Non-specific markers of inflammation in bare-nosed wombats (Vombatus ursinus) with sarcoptic mange.

Sarcoptic mange, caused by epidermal infection with Sarcoptes scabiei, negatively impacts the health, welfare, and local abundance of bare-nosed wombats (Vombatus ursinus) in Australia. Improved understanding of the host immune response to disease and its contribution to pathophysiology could be used to inform management actions for this species in and ex situ. To evaluate the immune response of bare-nosed wombats to sarcoptic mange, we validated three assays (haptoglobin, agarose gel electrophoresis, and micro-erythrocyte sedimentation rate) measuring non-specific markers of inflammation using serum samples from free-living wombats from Tasmania (n = 33). We then analysed correlations between the assay results for each non-specific marker of inflammation and wombat's sarcoptic mange scores, and performed histopathological examinations to investigate association of the acute phase response with systemic amyloidosis. We present evidence that haptoglobin and erythrocyte sedimentation rate increased, and albumin decreased, in association with sarcoptic mange scores. This research demonstrates links between the acute phase response and sarcoptic mange severity in bare-nosed wombats, highlighting the utility of non-specific markers of inflammation for aiding assessment of the systemic effects of mange. Showing the value of agarose gel electrophoresis, we also identified specific acute phase proteins warranting future evaluation and found evidence of an immunoglobulin response in mange-affected wombats, revealed by increasing γ-globulins in association with apparent disease severity. Meanwhile, owing to its relatively low resource requirements and rapidity, the erythrocyte sedimentation rate assay may be useful as a point-of-care test to support therapeutic decisions in the field. Our methods and findings are likely to be applicable to a range of other clinical and population health scenarios in captive and free-living wombats, and species impacted by sarcoptic mange globally.

Age, sex and breed effect on laboratory parameters in natural Babesia canis infection.

We tested the hypothesis that age, breed, and sex are related to hematology, biochemistry, acute phase proteins (APPs), seroreactivity and level of parasitemia in dogs with an acute phase response (APR) due to Babesia canis infection. The study enrolled 61 privately owned dogs that naturally acquired B. canis infection. Groups were formed according to the age: young dogs less than one year, and adult dogs more than one year old. Moreover, the group of males was compared to females and purebred to mixed breed dogs. Seroreactivity was tested with immunofluorescence antibody test, level of parasitemia with real-time polymerase chain reaction (real-time PCR), hematology, and biochemistry with automatic analyzers, serum amyloid A with enzyme-linked immunosorbent assay, fibrinogen with heat precipitation and ceruloplasmin and paraoxonase-1 with manual spectrophotometric methods. For protein separation agarose gel electrophoresis was used. The main changes in the whole population of B. canis-infected dogs were fever, pancytopenia, and change in APPs level. One-third of young, and 96% of adult dogs were seropositive (P < 0.001). The level of parasitemia was higher in the young dogs (P < 0.001). Erythroid lineage parameters (P < 0.01), and leukocytes (P < 0.05) were lower in the young, when compared to the adult dogs. Young dogs had lower total globulins (P < 0.001), ß- and γ-globulins (P < 0.001), and higher α-globulins (P = 0.022) than adult dogs. Young dogs had higher concentrations of phosphate (P = 0.003) and cholesterol (P < 0.001) and lower amylase (P = 0.014) and lipase activity (P = 0.020) than adult ones. Male dogs had lower neutrophil count than females (P = 0.035), and purebred dogs had more band neutrophils than mixed breed dogs (P = 0.004). In conclusion, dogs with natural Babesia canis infection at a young age have more severe anemia and APR including leukopenia than adults. Male and purebred dogs might also have more severe APR than females and mix-breeds, as they have more pronounced changes related to the myeloid lineage.

UGT1A1*28 detection using high-resolution agarose gel electrophoresis.

A new UGT1A1*28 detection method combining PCR and high-resolution agarose gel electrophoresis was developed. The viability of this method was demonstrated on 15 healthy adult volunteers. Subjects included 13 wild type homozygotes (86.7 %), 2 heterozygotes (13.3 %), and no mutant type homozygotes (0 %). The new UGT1A1*28 detection method results were fully consistent with DNA sequencing. PCR and agarose gel electrophoresis are common techniques with high-resolution agarose gels available commercially. These results support the clinical viability of this method potentially reducing UGT1A1*28 diagnosis complexity and cost.

In Vitro Determination of Temperature-Dependent DNA Binding of the Evening Complex Using Electrophoretic Mobility Shift Assays.

Electrophoretic mobility shift assays (EMSAs) of DNA-binding proteins and labeled DNA allow the qualitative and quantitative characterization of protein-DNA complex formation using native (nondenaturing) polyacrylamide or agarose gel electrophoresis. By varying the incubation temperature of the protein-DNA binding reaction and maintaining this temperature during electrophoresis, temperature-dependent protein-DNA interactions can be investigated. Here, we provide examples of the binding of a transcriptional repressor complex called the Evening Complex, comprising the DNA-binding protein LUX ARRYTHMO (LUX), the scaffold protein EARLY FLOWERING 3 (ELF3), and the adapter protein ELF4, to its cognate DNA and demonstrate direct detection and visualization of thermoresponsive binding in vitro. As negative controls we use the LUX DNA-binding domain and LUX full length protein, which do not exhibit temperature-dependent DNA binding.

Convection-Enhanced Drug Delivery: Experimental and Analytical Studies of Infusion Behavior in an In Vitro Brain Surrogate.

Convection-enhanced drug delivery (CED) directly infuses drugs with a large molecular weight toward target cells as a therapeutic strategy for neurodegenerative diseases and brain cancers. Despite the success of many previous in vitro experiments on CED, challenges still remain. In particular, a theoretical predictive model is needed to form a basis for treatment planning, and developing such a model requires well-controlled injection tests that can rigorously capture the convective (advective) and diffusive transport of an infusate. For this purpose, we investigated the advection-diffusion transport of an infusate (bromophenol blue solution) in the brain surrogate (0.2% w/w agarose gel) at different injection rates, ranging from 0.25 to 4 µL/min, by closely monitoring changes in the color intensity, propagation distance, and injection pressures. One dimensional closed-form solution was examined with two variable sets, such as the mathematically calculated coefficient of molecular diffusion and average velocity, and the hydraulic dispersion coefficient and seepage velocity by the least squared method. As a result, the seepage velocity was greater than the average velocity to some extent, particularly for the later infusion times. The poroelastic deformation in the brain surrogate might lead to changes in porosity, and consequently, slight increases in the actual flow velocity as infusion continues. The limitation of efficiency of the single catheter was analyzed by dimensionless analysis. Lastly, this study suggests a simple but robust approach that can properly capture the convective (advective) and diffusive transport of an infusate in an in vitro brain surrogate via well-controlled injection tests.

Alzheimer's Disease: A Molecular Model and Implied Path to Improved Therapy.

Amyloid-associated neurodegenerative diseases, including Alzheimer's disease (AD), are characterized by the in-brain accumulation of ß-sheet structured protein aggregates called amyloids. However, neither a disease model nor therapy is established. We review past data and present new, preliminary data and opinions to help solve this problem. The following is the data-derived model/hypothesis. (1) Amyloid-forming proteins have innate immunity functions implemented by conversion to another sheet conformation, α-sheet. (2) In health, α-sheet structured, amyloid-forming proteins inactivate microbes by co-assembly with microbe α-sheets. Amyloid-forming proteins then undergo α-to-ß-sheet conversion. (3) In disease, α-sheet-structured, amyloid-forming proteins over-accumulate and are neuron-toxic. This hypothesis includes formation by virus capsid subunits of α-sheets. In support, we find that 5-10 mM methylene blue (MB) at 54 °C has a hyper-expanding, thinning effect on the phage T4 capsid, as seen by negative stain- and cryo-electron microscopy after initial detection by native gel electrophoresis (AGE). Given the reported mild anti-AD effect of MB, we propose the following corollary hypothesis. (1) Anti-AD MB activity is, at least in part, caused by MB-binding to amyloid α-sheet and (2) MB induces the transition to α-sheet of T4 capsid subunits. We propose using AGE of drug incubated T4 to test for improved anti-AD activity.

A rapid, inexpensive and effective method for the efficient isolation of genomic DNA from Gram-negative bacteria.

Currently, there are several protocols to extract bacterial DNA based on different principles. However, the quantity and the quality of the DNA obtained by each method are highly variable and microorganism dependent. In most of these classical crude methods, highly toxic and hazardous organic solvents such as phenol and chloroform are used for deproteinization, whereas in certain protocols, expensive enzymes including RNases and Proteinases are used. This study was designed to introduce a simple, rapid, inexpensive and effective genomic DNA isolation procedure for Gram-negative bacteria, without the usage of toxic chemicals and costly enzymes. This novel method was compared with another classical method known as the salting-out method, which uses proteinase-K. Concentration and yield of the extracted DNA were determined by gel electrophoresis by comparing the gel band intensity of the sample DNA to that of a DNA quantitation standard and by the Quantus™ fluorometer. According to the results, the yield of extracted DNA was higher in the novel method compared to the salting-out method. Moreover, the entire process was accomplished in less than 2 h with the novel method. Purity and integrity of extracted genomic DNA by both methods were similar. In addition, the quality of DNA was determined using Multicopy Associated Filamentation (MAF) gene amplification by polymerase chain reaction (PCR). Thus, the described technique is non-toxic, less time and fund consuming, efficient and a well-suited method for routine DNA isolation from Gram negative bacteria.

Different behavior of Ferguson plot between agarose and polyacrylamide gels.

In this study, we conducted Ferguson plot analyses using both agarose and polyacrylamide gels in native electrophoresis and SDS-PAGE. The results revealed intriguing differences in the behavior of bovine serum albumin (BSA) and other model proteins. Specifically, BSA exhibited Ferguson plot slopes that were dependent on the oligomer size in agarose native gel electrophoresis, while such size-dependent behavior was not observed in native-PAGE or SDS-PAGE. These findings suggest that Ferguson plot analysis is a suitable approach when using agarose gel under the electrophoretic conditions employed in this study. Furthermore, our investigation extended to model proteins with acidic isoelectric points and larger molecular weights, namely Ferritin and caseinolytic peptidase B (ClpB). Notably, these proteins displayed distinct Ferguson plot slopes when subjected to agarose gel electrophoresis. Intriguingly, when polyacrylamide gel was employed, ClpB exhibited multiple bands, each with its unique Ferguson plot slope, deviating from the expected behavior based on molecular size. This divergence in Ferguson plot characteristics between agarose and polyacrylamide gels points to an interesting and complex interplay between protein properties and gel electrophoresis conditions.

Establishment of qualitative human immunodeficiency virus type 1 nucleic acid amplification test as an adjunct confirmatory test in low-prevalence areas and small- and medium-sized diagnostic laboratories.

Two simple and inexpensive in-house qualitative human immunodeficiency virus type 1 nucleotide amplification tests (HIV-1 NATs) were established as adjunct confirmatory HIV test for HIV antigen (Ag)-positive specimens identified from HIV screening test and for patients with indeterminate or negative HIV antibody (Ab) confirmatory test results. The limit of detection was <1000 copies/mL, which is lower than that of the HIV Ag/Ab combination assay. One test using QL1 detected all 11 HIV-1 subtypes/circulating recombinant forms/group samples with almost equal analytical sensitivity, and the other test, using QL2, also detected all, except for two group O samples. In the examination of 28 HIV-1 Ag-positive samples using Determine HIV Early Detect, 27 samples were reactive and one HIV-1 Ag-pseudo-positive sample was non-reactive using both methods. These in-house qualitative HIV-1 NATs are useful for confirming HIV-1 Ag-positive cases and excluding HIV-1 Ag false-positive cases in areas with low HIV prevalence and small- and medium-sized diagnostic laboratories.

Use of therapeutic plasma exchange to remove lipoprotein X in a patient with vanishing bile duct syndrome presenting with cholestasis, pseudohyponatremia, and hypercholesterolemia: A case report and review of literature.

INTRODUCTION: Lipoprotein X (Lp-X) is an abnormal lipoprotein found in multiple disease conditions, including liver dysfunction and cholestasis. High Lp-X concentrations can interfere with some laboratory testing that may result in spurious results. The detection of Lp-X can be challenging, and there is currently a lack of consensus regarding the management of Lp-X other than treating the underlying disease. CASE PRESENTATION: A 42-year-old female with Hodgkin's lymphoma treated with dexamethasone, high dose cytarabine and cisplatin and vanishing bile duct syndrome confirmed by liver biopsy presented with cholestasis, pseudohyponatremia (sodium, 113 mmol/L; reference range 136-146 mmL/L; serum osmolality, 303 mOsm/kg), and hypercholesterolemia (> 2800 mg/dL, reference range < 200 mg/dL). Lp-X was confirmed by lipoprotein electrophoresis (EP). Although she did not manifest any specific signs or symptoms, therapeutic plasma exchange (TPE) was initiated based on laboratory findings of extreme hypercholesterolemia, spuriously abnormal serum sodium, and HDL values, and the potential for short- and long-term sequelae such as hyperviscosity syndrome, xanthoma, and neuropathy. During the hospitalization, she was treated with four 1.0 plasma volume TPE over 6 days using 5% albumin for replacement fluid. After the first TPE, total cholesterol (TC) decreased to 383 mg/dL and sodium was measured at 131 mmol/L. The patient was transitioned into outpatient maintenance TPE to eliminate the potential of Lp-X reappearance while the underlying disease was treated. Serial follow-up laboratory testing with lipoprotein EP showed the disappearance of Lp-X after nine TPEs over a 10-week period. LITERATURE REVIEW: There are seven and four case reports of Lp-X treated with TPE and lipoprotein apheresis (LA), respectively. While all previous case reports showed a reduction in TC levels, none had monitored the disappearance of Lp-X after completing a course of therapeutic apheresis. CONCLUSION: Clinicians should have a heightened suspicion for the presence of abnormal Lp-X in patients with cholestasis, hypercholesterolemia, and pseudohyponatremia. Once Lp-X is confirmed by lipoprotein EP, TPE should be initiated to reduce TC level and remove abnormal Lp-X. Most LA techniques are not expected to be beneficial since Lp-X lacks apolipoprotein B. Therefore, we suggest that inpatient course of TPE be performed every other day until serum sodium, TC and HDL levels become normalized. Outpatient maintenance TPE may also be considered to keep Lp-X levels low while the underlying disease is treated. Serum sodium, TC, and HDL levels should be monitored while on maintenance TPE.

Rational design of agarose/dextran composite microspheres with tunable core-shell microstructures for chromatographic application.

A new type of core-shell microsphere was prepared by a pre-crosslinking method, consisting of cross-linked agarose microspheres as the core and agarose-dextran as the shell. After optimizing the preparation process, the microspheres with a uniform particle size were obtained and characterized using cryo-scanning electron microscopy to determine their surface and cross-sectional morphology. Results from flow rate-pressure and chromatographic performance tests showed that the core-shell agarose microspheres were supported by the core microspheres and composed of composite polysaccharides, forming an interpenetrating polymer network structure as a hard shell. The core-shell agarose microspheres showed a 300.5 % increase in linear flow rate compared to composite polysaccharide microspheres prepared from shell materials and a 141.5 % increase compared to 6 % agarose microspheres. Additionally, the large pore structure of the shell combined with the fine pore structure of the core improved the material separation efficiency in the range of 0.1-2000 kDa. These findings suggest that core-shell natural polysaccharide microspheres have great potential as a separation chromatographic medium.

3, 5-Dihydroxy 4', 7-dimethoxyflavone-DNA interaction study for nucleic acid detection and differential cell staining.

The present study is focused on application of a natural compound, 3, 5-dihydroxy 4', 7-dimethoxyflavone (DHDM) from a medicinal plant Alpinia nigra for nucleic acid detection and differential cell staining. DHDM was found to interact with nucleic acid and forms complex, which was investigated for various applications. It was successfully utilized to visualize plasmid, genomic, and ds-linear DNA in agarose gel electrophoresis without affecting the DNA mobility in the gel. Fluorescence of DHDM increased several fold upon binding to dsDNA. Photostability of the compound was assessed and showed photobleaching effect that decreased gradually over time. Application of the compound was further extended to differential cell staining. When observed in fluorescence microscope, DHDM stained the dead cells and differentiated them from live cells in the case of bacterial, yeast, and mammalian cells. Higher concentration of the compound was found to be less cytotoxic to cancerous cells. Nucleic acid staining dyes like Ethidium bromide (EtBr), Propidium iodide (PI), etc. are carcinogens and environmental pollutants and therefore DHDM a natural compound, is a major benefit and thus can serve as an alternative to the current dyes.

Drug release from PLGA microparticles can be slowed down by a surrounding hydrogel.

This study aimed to evaluate and better understand the potential impact that a layer of surrounding hydrogel (mimicking living tissue) can have on the drug release from PLGA microparticles. Ibuprofen-loaded microparticles were prepared with an emulsion solvent extraction/evaporation method. The drug loading was about 48%. The surface of the microparticles appeared initially smooth and non-porous. In contrast, the internal microstructure of the particles exhibited a continuous network of tiny pores. Ibuprofen release from single microparticles was measured into agarose gels and well-agitated phosphate buffer pH 7.4. Optical microscopy, scanning electron microscopy, differential scanning calorimetry, X-ray powder diffraction, and X-ray µCT imaging were used to characterize the microparticles before and after exposure to the release media. Importantly, ibuprofen release was much slower in the presence of a surrounding agarose gel, e.g., the complete release took two weeks vs. a few days in well agitated phosphate buffer. This can probably be attributed to the fact that the hydrogel sterically hinders substantial system swelling and, thus, slows down the related increase in drug mobility. In addition, in this particular case, the convective flow in agitated bulk fluid likely damages the thin PLGA layer at the microparticles' surface, giving the outer aqueous phase more rapid access to the inner continuous pore network: Upon contact with water, the drug dissolves and rapidly diffuses out through a continuous network of water-filled channels. Without direct surface access, most of the drug "has to wait" for the onset of substantial system swelling to be released.

Serum Protein Concentration and Serum Protein Fractions in Bottlenose Dolphins (Tursiops truncatus) under Human Care Using Agarose Gel Electrophoresis.

Serum protein electrophoresis (SPE) is the most used and reliable method to determine the percentage of serum protein subfractions. The interpretation of the kinetics of total proteins and albumin and globulin fractions is receiving increased attention in wild animals, as well as in domestic animals, due to the possibility of identifying typical pathologic patterns. However, the interpretation of these data had to be performed in light of an appropriate method-and species- specific reference intervals (RIs). In marine mammals, as well as other non-domestic species, specific attention should also be given to the different environment (free ranging vs. human managed) and the associated different exposure to environmental stimuli. The aim of this report was to establish RIs for the serum protein fractions evaluated using agarose gel electrophoresis (AGE) in bottlenose dolphins under human care. Peripheral blood samples were collected from 40 bottlenose dolphins during standard veterinary procedures to evaluate their health status. Total protein concentration was determined using the biuret method while AGE was performed using an automated system. A pooled dolphin's serum sample was used to determine the intra-assay and inter-assay imprecision of AGE. The RIs were calculated using an Excel spreadsheet with the Reference Value Advisor set of macroinstructions. The intra and inter-assay imprecisions were 1.2% and 2.5%, respectively, for albumin; 2.9% and 5.7%, respectively, for α-globulins; 3.8% and 4.0%, respectively, for ß-globulins; and 3.4% and 4.8%, respectively, for γ-globulins. The total protein, albumin, α-globulin, ß-globulin, and γ-globulin concentrations were 65.5 ± 5.4 g/L, 45.5 ± 4.9 g/L, 8.0 ± 1.0 g/L, 5.0 ± 2.0 g/L, and 7.0 ± 2.0 g/L, respectively. We established the RIs for the total protein and serum protein fractions using AGE in bottlenose dolphins under human care.

A Pilot Study Investigating Plasma Protein Electrophoresis in One Anuran and Six Urodelan Species.

As threats to amphibian health increase, there is a growing need for diagnostic tools to assess and monitor their health status. Plasma protein electrophoresis has proven to be useful in other nonmammalian species. It enables quantification of protein fractions in plasma that may be altered in various disease processes, and is therefore useful in narrowing down differential diagnoses and detecting inflammation, in combination with other modalities such as biochemical and hematologic testing. The amphibian electrophoretogram must be defined before baseline reference intervals are obtained across species. Agarose gel electrophoresis was performed on plasma samples collected from presumed clinically normal individuals of one anuran and six urodelans: Osteopilus septentrionalis (n=2), Gyrinophilus porphyriticus (n=1), Notophthalmus viridescens (n=1), Eurycea guttolineata (n=2), Amphiuma tridactylum (n=2), Cryptobranchus alleganiensis (n=5), and Siren lacertina (n=6). The electrophoretograms varied in number of fractions between each species; however, the number of fractions was consistent within a species. An albumin migrating fraction was consistently observed in all species. A prealbumin migrating fraction was identified in species that primarily use organs other than skin for respiration. This study provides preliminary examples of a normal plasma protein electrophoretogram for seven amphibian species. Further studies quantifying reference intervals and identification of protein fractions will help establish protein electrophoresis as a useful tool in amphibian health investigations.

An efficient extraction method for short single-stranded DNA from agarose gels in aptamer screening.

With the rapid advancements in aptamer screening, the efficient extraction of short single-stranded DNA (ssDNA) from agarose gel has become a new requirement. However, the currently available products are primarily designed for double-stranded DNA (dsDNA) and exhibit limited efficacy when applied to the extraction of short ssDNA. In this study, we successfully developed a novel method based on amino-modified silica-coated magnetic particles (ASMPs) for the extraction of short ssDNA from agarose gel. The gel slices containing short ssDNA were subjected to centrifugation in a spin column/centrifugation tube assembly with silica wool, followed by the adsorption using ASMPs. Subsequently, reagents containing phosphate groups were employed to desorb ssDNA from the surface of ASMPs. Through optimization of each step, we realized remarkable efficiency in the extraction of short ssDNA. To assess the efficacy of our method, we utilized it in aptamer screening. The results demonstrated that our method outperformed three commercially available DNA gel extraction products (Q-kit, S-kit, and V-kit). The relative recovery rates of all methods were as follows: M-dNTP (100.00 %) > M-BB (63.38 %) > Q-kit (46.64 %) > S-kit (15.98 %) > V-kit (0.38 %). The results strongly suggest that the developed method holds promise for short ssDNA extraction from agarose gel.

Long-Range Polymerase Chain Reaction.

Polymerase chain reaction (PCR) is a laboratory technique used to amplify a targeted region of DNA, demarcated by a set of oligonucleotide primers. Long-range PCR is a form of PCR optimized to facilitate the amplification of large fragments. Using the adapted long-range PCR protocol described in this chapter, we were able to generate PCR products of 6.6, 7.2, 13, and 20 kb from human genomic DNA samples. For some of the long PCRs, successful amplification was not possible without the use of PCR enhancers. Thus, we also evaluated the impact of some enhancers on long-range PCR and included the findings as part of this updated chapter.

Yield Gel via Quantitative Gel Electrophoresis.

Quantitative gel electrophoresis, also referred to as yield gel via gel electrophoresis, is an early quantification method that was developed to provide an estimate of the quality and the quantity of DNA extracted from evidence or reference samples. To conduct quantitative gel electrophoresis, an agarose gel that is combined with a nucleic acid gel stain is prepared. The gel stain intercalates between double-stranded DNA and can be visualized using UV light. DNA extract samples, along with DNA standards (ranging from 250 to 5 ng), and a 1 KB ladder are combined with a 6X loading dye and loaded on the agarose gel. Voltage is applied to facilitate DNA migration through the gel from the negative to the positive electrode, separating DNA fragments by size. After electrophoresis is complete, the results are visualized using UV light, and an image is captured for analysis. High-quality and -quantity DNA should contain a compact band comparable to that of the high molecular weight standards and ladder, with some smearing down the sample well. If a DNA extract sample does not produce a compact band and presents with only a smear, this is an indication that DNA degradation has occurred. This chapter provides instructions on how to successfully prepare an agarose gel, load DNA extract samples and corresponding controls, appropriately set up and run quantitative gel electrophoresis, interpret the results, and ensure comprehension of the method so troubleshooting can be performed if needed.

Use of a Waste-Derived Linde Type-A Immobilized in Agarose for the Remediation of Water Impacted by Coal Acid Mine Drainage at Pilot Scale.

A new adsorbent based on an immobilized waste-derived LTA zeolite in agarose (AG) has proven to be an innovative and efficient alternative for removing metallic contaminants from water impacted by acid mine drainage (AMD) because the immobilization prevents the solubilization of the zeolite in acidic media and eases its separation from the adsorbed solution. A pilot device was developed containing slices of the sorbent material [AG (1.5%)-LTA (8%)] to be used in a treatment system under an upward continuous flow. High removals of Fe2+ (93.45%), Mn2+ (91.62%), and Al3+ (96.56%) were achieved, thus transforming river water heavily contaminated by metallic ions into water suitable for non-potable use for these parameters, according to Brazilian and/or FAO standards. Breakthrough curves were constructed and the corresponding maximum adsorption capacities (mg/g) (Fe2+, 17.42; Mn2+, 1.38; Al3+, 15.20) calculated from them. Thomas mathematical model was well fitted to the experimental data, indicating the participation of an ion-exchange mechanism in the removal of the metallic ions. The pilot-scale process studied, in addition to being highly efficient in removing metal ions at toxic levels in AMD-impacted water, is linked to the sustainability and circular economy concepts, due to the use as an adsorbent of a synthetic zeolite derived from a hazardous aluminum waste.

CRISPR gel: A one-pot biosensing platform for rapid and sensitive detection of HIV viral RNA.

CRISPR technology has recently emerged as a powerful biosensing tool for sensitive and specific nucleic acid detection when coupled with isothermal amplification (e.g., recombinase polymerase amplification (RPA)). However, it remains a challenge to incorporate isothermal amplification into CRISPR detection in a one-pot system due to their poor compatibility. Here, we developed a simple CRISPR gel biosensing platform for human immunodeficiency virus (HIV) RNA detection by combining reverse transcription-recombinase polymerase amplification (RT-RPA) reaction solution with a CRISPR gel. In our CRISPR gel biosensing platform, CRISPR-Cas12a enzymes are embedded into the agarose gel, providing a spatially separated but connected reaction interface with the RT-RPA reaction solution. During isothermal incubation, the RT-RPA amplification occurs initially on the CRISPR gel. When RPA products are sufficiently amplified and reach the CRISPR gel, the CRISPR reaction occurs in the whole tube. With the CRISPR gel biosensing platform, we successfully detected down to 30 copies of HIV RNA per test within 30 min. Furthermore, we validated its clinical utility by detecting HIV clinical plasma samples, achieving superior performance compared with the real-time RT-PCR method. Thus, our one-pot CRISPR gel biosensing platform demonstrates great potential for rapid and sensitive molecular detection of HIV and other pathogens at the point of care.