Understanding the invisible hands of sample preparation for cryo-EM.

Cryo-electron microscopy (cryo-EM) is rapidly becoming an attractive method in the field of structural biology. With the exploding popularity of cryo-EM, sample preparation must evolve to prevent congestion in the workflow. The dire need for improved microscopy samples has led to a diversification of methods. This Review aims to categorize and explain the principles behind various techniques in the preparation of vitrified samples for the electron microscope. Various aspects and challenges in the workflow are discussed, from sample optimization and carriers to deposition and vitrification. Reliable and versatile specimen preparation remains a challenge, and we hope to give guidelines and posit future directions for improvement.

Cryo-EM structures from sub-nl volumes using pin-printing and jet vitrification.

The increasing demand for cryo-electron microscopy (cryo-EM) reveals drawbacks in current sample preparation protocols, such as sample waste and lack of reproducibility. Here, we present several technical developments that provide efficient sample preparation for cryo-EM studies. Pin printing substantially reduces sample waste by depositing only a sub-nanoliter volume of sample on the carrier surface. Sample evaporation is mitigated by dewpoint control feedback loops. The deposited sample is vitrified by jets of cryogen followed by submersion into a cryogen bath. Because the cryogen jets cool the sample from the center, premounted autogrids can be used and loaded directly into automated cryo-EMs. We integrated these steps into a single device, named VitroJet. The device's performance was validated by resolving four standard proteins (apoferritin, GroEL, worm hemoglobin, beta-galactosidase) to ~3 Å resolution using a 200-kV electron microscope. The VitroJet offers a promising solution for improved automated sample preparation in cryo-EM studies.

An Improved Enzyme-Linked Immunoassay for the Detection of Leptospira-Specific Antibodies.

Leptospirosis is a neglected zoonotic disease with worldwide endemicity and continues to be a significant public health burden on resource-limited populations. Previously, we produced three highly purified recombinant antigens (rLipL32, rLipL41, and rLigA-Rep) and evaluated their performance of detecting Leptospira-specific antibodies in enzyme-linked immunosorbent assay (ELISA) as compared with the microscopic agglutination test (MAT). The overall sensitivity of this assay approached 90%. Recently, another recombinant antigen (rLigB-Rep) was prepared. We tested each individual antigen and a 1:1:1:1 mixture of these four antigens for the detection of Leptospira-specific antibodies in ELISA. The performance of these recombinant antigens was evaluated with a much larger febrile patient panel (337 MAT-confirmed positive sera and 92 MAT-negative sera from febrile patients). Combining the detection results of immunoglobulin M and immunoglobulin G from these four individual antigens, the overall sensitivity was close to 90% but the specificity was only 66%, based on the MAT reference method. The overall sensitivity and specificity of the four-antigen mixture were 82% and 86%, respectively. The mixture of four antigens also exhibited a broader reactivity with MAT-positive samples of 18 serovars from six major pathogenic Leptospira species. Given the limitations of MAT, the data were further analyzed by Bayesian latent class model, showing that ELISA using a 1:1:1:1 mixture still maintained high sensitivity (79%) and specificity (88%) as compared with the sensitivity (90%) and specificity (83%) of MAT. Therefore, ELISA using a mixture of these four antigens could be a very useful test for seroprevalence studies.

The Mechanical Contribution of Vimentin to Cellular Stress Generation.

Contractile stress generation by adherent cells is largely determined by the interplay of forces within their cytoskeleton. It is known that actin stress fibers, connected to focal adhesions, provide contractile stress generation, while microtubules and intermediate filaments provide cells compressive stiffness. Recent studies have shown the importance of the interplay between the stress fibers and the intermediate filament vimentin. Therefore, the effect of the interplay between the stress fibers and vimentin on stress generation was quantified in this study. We hypothesized that net stress generation comprises the stress fiber contraction combined with the vimentin resistance. We expected an increased net stress in vimentin knockout (VimKO) mouse embryonic fibroblasts (MEFs) compared to their wild-type (vimentin wild-type (VimWT)) counterparts, due to the decreased resistance against stress fiber contractility. To test this, the net stress generation by VimKO and VimWT MEFs was determined using the thin film method combined with sample-specific finite element modeling. Additionally, focal adhesion and stress fiber organization were examined via immunofluorescent staining. Net stress generation of VimKO MEFs was three-fold higher compared to VimWT MEFs. No differences in focal adhesion size or stress fiber organization and orientation were found between the two cell types. This suggests that the increased net stress generation in VimKO MEFs was caused by the absence of the resistance that vimentin provides against stress fiber contraction. Taken together, these data suggest that vimentin resists the stress fiber contractility, as hypothesized, thus indicating the importance of vimentin in regulating cellular stress generation by adherent cells.

Lateral Flow Rapid Test for Accurate and Early Diagnosis of Scrub Typhus: A Febrile Illness of Historically Military Importance in the Pacific Rim.

Scrub typhus (ST) is an infection caused by Orientia tsutsugamushi. Historically, ST was ranked as the second most important arthropod-borne medical problem only behind malaria during World War II and the Vietnam War. The disease occurs mainly in Southeast Asia and has been shown to emerge and reemerge in new areas, implying the increased risk for U.S. military and civilian personnel deployed to these regions. ST can effectively be treated by doxycycline provided the diagnosis is made early, before the development of severe complications. Scrub Typhus Detect is a lateral flow rapid test based on a mixture of recombinant 56-kDa antigens with broad reactivity. The performance of this prototype product was evaluated against indirect immunofluorescence assay, the serological gold standard. Using 249 prospectively collected samples from Thailand, the sensitivity and specificity for IgM was found to be 100% and 92%, respectively, suggesting a high potential of this product for clinical use. This product will provide a user friendly, rapid, and accurate diagnosis of ST for clinicians to provide timely and accurate treatments of deployed personnel.

Development of Lyophilized Loop-Mediated Isothermal Amplification Reagents for the Detection of Leptospira.

Leptospirosis is considered to be the most widespread zoonosis. This worldwide emerging infectious disease is caused by the pathogenic species belonging to the genus Leptospira. Polymerase chain reaction (PCR)-based diagnostic assays have been developed for detecting Leptospira DNA in cell cultures and clinical samples. Because PCR requires specialized equipment and extensive end-user training, it is not suitable for routine work in resource-limited areas. We have developed a loop-mediated isothermal amplification (LAMP) assay to detect the presence of Leptospira in patient, animal and environmental samples using lyophilized reagents at a single temperature of around 63°C with a heating block. The sensitivity of this LAMP assay is very similar to the PCR method. The amplified DNA products can be visualized with the naked eyes using hydroxy naphthol blue or detected by the fluorescence signal of SYBR green dye in the reaction when an ultraviolet lamp or compact fluorescence tube scanner is used. This LAMP assay is simple, rapid, and can be performed with a water bath or heating block. The lyophilized LAMP reagents are stable for 3 months when stored at 4°C and 1 month when stored at 25°C, respectively. It is ideal for resource-limited settings where leptospirosis is endemic.

Highly Sensitive Loop-Mediated Isothermal Amplification for the Detection of Leptospira.

Leptospirosis is a worldwide zoonosis caused by an infection with the pathogenic species of Leptospira. We have developed a loop-mediated isothermal amplification (LAMP) assay to detect the DNA of Leptospira spp. Six sets of primers targeting the gene of the subsurface protein, lipL32, were evaluated for their detection sensitivity. The best primer set detected less than 25 copies of lipL32 per reaction of both plasmid DNA template and purified leptospiral genomic DNA. By combining primers targeting lipL32 with the previously published primer set targeting lipL41, the sensitivity of the assay was improved to 12 copies of L. interrogans. The specificity of the LAMP assay was evaluated by using the genomic DNA from other clinically encountered bacterial species such as different strains of Orientia tsutsugamushi, Rickettsia typhi, Rickettsia conorii, Rickettsia rickettsii, Coxiella burnetii, and Bartonella bacilliformis. These genomic DNA samples were all negative in our LAMP assay. The sensitivity of the LAMP assay was very similar to that of quantitative real time PCR. Several detection methods for the amplified product of LAMP assay were performed to demonstrate the simplicity of the assay. In summary, our results have suggested that this assay is rapid, robust, and easy to perform and has the potential to be used in endemic locations.