Spatial transcriptomics prediction from histology jointly through Transformer and graph neural networks.

The rapid development of spatial transcriptomics allows the measurement of RNA abundance at a high spatial resolution, making it possible to simultaneously profile gene expression, spatial locations of cells or spots, and the corresponding hematoxylin and eosin-stained histology images. It turns promising to predict gene expression from histology images that are relatively easy and cheap to obtain. For this purpose, several methods are devised, but they have not fully captured the internal relations of the 2D vision features or spatial dependency between spots. Here, we developed Hist2ST, a deep learning-based model to predict RNA-seq expression from histology images. Around each sequenced spot, the corresponding histology image is cropped into an image patch and fed into a convolutional module to extract 2D vision features. Meanwhile, the spatial relations with the whole image and neighbored patches are captured through Transformer and graph neural network modules, respectively. These learned features are then used to predict the gene expression by following the zero-inflated negative binomial distribution. To alleviate the impact by the small spatial transcriptomics data, a self-distillation mechanism is employed for efficient learning of the model. By comprehensive tests on cancer and normal datasets, Hist2ST was shown to outperform existing methods in terms of both gene expression prediction and spatial region identification. Further pathway analyses indicated that our model could reserve biological information. Thus, Hist2ST enables generating spatial transcriptomics data from histology images for elucidating molecular signatures of tissues.

Iso-E-Codelock: A Rebuilding-free Electrochemical Chip with a Customizable Decoding Probe for Real-Time and Portable Pathogen Diagnostics.

In order to realize portable pathogen diagnostics with easier quantitation, digitization and integration, we develop a ready-to-use electrochemical sensing strategy (Iso-E-Codelock) for real-time detection of isothermal nucleic acid amplification. Bridged by a branched DNA as codelock, the isothermal amplicon is transduced into increased current of an electrochemical probe, holding multiple advantages of high sensitivity, high selectivity, signal-on response, "zero" background and one-pot operation. Through a self-designed portable instrument (BioAlex PHE-T), the detection can be implemented on a multichannel microchip and output real-time amplification curves just like an expensive commercial PCR machine. The microchip is a rebuilding-free and disposable component. The branch codelock probe can be customized for different targets and designs. Such high performance and flexibility have been demonstrated utilizing four virus (SARS-CoV-2, African swine fever, FluA and FluB) genes as targets, and two branch (3-way and 4-way) DNAs as codelock probes.

CRISPR/Cas12a-Responsive Hydrogels for Conjugation-Free and Universal Indicator Release in Colorimetric Detection.

Recent advances have demonstrated the significant potential and advantages to repurpose existing point-of-care reactions/devices to realize portable detection of nonoriginal targets, e.g., pathogen genes. However, pursuing this aim usually requires protein indicator-nucleic acid conjugation via a covalent bond, which may bring drawbacks such as high cost, complicated procedure, and annoying component rebuilding. Herein, we developed a conjugation-free, effective, and universal detection platform called CRIs-gel (CRISPR/Cas12a-Responsive Indicators@RCA hydrogels). Various protein indicators are pre-encapsulated into the hydrogels made of effective and high-yield rolling circle amplification (RCA). Upon a targeting sequence binding with its antisense crRNA, CRISPR/Cas12a starts its trans-cleavage activity to crush the hydrogel, which may directly release the indicator for downstream readout. Two proteins, amylase (GA) and human chorionic gonadotropin (hCG), are successfully used as model indicators to trigger the downstream amylum-I2 color change and pregnancy test strip response. After coupling with upstream isothermal nucleic acid amplification, both portable readouts may detect as few as 2 copies/µL genetic sequences of influenza A virus (FluA), human papilloma virus (HPV), SARS-CoV-2, and influenza B virus (FluB). This conjugation-free CRIs-gel platform is thus simple, sensitive, and universal and can provide innovative insights for portable point-of-care testing (POCT) development.

Lego-Like Catalytic Hairpin Assembly Enables Controllable DNA-Oligomer Formation and Spatiotemporal Amplification in Single Molecular Signaling.

While the solid-state nanopore shows increasing potential during sensitive and label-free single molecular analysis, target concentration and signal amplification method is in urgent need. In this article, a solution via designing a model nucleic acid circuit reaction that can produce "Y" shape-structure three-way DNA oligomers with controllable size and polymerization degree is proposed. Such a so-called lego-like three-way catalytic hairpin assembly (LK-3W-CHA) can provide both concentration amplification (via CHA circuit) and programmable size control (via lego-like building mode) to enhance spatiotemporal resolution in single molecular sensing of solid-state nanopore. Oligomers containing 1-4 DNA three-way junctions (Y monomers, Y1-Y4) are designed in proof-of-concept experiments and applications. When the oligomers are applied to direct translocation measurements, Y2-Y4 can significantly increase the signal resolution and stability than that of Y1. Meanwhile, Y1 to Y4 can be used as the tags on the long DNA carrier to provide very legible secondary signals for specific identification, multiple assays, and information storage. Compared with other possible tags, Y1-Y4 provides higher signal density and amplitude, and quasi-linear "inner reference" for each other, which may provide more systematic, reliable, and controllable experimental results.

Base Pair Stacking Modulates Solid-State Charge Transport in DNA Hairpins.

Particular interest has been focused on modulation of solid-state charge transport (CT) in DNA. Nevertheless, it remains challenging to do so in a sensitive and predictive manner due to the lack of a definite relationship between DNA base pair stacking and DNA CT. The challenges can be mainly attributed to the ill-defined systems, which may lead to ambiguous and even contradictory conclusions. Here, we use DNA hairpins to construct the well-defined self-assembled monolayers. We reveal nearly positive-linear correlations between DNA conformation and CT in the DNA hairpins regulated with metal ion chelation and DNA sequence. The correlations have been confirmed by the solid-state current-voltage characteristics and circular dichroism in solution. The enhanced CT via metal ion chelated DNA hairpins is mainly from the improved DNA energy coupling to electrodes, not the almost unchanged energy barrier when Hg ion-induced DNA conformational switches toward the canonical B-form.

Multiplex detection of clinical pathogen nucleic acids via a three-way junction structure-based nucleic acid circuit.

Nucleic acid testing technology has made considerable progress in the last few years. However, there are still many challenges in the clinical application of multiple nucleic acid assays, such as how to ensure accurate results, increase speed and decrease cost. Herein, a three-way junction structure has been introduced to specifically translate analytes of loop-mediated isothermal amplification to a catalytic hairpin assembly. For different analyses, a well-optimized nucleic acid circuit can be directly applied to detection, through only one-component replacement, which only not avoids duplicate sequence design but also saves detection cost. Thanks to this design, multiple and logical analysis can be easily realized in a single reaction with ultra-high sensitivity and selectivity. In this paper, Mycoplasma pneumoniae and Streptococcus pneumoniae can be clearly distinguished from the clinical mixed sample with negative control or one analyte in one tube single fluorescence channel. The fair experimental results of actual clinical samples provide a strong support for the possibility of clinical application of this methodology.

A Duplex Polymerization Strategy for General, Programmable and High-Resolution Nanopore-Based Sensing.

Nanopore sensing is highly promising in single molecular analysis but their broad applications have been challenged by the limited strategies that can transduce a target-of-interest into a specific and anti-false/inference signal, especially for solid-state nanopores with relatively lower resolution and higher noise. Here we report a high-resolution signal-production concept named target-induced duplex polymerization strategy (DPS). Through linking the same or different duplex substrates (DSs) with a special linker (L) and an optional structure tag (ST), the DPS can generate target-specific DS polymers with highly controllable duration times, duration intervals and even distinguished secondary tagging currents. Experimentally, DPS mono-polymerization of single DS and co-polymerization of multiple DSs has verified the duration time of a DPS product is the sum of those for each DS monomer. Tetrahedron-DNA structures with different sizes are used as the STs to provide needle-like secondary peaks for further resolution enhancement and multiplex assay. With these examples DPS represents a general, programmable and advanced strategy that may simultaneously provide size-amplification, concentration amplification, and signal-specificity for molecular recognition. It is also promisingly in various applications regarding to single molecular investigation, such as polymerization degree, structure/side chain conformation, programmable multiplex decoding and information index.

A Rebuilding-Free Nucleic Acid Detection Strategy Enables Ultrasensitive Genotyping, N-in-1 Logic Screening and Accurate Multiplex Assay of Dangerous Pathogens.

Sensitive, rapid and low-cost nucleic acid detection is critical for controlling infectious pathogens. Here, we develop a ready-to-use and multimodal detection based on a rebuilding-free, ultrasensitive and selective strategy named dual hairpin ligation-induced isothermal amplification pro (DHLApro). Taking influenza A, influenza B, MERS-CoV, SARS-CoV-2 as model targets, we demonstrate DHLApro provides ≈zM level ultra-sensitivity, being equaling to 0.45 copy/µL in original sample. By simply changing the recognition module, a set of DHLApro components can be applied to a new target without performance loss. Moreover, DHLApro innovatively allows flexible logic/multiplex assay using one set of primer, for example, the "N pathogens-in-1" OR gate screening and accurate multi-channel multiplex assay. Compared with traditional methods, the cost of this logic/multiplex assay has been largely reduced and the cross-interference between the multiple primer sets is also avoided.

CLIPON: A CRISPR-Enabled Strategy that Turns Commercial Pregnancy Test Strips into General Point-of-Need Test Devices.

Desirable biosensing assays need to be sensitive, specific, cost-effective, instrument-free, and versatile. Herein we report a new strategy termed CLIPON (CRISPR and Large DNA assembly Induced Pregnancy strips for signal-ON detection) that can deliver these traits. CLIPON integrates a commercial pregnancy test strip (PTS) with four biological elements: the human chorionic gonadotropin (hCG), CRISPR-Cas12a, crRNA and cauliflower-like large-sized DNA assemblies (CLD). CLIPON uses the Cas12a/crRNA complex both to recognize a target of interest and to release CLD-bound hCG so that target presence can translate into a colorimetric signal on the PTS. We demonstrate the versatility of CLIPON through sensitive and specific detection of HPV genomic DNA, SARS-CoV-2 genomic RNA and adenosine. We also engineer a cell phone app and a hand-held microchip to achieve signal quantification. CLIPON represents an attractive option for biosensing and point-of-care diagnostics.

Establishment of Dual Hairpin Ligation-Induced Isothermal Amplification for Universal, Accurate, and Flexible Nucleic Acid Detection.

Isothermal amplifications have found their potentials in applications of portable nucleic acid diagnostics. However, there are still several certain deficiencies existing in the current amplification methods, including high false-positive signals, limited range of targets, difficult primer design, and so forth. Here, we report an effective solution via the development of dual hairpin ligation-induced isothermal amplification (DHLA) consisting of (1) the formation of a dual hairpin probe (DHP) based on sequence specific hybridization and ligation and (2) exponential isothermal amplification of DHP in the presence of polymerase and primers. Taking both microRNA and virus RNA as model targets, DHLA is proven to be accurate, flexible, and applicable to most deoxyribonucleic acid and ribonucleic acid targets ranging from ∼20 to hundreds of nt. The detection limit is down to the ∼aM level without a false-positive signal. More importantly, the whole detection can be directly applied to a new target via a slight change in the DHP sequence, without redesigning the primer set. This unique property not only simplifies the process for new reaction development but also enables flexible multiprobe strategies to achieve antidegradation analysis.

SARS-CoV-2 Point-of-Care (POC) Diagnosis Based on Commercial Pregnancy Test Strips and a Palm-Size Microfluidic Device.

Coronavirus diseases such as the coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), pose serious threats. Portable and accurate nucleic acid detection is still an urgent need to achieve on-site virus screening and timely infection control. Herein, we have developed an on-site, semiautomatic detection system, aiming at simultaneously overcoming the shortcomings suffered by various commercially available assays, such as low accuracy, poor portability, instrument dependency, and labor intensity. Ultrasensitive isothermal amplification [i.e., reverse transcription loop-mediated isothermal amplification (RT-LAMP)] was applied to generate intensified SARS-CoV-2 RNA signals, which were then transduced to portable commercial pregnancy test strips (PTSs) via ultraspecific human chorionic gonadotropin (hCG)-conjugated toehold-mediated strand exchange (TMSE) probes (hCG-P). The entire detection was integrated into a four-channel, palm-size microfluidic device, named the microfluidic point-of-care (POC) diagnosis system based on the PTS (MPSP) detection system. It provides rapid, cost-effective, and sensitive detection, of which the lowest concentration of detection was 0.5 copy/µL of SARS-CoV-2 RNA, regardless of the presence of other similar viruses, even highly similar severe acute respiratory syndrome coronavirus (SARS-CoV). The successful detection of the authentic samples from different resources evaluated the practical application. The commercial PTS provides a colorimetric visible signal, which is instrument- and optimization-free. Therefore, this MPSP system can be immediately used for SARS-CoV-2 emergency detection, and it is worthy of further optimization to achieve full automation and detection for other infectious diseases.

Low-Noise Nanopore Enables In-Situ and Label-Free Tracking of a Trigger-Induced DNA Molecular Machine at the Single-Molecular Level.

Solid-state nanopores have shown special high potential in a label-free molecular assay, structure identification, and target-index at the single-molecular level, even though frustrating electrical baseline noise is still one of the major factors that limit the spatial resolution and signaling reliability of solid-state nanopores, especially in small target detection. Here we develop a significant and easy-operating noise-reduction approach via mixing organic solvents with high dielectric constants into a traditional aqueous electrolyte. The strategy is generally effective for pores made of different materials, such as the most commonly used conical glass (CGN) or SiNx. While the mechanism should be multisourced, MD simulations suggest the noise reduction may partially arise from the even ionic distribution caused by the addition of higher dielectric species. Among all solvents experimentally tested, the two with the highest dielectric constants, formamide and methylformamide, exhibit the best noise reduction effect for target detection of CGN. The power spectral density at the low-frequency limit is reduced by nearly 3 orders with the addition of 20% formamide. Our work qualifies the reliability of solid-state nanopores into much subtler scales of detection, such as dsDNAs under 100 bp. As a practical example, bare CGN is innovatively employed to perform in-situ tracking of trigger-responsive DNA machine forming oligomers.

Homogeneous and Universal Detection of Various Targets with a Dual-Step Transduced Toehold Switch Sensor.

Toehold switch sensors represent a class of new advances that allow specific targets to trigger in situ expression of a protein reporter. Although they offer unique advantages of a label-free nature and high portability, they generally require repeated sequence design, high expenditure, and laborious optimization of toehold switch sequences according to different targets. To simplify the sensing process further and to improve its practicability, we innovatively introduce a dual-step pre-transduction upon traditional toehold switch sensor. Through two successive toehold-mediated strand-displacement reactions that are initiated, respectively, by a linear and an associative trigger, different DNAs, RNAs, or ligands of functional nucleic acids can be generally transduced into the input of one high-performance toehold switch sensor. This advance greatly increases the target range. Furthermore, the whole process is signal-on, homogeneous, and free of any requirements for complicated operations such as probe labeling, separation, and reconstruction of the toehold switch, being promising and practical even in portable or point-of-care assays.

Changes of pollen viability of ornamental plants after long-term preservation in a cryopreservation pollen bank.

This study determined the changes in pollen viability of 102 species/cultivars of ornamental plants (affiliated to 32 genera of 14 families) following long-term liquid nitrogen storage in a cryopreservation pollen bank. The goal was to provide information on the safety and stability of pollen cryopreservation technology. Fresh pollen at the time of storage was used as the control, and the study examined the pollen viability of ornamental plants cryopreserved for 8, 9, or 10 years. The results show that pollen of the 102 species/cultivars in the cryopreservation pollen bank retained viability ranging from 1% to 58%, After long-term storage there were changes in viability: 11.76% (12 species/cultivars) had increased viability, 16.67% (17 species/cultivars) had stable viability, and the viability of 71.57% (73 species/cultivars) showed a decreasing trend.

Adaption of a Solid-State Nanopore to Homogeneous DNA Organization Verification and Label-Free Molecular Analysis without Covalent Modification.

Recent advances have shown increasing designs of nucleic acid organizations via controlling the thermodynamics and kinetics of oligonucleotides. Nevertheless, deeper understanding and further applications of these DNA nanotechnologies are majorly hampered by the lack of effective analytical methodologies that are competent enough to investigate them. To deliver a potential solution, here we developed an innovative exploration that employed the emerging nanopore technique to characterize DNA organization at the single-molecule level and in completely homogeneous condition without covalent modification. With the help of counting and profiling the translocation-induced current drop of a DNA assembly structure passing through a conical glass nanopore (CGN), we have directly verified the formation of the individual double-helix concatemer generated from our model, hybridization chain reaction (HCR). Due to the ultrasensitivity of the nanopore technology, those concatemers that were difficult to observe on a conventional electrophoresis image were brought to light. The translocation duration time also provided the approximate length and folding information for the concatemers. These advantages were proven also applicable to structures with more sophisticated folding behaviors. Eventually, when coupling with an upstream reaction, CGN was further turned to a universal detector that was capable of even detecting other nucleic acid organization behaviors as well as targets that were unable to generate huge products. All of these results are expected to promote deeper study and applications of the nanopore technique in the field of nucleic acid nanotechnology.

Coupling Sensitive Nucleic Acid Amplification with Commercial Pregnancy Test Strips.

The detection of nucleic acid biomarkers for point-of-care (POC) diagnostics is currently limited by technical complexity, cost, and time constraints. To overcome these shortcomings, we have combined loop-mediated isothermal amplification (LAMP), programmable toehold-mediated strand-exchange signal transduction, and standard pregnancy test strips. The incorporation of an engineered hCG-SNAP fusion reporter protein (human chorionic gonadotropin-O6 -alkylguanine-DNA alkyltransferase) led to LAMP-to-hCG signal transduction on low-cost, commercially available pregnancy test strips. Our assay reliably detected as few as 20 copies of Ebola virus templates in both human serum and saliva and could be adapted to distinguish a common melanoma-associated SNP allele (BRAF V600E) from the wild-type sequence. The methods described are completely generalizable to many nucleic acid biomarkers, and could be adapted to provide POC diagnostics for a range of pathogens.

Engineering Signaling Aptamers That Rely on Kinetic Rather Than Equilibrium Competition.

During the past decade, aptasensors have largely been designed on the basis of the notion that ligand-modulated equilibration between aptamer conformations could be exploited for sensing. One implementation of this strategy has been to denature the aptamer with an antisense oligonucleotide, wait for dissociation of the antisense oligonucleotide, and stabilize the folded, signaling conformer with a ligand. However, there is a large kinetic barrier associated with releasing the oligonucleotide from the aptamer to again obtain an active, binding conformation. If the length of the antisense oligonucleotide is decreased to make dissociation from the aptamer more favorable, higher background signals are observed. To improve the general methodology for developing aptasensors, we have developed a novel and robust strategy for aptasensor design in which an oligonucleotide kinetically competes with the ligand for binding rather than having to be released from a stable duplex. While the oligonucleotide can induce conformational change, it initially chooses between the aptamer and a molecular beacon (MB), a process that does not require a lengthy pre-equilibration. Using an anti-ricin aptamer as a starting point, we developed a "competitive" aptasensor with a measured limit of detection (LOD) of 30 nM with an optical readout and as low as 3 nM for ricin toxin A-chain (RTA) detection on an electrochemical platform.

An exploratory cross-sectional study on the impact of education on perception of stigma by Chinese patients with schizophrenia.

BACKGROUND: Stigma is a major issue across various society and cultures, and few studies focus on the perception of stigma by Chinese patients with schizophrenia. In the current cross-sectional study, we sought to assess the extent of internalized stigma among outpatients with schizophrenia in China and to investigate whether education level correlated with the experience of stigma. METHODS: Outpatients with schizophrenia were evaluated using the brief psychosis rating scale (BPRS), the positive and negative syndrome scale (PANSS), the clinical global impression-severity of illness (CGI-SI) scale and the Stigma Scale for Mental Illness (SSMI 2C). Patients were categorized into the high education and low education group according to their educational levels. RESULTS: One hundred thirty-three subjects were included in the study. Their mean course of illness was 4.32 ± 6.14 years (range, 1 month to 15 years). Their mean BPRS score was 19.87 ± 5.46, their mean PANSS score was 44.11 ± 13.1, and their mean CGI-SI score was 2.22 ± 0.81. In addition, the mean SSMI 2C score of the high education group (7.15 ± 0.98) was markedly higher than that of the low education group (5.75 ± 0.79, P < 0.05). The mean domain I score of the high education group (2.30 ± 0.76) was comparable to that of the low education group (2.07 ± 0.78, P > 0.05). The mean domain II score of the high education group (2.42 ± 0.96) was markedly higher than that of the low education group (2.01 ± 0.79, P < 0.05). Moreover, the mean domain III score of the high education group (2.43 ± 0.79) was significantly higher than that of the low education group (1.67 ± 0.77, P < 0.05). CONCLUSIONS: Education level impacts on the perception of stigma by patients with schizophrenia and more psycho-education should be done to improve patients' knowledge about schizophrenia.

Heatstroke induces liver injury via IL-1ß and HMGB1-induced pyroptosis.

BACKGROUND & AIMS: Liver injury is a common complication of heat stroke (HS), and often constitutes a direct cause for patient death. The cellular and molecular mechanism underlying HS-induced liver injury remains unclear. Recent evidence indicates that inflammasome plays an important role in mediating sterile inflammation triggered by tissue damage. Using a rat HS model, we identified a novel mechanism by which inflammasome-dependent interleukin-1ß (IL-1ß) activation and hepatocyte pyroptosis mediate HS-induced liver injury. METHODS: To induce HS, rats were subjected to heat exposure. Inhibition of inflammasomes was achieved by RNA silencing and pharmacologic inhibitor prior to heat exposure. Inflammasome assembly, caspase-1 activation, histological changes, as well as serum levels of liver enzymes were measured. RESULTS: We demonstrated that the onset of HS activated inflammasome in the liver as evidenced by increased capase-1 activity and the association of inflammasome components NOD-like receptor family pyrin domain containing 3 (Nlrp3) and apoptosis speck-like protein containing a caspase-recruitment domain (ASC); and the activated inflammasome, in turn, induced IL-1ß activation and hepatocyte pyroptosis, and subsequent augmented liver injury. HS-induced hepatocyte inflammasome activation seems to be high-mobility group box 1 (HMGB1) dependent. Inhibition of Nlrp3, caspase-1, or HMGB1 prevented HS-induced liver inflammation and ameliorated liver injury. CONCLUSIONS: These findings demonstrate an important role of HMGB1 in mediating inflammasome activation in the development of liver injury following HS, and suggest that targeting inflammasome may represent a novel therapeutic strategy to limit cell death and prevent liver failure after HS.

Robust strand exchange reactions for the sequence-specific, real-time detection of nucleic acid amplicons.

Loop-mediated isothermal amplification (LAMP) of DNA is a powerful isothermal nucleic acid amplification method that can generate upward of 10(9) copies from less than 100 copies of template DNA within an hour. Unfortunately, although the amplification reactions are extremely powerful, real-time and specific detection of LAMP products remains analytically challenging. In order to both improve the specificity of LAMP detection and to make readout simpler and more reliable, we have replaced the intercalating dye typically used for monitoring in real-time fluorescence with a toehold-mediated strand exchange reaction termed one-step strand displacement (OSD). Due to the inherent sequence specificity of toehold-mediated strand exchange, the OSD reporter could successfully distinguish side products from true amplicons arising from templates corresponding to the biomedically relevant M. tuberculosis RNA polymerase (rpoB) and the melanoma-related biomarker BRAF. OSD allowed the Yes/No detection of rpoB in a complex mixture such as synthetic sputum and also demonstrated single nucleotide specificity in Yes/No detection of a mutant BRAF allele (V600E) in the presence of 20-fold more of the wild-type gene. Real-time detection of different genes in multiplex LAMP reactions also proved possible. The development of simple, readily designed, modular equivalents of TaqMan probes for isothermal amplification reactions should generally improve the applicability of these reactions and may eventually assist with the development of point-of-care tests.