Increased Cerebrospinal Fluid Adenosine 5'-Triphosphate Levels in Patients with Guillain-Barré Syndrome and Chronic Inflammatory Demyelinating Polyneuropathy.

Background: Extracellular adenosine 5'-triphosphate (ATP) acts as a signaling molecule in the peripheral nerves, regulating myelination after nerve injury. The present study examined whether the cerebrospinal fluid (CSF) ATP levels in patients with Guillain-Barré syndrome (GBS) and chronic inflammatory demyelinating polyneuropathy (CIDP) are related to disease severity. Methods: CSF ATP levels in 13 patients with GBS and 18 patients with CIDP were compared with those in a control group of 16 patients with other neurological diseases (ONDs). In patients with CIDP, CSF ATP levels were compared before and after treatment. The correlations between CSF ATP levels and other factors, including clinical data and CSF protein levels, were also evaluated. Results: Median CSF ATP levels were significantly higher in patients with GBS and CIDP than in those with ONDs. When patients with CIDP were classified into two groups depending on their responsiveness to immunotherapy, median CSF ATP levels were significantly higher in good responders than in ONDs. CSF ATP levels tended to decrease after treatment in patients with CIDP. In patients with CIDP, there is a negative correlation between CSF ATP and CSF protein levels. Conclusions: CSF ATP levels were increased in patients with GBS and CIDP. In particular, CSF ATP levels tended to decrease following treatment in patients with CIDP. CSF ATP levels may be useful biomarkers for the diagnosis or monitoring of therapeutic effects in patients with GBS and CIDP.

Melting temperature mapping method using imperfect-match linear long probes.

Identifying pathogenic microorganisms as early as possible is critical for selecting the appropriate antimicrobial therapy in infected patients. We previously reported the development of the Tm mapping method for identifying a broad range of pathogenic bacteria within 3 h of blood collection. However, the Tm mapping identification requires an analytical instrument with a tube-to-tube variation of no more than 0.1 °C, so we can only use a few instruments that have such high thermal accuracy. To address the problem, we developed the improved Tm mapping method using imperfect-match linear long quenching probes (IMLL Q-probes). Using IMLL Q-probes, almost all commercially available analytical instruments can be used for the Tm mapping method. Some bacterial species cannot be narrowed down to one species, but they can at least be narrowed down to the genus level. The Tm mapping method using IMLL Q-probes is useful for deciding on antimicrobial therapy in infected patients.

Establishing Monoclonal Gammopathy of Undetermined Significance as an Independent Pre-Disease State of Multiple Myeloma Using Raman Spectroscopy, Dynamical Network Biomarker Theory, and Energy Landscape Analysis.

Multiple myeloma (MM) is a cancer of plasma cells. Normal (NL) cells are considered to pass through a precancerous state, such as monoclonal gammopathy of undetermined significance (MGUS), before transitioning to MM. In the present study, we acquired Raman spectra at three stages-834 NL, 711 MGUS, and 970 MM spectra-and applied the dynamical network biomarker (DNB) theory to these spectra. The DNB analysis identified MGUS as the unstable pre-disease state of MM and extracted Raman shifts at 1149 and 1527-1530 cm-1 as DNB variables. The distribution of DNB scores for each patient showed a significant difference between the mean values for MGUS and MM patients. Furthermore, an energy landscape (EL) analysis showed that the NL and MM stages were likely to become stable states. Raman spectroscopy, the DNB theory, and, complementarily, the EL analysis will be applicable to the identification of the pre-disease state in clinical samples.

Novel rapid method for identifying and quantifying pathogenic bacteria within four hours of blood collection.

Sepsis is life-threatening organ dysfunction and is considered a major cause of health loss. However, since the current biomarkers of sepsis reflect the host's immune response to microorganisms, they would inevitably cause a time-lag. This means that there is still no truly reliable biomarker of sepsis. In the present study, we developed a novel method for identifying and quantifying unknown pathogenic bacteria within four hours of sample collection. The most important point of this study is that the novel method can be used to determine the number of bacteria in a sample as a novel biomarker of infectious diseases. Indeed, based on the number of bacteria, we were able to accurately estimate the severity of microbial infection. Furthermore, using the time-dependent changes in the number of bacteria, we were able to monitor the therapeutic effect accurately. The rapid identification and quantification of bacteria may change our approach to medical care.

New Possibilities for Evaluating the Development of Age-Related Pathologies Using the Dynamical Network Biomarkers Theory.

Aging is the slowest process in a living organism. During this process, mortality rate increases exponentially due to the accumulation of damage at the cellular level. Cellular senescence is a well-established hallmark of aging, as well as a promising target for preventing aging and age-related diseases. However, mapping the senescent cells in tissues is extremely challenging, as their low abundance, lack of specific markers, and variability arise from heterogeneity. Hence, methodologies for identifying or predicting the development of senescent cells are necessary for achieving healthy aging. A new wave of bioinformatic methodologies based on mathematics/physics theories have been proposed to be applied to aging biology, which is altering the way we approach our understand of aging. Here, we discuss the dynamical network biomarkers (DNB) theory, which allows for the prediction of state transition in complex systems such as living organisms, as well as usage of Raman spectroscopy that offers a non-invasive and label-free imaging, and provide a perspective on potential applications for the study of aging.

Duplex loop-mediated isothermal amplification assay for simultaneous detection of human and human male DNA.

OBJECTIVE: Screening of human and human male DNA is necessary for forensic DNA analyses. Although quantitative real-time PCR (qPCR) is commonly used for detecting and quantifying these DNA targets, its use as a screening tool is time-consuming and labor-intensive. To streamline and simplify the screening process, we aimed to develop a duplex loop-mediated isothermal amplification (LAMP) assay capable of simultaneously detecting human and human male DNA in a single tube. We assessed the duplex LAMP assay for forensic application. RESULTS: For our duplex LAMP assay, we have utilized two fluorescent probes with HEX and FAM fluorophores to specifically detect human and human male DNA, respectively. The HEX (human target) signal was detected from both the male and female DNA samples, and the FAM (male target) signal was detected from only the male DNA sample. This assay has a sensitivity of 10-1 pg of DNA for both targets. Additionally, we successfully detected the two targets in the DNA samples extracted from forensically relevant body fluids, including blood, saliva, semen, and vaginal secretions.

Predictive value of cell population data with Sysmex XN-series hematology analyzer for culture-proven bacteremia.

Background: Cell population data (CPD) parameters related to neutrophils, such as fluorescent light intensity (NE-SFL) and fluorescent light distribution width index (NE-WY), have emerged as potential biomarkers for sepsis. However, the diagnostic implication in acute bacterial infection remains unclear. This study assessed the diagnostic value of NE-WY and NE-SFL for bacteremia in patients with acute bacterial infections, and those associations with other sepsis biomarkers. Methods: Patients with acute bacterial infections were enrolled in this prospective observational cohort study. For all patients, a blood sample, with at least two sets of blood cultures, were collected at the onset of infection. Microbiological evaluation included examination of the blood bacterial load using PCR. CPD was assessed using Automated Hematology analyzer Sysmex series XN-2000. Serum levels of procalcitonin (PCT), interleukin-6 (IL-6), presepsin, and CRP were also assessed. Results: Of 93 patients with acute bacterial infection, 24 developed culture-proven bacteremia and 69 did not. NE-SFL and NE-WY were significantly higher in patients with bacteremia than in those without bacteremia (p < 0.005, respectively), and were significantly correlated with the bacterial load determined by PCR (r = 0.384 and r = 0.374, p < 0.005, respectively). To assess the diagnostic value for bacteremia, receiver operating characteristic curve analysis was used. NE-SFL and NE-WY showed an area under the curve of 0.685 and 0.708, respectively, while those of PCT, IL-6, presepsin, and CRP were 0.744, 0.778, 0.685, and 0.528, respectively. Correlation analysis showed that the levels of NE-WY and NE-SFL were strongly correlated with PCT and IL-6 levels. Conclusion: This study demonstrated that NE-WY and NE-SFL could predict bacteremia in a manner that may be different from that of other indicators. These findings suggest there are potential benefits of NE-WY/NE-SFL in predicting severe bacterial infections.

Sperm mRNA screening by reverse transcription-recombinase polymerase amplification (RT-RPA) assay: Decision-making for differential extraction.

The presence of sperm cells is an indicator for differential extraction on sexual assault samples. In general, sperm cells are identified by microscopic analysis; however, this conventional method takes time and effort, even for trained personnel. Here, we present a reverse transcription-recombinase polymerase amplification (RT-RPA) assay targeting sperm mRNA marker (PRM1). The RT-RPA assay requires only 40 min for PRM1 detection and demonstrates a sensitivity of 0.1 µL of semen. Our results indicate that the RT-RPA assay may be a rapid, simple, and specific strategy for screening sperm cells in sexual assault samples.

Periostin activates distinct modules of inflammation and itching downstream of the type 2 inflammation pathway.

Atopic dermatitis (AD) is a chronic relapsing skin disease accompanied by recurrent itching. Although type 2 inflammation is dominant in allergic skin inflammation, it is not fully understood how non-type 2 inflammation co-exists with type 2 inflammation or how type 2 inflammation causes itching. We have recently established the FADS mouse, a mouse model of AD. In FADS mice, either genetic disruption or pharmacological inhibition of periostin, a downstream molecule of type 2 inflammation, inhibits NF-κB activation in keratinocytes, leading to downregulating eczema, epidermal hyperplasia, and infiltration of neutrophils, without regulating the enhanced type 2 inflammation. Moreover, inhibition of periostin blocks spontaneous firing of superficial dorsal horn neurons followed by a decrease in scratching behaviors due to itching. Taken together, periostin links NF-κB-mediated inflammation with type 2 inflammation and promotes itching in allergic skin inflammation, suggesting that periostin is a promising therapeutic target for AD.

Loop-mediated isothermal amplification assay for fluorescence analysis and lateral flow detection of male DNA.

Male DNA screening is important in forensic investigations, such as sexual assault cases. Although quantitative real-time PCR is a robust method for detection of male DNA, it is time-consuming and labor-intensive. We herein report the development of a male DNA-targeted loop-mediated isothermal amplification (LAMP) assay that can be used for both laboratory-based fluorescence analysis and on-site lateral flow detection. The two detection systems are independent, but we streamlined the reaction before the detection by introducing a fluorescence probe and biotin-labeled primer into a single reaction. This allowed the evaluation of fluorescence signal followed by lateral flow detection. Both the fluorescence and lateral flow analyses detected as low as 10 pg of male DNA. We also integrated an alkaline lysis method with our LAMP assay. The direct assay successfully detected male DNA from forensic samples without purification. The workflow requires only <40 min for fluorescence analysis and <45 min for lateral flow detection. Furthermore, when combined with a lateral flow strip, this workflow does not require any sophisticated instruments. These findings suggest that our assay is a promising strategy for on-site male DNA screening as well as laboratory-based testing.

Improved reverse transcription-recombinase polymerase amplification assay for blood mRNA screening: comparison with one-step RT-qPCR assay.

mRNA profiling is effective for body fluid identification because of its sensitivity, specificity, and multiplexing capability. Body fluid mRNA markers can typically be detected using RT-qPCR, RT-PCR followed by capillary electrophoresis, or targeted RNA sequencing. However, due to the multiple handling steps involved, the analysis of many forensic samples using these methods requires time and effort. Here, we describe a rapid and simple method for detecting the blood mRNA marker hemoglobin ß (HBB), intended for use in screening before definitive blood identification. We employed a reverse transcription-recombinase polymerase amplification (RT-RPA) assay that can detect target mRNA within 20 min in a single tube. For comparison, we used a one-step RT-qPCR assay. We optimized the RT-RPA assay and found that it could detect HBB from 10-3-10-4 ng of leukocyte RNA and approximately 10-3 µL of blood. The sensitivity was 10-fold lower than that of the one-step RT-qPCR assay but higher than that of the comprehensive analysis methods for definitive blood identification. Thus, the rapidity and sensitivity of the RT-RPA assay support its use as a screening tool. We also found that the RT-RPA assay was highly tolerant to common inhibitors such as humic acid, hematin, tannic acid, and melanin. Considering the inhibitor tolerability, we integrated a simple lysis method (addition of TCEP/EDTA and heating at 95 °C for 5 min) without the RNA purification process into the RT-RPA assay. This direct assay successfully detected HBB in crude blood samples. Our findings suggest that the RT-RPA assay for HBB is a promising strategy for mRNA-based blood screening.

Application of the Dynamical Network Biomarker Theory to Raman Spectra.

The dynamical network biomarker (DNB) theory detects the early warning signals of state transitions utilizing fluctuations in and correlations between variables in complex systems. Although the DNB theory has been applied to gene expression in several diseases, destructive testing by microarrays is a critical issue. Therefore, other biological information obtained by non-destructive testing is desirable; one such piece of information is Raman spectra measured by Raman spectroscopy. Raman spectroscopy is a powerful tool in life sciences and many other fields that enable the label-free non-invasive imaging of live cells and tissues along with detailed molecular fingerprints. Naïve and activated T cells have recently been successfully distinguished from each other using Raman spectroscopy without labeling. In the present study, we applied the DNB theory to Raman spectra of T cell activation as a model case. The dataset consisted of Raman spectra of the T cell activation process observed at 0 (naïve T cells), 2, 6, 12, 24 and 48 h (fully activated T cells). In the DNB analysis, the F-test and hierarchical clustering were used to detect the transition state and identify DNB Raman shifts. We successfully detected the transition state at 6 h and related DNB Raman shifts during the T cell activation process. The present results suggest novel applications of the DNB theory to Raman spectra ranging from fundamental research on cellular mechanisms to clinical examinations.

Purulent lymphadenitis due to Halomonas hamiltonii: a case report.

Halomonas hamiltonii is a gram-negative rod bacterium isolated from highly saline environments. H. hamiltonii has rarely been reported as a human pathogen. Herein, we present the first case report of a purulent lymphadenitis caused by H. hamiltonii worldwide. The patient was a previously healthy girl aged 1 year who was referred to our hospital for left axillary lymphadenitis. Although oral amoxicillin was administered, lymphadenitis did not improve, and an abscess developed. After incision and drainage, the abscess was reduced. No recurrence of lymphadenitis was observed. The pus culture was negative. However, the 16S ribosomal DNA was amplified by the melting temperature mapping method. The amplified 16S ribosomal DNA sequence revealed 99.7% identity of H. hamiltonii. To the best of our knowledge, this is the first case of H. hamiltonii infection in a lymph node. This pathogen should be considered when diagnosing purulent lymphadenitis in healthy patients with lymphadenopathy of unknown origin.

Prospective Study of the Detection of Bacterial Pathogens in Pediatric Clinical Specimens Using the Melting Temperature Mapping Method.

The melting temperature (Tm) mapping method is a novel technique that uses seven primer sets without sequencing to detect dominant bacteria. This method can identify pathogenic bacteria in adults within 3 h of blood collection without using conventional culture methods. However, no studies have examined whether pathogenic bacteria can be detected in clinical specimens from pediatric patients with bacterial infections. Here, we designed a new primer set for commercial use, constructed a database with more bacterial species, and examined the agreement rate of bacterial species in vitro. Moreover, we investigated whether our system could detect pathogenic bacteria from pediatric patients using the Tm mapping method and compared the detection rates of the Tm mapping and culture methods. A total of 256 pediatric clinical specimens from 156 patients (94 males and 62 females; median age, 2 years [<18 years of age]) were used. The observed concordance rates between the Tm mapping method and the culture method for both positive and negative samples were 76.4% (126/165) in blood samples and 79.1% (72/91) in other clinical specimens. The Tm mapping detection rate was higher than that of culture using both blood and other clinical specimens. In addition, using the Tm mapping method, we identified causative bacteria in pediatric clinical specimens quicker than when using blood cultures. Hence, the Tm mapping method could be a useful adjunct for diagnosing bacterial infections in pediatric patients and may be valuable in antimicrobial stewardship for patients with bacterial infections, especially in culture-negative cases. IMPORTANCE This study provides novel insights regarding the use of the melting temperature (Tm) mapping method to identify the dominant bacteria in samples collected from pediatric patients. We designed a new set of primers for commercial use and developed a database of different bacteria that can be identified using these primers. We show that the Tm mapping method could identify bacteria from blood samples and other clinical specimens. Moreover, we provide evidence that the Tm mapping method has a higher detection rate than that of the culture-based methods and can achieve a relatively high agreement rate. We believe that our study makes a significant contribution to this field because rapid identification of the source of bacterial infections can drastically improve patient outcomes and impede the development of antibiotic-resistant bacteria.

Novel super-neutralizing antibody UT28K is capable of protecting against infection from a wide variety of SARS-CoV-2 variants.

Many potent neutralizing SARS-CoV-2 antibodies have been developed and used for therapies. However, the effectiveness of many antibodies has been reduced against recently emerging SARS-CoV-2 variants, especially the Omicron variant. We identified a highly potent SARS-CoV-2 neutralizing antibody, UT28K, in COVID-19 convalescent individuals who recovered from a severe condition. UT28K showed efficacy in neutralizing SARS-CoV-2 in an in vitro assay and in vivo prophylactic treatment, and the reactivity to the Omicron strain was reduced. The structural analyses revealed that antibody UT28K Fab and SARS-CoV-2 RBD protein interactions were mainly chain-dominated antigen-antibody interactions. In addition, a mutation analysis suggested that the emergence of a UT28K neutralization-resistant SARS-CoV-2 variant was unlikely, as this variant would likely lose its competitive advantage over circulating SARS-CoV-2. Our data suggest that UT28K offers potent protection against SARS-CoV-2, including newly emerging variants.

Rapid and direct detection of male DNA by recombinase polymerase amplification assay.

Screening of male DNA is important in forensic investigations, especially sexual assault cases. Quantitative real-time polymerase chain reaction (qPCR) is widely used for the detection of male DNA. However, the use of this technique as a screening tool is time-consuming and labor-intensive. In this study, we established a recombinase polymerase amplification (RPA) assay targeting the multicopy loci on the Y-chromosome for the rapid detection of male DNA (referred to as Y-RPA). The Y-RPA assay was able to detect male DNA in less than 20 min with a sensitivity of 0.025-0.005 ng/µL. Additionally, the Y-RPA assay was highly tolerant to inhibitors; male DNA was detectable in the presence of up to 1000 ng/µL humic acid, 250 µM indigo carmine, and 500 µM hematin. Then, considering its tolerance to inhibitors, we examined the feasibility of the direct Y-RPA assay. The alkaline lysis protocol (addition of sodium hydroxide and heating at 95 °C for 5 min) was employed for preparing the DNA template. The Y-RPA assay successfully detected male DNA using crude DNA extracted from blood, saliva, and semen samples. This approach enabled the screening of male DNA within approximately 30 min (5 min for lysis and 20 min for Y-RPA). These findings suggest that the Y-RPA assay is a promising screening tool for the rapid, simple, and efficient detection of male DNA.

Rapid detection of blood and semen mRNA markers by reverse transcription-recombinase polymerase amplification.

Body fluid identification is crucial for crime scene reconstruction. Recently, messenger RNA (mRNA) profiling has been an effective approach for body fluid identification. In general, mRNA is detected by real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) or end-point RT-PCR; however, these conventional methods are time-consuming and require extensive sample processing. Therefore, we developed a rapid and simple method for the detection of blood and semen mRNA markers by reverse transcription-recombinase polymerase amplification (RT-RPA). First, we screened mRNA markers for blood and semen and selected hemoglobin beta (HBB) and protamine 1 (PRM1), respectively, based on amplification specificity. Under optimized conditions, our RT-RPA assay detected HBB and PRM1 mRNAs within 20 min at a constant temperature of 42 °C. The detection limits for the assay were 0.01 ng/µL leukocyte RNA for HBB and 0.2 ng/µL semen RNA for PRM1. In addition, our RT-RPA assay exhibited high specificity and accuracy for HBB and PRM1 mRNA detection from mixed samples. Furthermore, as RPA has been reported to possess inhibitor tolerance, we evaluated the feasibility of direct RT-RPA for HBB mRNA detection. This direct approach reduced the number of processing steps and time required for template preparation and enabled the successful detection of HBB mRNA within 45 min from sample preparation. These findings suggest that RT-RPA is a useful method for mRNA-based blood and semen identification.