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Many natural saponins demonstrate immunostimulatory adjuvant activities, but they also have some inherent drawbacks that limit their clinical use. To overcome these limitations, extensive structure-activity-relationship (SAR) studies have been conducted. The SAR studies of QS-21 and related saponins reveal that their respective fatty side chains are crucial for potentiating a strong cellular immune response. Replacing the hydrolytically unstable ester side chain in the C28 oligosaccharide domain with an amide side chain in the same domain or in the C3 branched trisaccharide domain is a viable approach for generating robust semisynthetic saponin immunostimulants. Given the striking resemblance of natural momordica saponins (MS) I and II to the deacylated Quillaja Saponaria (QS) saponins (e.g., QS-17, QS-18, and QS-21), incorporating an amide side chain into the more sustainable MS, instead of deacylated QS saponins, led to the discovery of MS-derived semisynthetic immunostimulatory adjuvants VSA-1 and VSA-2. This review focuses on the authors' previous work on SAR studies of QS and MS saponins.
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Plasma membrane (PM)-associated abscisic acid (ABA) signal transduction is an important component of ABA signaling. The C2-domain ABA-related (CAR) proteins have been reported to play a crucial role in recruiting ABA receptor PYR1/PYL/RCAR (PYLs) to the PM. However, the molecular details of the involvement of CAR proteins in membrane-delimited ABA signal transduction remain unclear. For instance, where this response process takes place and whether any additional members besides PYL are taking part in this signaling process. Here, the GUS-tagged materials for all Arabidopsis CAR members were used to comprehensively visualize the extensive expression patterns of the CAR family genes. Based on the representativeness of CAR1 in response to ABA, we determined to use it as a target to study the function of CAR proteins in PM-associated ABA signaling. Single-particle tracking showed that ABA affected the spatiotemporal dynamics of CAR1. The presence of ABA prolonged the dwell time of CAR1 on the membrane and showed faster lateral mobility. Surprisingly, we verified that CAR1 could directly recruit hypersensitive to ABA1 (HAB1) and SNF1-related protein kinase 2.2 (SnRK2.2) to the PM at both the bulk and single-molecule levels. Furthermore, PM localization of CAR1 was demonstrated to be related to membrane microdomains. Collectively, our study revealed that CARs recruited the three main components of ABA signaling to the PM to respond positively to ABA. This study deepens our understanding of ABA signal transduction.
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Ácido Abscísico , Proteínas de Arabidopsis , Arabidopsis , Membrana Celular , Proteínas Serina-Treonina Quinases , Transdução de Sinais , Ácido Abscísico/metabolismo , Arabidopsis/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Membrana Celular/metabolismo , Regulação da Expressão Gênica de Plantas , Reguladores de Crescimento de Plantas/metabolismo , Plantas Geneticamente Modificadas , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genéticaRESUMO
A flow-cell offers many advantages for single-molecule studies. But, its merit as a quantitative single-molecule tool has long been underestimated. In this work, we developed a gas-pumped fully calibrated flow-cell system combined with fluorescence imaging for simultaneous single-molecule force measurement and visualization. Such a flow-cell system has considered the hydrodynamic drags on biomolecules and hence can apply and measure force up to more than 100 pN in sub-pN precision with an ultra-high force stability (force drift <0.01 pN in 10 minutes) and tuning accuracy (â¼0.04 pN). Meanwhile, it also allows acquiring force signals and fluorescence images at the same time, parallelly tracking hundreds of protein motors in real time as well as monitoring the conformational changes of biomolecules under a well-controlled force, as demonstrated by a series of single-molecule experiments in this work, including the studies of DNA overstretching dynamics, transcription under force and DNA folding/unfolding dynamics. Interesting findings, such as the very tight association of single-stranded binding (SSB) proteins with ssDNA and the reversed transcription, have also been made. These results together lay down an essential foundation for a flow-cell to be used as a versatile, quantitative and high-throughput tool for single-molecule manipulation and visualization.
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DNA de Cadeia Simples , DNA , DNA/química , Nanotecnologia , Proteínas , Imagem ÓpticaRESUMO
In underwater acoustic target recognition, there is a lack of massive high-quality labeled samples to train robust deep neural networks, and it is difficult to collect and annotate a large amount of base class data in advance unlike the image recognition field. Therefore, conventional few-shot learning methods are difficult to apply in underwater acoustic target recognition. In this report, following advanced self-supervised learning frameworks, a learning framework for underwater acoustic target recognition model with few samples is proposed. Meanwhile, a semi-supervised fine-tuning method is proposed to improve the fine-tuning performance by mining and labeling partial unlabeled samples based on the similarity of deep features. A set of small sample datasets with different amounts of labeled data are constructed, and the performance baselines of four underwater acoustic target recognition models are established based on these datasets. Compared with the baselines, using the proposed framework effectively improves the recognition effect of four models. Especially for the joint model, the recognition accuracy has increased by 2.04% to 12.14% compared with the baselines. The model performance on only 10 percent of the labeled data can exceed that on the full dataset, effectively reducing the dependence of model on the number of labeled samples. The problem of lack of labeled samples in underwater acoustic target recognition is alleviated.
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Drought is one of the key environmental factors affecting the growth and yield potential of rice. Grain shape, on the other hand, is an important factor determining the appearance, quality, and yield of rice grains. Here, we re-sequenced 275 Xian accessions and then conducted a genome-wide association study (GWAS) on six agronomic traits with the 404,411 single nucleotide polymorphisms (SNPs) derived by the best linear unbiased prediction (BLUP) for each trait. Under two years of drought stress (DS) and normal water (NW) treatments, a total of 16 QTLs associated with rice grain shape and grain weight were detected on chromosomes 1, 2, 3, 4, 5, 7, 8, 11, and 12. In addition, these QTLs were analyzed by haplotype analysis and functional annotation, and one clone (GSN1) and five new candidate genes were identified in the candidate interval. The findings provide important genetic information for the molecular improvement of grain shape and weight in rice.
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Introduction: Saline-alkali stress is one of the main abiotic factors limiting rice production worldwide. With the widespread use of rice direct seeding technology, it has become increasingly important to improve rice saline-alkali tolerance at the germination stage. Methods: To understand the genetic basis of saline-alkali tolerance and facilitate breeding efforts for developing saline-alkali tolerant rice varieties, the genetic basis of rice saline-alkali tolerance was dissected by phenotyping seven germination-related traits of 736 diverse rice accessions under the saline-alkali stress and control conditions using genome-wide association and epistasis analysis (GWAES). Results: Totally, 165 main-effect quantitative trait nucleotides (QTNs) and 124 additional epistatic QTNs were identified as significantly associated with saline-alkali tolerance, which explained a significant portion of the total phenotypic variation of the saline-alkali tolerance traits in the 736 rice accessions. Most of these QTNs were located in genomic regions either harboring saline-alkali tolerance QTNs or known genes for saline-alkali tolerance reported previously. Epistasis as an important genetic basis of rice saline-alkali tolerance was validated by genomic best linear unbiased prediction in which inclusion of both main-effect and epistatic QTNs showed a consistently better prediction accuracy than either main-effect or epistatic QTNs alone. Candidate genes for two pairs of important epistatic QTNs were suggested based on combined evidence from the high-resolution mapping plus their reported molecular functions. The first pair included a glycosyltransferase gene LOC_Os02g51900 (UGT85E1) and an E3 ligase gene LOC_Os04g01490 (OsSIRP4), while the second pair comprised an ethylene-responsive transcriptional factor, AP59 (LOC_Os02g43790), and a Bcl-2-associated athanogene gene, OsBAG1 (LOC_Os09g35630) for salt tolerance. Detailed haplotype analyses at both gene promoter and CDS regions of these candidate genes for important QTNs identified favorable haplotype combinations with large effects on saline-alkali tolerance, which can be used to improve rice saline-alkali tolerance by selective introgression. Discussion: Our findings provided saline-alkali tolerant germplasm resources and valuable genetic information to be used in future functional genomic and breeding efforts of rice saline-alkali tolerance at the germination stage.
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To study the corrosion mechanism of high-nitrogen steel welds, this study investigated the effects of laser outputs on the corrosion behavior of high-nitrogen steel hybrid welded joints in hybrid laser-arc welding. The relationship between the ferrite content and laser output was characterized. The ferrite content increased with the increase in the laser power. The corrosion phenomenon first occurred at the two-phase interface, thereby forming corrosion pits. Ferritic dendrites were first corroded to form dendritic corrosion channels. Furthermore, first-principles calculations were performed to investigate the properties of the austenite and ferrite content. The work function and surface energy indicated that solid-solution nitrogen austenite exhibits a higher surface structural stability than austenite and ferrite. This study provides useful information for high-nitrogen steel weld corrosion.
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BACKGROUND: Most intracranial aneurysms (IAs) will be abnormal bulges on the walls of intracranial arteries that result from the dynamic interaction of geometric morphology, hemodynamics, and pathophysiology. Hemodynamics plays a key role in the origin, development, and rupture of IAs. In the past, hemodynamic studies of IAs were mostly based on the rigid wall hypothesis of computational fluid dynamics, and the influence of arterial wall deformation was ignored. We used fluid-structure interaction (FSI) to study the features of ruptured aneurysms, because it can solve this problem very well and the simulation will be more realistic. METHODS: A total of 12 IAs, 8 ruptured and 4 unruptured, at the middle cerebral artery bifurcation were studied using FSI to better identify the characteristics of ruptured IAs. We studied the differences in the hemodynamic parameters, including the flow pattern, wall shear stress (WSS), oscillatory shear index (OSI), and displacement and deformation of the arterial wall. RESULTS: Ruptured IAs had a larger low WSS area and more complex, concentrated, and unstable flow. Also, the OSI was higher. In addition, the displacement deformation area at the ruptured IA was more concentrated and larger. CONCLUSIONS: A large aspect ratio; a large height/width ratio; complex, unstable, and concentrated flow patterns with small impact areas; a large low WSS region; large WSS fluctuation, high OSI; and large displacement of the aneurysm dome could be risk factors associated with aneurysm rupture. If similar cases are encountered when simulation is used in the clinic, priority should be given to diagnosis and treatment.
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Aneurisma Roto , Aneurisma Intracraniano , Humanos , Aneurisma Intracraniano/complicações , Artéria Cerebral Média , Aneurisma Roto/complicações , Fatores de Risco , Hemodinâmica/fisiologia , Hidrodinâmica , Estresse MecânicoRESUMO
A method for detecting the surface defects of high reflection objects using phase deflection is proposed. The abrupt change in the surface gradient at the defect leads to the change in the fringe phase. Therefore, Gray code combined with a four-step phase-shift method was employed to obtain the surface gradients to characterize the defects. Then, through the double surface illumination model, the relationship between illumination intensity and phase was established. The causes of periodic error interference were analyzed, and the method of adjusting the fringe width to eliminate it was proposed. Finally, experimental results showed the effectiveness of the proposed method.
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Telomeres, which are located at the ends of eukaryotic chromosomes, are crucial for genomic maintenance. Most telomeric DNA is composed of tandemly repeated guanine (G)-rich sequences, which form G-quadruplexes (G4s). The structures and dynamics of telomeric G4s are essential for telomere functioning and helpful for G4-based biosensing. However, they are far from being understood, especially for plants. In this contribution, the folding, environment-induced G4 dynamics, and protein-catalyzed unfolding of plant telomeric G4s were comprehensively studied. It was found that diverse plant telomeric sequences from land plants to green algae could fold into G4 structures. In addition, 5'-proximal ssDNA but not 3'-proximal ssDNA drove conversion of anti-parallel G4 structures to parallel structures, and both 5' and 3' ssDNA decreased the stability of G4s in dilute solution. Furthermore, molecular crowding promoted the formation of parallel structures for three-layer but not for two-layer G4s, and increased the stability of all selected G4s. Finally, AtRecQ2 helicase resolved the stable parallel structure of typical plant telomeric G4 in crowded solution, but ssDNA binding protein AtRPA did not. Furthermore, AtRecQ2 unwound the structure more efficiently in the presence of AtRPA. The results may expand our understanding on the structures and dynamics of plant telomeric G4s.
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Quadruplex G , DNA/química , DNA Helicases/metabolismo , Telômero/metabolismo , DNA de Cadeia SimplesRESUMO
Plasmodium vivax and Plasmodium falciparum cases have opposite trends in Anhui China in the past decade. Long term and seasonal trends in the transmission rate of P. falciparum in Africa has been well studied, however that of P. vivax transmitted by Anopheles sinensis in China has not been investigated. There is a lot of work on the relationship between P. vivax cases and climatic factors in China, with sometimes contradicting results. However, how climatic factors affect transmission rate of P. vivax in China is unknown. We used Anhui province as an example to analyze the recent transmission dynamics where two types of malaria have been reported with differing etiologies. We examined breakpoints of the P. vivax and P. falciparum malaria long term dynamics in the recent decade. For locally transmitted P. vivax malaria, we analyzed the transmission rate and its seasonality using the combined human and mosquitos SIR-SI model with time-varied mosquito biting rate. We identified the effects of meteorological factors on the seasonality in transmission rate using a GAM model. For the imported P. falciparum malaria, we analyzed the potential reason for the observed increase in cases. The breakpoints of P. vivax and P. falciparum dynamics happened in a same year, 2010. The seasonality in the transmission rate of P. vivax malaria was high (42.4%) and was linearly associated with temperature and nonlinearly with rainfall. The abrupt increase in imported P. falciparum cases after the breakpoint was significantly related to the increased annual Chinese investment in Africa. Under the conditions of the existing vectors of malaria, long-term trends in climatic factors, and increasing trend in migration to/from endemic areas and imported malaria cases, we should be cautious of the possibility of the reestablishment of malaria in regions where it has been eliminated or the establishment of other vector-borne diseases.
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VSA-1 is a semisynthetic saponin adjuvant prepared from naturally occurring Momordica saponin and capable of stimulating antigen-specific humoral and cellular immune responses. Its immunostimulating activity in enhancing the immune responses induced by the clinical glycoconjugate pneumococcal vaccine PCV13 is compared with QS-21 in female BALB/c mice. Both VSA-1 and QS-21 boosted IgG and opsonic antibodies titers against seven selected serotypes, including serotypes 3, 14, and 19A that are involved in most PCV13 breakthroughs. Since VSA-1 is much more accessible and of lower toxicity than QS-21, it can be a practical saponin immunostimulant to be included in a new glycoconjugate pneumococcal vaccine formulation.
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Vacinas Pneumocócicas , Saponinas , Animais , Camundongos , Feminino , Adjuvantes Imunológicos/farmacologia , Adjuvantes Farmacêuticos , Imunoglobulina G , Saponinas/farmacologiaRESUMO
VSA-2 is a recently developed semisynthetic saponin immunostimulant. It is prepared by incorporating a terminal-functionalized side chain to the branched trisaccharide domain at the C3 position of Momordica saponin II (MS II) isolated from the seeds of perennial Momordica cochinchinensis Spreng. Direct comparison of VSA-2 and the clinically proven saponin adjuvant QS-21 shows that VSA-2 is comparable to QS-21 in enhancing humoral and cellular immune responses. Structure-activity relationship studies show that structural changes in the side chain have a significant impact on saponins' adjuvant activity. However, with the VSA-2 molecular framework intact, the new VSA-2 analogues with various substitution(s) at the terminal benzyl group of the side chain retain the ability of potentiating antigen-specific humoral and cellular responses.
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Momordica , Saponinas , Momordica/química , Adjuvantes de Vacinas , Saponinas/química , Adjuvantes Imunológicos/farmacologia , Adjuvantes Imunológicos/química , Relação Estrutura-AtividadeRESUMO
I-motifs are four-strand noncanonical secondary structures formed by cytosine (C)-rich sequences in living cells. The structural dynamics of i-motifs play essential roles in many cellular processes, such as telomerase inhibition, DNA replication, and transcriptional regulation. In cells, the structural dynamics of the i-motif can be modulated by the interaction of poly(C)-binding proteins (PCBPs), and the interaction is closely related to human health, through modulating the transcription of oncogenes and telomere stability. Therefore, the mechanisms of how PCBPs interact with i-motif structures are fundamentally important. However, the underlying mechanisms remain elusive. I-motif structures in the promoter of the c-MYC oncogene can be unfolded by heterogeneous nuclear ribonucleoprotein K (hnRNP K), a PCBP, to activate its transcription. Here, we selected this system as an example to comprehensively study the unfolding mechanisms. We found that the promoter sequence containing 5 C-runs preferred folding into type-1245 to type-1234 i-motif structures based on their folding stability, which was further confirmed by single-molecule FRET. In addition, we first revealed that the c-MYC i-motif structure was discretely resolved by hnRNP K through two intermediate states, which were assigned to the opposite hairpin and neighboring hairpin, as further confirmed by site mutations. Furthermore, we found all three KH (hnRNP K homology) domains of hnRNP K could unfold the c-MYC i-motif structure, and KH2 and KH3 were more active than KH1. In conclusion, this study may deepen our understanding of the interactions between i-motifs and PCBPs and may be helpful for drug development.
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Proteínas de Transporte , Ribonucleoproteínas Nucleares Heterogêneas Grupo K , Humanos , Ribonucleoproteínas Nucleares Heterogêneas Grupo K/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo K/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Ligação a RNA/metabolismo , DNA/metabolismo , Estrutura Secundária de ProteínaRESUMO
Background: Metagenomic next-generation sequencing (mNGS) is increasingly being used to detect pathogens directly from clinical specimens. However, the optimal application of mNGS and subsequent result interpretation can be challenging. In addition, studies reporting the use of mNGS for the diagnosis of invasive fungal infections (IFIs) are rare. Objective: We critically evaluated the performance of mNGS in the diagnosis of pulmonary IFIs, by conducting a multicenter retrospective analysis. The methodological strengths of mNGS were recognized, and diagnostic cutoffs were determined. Methods: A total of 310 patients with suspected pulmonary IFIs were included in this study. Conventional microbiological tests (CMTs) and mNGS were performed in parallel on the same set of samples. Receiver operating characteristic (ROC) curves were used to evaluate the performance of the logarithm of reads per kilobase per million mapped reads [lg(RPKM)], and read counts were used to predict true-positive pathogens. Result: The majority of the selected patients (86.5%) were immunocompromised. Twenty species of fungi were detected by mNGS, which was more than was achieved with standard culture methods. Peripheral blood lymphocyte and monocyte counts, as well as serum albumin levels, were significantly negatively correlated with fungal infection. In contrast, C-reactive protein and procalcitonin levels showed a significant positive correlation with fungal infection. ROC curves showed that mNGS [and especially lg(RPKM)] was superior to CMTs in its diagnostic performance. The area under the ROC curve value obtained for lg(RPKM) in the bronchoalveolar lavage fluid of patients with suspected pulmonary IFIs, used to predict true-positive pathogens, was 0.967, and the cutoff value calculated from the Youden index was -5.44. Conclusions: In this study, we have evaluated the performance of mNGS-specific indicators that can identify pathogens in patients with IFIs more accurately and rapidly than CMTs, which will have important clinical implications.
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Infecções Fúngicas Invasivas , Pneumopatias Fúngicas , Micoses , Pneumonia , Proteína C-Reativa , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Infecções Fúngicas Invasivas/diagnóstico , Pneumopatias Fúngicas/diagnóstico , Pneumopatias Fúngicas/microbiologia , Metagenômica/métodos , Pneumonia/microbiologia , Pró-Calcitonina , Estudos Retrospectivos , Sensibilidade e Especificidade , Albumina SéricaRESUMO
Background: In an initial trauma evaluation, computed tomography of the head (CTH) is performed to assess for life-threatening intracranial injury. Given the high incidence of concomitant facial injuries, many facial fractures are diagnosed incidentally during this evaluation. Although maxillofacial CT (CTMF) is widely accepted as the most sensitive method for evaluating facial fractures, it is often excluded from the initial survey. Failure to obtain dedicated imaging can lead to increased costs related to a missed or delayed facial fracture diagnosis. Our study investigates the location and type of missed facial fractures on CTH by reviewing imaging data from patients who presented at a level 1 trauma center and underwent both CTH and CTMF. Methods: A retrospective review of all facial fractures diagnosed at a single institution from 2002 through 2016 was conducted. Inclusion criteria included adults aged 18 years or older who received CTH and then subsequent CTMF. Patients who had either CTH or CTMF only or combined CTH/CTMF were excluded. The facial fractures were further subdivided by location. Results: There were 501 patients with 1743 total facial fractures. CTH successfully identified 788 (45.21%) fractures, versus 1743 (100%) for CTMF. The most common fractures, in both cohorts, were nasal bone (15.7%) and orbital floor (12.8%) fractures. Using CTMF to identify missed fractures on CTH, significant differences were noted in the following locations: anterior table frontal sinus, medial/lateral pterygoid, maxillary sinus, lateral orbital wall, zygomatic arch, palate, and all types of mandible fractures excluding the mandibular condyle. Conclusions: CTH for initial trauma evaluation often misses facial fractures. CTH alone was only sufficient in detecting posterior frontal sinus, orbital (excluding lateral wall), and mandibular condyle fractures. In patients with suspected facial injury, dedicated imaging should be performed to detect the location and extent of injury because CTH inadequately identifies most facial fractures.
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RNA G-quadruplexes (rG4s) are noncanonical RNA secondary structures formed by guanine (G)-rich sequences. These complexes play important regulatory roles in both animals and plants through their structural dynamics and are closely related to human diseases and plant growth, development, and adaption. Thus, studying the structural dynamics of rG4s is fundamentally important; however, their folding pathways and their unfolding by specialized helicases are not well understood. In addition, no plant rG4-specialized helicases have been identified. Here, using single-molecule FRET, we experimentally elucidated for the first time the folding pathway and intermediates, including a G-hairpin and G-triplex. In addition, using proteomics screening and microscale thermophoresis, we identified and validated five rG4-specialized helicases in Arabidopsis thaliana. Furthermore, DExH1, the ortholog of the famous human rG4 helicase RHAU/DHX36, stood out for its robust rG4 unwinding ability. Taken together, these results shed light on the structural dynamics of plant rG4s.
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Quadruplex G , Animais , Catálise , RNA Helicases DEAD-box/metabolismo , DNA Helicases/metabolismo , Humanos , RNA/metabolismoRESUMO
OBJECTIVES: Malaria is a vector-borne disease that remains a serious public health problem due to its climatic sensitivity. Accurate prediction of malaria re-emergence is very important in taking corresponding effective measures. This study aims to investigate the impact of climatic factors on the re-emergence of malaria in mainland China. DESIGN: A modelling study. SETTING AND PARTICIPANTS: Monthly malaria cases for four Plasmodium species (P. falciparum, P. malariae, P. vivax and other Plasmodium) and monthly climate data were collected for 31 provinces; malaria cases from 2004 to 2016 were obtained from the Chinese centre for disease control and prevention and climate parameters from China meteorological data service centre. We conducted analyses at the aggregate level, and there was no involvement of confidential information. PRIMARY AND SECONDARY OUTCOME MEASURES: The long short-term memory sequence-to-sequence (LSTMSeq2Seq) deep neural network model was used to predict the re-emergence of malaria cases from 2004 to 2016, based on the influence of climatic factors. We trained and tested the extreme gradient boosting (XGBoost), gated recurrent unit, LSTM, LSTMSeq2Seq models using monthly malaria cases and corresponding meteorological data in 31 provinces of China. Then we compared the predictive performance of models using root mean squared error (RMSE) and mean absolute error evaluation measures. RESULTS: The proposed LSTMSeq2Seq model reduced the mean RMSE of the predictions by 19.05% to 33.93%, 18.4% to 33.59%, 17.6% to 26.67% and 13.28% to 21.34%, for P. falciparum, P. vivax, P. malariae, and other plasmodia, respectively, as compared with other candidate models. The LSTMSeq2Seq model achieved an average prediction accuracy of 87.3%. CONCLUSIONS: The LSTMSeq2Seq model significantly improved the prediction of malaria re-emergence based on the influence of climatic factors. Therefore, the LSTMSeq2Seq model can be effectively applied in the malaria re-emergence prediction.
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Aprendizado Profundo , Malária Falciparum , Malária , China/epidemiologia , Mudança Climática , Humanos , Malária/epidemiologiaRESUMO
Single-molecule imaging is emerging as a revolutionary approach to studying fundamental questions in plants. However, compared with its use in animals, the application of single-molecule imaging in plants is still underexplored. Here, we review the applications, advantages, and challenges of single-molecule fluorescence imaging in plant systems from the perspective of methodology. Firstly, we provide a general overview of single-molecule imaging methods and their principles. Next, we summarize the unprecedented quantitative details that can be obtained using single-molecule techniques compared to bulk assays. Finally, we discuss the main problems encountered at this stage and provide possible solutions.