The procedure described here provides instructions for detection of Cryptosporidium recovered from large-volume water samples. Water samples are collected by dead-end ultrafiltration in the field and ultrafilters are processed in a laboratory. Microbes recovered from the filters are further concentrated and subjected to Cryptosporidium isolation or nucleic acid extraction methods for the detection of Cryptosporidium oocysts or Cryptosporidium DNA.
Networks of protein—protein interactions PPI constitute either stable or transient complexes in every cell. Most of the cellular complexes keep their function, and therefore stay similar, during evolution.
The evolutionary constraints preserve most. The evolutionary constraints preserve most cellular functions via preservation of protein structures and interactions. The evolutionary conservation information is utilized in template-based approaches, like protein structure modeling or docking. Here we use the combination of the template-free docking method with conservation-based selection of the best docking model using our newly developed COZOID tool.
We describe a step-by-step protocol for visual selection of docking models, based on their similarity to the original protein complex structure. Using the COZOID tool, we first analyze contact zones of the original complex structure and select contact amino acids for docking restraints. Then we model and dock the homologous proteins. Finally, we utilize different analytical modes of our COZOID tool to select the docking models most similar to the original complex structure.
The perineuronal net PNN is a specialized extracellular matrix structure that surrounds subpopulations of neurons in the central nervous system CNS. The appearance of PNNs on the cell surface marks the closure of the critical period during. The appearance of PNNs on the cell surface marks the closure of the critical period during development and has been observed to reduce synaptic plasticity.
Perineuronal nets comprise hyaluronan, chondroitin sulfate proteoglycans CSPGs , link proteins, tenascin-R, and other components, some of which are substrates for a disintegrin-like and metalloprotease domain with thrombospondin type 1 motifs ADAMTS proteases. Depending on which part of the CNS is studied, the PNNs may be observed surrounding the soma, or both the soma and proximal dendrites.
The most robust marker for PNN is a lectin called Wisteria floribunda agglutinin. Snake and spider envenomation have a considerable impact on public health. Their pathology is induced by a variety of toxins composing the venom which induce cytotoxicity to cells of different organs by several cell death pathways. Described in this.
Described in this chapter are methods in vitro used to assess venoms and toxin-induced cell death using mammalian cell cultures. The chapter is divided into five sections: 1 a brief overview of in vitro cytotoxicity and categories of cell death induced by venoms and toxins; 2 a common method to measure necrotic cell death using lactate dehydrogenase LDH release; 3 a flow cytometry method that simultaneously measures necrosis and apoptosis; 4 measurements of nuclear morphology; and 5 measurements of the autophagy following microtubule-associated protein light chain 3 LC3 expression, by immunoblotting and by fluorescence microscopy of LC3-positive vesicles, to assess the levels of autophagosomes.
Antibody Data Search Beta. Publication Year. Invalid publishing year. Video available Bars represent standard deviation errors. A population dynamics analysis was conducted on all 17 plants, namely the 11 blood oranges and 6 Murcott mandarins under double-, single- or non-infection conditions as mentioned above. The population size of HSVd was larger than that of CEVd during the first year; in subsequent years, however, the two viroids had similar population sizes Fig. CEVd titers increased steadily from the winter of through , while HSVd titers generally followed the same pattern but decreased in The populations of both viroids obviously decreased in the spring and summer seasons of The titers of both viroids reached their highest concentrations of the 3-year study in the winter of Fig.
Overview of 3-year population dynamics of CEVd and HSVd in 17 citrus plants from an infected field over 12 spring, summer, fall and winter 3-month periods. The statistical analysis incorporated 0. The blue and red bars indicate standard deviation errors. The green bars correspond to monthly average temperatures MAT during each 3-month period. Titer levels of the two viroids were significantly positively correlated in rootstock bark and leaves of blood orange; a similar correlation in titer levels was observed in rootstock bark and roots of Murcott mandarin.
Viroid titer levels were not correlated in any of the remaining tissues in either citrus cultivar.
Transmission electron microscopy DNA sequencing
In contrast, the two viroids were not correlated in blood orange under high temperature conditions. Significant correlations were generally observed in roots and rootstock bark of Murcott mandarins in all three temperature groups, whereas correlations were only observed in rootstock bark of blood oranges under medium temperatures. Compared with the CEVd population under single-infection conditions, the CEVd population under double infection increased significantly in size during half of the 12 monitored seasons Fig.
There was only one season in which the HSVd population under double infection increased significantly compared with the population under single infection Fig. The doubly infected citrus trees consisted of five blood oranges and four Murcott mandarins. CEVd singly infected citrus trees comprised two blood oranges, and HSVd singly infected citrus trees were represented by two blood oranges and two Murcott mandarins. The y-axis corresponds to log 10 -transformed copy numbers of each viroid and the bars represent standard deviation errors. To evaluate the distributions of the two positively correlated viroids in four citrus tissues, DIG-labeled riboprobes of CEVd and HSVd were separately used for detection.
We observed that the two viroids occupied similar locations in continuous sections of the four examined tissues. In root tissues, both viroids localized mostly in the endodermis and pericycle Fig. In rootstock bark tissues, the viroids were detected in cortical cells near the cork cambium Fig. Both viroids occupied outer cortical and phloem cells in twig bark tissues Fig. Three citrus tissues with high viroid titers roots, rootstock bark and twig bark were used to evaluate subcellular distributions of the two location-correlated viroids.
Our analysis revealed that the two viroids were generally present in the nucleoplasm, vacuoles, cytoplasm, plasma membranes and cell walls of all three tissues. In roots, the two viroids were mostly present in the vacuoles and nucleoplasm Fig.
In rootstock bark, both viroids were more densely distributed in the nucleoplasm, with HSVd found in the cell walls but not in the vacuoles Fig. In twig bark, massively intense HSVd signals were observed in the cytoplasm; similarly, CEVd could also be detected in the cytoplasm Fig. No obvious difference in viroid subcellular localization was found between the two citrus cultivars. Both viroids were concentrated mostly in the nucleoplasm, vacuoles and cytoplasm rather than in the other subcellular compartments. The locations of CEVd and HSVd in three tissues of blood oranges and Murcott mandarins were detected by in situ hybridization with digoxigenin DIG -labeled and biotinylated anti-sense riboprobes, respectively.
HSVd biotin-labeled probes were detected with nm colloidal streptavidin-gold from Streptomyces avidinii. Ultrathin sections of viroid-negative roots a , rootstock bark d and twig bark g hybridized with the same probes revealed neither CEVd nor HSVd signals in the nucleus nor in any other subcellular structures. Most of the probe signals were associated with the nucleus and present near the plasma membrane and cytoplasm. The viroid signals were associated with the vacuole and cell wall. The probes were associated with the nucleoplasm or cytoplasm. The viroid signals were associated with cell walls, cytoplasm and the nucleoplasm.
The probes were associated with the cytoplasm and cell walls. The viroid signals were associated with the cytoplasm. We have previously surveyed blood oranges and Murcott mandarins in Taiwan and confirmed the frequent occurrence of simultaneous CEVd and HSVd infections [ 15 ]. The infected hosts exhibited only typical exocortis symptoms, thus increasing the difficulty of measuring the interaction between the two viroids.
In addition, previous research has detailed the distributions of viroids in host plants [ 10 — 12 ], but the association between symptom expression and viroid location is unclear. In this study, we therefore used molecular, statistical and in situ hybridization methods to look for correlations between the two viroids and to determine the relationship between their interaction and distribution. According to our seasonal investigation of the two citrus cultivars grafted onto susceptible Rangpur lime rootstock, viroid titers were higher in underground parts than in aerial portions, and the titer of HSVd was also higher than that of CEVd.
In general, viroid populations ranged from 10 2 to 10 4 copy numbers per microliter of total RNA extract. Murcott mandarins were more susceptible than blood oranges to infection. Accumulations of HSVd were nearly fold higher than those of CEVd in Murcott mandarins; this suggests that unknown factors in Murcott mandarin contributed to the higher accumulated titers, even though the two viroids likely use the same replication mechanism. The titer of HSVd in Murcott mandarins was almost doubles that in blood oranges, indicating different interactions between this viroid and the two citrus cultivars.
The reason for the obvious decrease in CEVd titers in twig bark and leaves of Murcott mandarins is not clear. Additional research may help explain the uneven distributions in different tissues, and, in particular, why the viroids could barely be detected in leaves. With the complete sequencing of the draft genome of sweet orange [ 16 ], the interaction between a citrus host and its viroid pathogens may be further studied at the molecular level.
Whether antagonistic or synergistic, the interaction between two viroids depends on multiple factors, including pathogenicity, host susceptibility, and competition for resources. The defined nature of the interaction between two plant viruses depends on the extent of symptoms and pathogen titers [ 17 ]; this is probably true for viroids as well. The HSVd isolates in Taiwan were non-cachexia variants, but with no differences observed in exocortis symptoms of co-infected citrus plants compared with those infected only with CEVd. A previous long-term investigation carried out from to similarly revealed no significant differences in yields or other growth parameters when CEVd and HSVd co-infected Commune clementine grafted onto Pomeroy trifoliate orange [ 8 ].
As a consequence, dynamic titers are by necessity the major indicators available to confirm viroid interactions in citrus. As shown in Fig. We speculate that this difference is due to the small number of doubly infected citrus plants sampled. Furthermore, various combinations of mild and severe CEVd and HSVd isolates should be considered to test for different degrees of interaction among them. Surprisingly, CEVd and HSVd do not appear to compete for resources even though they are both in the family Pospiviroidae and have many biological properties in common.
This situation is counter-intuitive because similar pathogens usually compete for the same resources when colonizing the same space. We were unable to discern the mechanism of interaction between the two viroids at macroscopic or microscopic levels. To explain this phenomenon, we hypothesize that CEVd and HSVd use different host cell resources under specific temperature conditions to fulfill their biological functions.
Alternatively, each viroid may rely on an unknown mechanism to interact with the other one in a positive fashion. Additional research involving proteomic or transcriptomic analysis of viroid co-infected citrus should be carried out under different temperature conditions [ 19 , 20 ]. Previous studies have shown that viroids in the family Pospiviroidae replicate in the nucleus, are distributed to other organelles, and spread via phloem, resulting in systemic infection [ 11 ].
However, no studies have focused on the distributions of interacting viroids and whether their distributions are complimentary or antagonistic. Our investigation has confirmed that CEVd and HSVd are both concentrated in endodermal cells and the pericycle of root tissues.
In a previous study in tomato, PSTVd was similarly detected in the outer part of the central cylinder containing the endodermis, pericycle and vascular tissue [ 21 ]. Our study is the first to identify the location of two viroids in bark exhibiting classical exocortis symptoms. We have shown that the two viroids are concentrated only in cortical cells near the cork cambium and not in inner cortical cells.
We postulate that the specific distributions of the viroids may be related to an unknown pathogenicity mechanism that controls exocortis formation. To our knowledge, our study is the first to simultaneously detect a pair of interacting viroids in two citrus cultivars. Statistically supported positive correlations were uncovered between the two viroids in rootstock bark and leaves of blood oranges and in roots and rootstock bark of Murcott mandarins. The regions without any tissue showed no non-specific amplification. The results are shown in Supplementary Figs.
Note that the tissue boundaries are maintained throughout the reaction. As a final specificity test of the on-chip assay, we loaded cancer and non-cancer mouse skeletal muscle tissue tissue on the same chip and performed the RT-LAMP reaction. Only the cancerous tissue amplified validating the specificity of our assay.
Note the amplification occurs only for the cancerous tissue. In this setting, only cancer epithelium are expected to express TOP2A. FTIR provides a tissue-level view of the sample without the use of dyes or other reagents that are known to degrade RNA 22 , While IR imaging is usually performed on specialized substrates, here we made a small modification to make it compatible with our silicon microchips.
As shown in Supplementary Fig.
The results clearly demonstrate that only cancerous regions amplified. To visualize the spatial heterogeneity in TOP2A mRNA across the tissue, we plotted the fraction of cancerous tissue per well color bar against spatially mapped threshold times to create a 4-dimensional plot Supplementary Fig. Taking a given fraction of cancerous tissue per well as a reference for the starting sample amount in wells, the heterogeneity in TOP2A expression is evident as variation in threshold times in those wells as seen in Supplementary Fig. It can be observed that wells with none to negligible fraction of cancerous tissue dark red either do not amplify or have very high threshold times, whereas regions with high fraction of cancerous tissue yellow tend to show lower threshold times.
This turnaround time compared very favorably against the over 2 days required to perform the mFISH experiments, and that is without taking into account the considerable time that was required to capture and process mFISH images. These important advantages make microchip amplification a suitable technique for a wide range of research and clinical applications. The spatial pattern of TOP2A expression is similar between the two assay types. The technique presented here can be tuned to perform quantitative spatially mapped nucleic acid analysis of any tissue sample type on a simple hot plate and a fluorescence reader.
It can also be integrated into a completely portable setup using a smartphone and an in-built heater making the technique accessible even to labs without a microscope Our technique allows analysis of small-to-large tissue regions without any cross-talk between individual tissue pixels. This is important considering that commercial solutions for spotting arrays of nanodroplets cannot spot picoliters of volume in close spacing, have large dead volumes, and suffer from long sample loading times serial loading of wells would take several hours 27 , Our technique can be scaled to fill larger arrays with millions of wells using the same principle in a matter of minutes.
Our technique, which can be easily performed in routine practice, has many important clinical and biological applications such as understanding tumor heterogeneity, predicting patient outcomes, and post-operative characterization of surgical margins As such, we predict that microchip amplification will find a wide range of applications in clinical and research settings.
LNCaP cells were first incubated and grown to confluence. The mice were then monitored daily for the presence of tumors. Ten microliter template of the appropriate concentration and 1.
Pixelated spatial gene expression analysis from tissue | Nature Communications
All the off-chip LAMP tests were carried out in 0. Fluorescence data were recorded after each cycle of the reaction. Triplicates were done for each reaction. The same process was repeated on the polished size of the wafer. The photoresist on the shiny side was patterned using an EVG aligner with a high-resolution transparency mask FineLine Imaging. The unprotected silicon oxide was etched in buffered oxide etchant VWR to reveal the underlying bare silicon. To render a positive charge on well surfaces for tissue adhesion, silicon chips were silanized with functional groups using APTES.
The chips were dipped in a glass jar containing 0. The chips were then dipped five times in a separate vessel containing distilled water. This step was repeated three more times with the water being replaced between each step. The silanized chips were stored in a desiccator, and were used within 15 days of silanization. A standard acetone fixation protocol was followed to fix the tissue onto the chip. After the incubation step, the chip underwent a series of wash steps.
First, half of the acetone was discarded from the petri dish and an equal amount of cold phosphate buffered saline PBS Fisher Scientific was poured into the Petri dish to replace the acetone. This was followed by a rinse with DEPC-treated water at room temperature for one minute. The chip was placed on a Petri dish with Proteinase K at a concentration of 7. After degassing, the chip was dipped in mineral oil, and an air pressure is applied at an angle to shear off and remove excess reagents from top of the wells. The chip was then placed on a copper bowl and placed on a hotplate under a fluorescent microscope to perform the on-chip RT-LAMP reactions.
For on-chip reactions, the raw fluorescent intensity on-chip was extracted from each well and was plotted against time to generate the raw fluorescence curves.
Polymerase Chain Reaction Testing: Selected Indications
Each raw amplification curve was fitted to a sigmoidal curve using a four-point parameter modeling Supplementary Fig. The following equation was used for the analysis:. The positive and negative wells were differentiated on the basis of the R 2 value of the sigmoidal fit and the parameters a and x 0. The threshold time was taken as the point of inflection. The sample was imaged in the reflection—absorption mode.
Data were water vapor corrected and used for classification using the previously described Bayesian approach, metrics, and algorithms for prostate tissue. Stephen J. Experimental procedures for FISH staining were reported previously 30 and provided by the probe supplier. The immunostaining IHC experiment was performed at the Mayo Pathology Research Core and followed our previously published protocol 31 with minor modifications. This system includes the hydrogen peroxidase block, secondary antibody polymer, DAB, and hematoxylin. Slides were dehydrated in increasing concentrations of ethyl alcohol and xylene prior to permanent coverslipping in xylene-based media.
Espina, V. Laser-capture microdissection. Armani, M. Bagasra, O. Moffitt, J. High-performance multiplexed fluorescence in situ hybridization in culture and tissue with matrix imprinting and clearing. Natl Acad. USA , Femino, A. Visualization of single RNA transcripts in situ. Science , — Raj, A. Imaging individual mRNA molecules using multiple singly labeled probes.