Dibutyl phthalate released by solitary female locusts mediates sexual communication at low density.

Sex pheromones play a crucial role in mate location and reproductive success. Insects face challenges in finding mates in low-density environments. The population dynamics of locusts vary greatly, ranging from solitary individuals to high-density swarms, leading to multiple-trait divergence between solitary and gregarious phases. However, differences in sexual communication between solitary and gregarious locusts have not been sufficiently explored. Herein, we found that solitary locusts but not gregarious ones heavily rely on a single compound, dibutyl phthalate (DBP), for sexual communication. DBP is abundantly released by solitary female locusts and elicits strong attraction of male solitary and gregarious locusts. Solitary adult males display much higher electrophysiological responses to DBP than adult females. Additionally, LmigOr13 was identified as the DBP-specific odorant receptor expressed in neurons housed in basiconic sensilla. Male LmigOr13-/- mutants generated by CRISPR/Cas9 have low electrophysiological responses and behavioral attraction to DBP in both laboratory and field cage experiments. Notably, the attractiveness of DBP to male locusts becomes more evident at lower population densities imposed by controlling the cage size. This finding sheds light on the utilization of a sex pheromone to promote reproductive success in extremely low-density conditions and provides important insights into alternative approaches for population monitoring of locusts.

4-Vinylanisole is an aggregation pheromone in locusts.

Locust plagues threaten agricultural and environmental safety throughout the world1,2. Aggregation pheromones have a crucial role in the transition of locusts from a solitary form to the devastating gregarious form and the formation of large-scale swarms3,4. However, none of the candidate compounds reported5-7 meet all the criteria for a locust aggregation pheromone. Here, using behavioural assays, electrophysiological recording, olfactory receptor characterization and field experiments, we demonstrate that 4-vinylanisole (4VA) (also known as 4-methoxystyrene) is an aggregation pheromone of the migratory locust (Locusta migratoria). Both gregarious and solitary locusts are strongly attracted to 4VA, regardless of age and sex. Although it is emitted specifically by gregarious locusts, 4VA production can be triggered by aggregation of four to five solitary locusts. It elicits responses specifically from basiconic sensilla on locust antennae. We also identified OR35 as a specific olfactory receptor of 4VA. Knockout of OR35 using CRISPR-Cas9 markedly reduced the electrophysiological responses of the antennae and impaired 4VA behavioural attractiveness. Finally, field trapping experiments verified the attractiveness of 4VA to experimental and wild populations. These findings identify a locust aggregation pheromone and provide insights for the development of novel control strategies for locusts.

Rapid detection of pathogenic fungi from coastal population with respiratory infections using microfluidic chip technology.

BACKGROUND: Currently, culture methods are commonly used in clinical tests to detect pathogenic fungi including Candida spp. Nonetheless, these methods are cumbersome and time-consuming, thereby leading to considerable difficulties in diagnosis of pathogenic fungal infections, especially in situations that respiratory samples such as alveolar lavage fluid and pleural fluid contain extremely small amounts of microorganisms. The aim of this study was to elucidate the utility and practicality of microfluidic chip technology in quick detection of respiratory pathogenic fungi. METHODS: DNAs of clinical samples (mainly derived from sputa, alveolar lavage fluid, and pleural fluid) from 64 coastal patients were quickly detected using microfluidic chip technology with 20 species of fungal spectrum and then validated by Real-time qPCR, and their clinical baseline data were analyzed. RESULTS: Microfluidic chip results showed that 36 cases infected with Candida spp. and 27 cases tested negative for fungi, which was consistent with Real-time qPCR validation. In contrast, only 16 cases of fungal infections were detected by the culture method; however, one of the culture-positive samples tested negative by microfluidic chip and qPCR validation. Moreover, we found that the patients with Candida infections had significantly higher rates of platelet count reduction than fungi-negative controls. When compared with the patients infected with C. albicans alone, the proportion of males in the patients co-infected with multiple Candidas significantly increased, while their platelet counts significantly decreased. CONCLUSIONS: These findings suggest that constant temperature amplification-based microfluidic chip technology combined with routine blood tests can increase the detection speed and accuracy (including sensitivity and specificity) of identifying respiratory pathogenic fungi.

Juvenile hormone suppresses aggregation behavior through influencing antennal gene expression in locusts.

Animals often exhibit dramatically behavioral plasticity depending on their internal physiological state, yet little is known about the underlying molecular mechanisms. The migratory locust, Locusta migratoria, provides an excellent model for addressing these questions because of their famous phase polyphenism involving remarkably behavioral plasticity between gregarious and solitarious phases. Here, we report that a major insect hormone, juvenile hormone, is involved in the regulation of this behavioral plasticity related to phase change by influencing the expression levels of olfactory-related genes in the migratory locust. We found that the treatment of juvenile hormone analog, methoprene, can significantly shift the olfactory responses of gregarious nymphs from attraction to repulsion to the volatiles released by gregarious nymphs. In contrast, the repulsion behavior of solitarious nymphs significantly decreased when they were treated with precocene or injected with double-stranded RNA of JHAMT, a juvenile hormone acid O-methyltransferase. Further, JH receptor Met or JH-response gene Kr-h1 knockdown phenocopied the JH-deprivation effects on olfactory behavior. RNA-seq analysis identified 122 differentially expressed genes in antennae after methoprene application on gregarious nymphs. Interestingly, several olfactory-related genes were especially enriched, including takeout (TO) and chemosensory protein (CSP) which have key roles in behavioral phase change of locusts. Furthermore, methoprene application and Met or Kr-h1 knockdown resulted in simultaneous changes of both TO1 and CSP3 expression to reverse pattern, which mediated the transition between repulsion and attraction responses to gregarious volatiles. Our results suggest the regulatory roles of a pleiotropic hormone in locust behavioral plasticity through modulating gene expression in the peripheral olfactory system.

CRISPR/Cas9-mediated genome editing induces exon skipping by complete or stochastic altering splicing in the migratory locust.

BACKGROUND: The CRISPR/Cas9 system has been widely used to generate gene knockout/knockin models by inducing frameshift mutants in cell lines and organisms. Several recent studies have reported that such mutants can lead to in-frame exon skipping in cell lines. However, there was little research about post-transcriptional effect of CRISPR-mediated gene editing in vivo. RESULTS: We showed that frameshift indels also induced complete or stochastic exon skipping by deleting different regions to influence pre-mRNA splicing in vivo. In the migratory locust, the missing 55 bp at the boundary of intron 3 and exon 4 of an olfactory receptor gene, LmigOr35, resulted in complete exon 4 skipping, whereas the lacking 22 bp in exon 4 of LmigOr35 only resulted in stochastic exon 4 skipping. A single sgRNA induced small insertions or deletions at the boundary of intron and exon to disrupt the 3' splicing site causing completely exon skipping, or alternatively induce small insertions or deletions in the exon to stochastic alter splicing causing the stochastic exon skipping. CONCLUSIONS: These results indicated that complete or stochastic exon skipping could result from the CRISPR-mediated genome editing by deleting different regions of the gene. Although exon skipping caused by CRISPR-mediated editing was an unexpected outcome, this finding could be developed as a technology to investigate pre-mRNA splicing or to cure several human diseases caused by splicing mutations.

CRISPR/Cas9-mediated genome editing induces gene knockdown by altering the pre-mRNA splicing in mice.

BACKGROUND: Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein 9 (CRISPR/Cas9) has been wildly used to generate gene knockout models through inducing indels causing frame-shift. However, there are few studies concerning the post-transcript effects caused by CRISPR-mediated genome editing. RESULTS: In the present study, we showed that gene knockdown model also could be generated using CRISPR-mediated gene editing by disrupting the boundary of exon and intron in mice (C57BL/6 J). CRISPR induced indel at the boundary of exon and intron (5' splice site) caused alternative splicing and produced multiple different mRNAs, most of these mRNAs introduced premature termination codon causing down expression of the gene. CONCLUSIONS: These results showed that alternative splicing mutants were able to generate through CRISPR-mediated genome editing by deleting the boundary of exon and intron causing disruption of 5' splice site. Although alternative splicing was an unexpected outcome, this finding could be developed as a technology to generate gene knockdown models or to investigate pre-mRNA splicing.

Identification and functional analysis of olfactory receptor family reveal unusual characteristics of the olfactory system in the migratory locust.

Locusts represent the excellent model of insect olfaction because the animals are equipped with an unusual olfactory system and display remarkable density-dependent olfactory plasticity. However, information regarding receptor molecules involved in the olfactory perception of locusts is very limited. On the basis of genome sequence and antennal transcriptome of the migratory locust, we conduct the identification and functional analysis of two olfactory receptor families: odorant receptors (ORs) and ionotropic receptors (IRs). In the migratory locust, there is an expansion of OR family (142 ORs) while distinctly lower number of IR genes (32 IRs) compared to the repertoires of other insects. The number of the locust OR genes is much less than that of glomeruli in antennal lobe, challenging the general principle of the "one glomerulus-one receptor" observed in other insects. Most OR genes are found in tandem arrays, forming two large lineage-specific subfamilies in the phylogenetic tree. The "divergent IR" subfamily displays a significant contraction, and most of the IRs belong to the "antennal IR" subfamily in the locust. Most ORs/IRs have olfactory-specific expression while some broadly- or internal-expressed members are also found. Differing from holometabolous insects, the migratory locust contains very similar expression profiles of ORs/IRs between nymph and adult stages. RNA interference and behavioral assays indicate that an OR-based signaling pathway, not IR-based, mediates the attraction of locusts to aggregation pheromones. These discoveries provide insights into the unusual olfactory system of locusts and enhance our understanding of the evolution of insect olfaction.

Analysis of early hypertension and clinical outcome with bevacizumab: results from seven phase III studies.

BACKGROUND: Hypertension is associated with antivascular endothelial growth factor treatment, but the clinical implications of hypertension are uncertain. To assess the prognostic and predictive value of bevacizumab-related hypertension, a comprehensive analysis of whether hypertension and efficacy outcomes are associated was conducted on seven company-sponsored placebo-controlled phase III studies of bevacizumab. METHODS: Patient-specific data were available from 6,486 patients with metastatic colorectal, breast, non-small cell lung, pancreatic, and renal cell cancers. Primary hypertension endpoint was a blood pressure (BP) increase of >20 mmHg systolic or >10 mmHg diastolic within the first 60 days of treatment. Additional endpoints included other predefined thresholds of change in BP and severity of hypertension graded using the National Cancer Institute's Common Terminology Criteria for Adverse Events. To analyze the general prognostic importance of an early BP increase, multivariate Cox regression models were used to assess the correlation between BP changes and progression-free (PFS) and overall survival (OS) outcomes in the control groups. To analyze whether early BP increases could predict for benefit from bevacizumab, similar analyses were conducted in the bevacizumab-treated and control groups. RESULTS: In six of seven studies, early BP increase was neither predictive of clinical benefit from bevacizumab nor prognostic for the course of the disease. For study AVF2107g, early increased BP was associated with longer PFS and OS times in the bevacizumab group but shorter OS time in the control group. CONCLUSIONS: Early treatment-related BP increases do not predict clinical benefit from bevacizumab based on PFS or OS outcomes. BP increases do not appear to have general prognostic importance for patients with advanced cancer.

Clinical anatomy related to the hepatic veins for right lobe living donor liver transplantation.

The complexity of liver reconstruction has limited partial right lobe living donor liver transplantation. It is largely due to the difficulty of dealing with the middle hepatic vein. We sought to define the anatomic features of hepatic veins. Forty-one fresh adult livers, 43 formalin-fixed adult cadaver livers, and 91 adult liver corrosion casts were used for the study. We determined the number of branches, the maximum diameter, the whole length, the extrahepatic length of the hepatic veins, and the deviation of the middle hepatic vein from the main portal fissure. Nakamura and Tsuzuki's classification of hepatic vein types was used. Type A, B, and C accounted for 59.4, 27.8, and 12.8% of all specimens in this study, respectively. The middle and left hepatic veins formed a common trunk in 60.3% of the specimens, and the length of the common trunk was 1.12 ± 0.62 cm. The degree of deviation to the right of the middle hepatic vein from the main portal fissure was 14.11° ± 12.65°. The frequency of hepatic vein types and the degree of deviation to the right of the middle hepatic vein in this study is markedly different from that reported in other literature. The anatomic features of the hepatic veins in this study suggest that right lobe living donor liver transplantation is more suitable for Chinese.

Vascular exclusion by preserving tumor-contralateral branch of hepatic artery in hepatectomy in treatment liver cancer with cirrhosis.

BACKGROUND/AIMS: To investigate the effectiveness and safety of vascular exclusion by preserving tumor-contralateral branch of hepatic artery in hepatectomy in treatment liver cancer with cirrhosis. METHODOLOGY: The clinical data of 10 cases treated with hepatectomy for liver cancer were analyzed retrospectively. Vascular exclusion by preserving tumor-contralateral branch of hepatic artery was applied to control bleeding. Blood loss, operative time and postoperative hepatic function were observed. RESULTS: The average blood loss was 515mL, the operative time was 191 minutes and the mean time of exclusion was 30.20 minutes. There is no significant difference between hepatic function (serum total bilirubin, alanine transaminase) of postoperative day 7 and that of pre-operation. CONCLUSIONS: Vascular exclusion by preserving tumor-contralateral branch of hepatic artery could effectively control bleeding and preserve hepatic function and is proved to be applicable to patients of liver cancer with cirrhosis.

Hydrogen-oxygen therapy alleviates clinical symptoms in twelve patients hospitalized with COVID-19: A retrospective study of medical records.

ABSTRACT: A global public health crisis caused by the 2019 novel coronavirus disease (COVID-19) leads to considerable morbidity and mortality, which bring great challenge to respiratory medicine. Hydrogen-oxygen therapy contributes to treat severe respiratory diseases and improve lung functions, yet there is no information to support the clinical use of this therapy in the COVID-19 pneumonia.A retrospective study of medical records was carried out in Shishou Hospital of Traditional Chinese Medicine in Hubei, China. COVID-19 patients (aged ≥ 30 years) admitted to the hospital from January 29 to March 20, 2020 were subjected to control group (n = 12) who received routine therapy and case group (n = 12) who received additional hydrogen-oxygen therapy. The clinical characteristics of COVID-19 patients were analyzed. The physiological and biochemical indexes, including immune inflammation indicators, electrolytes, myocardial enzyme profile, and functions of liver and kidney, were examined and investigated before and after hydrogen-oxygen therapy.The results showed significant decreases in the neutrophil percentage and the concentration and abnormal proportion of C-reactive protein in COVID-19 patients received additional hydrogen-oxygen therapy.This novel therapeutic may alleviate clinical symptoms of COVID-19 patients by suppressing inflammation responses.

Aggregation pheromone 4-vinylanisole promotes the synchrony of sexual maturation in female locusts.

Reproductive synchrony generally occurs in various group-living animals. However, the underlying mechanisms remain largely unexplored. The migratory locust, Locusta migratoria, a worldwide agricultural pest species, displays synchronous maturation and oviposition when forms huge swarm. The reproductive synchrony among group members is critical for the maintenance of locust swarms and population density of next generation. Here, we showed that gregarious female locusts displayed more synchronous sexual maturation and oviposition than solitarious females and olfactory deficiency mutants. Only the presence of gregarious male adults can stimulate sexual maturation synchrony of female adults. Of the volatiles emitted abundantly by gregarious male adults, the aggregation pheromone, 4-vinylanisole, was identified to play key role in inducing female sexual maturation synchrony. This maturation-accelerating effect of 4-vinylanisole disappeared in the females of Or35-/- lines, the mutants of 4-vinylanisole receptor. Interestingly, 4-vinylanisole displayed a time window action by which mainly accelerates oocyte maturation of young females aged at middle developmental stages (3-4 days post adult eclosion). We further revealed that juvenile hormone/vitellogenin pathway mediated female sexual maturation triggered by 4-vinylanisole. Our results highlight a 'catch-up' strategy by which gregarious females synchronize their oocyte maturation and oviposition by time-dependent endocrinal response to 4-vinylanisole, and provide insight into reproductive synchrony induced by olfactory signal released by heterosexual conspecifics in a given group.

Since 2019, a plague of flying insects known as migratory locusts has been causing extensive damage to crops in East Africa. Migratory locusts sometimes live a solitary lifestyle but, if environmental conditions allow, they form large groups containing millions of individuals known as swarms that are responsible for causing locust plagues.Locusts are able to maintain such large swarms because they can aggregate and synchronize. When they live in swarms, individual locusts produce odors that are sensed by other individuals in the group. For example, an aggregation pheromone, called 4-vinylanisole, is known to help keep large groups of locusts together. However, it is less clear how odors synchronize the reproductive cycles of the females in a swarm so that they are ready to mate with males and lay their eggs at the same time. To address this question, Chen et al. examined when female locusts reached sexual maturity after they were exposed to odors produced by other locusts living alone or in groups. The experiments found that only 4-vinylanisole, which was abundantly released by adult male locusts living in groups, stimulated female locusts to reach sexual maturity at the same time. This odor increased the levels of a hormone known as juvenile hormone in less-developed females to help them reach sexual maturity sooner. These findings demonstrate that when migratory locusts are living in swarms, male locusts promote the female locusts to reach sexual maturity at the same time by promoting less-developed females to 'catch up' with other females in the group. A next step will be to investigate the neural and molecular mechanisms underlying the 'catch up' effect induced by 4-vinylanisole.

A portable, integrated analyzer for microfluidic - based molecular analysis.

A portable, fully automated analyzer that provides actuation and flow control to a disposable, self-contained, microfluidic cassette ("chip") for point-of-care, molecular testing is described. The analyzer provides mechanical actuation to compress pouches that pump liquids in the cassette, to open and close diaphragm valves for flow control, and to induce vibrations that enhance stirring. The analyzer also provides thermal actuation for the temperature cycling needed for polymerase chain reaction (PCR) amplification of nucleic acids and for various drying processes. To improve the temperature uniformity of the PCR chamber, the system utilizes a double-sided heating/cooling scheme with a custom feedforward, variable, structural proportional-integral-derivative (FVSPID) controller. The analyzer includes a programmable central processing unit that directs the sequence and timing of the various operations and that is interfaced with a computer. The disposable cassette receives a sample, and it carries out cell lysis, nucleic acid isolation, concentration, and purification, thermal cycling, and either real time or lateral flow (LF) based detection. The system's operation was demonstrated by processing saliva samples spiked with B. cereus cells. The amplicons were detected with a lateral flow assay using upconverting phosphor reporter particles. This system is particularly suited for use in regions lacking centralized laboratory facilities and skilled personnel.

A Portable Analyzer for Pouch-Actuated, Immunoassay Cassettes.

A portable, small footprint, light, general purpose analyzer (processor) to control the flow in immunoassay cassettes and to facilitate the detection of test results is described. The durable analyzer accepts disposable cassettes that contain pouches and reaction chambers for various unit operations such as hydration of dry reagents, stirring, and incubation. The analyzer includes individually controlled, linear actuators to compress the pouches in the cassette, which facilitates the pumping and mixing of sample and reagents, and to close diaphragm-based valves for flow control. The same types of actuators are used to compress pouches and actuate valves. The analyzer also houses a compact OEM scanner/reader to excite fluorescence and detect emission from labels. The analyzer is hydraulically isolated from the cassette, reducing the possibility of cross-contamination. The analyzer facilitates programmable, automated execution of a sequence of operations such as pumping and valving in a timely fashion, reducing the level of expertise required from the operator and the possibility for errors. The analyzer's design is modular and expandable to accommodate cassettes of various complexities and additional functionalities. In this paper, the utility of the analyzer has been demonstrated with the execution of a simple, consecutive, lateral flow assay of a model biological system and the test results were detected with up converting phosphor labels that are excited at infrared frequencies and emit in the visible spectrum.

A large volume, portable, real-time PCR reactor.

A point-of-care, diagnostic system incorporating a portable thermal cycler and a compact fluorescent detector for real-time, polymerase chain reaction (PCR) on disposable, plastic microfluidic reactors with relatively large reaction volume (ranging from 10 µL to 100 µL) is described. To maintain temperature uniformity and a relatively fast temperature ramping rate, the system utilizes double-sided heater that features a master, thermoelectric element and a thermal waveguide connected to a second thermoelectric element. The waveguide has an aperture for optical coupling between a miniature, fluorescent reader and the PCR reaction chamber. The temperature control is accomplished with a modified, feedforward, variable structural proportional-integral-derivative controller. The temperature of the liquid in the reaction chamber tracks the set-point temperature with an accuracy of ± 0.1 °C. The transition times from one temperature to another are minimized with controllable overshoots (< 2 °C) and undershoots (< 5 °C). The disposable, single-use PCR chip can be quickly inserted into a thermal cycler/reader unit for point-of-care diagnostics applications. The large reaction chamber allows convenient pre-storing of dried, paraffin-encapsulated PCR reagents (polymerase, primers, dNTPs, dyes, and buffers) in the PCR chamber. The reagents are reconstituted "just in time" by heating during the PCR process. The system was tested with viral and bacterial nucleic acid targets.

An integrated, self-contained microfluidic cassette for isolation, amplification, and detection of nucleic acids.

A self-contained, integrated, disposable, sample-to-answer, polycarbonate microfluidic cassette for nucleic acid-based detection of pathogens at the point of care was designed, constructed, and tested. The cassette comprises on-chip sample lysis, nucleic acid isolation, enzymatic amplification (polymerase chain reaction and, when needed, reverse transcription), amplicon labeling, and detection. On-chip pouches and valves facilitate fluid flow control. All the liquids and dry reagents needed for the various reactions are pre-stored in the cassette. The liquid reagents are stored in flexible pouches formed on the chip surface. Dry (RT-)PCR reagents are pre-stored in the thermal cycling, reaction chamber. The process operations include sample introduction; lysis of cells and viruses; solid-phase extraction, concentration, and purification of nucleic acids from the lysate; elution of the nucleic acids into a thermal cycling chamber and mixing with pre-stored (RT-)PCR dry reagents; thermal cycling; and detection. The PCR amplicons are labeled with digoxigenin and biotin and transmitted onto a lateral flow strip, where the target analytes bind to a test line consisting of immobilized avidin-D. The immobilized nucleic acids are labeled with up-converting phosphor (UCP) reporter particles. The operation of the cassette is automatically controlled by an analyzer that provides pouch and valve actuation with electrical motors and heating for the thermal cycling. The functionality of the device is demonstrated by detecting the presence of bacterial B.Cereus, viral armored RNA HIV, and HIV I virus in saliva samples. The cassette and actuator described here can be used to detect other diseases as well as the presence of bacterial and viral pathogens in the water supply and other fluids.

A PCR reactor with an integrated alumina membrane for nucleic acid isolation.

Recently, there has been a growing interest in point-of-care devices capable of detecting nucleic acids (NA) in clinical and environmental samples. Nucleic acid detection requires, however, various sample preparation steps that complicate device operation. An attractive remedy is to integrate many, if not all, sample preparation operations and nucleic acid amplification into a single reaction chamber. A microfluidic chip that integrates, in a single chamber, polymerase chain reaction (PCR) amplification with solid-phase extraction of nucleic acids using a nanoporous, aluminium oxide membrane (AOM) is described. Samples suspected of containing target bacteria and/or viruses are mixed with lysis agents and a chaotropic salt and loaded into a plastic chip housing a nanoporous, aluminium oxide membrane. The nucleic acids in the lysate bind to the membrane. The membrane is then washed, the chamber is filled with the PCR reaction reagents, and the chamber's temperature is cycled to amplify the captured nucleic acids and produce detectable products. Both DNA and RNA (with reverse-transcription) isolation and amplification are demonstrated. Due to the dry membrane's high resistance to liquid flow, a specialized flow control system was devised to facilitate sample introduction and membrane washing.

An automated, pre-programmed, multiplexed, hydraulic microvalve.

An automated, pre-programmed, multiplexed hydraulic valve actuator is described. The valve is membrane-based and normally open. In contrast to the membrane-based pneumatic valve, the hydraulic valve uses hydraulic liquid to exert the control pressure. The line pressure is controlled with a roller moving over a prefabricated topology. Multiple rollers, each traversing its own track, are assembled into a single carriage, which can be actuated either manually or with a single computer-controlled motor. A valve manifold and roller actuators are designed, fabricated, and tested to demonstrate three-way valve actuation in a pre-determined sequence. The performance of the valve is evaluated and the utility of the valve in the operation of a micro thermal cycler was demonstrated. Hydraulic controllers of the type described here can be operated either manually or under computer control and provide an inexpensive means of controlling flow in lab-on-a-chip devices.

A timer-actuated immunoassay cassette for detecting molecular markers in oral fluids.

An inexpensive, hand-held, point-of-care, disposable, self-contained immunoassay cassette comprised of air pouches for pumping, a metering chamber, reagents storage chambers, a mixer, and a lateral flow strip was designed, constructed, and tested. The assay was carried out in a consecutive flow format. The detection was facilitated with up-converting phosphor (UCP) reporter particles. The automated, timely pumping of the various reagents was driven by a spring-loaded timer. The utility of the cassette was demonstrated by detecting antibodies to HIV in saliva samples and further evaluated with a non-contagious, haptenized DNA assay. The cassette has several advantages over dip sticks such as sample preprocessing, integrated storage of reagents, and automated operation that reduces operator errors and training. The cassette and actuator described herein can readily be extended to detect biomarkers of other diseases in body fluids and other fluids at the point of care. The system is particularly suitable for resource-poor countries, where funds and trained personnel are in short supply.