Short Probes Research Articles

Towards in vivo Bronchoscopic Functional CFTR Assessment using a Short Circuit Current Measurement Probe.

The epithelial lining of luminal organs provides an immune barrier against external factors and regulates transport of nutrients, ions, and water into the body. Several conditions are associated with a breakdown or dysfunction of the epithelial lining. Short circuit current (Isc) measurement using a bulky, expensive, and hard to deploy system known as the Ussing chamber is the gold standard for evaluation of epithelial transport function but requires tissue excision. We demonstrated the ability of the Isc probe to measure Isc in normal wild type (WT) versus reduced CFTR function knockout (KO) rats as a relevant animal model for testing ion channel function.

Use of high-resolution fluorescence in situ hybridization for fast and robust detection of SARS-CoV-2 RNAs

Early, rapid, and accurate diagnostic tests play critical roles not only in the identification/management of individuals infected by SARS-CoV-2, but also in fast and effective public health surveillance, containment, and response. Our aim has been to develop a fast and robust fluorescence in situ hybridization (FISH) detection method for detecting SARS-CoV-2 RNAs by using an HEK 293 T cell culture model. At various times after being transfected with SARS-CoV-2 E and N plasmids, HEK 293 T cells were fixed and then hybridized with ATTO-labeled short DNA probes (about 20 nt). At 4 h, 12 h, and 24 h after transfection, SARS-CoV-2 E and N mRNAs were clearly revealed as solid granular staining inside HEK 293 T cells at all time points. Hybridization time was also reduced to 1 h for faster detection, and the test was completed within 3 h with excellent results. In addition, we have successfully detected 3 mRNAs (E mRNA, N mRNA, and ORF1a (−) RNA) simultaneously inside the buccal cells of COVID-19 patients. Our high-resolution RNA FISH might significantly increase the accuracy and efficiency of SARS-CoV-2 detection, while significantly reducing test time. The method can be conducted on smears containing cells (e.g., from nasopharyngeal, oropharyngeal, or buccal swabs) or smears without cells (e.g., from sputum, saliva, or drinking water/wastewater) for detecting various types of RNA viruses and even DNA viruses at different timepoints of infection.

Effects of ipsilateral, contralateral, and bilateral noise precursors on psychoacoustical tuning curves in humans

Cochlear tuning and hence auditory frequency selectivity are thought to change in noisy environments by activation of the medial olivocochlear reflex (MOCR). In humans, auditory frequency selectivity is often assessed using psychoacoustical tuning curves (PTCs), a plot of the level required for pure-tone maskers to just mask a fixed-level pure-tone probe as a function of masker frequency. Sometimes, however, the stimuli used to measure a PTC are long enough that they can activate the MOCR by themselves and thus affect the PTC. Here, PTCs for probe frequencies of 500 Hz and 4 kHz were measured in forward masking using short maskers (30 ms) and probes (10 ms) to minimize the activation of the MOCR by the maskers or the probes. PTCs were also measured in the presence of long (300 ms) ipsilateral, contralateral, and bilateral broadband noise precursors to investigate the effect of the ipsilateral, contralateral, and bilateral MOCR on PTC tuning. Four listeners with normal hearing participated in the experiments. At 500 Hz, ipsilateral and bilateral precursors sharpened the PTCs by decreasing the thresholds for maskers with frequencies at or near the probe frequency with minimal effects on thresholds for maskers remote in frequency from the probe. At 4 kHz, by contrast, ipsilateral and bilateral precursors barely affected thresholds for maskers near the probe frequency but broadened PTCs by reducing thresholds for maskers far from the probe. Contralateral precursors barely affected PTCs. An existing computational model was used to interpret the results. The model suggested that despite the apparent differences, the pattern of results is consistent with the ipsilateral and bilateral MOCR inhibiting the cochlear gain similarly at the two probe frequencies and more strongly than the contralateral MOCR.

Polymer composite coatings subjected to sliding wear under simulated lunar temperature and dust conditions

Due to the detrimental effect of lunar dust on tribological components, it is imperative to develop bearing materials and surfaces which can survive and mitigate dust at the interface while operating under lunar environmental conditions. This issue is poised to become more significant in the near future, with lunar exploration evolving beyond the short probing missions of the Apollo era. Therefore, this study aims to investigate the combined effect of temperature and lunar dust on the tribological performance of aromatic thermosetting copolyester (ATSP) and polyether ether ketone (PEEK) -based polymer composite coatings from −150 to 110 °C. The experiments were conducted using a flat pin-on-disk configuration under dry sliding conditions, sliding speed of 0.25 m/s, 17.5 MPa contact pressure, and nitrogen gas environment. It was found that metal-on-polymer coating sliding results in dust embedment and accumulation on the softer material, which yields high friction and two-body abrasive wear. The chemical analysis of transfer film on the metal pin surface indicated a mixture of dust and polymer phases, which increased with increasing temperature, and thus resulted in deteriorated tribological performance. Self-mating polymer coatings yielded lower friction coefficient and wear due to a better dust mitigation resulting in three-body wear at the interface. The ATSP-based coatings showed higher wear resistance at all temperatures, particularly under self-mating sliding condition.

Aphids May Facilitate the Spread of Sclerotinia Stem Rot in Oilseed Rape by Carrying and Depositing Ascospores.

Aphids and Sclerotinia stem rot in oilseed rape are often studied in isolation, and their relationship is rarely explored. Our field studies have revealed a significant positive correlation between the number of aphids and the incidence of Sclerotinia stem rot. Hence, starting with the colonizing stages of the two pests, Breveroryne brassicae was assessed for its potential to acquire, transmit, and inoculate Sclerotinia sclerotiorum by being sprayed with an ascospore suspension. Moreover, distinctions in aphid feeding behavior were examined between aphids on inoculated/uninoculated winter and spring oilseed rape plants or aphids, both with and without S. sclerotiorum ascospores, using electropenetrography (EPG). The results showed that aphid feeding followed by dropping ascospore suspension significantly increased the incidence of S. sclerotiorum. Ascospores were able to adhere to aphids and were carried by aphids to healthy plants, causing disease. The results of the EPG analysis indicated that aphid feeding behavior was significantly altered in all leaf tissue levels following infection with S. sclerotiorum. Specifically, aphids initiated their first puncture significantly sooner, began probing mesophyll cells earlier, had a significantly shorter pathway duration, and secreted saliva more frequently but reduced salivation prior to feeding and ingestion compared to aphids feeding on uninfected oilseed rape. Additionally, the feeding behavior of aphids carrying ascospores was markedly different from that of aphids not carrying ascospores, implying that ascospores directly influence aphid feeding behavior but that this influence appeared to be beneficial only for S. sclerotiorum infection. Aphids carrying ascospores started to puncture cells more quickly, with a significant increase in the frequency and duration of short probes and cell punctures, shortened pathway durations, and reduced salivation before feeding compared to aphids not carrying ascospores. It is clear that there is an interaction between aphids and S. sclerotiorum. The impact of S. sclerotiorum on aphid feeding behavior is directional, favoring the spread of the fungus.

A sensitive and scalable fluorescence anisotropy single stranded RNA targeting approach for monitoring riboswitch conformational states.

The capacity of riboswitches to undergo conformational changes in response to binding their native ligands is closely tied to their functional roles and is an attractive target for antimicrobial drug design. Here, we established a probe-based fluorescence anisotropy assay to monitor riboswitch conformational switching with high sensitivity and throughput. Using the Bacillus subtillis yitJ S-Box (SAM-I), Fusobacterium nucleatum impX RFN element of (FMN)and class-I cyclic-di-GMP from Vibrio cholerae riboswitches as model systems, we developed short fluorescent DNA probes that specifically recognize either ligand-free or -bound riboswitch conformational states. We showed that increasing concentrations of native ligands cause measurable and reproducible changes in fluorescence anisotropy that correlate with riboswitch conformational changes observed by native gel analysis. Furthermore, we applied our assay to several ligand analogues and confirmed that it can discriminate between ligands that bind, triggering the native conformational change, from those that bind without causing the conformational change. This new platform opens the possibility of high-throughput screening compound libraries to identify potential new antibiotics that specifically target functional conformational changes in riboswitches.

On-Site Fluorescent Detection of Sepsis-Inducing Bacteria using a Graphene-Oxide CRISPR-Cas12a (GO-CRISPR) System.

Sepsis is an extremely dangerous medical condition that emanates from the body's response to a pre-existing infection. Early detection of sepsis-inducing bacterial infections can greatly enhance the treatment process and potentially prevent the onset of sepsis. However, current point-of-care (POC) sensors are often complex and costly or lack the ideal sensitivity for effective bacterial detection. Therefore, it is crucial to develop rapid and sensitive biosensors for the on-site detection of sepsis-inducing bacteria. Herein, we developed a graphene oxide CRISPR-Cas12a (GO-CRISPR) biosensor for the detection of sepsis-inducing bacteria in human serum. In this strategy, single-stranded (ssDNA) FAM probes were quenched with single-layer graphene oxide (GO). Target-activated Cas12a trans-cleavage was utilized for the degradation of the ssDNA probes, detaching the short ssDNA probes from GO and recovering the fluorescent signals. Under optimal conditions, we employed our GO-CRISPR system for the detection of Salmonella Typhimurium (S. Typhimurium) with a detection sensitivity of as low as 3 × 103 CFU/mL in human serum, as well as a good detection specificity toward other competing bacteria. In addition, the GO-CRISPR biosensor exhibited excellent sensitivity to the detection of S. Typhimurium in spiked human serum. The GO-CRISPR system offers superior rapidity for the detection of sepsis-inducing bacteria and has the potential to enhance the early detection of bacterial infections in resource-limited settings, expediting the response for patients at risk of sepsis.

Northern blotting of endogenous full-length human-specific LINE-1 RNA.

LINE-1 belongs to a family of DNA elements that move to new locations in the genome in a process called "retrotransposition." This is achieved by a copy-and-paste mechanism with the aid of an RNA intermediate. The full-length LINE-1 is responsible for most retrotransposition activity in the human genome. Detecting the active LINE-1 RNA at the endogenous level is challenging due to its small percentage among inactive copies and its different forms of transcripts. Here, we describe a method of designing RNA probes to detect active LINE-1 by northern blotting and use optimized conditions and tools to make the detection practical. This method uses a classical long RNA probe and provides an alternative way to detect LINE-1 RNA using multiple short RNA probes.

Исследование импульсного режима работы пьезопреобразователя иммерсионного дефектоскопа

Purpose of the work: To evaluate the influence of the shape of a complex electrical signal at the input of a piezoelectric transducer on the duration of the acoustic signal emitted by it. Materials and methods: Theoretical modeling using the d'Alembert method for a one-sided loaded piezoelectric plate was used. Results: The dynamics of changes in the probing signal depending on the moment of supply of a correctly formed compensating half-cycle has been studied. A comparison was made of the shapes of the probing signals for the cases of the presence and absence of a damper on the back side of the plate. The possibility of obtaining signals of various durations is shown. It has been established that when a complex electrical signal is applied to the piezoelectric element of a damped probe, a probing signal is generated with a higher level of oscillations (with incomplete compensation) than for an undamped transducer. Conclusion: A plate-type piezoelectric transducer is considered, designed to emit short probing signals, which is achieved by exciting it with electrical signals of complex shapes. The problem was solved by the d'Alembert method for a one-sided loaded piezoelectric plate. The aquatic environment was chosen as the acoustic load. The plate is considered in two versions – with and without a damper.

Inserting a Neuropixels probe into awake monkey cortex: two probes, two methods

BackgroundNeuropixels probes have revolutionized neurophysiological studies in the rodent, but inserting these probes through the much thicker primate dura remains a challenge. New methodsHere we describe two methods we have developed for the insertion of two types of Neuropixels probes acutely into the awake macaque monkey cortex. For the fine rodent probe (Neuropixels 1.0, IMEC), which is unable to pierce native primate dura, we developed a dural-eyelet method to insert the probe repeatedly without breakage. For the thicker short NHP probe (Neuropixels NP1010), we developed an artificial dura system to insert the probe. Results and comparison with existing methodsWe have now conducted successful experiments in 3 animals across 7 recording chambers with the procedures described here and have achieved recordings with similar yields over several months in each case. ConclusionWe hope that our hardware, surgical preparation, methods for insertion and methods for removal of broken probe parts are of value to primate physiologists everywhere.

Discovery of Ligands for TRIM58, a Novel Tissue-Selective E3 Ligase.

Redirecting E3 ligases to neo-substrates, leading to their proteasomal disassembly, known as targeted protein degradation (TPD), has emerged as a promising alternative to traditional, occupancy-driven pharmacology. Although the field has expanded tremendously over the past years, the choice of E3 ligases remains limited, with an almost exclusive focus on CRBN and VHL. Here, we report the discovery of novel ligands to the PRY-SPRY domain of TRIM58, a RING ligase that is specifically expressed in erythroid precursor cells. A DSF screen, followed by validation using additional biophysical methods, led to the identification of TRIM58 ligand TRIM-473. A basic SAR around the chemotype was established by utilizing a competitive binding assay employing a short FP peptide probe derived from an endogenous TRIM58 substrate. The X-ray co-crystal structure of TRIM58 in complex with TRIM-473 gave insights into the binding mode and potential exit vectors for bifunctional degrader design.

Heteromultivalency enables enhanced detection of nucleic acid mutations.

Detecting genetic mutations such as single nucleotide polymorphisms (SNPs) is necessary to prescribe effective cancer therapies, perform genetic analyses and distinguish similar viral strains. Traditionally, SNP sensing uses short oligonucleotide probes that differentially bind the SNP and wild-type targets. However, DNA hybridization-based techniques require precise tuning of the probe's binding affinity to manage the inherent trade-off between specificity and sensitivity. As conventional hybridization offers limited control over binding affinity, here we generate heteromultivalent DNA-functionalized particles and demonstrate optimized hybridization specificity for targets containing one or two mutations. By investigating the role of oligo lengths, spacer lengths and binding orientation, we reveal that heteromultivalent hybridization enables fine-tuned specificity for a single SNP and dramatic enhancements in specificity for two non-proximal SNPs empowered by highly cooperative binding. Capitalizing on these abilities, we demonstrate straightforward discrimination between heterozygous cis and trans mutations and between different strains of the SARS-CoV-2 virus. Our findings indicate that heteromultivalent hybridization offers substantial improvements over conventional monovalent hybridization-based methods.

Tuning Plant Promoters Using a Simple Split Luciferase Method to Assess Transcription Factor-DNA Interactions.

Sequence features, including the affinity of binding motifs for their cognate transcription factors, are important contributors to promoter behavior. The ability to predictably recode affinity enables the development of synthetic promoters with varying levels of response to known cellular signals. Here we describe a luminescence-based microplate assay for comparing the interactions of transcription factors with short DNA probes. We then demonstrate how these data can be used to design synthetic plant promoters of varying strengths that respond to the same transcription factor.

Introduction to bioimaging‐based spatial multi‐omic novel methods

In this review, we introduced five different multiplex FISH methods used for image‐based spatial multi‐omics: seqFISH+, merFISH, DNA seqFISH+, DNA merFISH, and MINA. We provided a systematic collective perspective to review these FISH methods that could significantly benefit researchers on conducting their studies in the field. Our study provided an informative survey on these multiplex FISH methods. Therefore, this review would provide better understanding for researchers in the community to help them select the proper method, in order to understand the molecular mechanism in life sciences.BackgroundSpatial multi‐omics are demonstrated to be a powerful method to assist researchers on genetic studies. In this review, bioimaging‐based spatial multi‐omics techniques such as seqFISH+, merFISH, integrated DNA seqFISH+, DNA merFISH, and MINA are introduced along with each technique’s probe design, development, and imaging processes.ResultsseqFISH employed 4–5 fluorophores to barcode and conducted multiple rounds of hybridization, in order that mRNA can be identified through color‐coding. seqFISH+ added 60 pseudo‐color and distributed them equally into three channels to enhance imaging power, in order that i.e., 24,000 genes can be imaged in total. merFISH utilized 4 out 16 Hamming distance to innovatively provide a robust error‐detecting method. MINA, a methodology combining merFISH (multiplexed error‐robust fluorescence in situ hybridization) and chromosomal tracing, enabled multiplexed genomic architecture imaged in mammalian single cells. Optical reconstruction of chromatin architecture (ORCA) a method that could conduct DNA path tracing in nanoscale manner with kilobase resolution, an FISH variation that improved genetic resolution, enable high‐precision fiducial registration and sequential imaging, and utilized Oligopaint probe to hybridize the short genomic region ranging from 2 to 10 kilobase. ORCA then prescribes these short section primary probes with individual barcodes to attach fluorophore and to be imaged.ConclusionThis review concentrated on providing a comprehensive overview for these spatial‐multi‐omics techniques with the intention on helping researchers on selecting appropriate technique for their research.

Ultrafast Hidden Spin Polarization Dynamics of Bright and Dark Excitons in 2H-WSe_{2}.

We performed spin-, time- and angle-resolved extreme ultraviolet photoemission spectroscopy of excitons prepared by photoexcitation of inversion-symmetric 2H-WSe_{2} with circularly polarized light. The very short probing depth of XUV photoemission permits selective measurement of photoelectrons originating from the top-most WSe_{2} layer, allowing for direct measurement of hidden spin polarization of bright and momentum-forbidden dark excitons. Our results reveal efficient chiroptical control of bright excitons' hidden spin polarization. Following optical photoexcitation, intervalley scattering between nonequivalent K-K^{'} valleys leads to a decay of bright excitons' hidden spin polarization. Conversely, the ultrafast formation of momentum-forbidden dark excitons acts as a local spin polarization reservoir, which could be used for spin injection in van der Waals heterostructures involving multilayer transition metal dichalcogenides.

Blocker-dUThiophene poly tailing-based method for assessing methyl transferase activity.

In this report, we present a method for the selective and sensitive detection of methyl transferase activity. The method uses a dsDNA probe that contains C3 spacers and is coupled with dUThioTP-TdT polymerase-based poly-tailing. The short dsDNA probe is designed with C3 spacers at both 3' ends to prevent any type of tailing reaction. However, the probe contains a methyl transferase recognition sequence that can methylate adenosines in the palindromic part of both strands. When a specific DpnI endonuclease is introduced, it selectively cleaves the dsDNA probe such that both strands are methylated, unblocking the probe into two separate dsDNA forms with exposed 3' OH groups. This makes the probe susceptible to tailing in the presence of a TdT tailing polymerase. The unblocked probe is then subjected to fluorescent dUThioTP-based tailing, which produces a strong fluorescent signal that indicates the presence of methyl transferase activity. In the absence of methyl transferase, the probe remains in the blocked state and does not undergo fluorescence. This method has a limit of detection of 0.049 U/mL with good selectivity and the potential for accurate MTase analysis.

Brillouin-Scattering Induced Noise in DAS: A Case Study

In the paper, the effect of spontaneous Brillouin scattering (SpBS) is analyzed as a noise source in distributed acoustic sensors (DAS). The intensity of the SpBS wave fluctuates over time, and these fluctuations increase the noise power in DAS. Based on experimental data, the probability density function (PDF) of the spectrally selected SpBS Stokes wave intensity is negative exponential, which corresponds to the known theoretical conception. Based on this statement, an estimation of the average noise power induced by the SpBS wave is given. This noise power equals the square of the average power of the SpBS Stokes wave, which in turn is approximately 18 dB lower than the Rayleigh backscattering power. The noise composition in DAS is determined for two configurations, the first for the initial backscattering spectrum and the second for the spectrum in which the SpBS Stokes and anti-Stokes waves are rejected. It is established that in the analyzed particular case, the SpBS noise power is dominant and exceeds the powers of the thermal, shot, and phase noises in DAS. Accordingly, by rejecting the SpBS waves at the photodetector input, it is possible to reduce the noise power in DAS. In our case, this rejection is carried out by an asymmetric Mach-Zehnder interferometer (MZI). The rejection of the SpBS wave is most relevant for broadband photodetectors, which are associated with the use of short probing pulses to achieve short gauge lengths in DAS.

Modification of DNA by Genetic Engineering

Abstract: Genetic engineering modifies a living organism's DNA in a lab. Altering a single DNA base pair (A-T or C-G), deleting or inserting DNA, or employing all three approaches together may accomplish this purpose. Inserting a gene with a desired characteristic into a separate species' genome is one use of genetic engineering. Genetic modification has been utilized to develop cancer drugs, brewing yeasts, GM crops and animals, and other commercial and scientific items. Probe tests on genomic DNA and RNA can identify certain sequences. Short DNA probes may be detected using radioactive or fluorescent reagents. Gel electrophoresis separates different-sized nucleic acid fragments. Blotting transfers gel particles to nylon membranes. Radioactive or fluorescent probe sequences may identify trans-membrane nucleic acid components. Southern blotting transfers DNA to a nylon membrane, whereas northern blotting transfers RNA. Northern blots identify gene expression, whereas southern blots examine nucleotide patterns. Below is a reversed gel electrophoresis image. Gel electrophoresis shows the band is concentrated in two locations. After PCR amplification, the snapshot was taken. The band suggests a substantial quantity of plasmid DNA in the sample. The outside area also has a few more distinct bands. This means the bands are localized and accumulation is lesser.

Analysis of picosecond coherent anti-Stokes Raman spectra for gas-phase diagnostics

We present a hybrid frequency- and time-domain solution, applicable to the case of picosecond coherent anti-Stokes Raman scattering (CARS), for gas-phase diagnostics. A solution has been derived based on both physical arguments and four-wave mixing equations for picosecond CARS, with pulse durations that are comparable to the dephasing time scale for gas-phase Raman coherence—a regime where commonly employed solutions for impulsive (femtosecond) or cw (nanosecond) pump/Stokes forcing are not strictly valid. We present the ps-CARS spectrum in the form of incoherent sums of CARS intensity spectra, calculated from the fundamental solution for impulsive pump/Stokes Raman preparation. The solution was examined for temperatures from 1000–3000 K, for four plausible experimental configurations, with laser pulse durations of 50–150 ps, and probe pulse delays from −20 to 240 ps. Approximations based on cw and impulsive pump/Stokes preparation to fit picosecond CARS spectra at atmospheric pressure were examined and the relative thermometric accuracy and computational cost of these approximations were quantified for the case of a zero nonresonant CARS contribution, and a nonresonant susceptibility equal to 10% of the Raman-resonant value at the N2 bandhead. The nanosecond CARS approximation can result in large fitting errors when the probe pulse time delay is less than the probe pulse duration. Errors as large as 10–20% are observed in the fit temperatures for a zero picosecond probe pulse delay, when the nonresonant background is neglected, largely due to an inability of the time-independent cw model to capture transient frequency spread dephasing effects at the Q-branch bandhead. The inclusion of a nonresonant background results in 40–60% thermometry errors with a nanosecond model at a zero-probe delay. Time-dependent impulsive calculations used for femtosecond CARS better approximate the structure of the N2 bandhead, reducing temperature fitting errors to 5–10% at a short probe pulse delay. The impulsive approximation results in errors up to 10% at intermediate probe pulse delays, where the coherence of the pump and probe pulses leads to multiple terms in the picosecond CARS solution. Both approximations improve as the probe pulse delay exceeds the probe duration. The nanosecond approximation results in a 2–3% error, while the impulsive model results in differences of less than 1% in some cases. Fits to experimental data obtained using short, ∼60ps pulses at a zero probe time delay and longer 100 ps pulses at a substantial 200 ps delay are presented with accuracies of 1–3% in the fit temperature.

Nanoparticle-Coupled Single-Molecule Kinetic Fingerprinting for Enzymatic Activity Detection.

The sensitive and accurate detection of biomarkers plays an important role in clinical diagnosis and drug discovery. Currently, amplification-based methods for biomarker detection are widely explored. However, the key challenges of these methods are limited reproducibility and high background noise. To overcome these limitations, we develop a robust plasmonic nanoparticle-coupled single-molecule kinetic fingerprinting (PNP-SMKF) method to achieve ultrasensitive detection of protein kinase A (PKA). Transient binding of a short fluorescent probe with the genuine target produces a distinct kinetic signature that is completely different from that of the background signal, allowing us to recognize PKA sensitively. Importantly, integrating a plasmonic nanoparticle efficiently breaks the concentration limit of the imager strand for single-molecule imaging, thus achieving a much faster imaging speed. A limit of detection (LOD) of as low as 0.0005 U/mL is readily realized. This method holds great potential as a versatile platform for enzyme detection and inhibitor screening in the future.