Multiplex Detection of Foodborne Pathogens using 3D Nanostructure Swab and Deep Learning-Based Classification of Raman Spectra.

Proactive management of foodborne illness requires routine surveillance of foodborne pathogens, which requires developing simple, rapid, and sensitive detection methods. Here, a strategy is presented that enables the detection of multiple foodborne bacteria using a 3D nanostructure swab and deep learning-based Raman signal classification. The nanostructure swab efficiently captures foodborne pathogens, and the portable Raman instrument directly collects the Raman signals of captured bacteria. a deep learning algorithm has been demonstrated, 1D convolutional neural network with binary labeling, achieves superior performance in classifying individual bacterial species. This methodology has been extended to mixed bacterial populations, maintaining accuracy close to 100%. In addition, the gradient-weighted class activation mapping method is used to provide an investigation of the Raman bands for foodborne pathogens. For practical application, blind tests are conducted on contaminated kitchen utensils and foods. The proposed technique is validated by the successful detection of bacterial species from the contaminated surfaces. The use of a 3D nanostructure swab, portable Raman device, and deep learning-based classification provides a powerful tool for rapid identification (≈5 min) of foodborne bacterial species. The detection strategy shows significant potential for reliable food safety monitoring, making a meaningful contribution to public health and the food industry.

Electrospun Nanofibrous Membrane-Based Colorimetric Device for Rapid and Simple Screening of Amphetamine-Type Stimulants in Drinks.

With the growth of drug-facilitated crimes, prevention has become increasingly important. Although various drug detection technologies exist, most focus on postconsumption detection. However, the prevention of drug-facilitated crimes requires technology for the quick and easy detection of amphetamine-type stimulants (ATSs) before ingestion. Herein, drug screening kits (DSKs) were developed for the simple detection of ATSs in drinks. The DSKs consisted of polydiacetylene nanofiber-based paper sensors fabricated by electrospinning with 10,12-pentacosadiynoic acid (PCDA) and PCDA-dopamine as sensing materials that can bind ATSs via hydrogen bonding and π-π interactions. Dropping a drink on the DSK provided an immediate visual indication of the presence of ATSs. When ATSs were present in the drink, the color of the DSK clearly changed from blue to red, with the increase in red intensity being more than twofold greater than that observed when water alone was tested. Notably, the result could be confirmed by the naked eye without any analytical instrumentation. A color change indicating the presence of ATSs was successfully observed in various alcoholic and nonalcoholic drinks. These results indicate the potential of DSKs for preventing drug-facilitated crimes caused by unwanted drug intake.

FRET-based hACE2 receptor mimic peptide conjugated nanoprobe for simple detection of SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has led to a pandemic of acute respiratory disease, namely coronavirus disease (COVID-19). This disease threatens human health and public safety. Early diagnosis, isolation, and prevention are important to suppress the outbreak of COVID 19 given the lack of specific antiviral drugs to treat this disease and the emergence of various variants of the virus that cause breakthrough infections even after vaccine administration. Simple and prompt testing is paramount to preventing further spread of the virus. However, current testing methods, namely RT-PCR, is time-consuming. Binding of the SARS-CoV-2 spike (S) glycoprotein to human angiotensin-converting enzyme 2 (hACE2) receptor plays a pivotal role in host cell entry. In the present study, we developed a hACE2 mimic peptide beacon (COVID19-PEB) for simple detection of SARS-CoV-2 using a fluorescence resonance energy transfer system. COVID19-PEB exhibits minimal fluorescence in its ''closed'' hairpin structure; however, in the presence of SARS-CoV-2, the specific recognition of the S protein receptor-binding domain by COVID19-PEB causes the beacon to assume an ''open'' structure that emits strong fluorescence. COVID19-PEB can detect SARS-CoV-2 within 3 h or even 50 min and exhibits strong fluorescence even at low viral concentrations, with a detection limit of 4 × 103 plaque-forming unit/test. Furthermore, in SARS-CoV-2-infected patient samples confirmed using polymerase chain reaction, COVID19-PEB accurately detected the virus. COVID19-PEB could be developed as a rapid and accurate diagnostic tool for COVID-19.

Investigation of Flow Changes in Intracranial Vascular Disease Models Constructed with MRA Images.

This study aimed to develop a magnetic resonance imaging (MRI)-compatible flow delivery system and individualized models of circle of Willis (CoW), which include 50% and 100% blockage in internal carotid artery (ICA50 and ICA100), and 100% blockage in vertebral artery (VA100). Images were obtained using 3D time-of-flight and phase-contrast magnetic resonance angiography (MRA) sequences, and changes in velocity and flow direction at CoW models were analyzed. For the ICA50 and VA100 models, the flow was similar to that of the normal model. For the ICA 50 model, it was found that 50% blockage did not affect cerebral blood flow. For the VA100 model, decreased flow in the posterior cerebral artery and a change to the flow direction in the posterior communicating artery were found. For the ICA100 model, particularly, decreased flow in the ipsilateral middle and anterior cerebral arteries and a change to the flow direction in the ipsilateral anterior cerebral artery of the CoW were found. These results demonstrated that the flow system with various CoW disease models tailored to individual characteristics could be used to predict stroke onset more quickly. For the ICA50 and VA100 models, the possibility of cerebral infarction was significantly lower. On the other hand, for the ICA100 model, there was a high possibility of decreased flow, which could lead to cerebral infarction.

MRI-Compatible Microcirculation System Using Ultrasonic Pumps for Microvascular Imaging on 3T MRI.

The diagnosis of small vessel disease is attracting interest; however, it remains difficult to visualize the microvasculature using 3 Tesla (T) magnetic resonance imaging (MRI). Therefore, this study aimed to visualize the microvascular structure and measure a slow flow on 3T MRI. We developed a microcirculation system using piezoelectric pumps connected to small tubes (0.4, 0.5, 0.8, and 1.0 mm) and evaluated various MR sequences and imaging parameters to identify the most appropriate acquisition parameters. We found that the system could image small structures with a diameter of 0.5 mm or more when using a 1 m-long tube (maximal signal intensity of 241 in 1 mm, 199 in 0.8 mm, and 133 in 0.5 mm). We also found that the highest signal-to-noise ratio (SNR) appeared on 2-dimensional time-of-flight low-resolution imaging and that the flow velocity (10.03 cm/s) was similar to the actual velocity (11.01 cm/s in a flowmeter) when velocity encoding of 30 cm/s was used in a 0.8 mm-diameter tube. In conclusion, this study demonstrates that a microcirculation system can be used to image small vessels. Therefore, our results could serve as a basis for research on vessels' anatomical structure and pathophysiological function in small vessel disease.

Novel Technique to Measure Pulse Wave Velocity in Brain Vessels Using a Fast Simultaneous Multi-Slice Excitation Magnetic Resonance Sequence.

In this study, we proposed a novel pulse wave velocity (PWV) technique to determine cerebrovascular stiffness using a 3-tesla magnetic resonance imaging (MRI) to overcome the various shortcomings of existing PWV techniques for cerebral-artery PWV, such as long scan times and complicated procedures. The technique was developed by combining a simultaneous multi-slice (SMS) excitation pulse sequence with keyhole acquisition and reconstruction (SMS-K). The SMS-K technique for cerebral-artery PWV was evaluated using phantom and human experiments. In the results, common and internal carotid arteries (CCA and ICA) were acquired simultaneously in an image with a high temporal resolution-of 48 ms for one measurement. Vascular signals at 500 time points acquired within 30 s could generate pulse waveforms of CCA and ICA with 26 heartbeats, allowing for the detection of PWV changes over time. The results demonstrated that the SMS-K technique could provide more PWV information with a simple procedure within a short period of time. The procedural convenience and advantages of PWV measurements will make it more appropriate for clinical applications.

Brain activity during a working memory task in different postures: an EEG study.

While working is more comfortable in a supine position and healthier in a standing, most people work in a sitting. However, it is unclear whether there are differences in brain activity efficiency in different postures. Here, we, therefore, compared changes in brain activity across three different postures to determine the optimal posture for performing working memory tasks. Their effect on brain activity was examined using EEG signals together with the information of accuracy and reaction times during 2-back task in 24 subjects. Substantial differences in brain waves were observed at sitting and standing positions compared to the supine, especially in delta waves and frontal lobe, where is known to improve the modulation of brain activity efficiently. Brain efficiency was higher during standing and sitting than in a supine. These findings show that postural changes may affect the efficiency of brain activity during working memory tasks. Practitioner summary: Differences in brain efficiency between different postures during working memory tasks have not been explored. This study suggests that efficiency in several brain areas is higher during sitting and standing than in a supine position. This finding has important implications regarding workplace environments. Furthermore, this result would be useful to improve accomplishment and reduce negative effects of work posture. Abbreviations: EEG: electroencephalogram; PSQI: Pittsburgh sleep quality index; KSS: Karolinska sleepiness scale; FFT: fast fourier transform; ROI: region of interest; ANS: autonomic nervous system; Fp: prefrontal; AF: anterior frontal; frontal; Fz: midline frontal; temporal; central; Cz: midline central; P: parietal; Pz: midline parietal; O: occipital; Oz: midline occipital.

Anti-Wrinkle Benefits of Peptides Complex Stimulating Skin Basement Membrane Proteins Expression.

The dermal-epidermal junction (DEJ) provides a physical and biological interface between the epidermis and the dermis. In addition to providing a structural integrity, the DEJ also acts as a passageway for molecular transport. Based on the recently reported importance of the DEJ in skin aging, novel peptide derivatives have been tested for their effects on basement membrane (BM) protein expressions in cultured human epidermal keratinocytes. As a result, protein expressions of collagen XVII, laminin and nidogen were stimulated by the test peptide and peptides complex. Further ex vivo evaluation using excised human skin, confirmed that the topical application of the peptides complex significantly increased dermal collagen expression, as well as expressions of collagen XVII and laminin. Interestingly, while the origin of the laminin protein is epidermal keratinocytes, the immunohistochemical staining of skin showed that laminin was only detected in the uppermost layer of the dermis, which suggests a tight assembly of laminin protein onto the dermal side of the DEJ. These results suggest that a peptide complex could improve the structural properties of the DEJ through its ability to stimulate BM proteins. In order to evaluate the anti-wrinkle benefits of the peptide complex in vivo, a clinical study was performed on 22 healthy Asian female volunteers older than 40 years. As a result, significant improvements in skin wrinkles for all of the five sites were observed after two weeks, as assessed by skin topographic measurements. Collectively, these results demonstrate the anti-aging efficacy of the peptides complex.

Fabrication of Oligomeric Avidin Scaffolds for Valency-Controlled Surface Display of Functional Ligands.

Multivalent surface display of biomolecules is crucial to study and utilize multivalent biological interactions. However, precise valency control of surface-displayed ligands remains extremely difficult. Now a series of new oligomeric avidin proteins were fabricated that allow facile control of surface multivalency of biotinylated ligands. Naturally dimeric rhizavidin (RA) was engineered to form a mixture of oligomeric avidin assemblies, and discrete RA oligomers from the dimer to octamer of RA, were homogeneously prepared. These oligomeric avidins are in polygonal forms with expected numbers of stable biotin binding sites. Upon immobilization on low-density biotin-coated gold surfaces, RA dimer, trimer, and tetramer scaffolds provided accurate mean residual valencies of 2, 3, and 4, respectively, for biotinylated proteins. Valency-controlled display of antibody binding protein G on these RA surfaces showed clear valency-dependent enhancement of antibody capturing stability.

Crystal structures of two forms of the Acanthamoeba polyphaga mimivirus Rab GTPase.

Acanthamoeba polyphaga mimivirus (APMV) is a member of the family of giant viruses, harboring a 1,200 kbp genome within its 700 nm-diameter viral particle. The R214 gene of the APMV genome was recently shown to encode a homologue of the Rab GTPases, molecular switch proteins known to play a pivotal role in the regulation of membrane trafficking that were considered to exist only in eukaryotes. Herein, we report the first crystal structures of GDP- and GTP-bound forms of APMV Rab GTPase, both of which were determined at high resolution. An in-depth structural comparison of APMV Rab with each other and with mammalian Rab homologues led to an atomic-level elucidation of the inactive-active conformational change upon GDP/GTP exchange. APMV Rab GTPase exhibited considerable structural similarity to human Rab5, as previously predicted based on its amino acid sequence. However, it also contains unique structural features differentiating it from mammalian homologues, such as the functional substitution of a phenylalanine residue for the stabilization of the nucleotide's guanine base.

In vivo deletion of CAR resulted in high bone mass phenotypes in male mice.

Constitutive androstane receptor (CAR) was originally identified as xenobiotic sensor that regulates the expression of cytochrome P450 genes. However, recent studies suggest that this nuclear receptor is also involved in the regulation of energy metabolism including glucose and lipid homeostasis. This study investigated the role of CAR in the regulation of bone mass in vivo using CAR(-/-) mice. Endogenous mRNA expression of CAR was observed in both primary osteoblasts and osteoclast precursors. CAR(-/-) mice have exhibited significant increase in whole body bone mineral density (BMD) by 9.5% (P < 0.01) and 5.5% (P < 0.05) at 10 and 15 weeks of age, respectively, compared with WT mice in males. Microcomputed tomography analysis of proximal tibia demonstrated a significant increase in trabecular bone volume (62.7%), trabecular number (54.1%) in male CAR(-/-) mice compared with WT mice. However, primary culture of calvarial cells exhibited no significant changes in osteogenic differentiation potential between CAR(-/-) and WT. In addition, the number of tartrate-resistant acid-phosphatase positive osteoclasts in the femur and serum level of CTx was not different between CAR(-/-) and WT mice. The higher BMD and microstructural parameters were not observed in female mice. Interestingly, serum level of testosterone in male CAR(-/-) mice was 2.5-fold higher compared with WT mice and the mRNA expressions of Cyp2b9 and 2b10 in the liver, which regulate testosterone metabolism, were significantly down-regulated in male CAR(-/-) mice. Furthermore, the difference in BMD between CAR(-/-) and WT mice disappeared at 8 weeks after performing orchiectomy. CAR(-/-) mice also exhibited significant increase in serum 1,25(OH)2 D3 levels but Cyp 27B1 which converts 25(OH)D3 to 1,25(OH)2 D3 was significantly down-regulated compared to WT mice. These results suggest that in vivo deletion of CAR resulted in higher bone mass, which appears to be a result from reduced metabolism of testosterone due to down-regulation of Cyp2b.

Highly improved specificity for hybridization-based microRNA detection by controlled surface dissociation.

Poor specificity has been a lingering problem in many microRNA profiling methods, particularly surface hybridization-based methods such as microarrays. Here, we carefully investigated surface hybridization and dissociation processes of a number of sequentially similar microRNAs against nucleic acid capture probes. Single-base mismatched microRNAs were similarly hybridized to a complementary DNA capture probe and thereby poorly discriminated during conventional stringent hybridization. Interestingly, however, mismatched microRNAs showed significantly faster dissociation from the probe than the perfectly matched microRNA. Systematic analysis of various washing conditions clearly demonstrated that extremely high specificity can be obtained by releasing non-specific microRNAs from assay surfaces during a stringent and controlled dissociation step. For instance, compared with stringent hybridization, surface dissociation control provided up to 6-fold better specificity for Let-7a detection than for other Let-7 family microRNAs. In addition, a synthetically introduced single-base mismatch on miR206 was almost completely discriminated by optimized surface dissociation of captured microRNAs, while this mismatch was barely distinguished from target miR206 during stringent hybridization. Furthermore, a single dissociation condition was successfully used to simultaneously measure four different microRNAs with extremely high specificity using melting temperature-equalized capture probes. The present study on selective dissociation of surface bound microRNAs can be easily applied to various hybridization based detection methods for improved specificity.

Effect of Forward Head Posture on Resting State Brain Function.

Forward head posture (FHP) is a common postural problem experienced by most people. However, its effect on brain activity is still unknown. Accordingly, we aimed to observe changes in brain waves at rest to determine the effect of FHP on the nervous systems. A total of 33 computer users (Male = 17; Female = 16; age = 22.18 ± 1.88) were examined in both FHP and neutral posture. For each session, brain waves were measured for 5 min, and then muscle mechanical properties and cranio-vertebral angle (CVA) were measured. Changes in brain waves between the neutral posture and FHP were prominent in gamma waves. A notable increase was confirmed in the frontal and parietal lobes. That is, eight channels in the frontal lobe and all channels in the parietal lobe showed a significant increase in FHP compared to neutral posture. Additionally, FHP changes were associated with a decrease in CVA (p < 0.001), an increase in levator scapulae tone (Right, p = 0.014; Left, p = 0.001), and an increase in right sternocleidomastoid stiffness (p = 0.002), and a decrease in platysma elasticity (Right, p = 0.039; Left, p = 0.017). The change in CVA was found to have a negative correlation with the gamma activity (P7, p = 0.044; P8, p = 0.004). Therefore, increased gamma wave activity in FHP appears to be related to CVA decrease due to external force that was applied to the nervous system and cervical spine.

PoreGlow: A split green fluorescent protein-based system for rapid detection of Listeria monocytogenes.

Listeria monocytogenes, a severe foodborne pathogen causing severe diseases underscores the necessity for the development of a detection system with high specificity, sensitivity and utility. Herein, the PoreGlow system, based on split green fluorescent protein (GFP), was developed and assessed for the fast and accurate detection of L. monocytogenes. Split GFP-encapsulated liposomes were optimized for targeted analysis. The system utilizes listeriolysin O (LLO), a toxin produced by L. monocytogenes that enlarges the pores split GFP-encapsulated liposomes, to detect L. monocytogenes by measuring the fluorescent signal generated when the encapsulated GFP is released and reacted with the externally added fragment of the split GFP. The system exhibited a limit of detection of 0.17 µg/ml for LLO toxin and 10 CFU/mL for L. monocytogenes with high sensitivity and specificity and no cross-reactivity with other bacteria. The PoreGlow system is practical, rapid, and does not require sample pre-treatment, making it a promising tool for the early detection of L. monocytogenes in food products, which is crucial for preventing outbreaks and protecting public health.

On-site detection of methicillin-resistant Staphylococcus aureus (MRSA) utilizing G-quadruplex based isothermal exponential amplification reaction (GQ-EXPAR).

Methicillin-resistant Staphylococcus aureus (MRSA) has a high incidence in infectious hospitals and communities, highlighting the need for early on-site detection due to its resistance to methicillin antibiotics. The present study introduces a highly sensitive detection system for mecA, a crucial methicillin marker, utilizing an RCA-based isothermal exponential amplification reaction. The G-quadruplex-based isothermal exponential amplification reaction (GQ-EXPAR) method designs probes to establish G-quadruplex secondary structures incorporating thioflavin T for fluorescence. The system, unlike conventional genetic detection methods, works with portable isothermal PCR devices (isoQuark), facilitating on-site detection. A detection limit of 0.1 fmol was demonstrated using synthetic DNA, and effective detection was proven using thermal lysis. The study also validated the detection of targets swabbed from surfaces within bacterial 3D nanostructures using the GQ-EXPAR method. After applying complementary sequences to the padlock probe for the target, the GQ-EXPAR method can be used on various targets. The developed method could facilitate rapid and accurate diagnostics within MRSA strains.

Dual structure-switching aptamer-mediated signal amplification cascade for SARS-CoV-2 detection.

Since the outbreak of the novel severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) at the end of 2019, the spread of the virus has posed a significant threat to public health and the global economy. This work proposed a one-step, dual-structure-switching aptamer-mediated signal amplification cascade for rapid and sensitive detection of the SARS-CoV-2 nucleocapsid protein. This system consisted of two DNA aptamers with structure-switching functionality and fuel DNA, where a cascade of strand hybridization and displacement triggered fluorescence generation and signal amplification. This aptamer-based amplification cascade required neither an amplification stage using enzymes nor pre-processing steps such as washing, viral isolation, and gene extraction. The assay could distinguish SARS-CoV-2 from other respiratory viruses and detect up to 1.0 PFU/assay of SARS-CoV-2 within 30 min at room temperature. In 35 nasopharyngeal clinical samples, the assay accurately assessed 25 positive and 10 negative clinical swab samples, which were confirmed using quantitative polymerase chain reaction. The strategy reported herein can help detect newly emerging pathogens and biomarkers of various diseases in liquid samples. In addition, the developed detection system consisting of only DNA and fluorophores can be widely integrated into liquid biopsy platforms for disease diagnosis.

Hybrid CRISPR/Cas protein for one-pot detection of DNA and RNA.

Clustered regularly interspaced short palindromic repeats (CRISPR)-based diagnostics have emerged as next-generation molecular diagnostics. In CRISPR-based diagnostics, Cas12 and Cas13 proteins have been widely employed to detect DNA and RNA, respectively. Herein, we developed a novel hybrid Cas protein capable of detecting universal nucleic acids (DNA and RNA). The CRISPR/hybrid Cas system simultaneously recognizes both DNA and RNA, enabling the dual detection of pathogenic viruses in a single tube. Using wild-type (WT) and N501Y mutant severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as detection models, we successfully detected both virus strains with a detection limit of 10 viral copies per reaction without cross-reactivity. Furthermore, it is demonstrated the detection of WT SARS-CoV-2 and N501Y mutant variants in clinical samples by using the CRISPR/hybrid Cas system. The hybrid Cas protein is expected to be utilized in a molecular diagnostic method for infectious diseases, tissue and liquid biopsies, and other nucleic acid biomarkers.

ANCA: artificial nucleic acid circuit with argonaute protein for one-step isothermal detection of antibiotic-resistant bacteria.

Endonucleases have recently widely used in molecular diagnostics. Here, we report a strategy to exploit the properties of Argonaute (Ago) proteins for molecular diagnostics by introducing an artificial nucleic acid circuit with Ago protein (ANCA) method. The ANCA is designed to perform a continuous autocatalytic reaction through cross-catalytic cleavage of the Ago protein, enabling one-step, amplification-free, and isothermal DNA detection. Using the ANCA method, carbapenemase-producing Klebsiella pneumoniae (CPKP) are successfully detected without DNA extraction and amplification steps. In addition, we demonstrate the detection of carbapenem-resistant bacteria in human urine and blood samples using the method. We also demonstrate the direct identification of CPKP swabbed from surfaces using the ANCA method in conjunction with a three-dimensional nanopillar structure. Finally, the ANCA method is applied to detect CPKP in rectal swab specimens from infected patients, achieving sensitivity and specificity of 100% and 100%, respectively. The developed method can contribute to simple, rapid and accurate diagnosis of CPKP, which can help prevent nosocomial infections.

Conductive Thread-Based Immunosensor for Pandemic Influenza A (H1N1) Virus Detection.

Infectious agents such as viruses pose significant threats to human health, being transmitted via direct contact as well as airborne transmission without direct contact, thus requiring rapid detection to prevent the spread of infectious diseases. In this study, we developed a conductive thread-based immunosensor (CT-IS), a biosensor to easily detect the presence of airborne viruses. CT-IS utilizes an antibody that specifically recognizes the HA protein of the pandemic influenza A (pH1N1) virus, which is incorporated into the conductive thread. The antigen-antibody interaction results in increased strain on the conductive thread in the presence of the pH1N1 virus, resulting in increased electrical resistance of the CT-IS. We evaluated the performance of this sensor using the HA protein and the pH1N1 virus, in addition to samples from patients infected with the pH1N1 virus. We observed a significant change in resistance in the pH1N1-infected patient samples (positive: n = 11, negative: n = 9), whereas negligible change was observed in the control samples (patients not infected with the pH1N1 virus; negative). Hence, the CT-IS is a lightweight fiber-type sensor that can be used as a wearable biosensor by combining it with textiles, to detect the pH1N1 virus in a person's vicinity.

A portable smartphone-based colorimetric sensor that utilizes dual amplification for the on-site detection of airborne bacteria.

Over the past few years, infections caused by airborne pathogens have spread worldwide, infecting several people and becoming an increasingly severe threat to public health. Therefore, there is an urgent need for developing airborne pathogen monitoring technology for use in confined environments to enable epidemic prevention. In this study, we designed a colorimetry-based bacterial detection platform that uses a clustered regularly interspaced short palindromic repeat-associated protein 12a system to amplify signals and a urease enzyme to induce color changes. Furthermore, we have developed a smartphone application that can distinguish colors under different illumination conditions based on the HSV model and detect three types of disease-causing bacteria. Even synthetic oligomers of a few picomoles of concentration and genomic DNA of airborne bacteria smaller than several nanograms can be detected with the naked eye and using color analysis systems. Furthermore, in the air capture model system, the bacterial sample generated approximately a 2-fold signal difference compared with that in the control group. This colorimetric detection method can be widely applied for public safety because it is easy to use and does not require complex equipment.