventions.     

Data standards in diabetes patient registries

Widespread adoption of electronic health records (EHRs) and expansion of patient registries present opportunities to improve patient care and population health and advance translational research. However, optimal integration of patient registries with EHR functions and aggregation of regional registries to support national or global analyses will require the use of standards. Currently, there are no standards for patient registries and no content standards for health care data collection or clinical research, including diabetes research. Data standards can facilitate new registry development by supporting reuse of well-defined data elements and data collection systems, and they can enable data aggregation for future research and discovery. This article introduces standardization topics relevant to diabetes patient registries, addresses issues related to the quality and use of registries and their integration with primary EHR data collection systems, and proposes strategies for implementation of data standards in diabetes research and management.     

Malaria vaccines. Evaluation and implementation

Development of an effective malaria vaccine that could be deployed widely in endemic areas has proved to be more difficult than was anticipated, when the first clinical trials of malaria vaccines were conducted 30 years ago. Substantial progress has been made during the past few years and one candidate vaccine, RTS,S/AS02, shows promise. However, during the period that malaria vaccine development has been taking place, alternative methods of malaria control including insecticide treated bednets and intermittent preventive treatment in infancy and childhood have been developed and shown to be partially effective. Malaria vaccines will need to be more effective or more cost effective than these alternative control strategies or be able to provide substantial added value, when deployed with them if they are to be adopted widely.     

Malaria: current status of control,diagnosis, treatment,and a proposed agenda for research and development

Rolling back malaria is possible. Tools are available but they are not used. Several countries deploy, as their national malaria control treatment policy, drugs that are no longer effective. New and innovative methods of vector control, diagnosis, and treatment should be developed, and work towards development of new drugs and a vaccine should receive much greater support. But the pressing need, in the face of increasing global mortality and general lack of progress in malaria control, is research into the best methods of deploying and using existing approaches, particularly insecticide-treated mosquito nets, rapid methods of diagnosis, and artemisinin-based combination treatments. Evidence on these approaches should provide national governments and international donors with the cost-benefit information that would justify much-needed increases in global support for appropriate and effective malaria control.     

Malaria epidemiology and control in refugee camps and complex emergencies.

Owing to the breakdown of health systems, mass population displacements, and resettlement of vulnerable refugees in camps or locations prone to vector breeding, malaria is often a major health problem during war and the aftermath of war. During the initial acute phase of the emergency, before health services become properly established, mortality rates may rise to alarming levels. Establishing good case management and effective malaria prevention are important priorities for international agencies responsible for emergency health services. The operational strategies and control methods used in peacetime must be adapted to emergency conditions, and should be regularly re-assessed as social, political and epidemiological conditions evolve. During the last decade, research on malaria in refugee camps on the Pakistan-Afghanistan and Thailand-Burma borders has led to new methods and strategies for malaria prevention and case management, and these are now being taken up by international health agencies. This experience has shown that integration of research within control programmes is an efficient and dynamic mode of working that can lead to innovation and hopefully sustainable malaria control. United Nations' humanitarian and non-governmental agencies can play a significant part in resolving the outstanding research issues in malaria control.     

An integrative translational approach to study heart failure with preserved ejection fraction: a position paper from the Working Group on Myocardial Function of the European Society of Cardiology

As heart failure with preserved ejection fraction (HFpEF) rises to epidemic proportions, major steps in patient management and therapeutic development are badly needed. With the current position paper we seek to update our view on HFpEF as a highly complex systemic syndrome, from risk factors and mechanisms to long‐term clinical manifestations. We will revise recent advances in animal model development, experimental set‐ups and basic and translational science approaches to HFpEF research, highlighting their drawbacks and advantages. Directions are provided for proper model selection as well as for integrative functional evaluation from the in vivo setting to in vitro cell function testing. Additionally, we address new research challenges that require integration of higher‐order inter‐organ and inter‐cell communication to achieve a full systems biology perspective of HFpEF.     

Shaping acoustic fields as a toolset for microfluidic manipulations in diagnostic technologies

Ultrasonics offers the possibility of developing sophisticated fluid manipulation tools in lab-on-a-chip technologies. Here we demonstrate the ability to shape ultrasonic fields by using phononic lattices, patterned on a disposable chip, to carry out the complex sequence of fluidic manipulations required to detect the rodent malaria parasite Plasmodium berghei in blood. To illustrate the different tools that are available to us, we used acoustic fields to produce the required rotational vortices that mechanically lyse both the red blood cells and the parasitic cells present in a drop of blood. This procedure was followed by the amplification of parasitic genomic sequences using different acoustic fields and frequencies to heat the sample and perform a real-time PCR amplification. The system does not require the use of lytic reagents nor enrichment steps, making it suitable for further integration into lab-on-a-chip point-of-care devices. This acoustic sample preparation and PCR enables us to detect ca. 30 parasites in a microliter-sized blood sample, which is the same order of magnitude in sensitivity as lab-based PCR tests. Unlike other lab-on-a-chip methods, where the sample moves through channels, here we use our ability to shape the acoustic fields in a frequency-dependent manner to provide different analytical functions. The methods also provide a clear route toward the integration of PCR to detect pathogens in a single handheld system.     

Fc-receptors and immunity to malaria: from models to vaccines

The complexity and number of antigens (Ags) seen during an immune response has hampered the development of malaria vaccines. Antibodies (Abs) play an important role in immunity to malaria and their passive administration is effective at controlling the disease. Abs represent approximately 25% of all proteins undergoing clinical trials, and these 'smart biologicals' have undergone a major revival with the realization that Abs lie at the interface between innate and adaptive immunity. At least 18 Abs have FDA approval for clinical use and approximately 150 are in clinical trials, the majority for the treatment of cancer, allograft rejection or autoimmune disease. Despite these triumphs none are in development for malaria, principally because they are perceived as being too expensive for a disease mainly afflicting poor and marginalized populations. Although unlikely, at least in the foreseeable future, that Ab-based prophylaxis will be made available to the millions of people at risk from malaria, they may be incorporated into current vaccine approaches, since Abs act as correlates of protection in studies aimed at defining the best Ags to include in vaccines. Abs may also form the basis for novel vaccination strategies by targeting Ags to appropriate antigen presenting cells. Therefore, to develop the most efficacious vaccines it will be necessary to fully understand which Abs and Fc-receptors (FcRs) are best engaged for a positive outcome.     

Are Plasmodium falciparum parasites present in peripheral blood genetically the same as those sequestered in the tissues?

Since quinine does not inhibit the growth of Plasmodium falciparum ring stages or mature schizonts, parasites may continue to emerge from sequestration sites after starting treatment. We used polymerase chain reaction amplification of P. falciparum merozoite surface protein 1 (MSP-1) and MSP-2 alleles to distinguish genotypes infecting 58 children with severe malaria. To examine changes in parasite populations in peripheral blood over time, we compared changes in number and spectrum of genotypes in samples on admission to a hospital to those obtained up to 24 hours later. Thirty-four children lost genotypes, 21 retained genotypes, and 3 gained an extra P. falciparum genotype at one locus but not the other. The lack of novel genotypes emerging suggests that among children with severe malaria the dominant clones sequestered in deep organs are usually the same as those in peripheral circulation.     

Natural protection against severe Plasmodium falciparum malaria due to impaired rosette formation

Genes for two lethal diseases, thalassemia and sickle cell anemia, are favored by evolution because, in their heterozygous form, they protect against cerebral malaria. Rosette formation, the binding of uninfected red cells (RBCs) to Plasmodium falciparum-infected RBCs (PRBCs), has previously been found to be associated with cerebral malaria, the most important severe manifestation of P falciparum malaria. We show here that thalassemic RBCs and, under certain conditions, even hemoglobin S (HbS)-containing RBCs possess an impaired ability to bind to PRBCs, forming small and weak erythrocyte rosettes compared with rosettes formed by normal RBCs. This decreased rosetting ability is associated with the small size of the thalassemic RBCs and with distortion of the mechanical properties of HbS-containing RBCs. The impairment of rosette formation may hinder the development of cerebral malaria by abatement of sequestration.     

From malaria control to eradication: The WHO perspective

Efforts to control malaria have been boosted in the past few years with increased international funding and greater political commitment. Consequently, the reported malaria burden is being reduced in a number of countries throughout the world, including in some countries in tropical Africa where the burden of malaria is greatest. These achievements have raised new hopes of eradicating malaria. This paper summarizes the outcomes of a World Health Organization’s expert meeting on the feasibility of such a goal. Given the hindsight and experience of the Global Malaria Eradication Programme of the 1950s and 1960s, and current knowledge of the effectiveness of antimalarial tools and interventions, it would be feasible to effectively control malaria in all parts of the world and greatly reduce the enormous morbidity and mortality of malaria. It would also be entirely feasible to eliminate malaria from countries and regions where the intensity of transmission is low to moderate, and where health systems are strong. Elimination of malaria requires a re‐orientation of control activity, moving away from a population‐based coverage of interventions, to one based on a programme of effective surveillance and response. Sustained efforts will be required to prevent the resurgence of malaria from where it is eliminated. Eliminating malaria from countries where the intensity of transmission is high and stable such as in tropical Africa will require more potent tools and stronger health systems than are available today. When such countries have effectively reduced the burden of malaria, the achievements will need to be consolidated before a programme re‐orientation towards malaria elimination is contemplated. Malaria control and elimination are under the constant threat of the parasite and vector mosquito developing resistance to medicines and insecticides, which are the cornerstones of current antimalarial interventions. The prospects of malaria eradication, therefore, rest heavily on the outcomes of research and development for new and improved tools. Malaria control and elimination are complementary objectives in the global fight against malaria.     

Emergence of complex dynamics in a simple model of signaling networks

Various physical, social, and biological systems generate complex fluctuations with correlations across multiple time scales. In physiologic systems, these long-range correlations are altered with disease and aging. Such correlated fluctuations in living systems have been attributed to the interaction of multiple control systems; however, the mechanisms underlying this behavior remain unknown. Here, we show that a number of distinct classes of dynamical behaviors, including correlated fluctuations characterized by 1/f scaling of their power spectra, can emerge in networks of simple signaling units. We found that, under general conditions, complex dynamics can be generated by systems fulfilling the following two requirements, (i) a "small-world" topology and (ii) the presence of noise. Our findings support two notable conclusions. First, complex physiologic-like signals can be modeled with a minimal set of components; and second, systems fulfilling conditions i and ii are robust to some degree of degradation (i.e., they will still be able to generate 1/f dynamics).     

The path of malaria vaccine development: challenges and perspectives

Malaria is a life‐threatening disease caused by parasites of the Plasmodium genus. In many parts of the world, the parasites have developed resistance to a number of antimalarial agents. Key interventions to control malaria include prompt and effective treatment with artemisinin‐based combination therapies, use of insecticidal nets by individuals at risk and active research into malaria vaccines. Protection against malaria through vaccination was demonstrated more than 30 years ago when individuals were vaccinated via repeated bites by Plasmodium falciparum‐infected and irradiated but still metabolically active mosquitoes. However, vaccination with high doses of irradiated sporozoites injected into humans has long been considered impractical. Yet, following recent success using whole‐organism vaccines, the approach has received renewed interest; it was recently reported that repeated injections of irradiated sporozoites increased protection in 80 vaccinated individuals. Other approaches include subunit malaria vaccines, such as the current leading candidate RTS,S (consisting of fusion between a portion of the P. falciparum‐derived circumsporozoite protein and the hepatitis B surface antigen), which has been demonstrated to induce reasonably good protection. Although results have been encouraging, the level of protection is generally considered to be too low to achieve eradication of malaria. There is great interest in developing new and better formulations and stable delivery systems to improve immunogenicity. In this review, we will discuss recent strategies to develop efficient malaria vaccines.     

Establishment of a human hepatocyte line that supports in vitro development of the exo-erythrocytic stages of the malaria parasites Plasmodium falciparum and P. vivax

Our understanding of the biology of malaria parasite liver stages is limited because of the lack of efficient in vitro systems that support the exo-erythrocytic (EE) development of the parasite. We report the development of a new hepatocyte line (HC-04) from normal human liver cells. The HC-04 cells have proliferated in hormone-free medium for more than 200 passages. The cells were hyperdiploid, resembled liver parenchymal cells, and synthesized major liver-specific proteins and enzymes. Using Plasmodium falciparum and P. vivax sporozoites harvested from salivary glands of infected mosquitoes, we showed that HC-04 cells supported the complete EE development of these two most prevalent human malaria parasites. The EE parasites attained full maturation as shown by their infectivity to human erythrocytes. The infection rates of the liver cells were estimated to be 0.066% and 0.041% for P. falciparum and P. vivax, respectively. As the first human hepatocyte line known to support complete EE development of both P. falciparum and P. vivax, HC-04 will provide an experimental model that can be used for studying the biology of liver stage malaria parasites.     

Influence of socioeconomic factors on the treatment and prevention of malaria in pregnant and non-pregnant adolescent girls in Nigeria.

The influence of socioeconomic factors on the treatment and prevention of malaria was investigated in 45 pregnant and a control group of 47 non-pregnant adolescent girls in the rural community of Imesi-Ile, Nigeria. The study consisted of focus group discussions and a house-to-house survey. During the survey, clinical and anthropometric measurements were taken of the girls and they were assisted in completing a prepared questionnaire which sought information on their methods of treatment and prevention of malaria. The results showed a higher incidence of malaria parasitaemia, anaemia and fever episodes in the group of pregnant adolescent girls. Both groups of girls use identical methods for the prevention of malaria but only a few pregnant girls as compared to non-pregnant girls receive modern hospital treatment for malaria. In particular, pregnant unmarried girls were less likely to receive antenatal care, to use the local health centres for the treatment of malaria and to adopt appropriate measures for the prevention of malaria. This trend was not due to aversion to hospital treatment since most people in the community prefer modern treatment of malaria to traditional or religious methods of treatment. The low utilization of hospital treatment by the pregnant girls was found to be due to perceived high cost of treatment at the health centres and to lack of privacy. We conclude that a malaria treatment service that utilizes modern methods and is both cheap and confidential will be preferred by pregnant adolescent girls in Imesi-Ile.