Clinical Testing Methods Research Articles

Assessing Finger Flexor Pulley Injuries in Climbers: A Comprehensive Review of Clinical and Functional Testing Methods

Objectives: This systematic review addressed the following questions: what are the most recommended diagnostic criteria for pulley injuries in finger flexors among climbers? What is the best functional or classification clinical test for these injuries based on the available evidence? Materials and Methods: Following the PRISMA Statement checklist, a systematic literature review was conducted between August and September 2023, using a search on the PubMed database with a string of keywords and MeSH terms. The PEDro scale was used to analyze bias in the individual studies examined. Conclusions: Based on the exclusion criteria and research question, 14 articles with heterogeneous study designs were selected. Studies involving diagnosis through clinical examination or instrumental tests were analyzed. The data obtained provide an overview of different diagnostic and classification criteria for the injury under consideration. Ultrasounds remain the gold standard in diagnosing finger pulley injuries. The distance between the tendon and bone is the most used diagnostic criterion, with a distance greater than 2 mm corresponding to an A2 or A4 pulley injury. The clinical sign of bowstringing coincides with a multiple pulley injury involving A2, A3, and A4. Clinical signs, ultrasounds, and grip strength should be evaluated for a comprehensive diagnosis. A grip deficit of 41.4% corresponds to a pulley injury.

Viable Bacteria Assay of Helicobacter pylori by RT-qPCR Measurement of cgt Gene Expression Levels: Establishment and Application of a New Method

To establish a viable bacteria assay for Helicobacter pylori (H. pylori) by assessing the cgt gene expression, and to develop accordingly a rapid and novel testing method for clinical precision treatment. Viable bacteria count was determined in bacterial cultures. The transcriptional expression level of cgt (hp0421), the conserved gene that encodes cholesterol-α-glucosyltransferase (CGT) in H. pylori, was measured by RT-PCR. The correlation between the number of colonies and cgt gene transcription expression was analyzed and the regression model was constructed. The linear range, sensitivity, and specificity of the new method were examined accordingly. The bactericidal action of clarithromycin was assessed using this method to verify the performance of the method in determining clinical bacterial drug resistance. The Ct values of cgt for H. pylori colony counts of 102, 104, 106, and 108 CFU/mL were 29.67±0.14, 23.37±0.36, 17.65±0.37, and 11.38±0.39, respectively. In the range of 101-108 CFU/mL, the regression equation for cgt gene expression and viable bacterial counts determined by RT-qPCR was y=-0.3501x+12.49, with the correlation coefficient being R 2=0.9992 and the sensitivity being 101 CFU/mL, showing no cross-reaction with 13 other bacteria. The lg values of live H. pylori bacteria treated with clarithromycin at 0, 5, 10, 20, and 40 μg/mL for 12 h were 2.57±0.02, 2.45±0.01, 2.19±0.02, 1.91±0.07, and 1.33±0.05, respectively. The corresponding cgt gene Ct values were 27.76±0.09, 28.37±0.24, 29.51±0.14, 30.11±0.12, and 31.66±0.11. By applying the cgt gene expression in the equation, the estimated counts of viable bacteria were found to be 2.73±0.03, 2.52±0.08, 2.11±0.05, 1.89±0.02, and 1.33±0.04, showing no significant difference in statistical analysis (P>0.05). The method for assessing viable bacteria account by evaluating cgt gene expression in H. pylori was successfully established, significantly reducing the time required to determine viable bacteria count and providing a new method for clinical viable bacteria testing.

Optimized biomarker evaluation and molecular testing in the era of breast cancer precision medicine

ABSTRACT Ground breaking advances in medicine, driven in part by major technologic developments in molecular biology have led us to a new model for cancer care that has been termed personalized, or precision medicine. Precision medicine is a model for making medical decisions that employs an innovative clinical approach and advanced tumor testing methods that are tailored to understanding an individual patient’s tumor biology and the molecular drivers of their disease. This medical model includes a combination of diagnostic testing and specific treatment options that can be offered to patients at presentation and in theory throughout the course of their disease as new mutations arise with the development of disease recurrence. Although the precision medicine model offers incredible potential to transform cancer care, these advances are only meaningful when they reach the correct patients. The evolving paradigm of precision medicine is changing the practice of pathology, and the pathology community needs to be mindful of these changes because every tissue specimen represents a patient’s life, and those patients are depending on the pathology community to handle their tissue correctly. The diagnostic tests performed in the pathology laboratory for precision medicine are increasingly complex, and pathologists along with the entire laboratory and clinical communities need to take steps to ensure that the right diagnosis is given to the right patient to inform the right treatment options, at the right time, along every step of the continuum of care for cancer patients. While hormone receptors and human epidermal growth factor receptor 2 (HER2) overexpression and/or amplification have been the mainstay for risk-stratification, and treatment decision making in breast cancer since the early 2000’s, the seminal work on gene expression by Perou and colleagues in the early 2000’s opened the door for molecular testing in the prognostic and predictive assessment of breast cancer. Molecular testing is now part of the standard of care in the precision medicine model for breast cancer care. In this article, the reader will gain a better understanding of how the lack of standardization of pre-analytic factors has the potential to negatively impact the quality of the tissue specimen for downstream biomarker and molecular testing, which ultimately can negatively affect patient care. The reader will also gain insight into the current climate surrounding molecular testing in breast cancer.

Practical updates in clinical antiviral resistance testing.

The laboratory diagnosis of antiviral resistance is a quickly changing field due to new drug availability, the sunsetting of older drugs, the development of novel technologies, rapid viral evolution, and the financial/logistic pressures of the clinical laboratory. This mini-review summarizes the current state of clinically available antiviral resistance testing in the United States in 2024, covering the most commonly used test methods, mechanisms, and clinical indications for herpes simplex virus, cytomegalovirus, human immunodeficiency virus, influenza, hepatitis B virus, and hepatitis C virus drug resistance testing. Common themes include the move away from phenotypic to genotypic methods for first-line clinical testing, as well as uncertainty surrounding the clinical meaningfulness of minority variant detection as next-generation sequencing methods have become more commonplace.

Detection of Urea in Human Blood using Graphene-Based Amperometric Biosensor

Urea being an essential clinical analyte, its early and easy detection would be helpful in preventing various diseases. In this study, a graphene-based screen-printed enzymatic biosensor was fabricated for the blood urea detection by amperometric method. The electrode surface was modified with urease and glutamate dehydrogenase (Urs-GLDH) enzymes for sensitive and selective detection of urea with a wide detection range. The diffusion coefficient and the electron transfer rate constant of the screen-printed graphene electrode (SPGE) was found to be 8.53 × 10−7 cm2 s−1 and 7.58 × 10−5 cm s−1, respectively. The proposed graphene-based enzymatic urea biosensor can detect urea ranging from 2.5 to 30 mM with a sensitivity of 10.59 μA·mM−1·cm−2. The reliability of this enzymatic biosensor was confirmed by measuring urea in human blood and the results showed excellent agreement with the clinical testing method. Hence, the graphene-based biosensor described herein could provide rapid, sensitive, and selective detection of urea with low-cost and simple fabrication. In conclusion, this urea detection approach could be a promising method for developing feasible low-cost clinical diagnostic systems.

Development of Luminescent Biosensors for Calprotectin.

Calprotectin, a metal ion-binding protein complex, plays a crucial role in the innate immune system and has gained prominence as a biomarker for various intestinal and systemic inflammatory and infectious diseases, including inflammatory bowel disease (IBD) and tuberculosis (TB). Current clinical testing methods rely on enzyme-linked immunosorbent assays (ELISAs), limiting accessibility and convenience. In this study, we introduce the Fab-Enabled Split-luciferase Calprotectin Assay (FESCA), a novel quantitative method for calprotectin measurement. FESCA utilizes two new fragment antigen binding proteins (Fabs), CP16 and CP17, that bind to different epitopes of the calprotectin complex. These Fabs are fused with split NanoLuc luciferase fragments, enabling the reconstitution of active luciferase upon binding to calprotectin either in solution or in varied immobilized assay formats. FESCA's output luminescence can be measured with standard laboratory equipment as well as consumer-grade cell phone cameras. FESCA can detect physiologically relevant calprotectin levels across various sample types, including serum, plasma, and whole blood. Notably, FESCA can detect abnormally elevated native calprotectin from TB patients. In summary, FESCA presents a convenient, low-cost, and quantitative method for assessing calprotectin levels in various biological samples, with the potential to improve the diagnosis and monitoring of inflammatory diseases, especially in at-home or point-of-care settings.

Stability of Liposomal Membrane of Hemoglobin-Vesicles (Artificial Red Cells) for Over Years of Storage Evaluated Using Liquid Chromatography-Mass Spectrometry.

Hemoglobin-Vesicles (Hb-V) are artificial oxygen carriers encapsulating a purified and concentrated Hb solution in liposomes composed of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC), cholesterol, 1,5-O-dihexadecyl-N-succinyl-l-glutamate (DHSG), and 1,2-distearoyl-sn-glycero-3-phosphatidylethanolamine-N-poly(ethylene glycol) (PEG5000) (DSPE-PEG). The safety and efficacy of Hb-V have been studied extensively by both preclinical and clinical test methods. Deoxygenation of Hb-V prevents autoxidation of Hb and can extend its shelf life to 2 years at room temperature. However, the lipid components raise concerns about hydrolysis because Hb-V is dispersed in saline. For this study, we attempted to estimate the lipid degradation of long-term stored Hb-V using liquid chromatography-mass spectrometry. Analyses of lipid components extracted from the stored Hb-V showed that the degradation increased depending on the storage temperature. The calculated % remaining of intact lipids of Hb-V were 98.1% after 4 years and 90.4% after 7.2 years at 4 °C, 95.8% after 1 year and 86.7% after 2 years at 25 °C, and 85.6% after 6 months at 40 °C. The main degradation products were lyso-PC and palmitic acid which are hydrolyzed at the ester bond of DPPC. A few hydrolyzed products of DHSG and DSPE-PEG were also detected in Hb-V, but almost no degradation or oxidation products derived from cholesterol could be identified. A shear test of Hb-V at 1500 s-1 showed no significant increase in Hb leakage after storage of 2 years at 25 °C and 6 months at 40 °C. Lipid degradation products including free fatty acids would decrease the pH of the Hb-V dispersion and synergistically facilitate degradation, but it maintained pH 6.5 during 6 years at 4 °C, 2 years at 25 °C, and 3 months at 40 °C because of its high buffering capacity. These results indicate that the storage conditions for Hb-V are appropriate to minimize lipid degradation in the long term.

Comparative studies between humans and golden Syrian hamsters via thromboelastography.

Thromboelastography (TEG) is a widely utilized clinical testing method for real-time monitoring of platelet function and the thrombosis process. Lipid metabolism disorders are crucial risk factors for thrombosis. The lipid metabolism characteristics of hamsters resemble those of humans more closely than mice and rats, and their relatively large blood volume makes them suitable for studying the mechanisms of thrombosis related to plasma lipid mechanisms. Whole blood samples from golden Syrian hamsters and healthy humans were obtained following standard clinical procedures. TEG was employed to evaluate coagulation factor function, fibrinogen (Fib) function, platelet function, and the fibrinolytic system. The whole blood from hamster or healthy human was isolated following the clinical procedure, and TEG was employed to evaluate the coagulation factor function, Fib function, platelet function, and fibrinolytic system. Coagulation analysis used ACLTOP750 automatic coagulation analysis pipeline. Blood routine testing used XN-2000 automatic blood analyzer. TEG parameters revealed that hamsters exhibited stronger coagulation factor function than humans (reaction time [R], p = 0.0117), with stronger Fib function (alpha angle, p < 0.0001; K-time [K], p < 0.0001). Platelet function did not differ significantly (maximum amplitude [MA], p = 0.077). Hamsters displayed higher coagulation status than humans (coagulation index [CI], p = 0.0023), and the rate of blood clot dissolution in hamsters differed from that in humans (percentage lysis 30 min after MA, p = 0.02). Coagulation analysis parameters indicated that prothrombin time (PT) and activated partial thromboplastin time (APTT) were faster in hamsters than in humans (PT, p = 0.0014; APTT, p = 0.03), whereas the Fib content was significantly lower in hamsters than in humans (p < 0.0001). No significant difference was observed in thrombin time (p = 0.1949). In summary, TEG could be used to evaluate thrombosis and bleeding parameters in whole blood samples from hamsters. The platelet function of hamsters closely resembled that of humans, whereas their coagulation function was significantly stronger.

A population-based study of the trend in SARS-CoV-2 diagnostic modalities from the beginning of the pandemic to the Omicron surge in Kyoto City, Kyoto, Japan

BackgroundThe coronavirus disease 2019 (COVID-19) presents critical diagnostic challenges for managing the pandemic. We investigated the 30-month changes in COVID-19 testing modalities and functional testing sites from the early period of the pandemic to the most recent Omicron surge in 2022 in Kyoto City, Japan.MethodsThis is a retrospective-observational study using a local anonymized population database that included patients' demographic and clinical information, testing methods and facilities from January 2020 to June 2022, a total of 30 months. We computed the distribution of symptomatic presentation, testing methods, and testing facilities among cases. Differences over time were tested using chi-square tests of independence.ResultsDuring the study period, 133,115 confirmed COVID-19 cases were reported, of which 90.9% were symptomatic. Although nucleic acid amplification testing occupied 68.9% of all testing, the ratio of lateral flow devices (LFDs) rapidly increased in 2022. As the pandemic continued, the testing capability was shifted from COVID-19 designated facilities to general practitioners, who became the leading testing providers (57.3% of 99,945 tests in 2022).ConclusionsThere was a dynamic shift in testing modality during the first 30 months of the pandemic in Kyoto City. General practitioners increased their role substantially as the use of LFDs spread dramatically in 2022. By comprehending and documenting the evolution of testing methods and testing locations, it is anticipated that this will contribute to the establishment of an even more efficient testing infrastructure for the next pandemic.

Confirmatory structural validation and refinement of the Recurrent Urinary Tract Infection Symptom Scale.

To confirm the structural validity of the Recurrent Urinary Tract Infection Symptom Scale (RUTISS), determining whether a bifactor model appropriately fits the questionnaire's structure and identifying areas for refinement. Used in conjunction with established clinical testing methods, this patient-reported outcome measure addresses the urgent need to validate the patient perspective. A clinically and demographically diverse sample of 389 people experiencing recurrent UTI across 37 countries (96.9% female biological sex, aged 18-87 years) completed the RUTISS online. A bifactor graded response model was fitted to the data, identifying potential items for deletion if they indicated significant differential item functioning (DIF) based on sociodemographic characteristics, contributed to local item dependence or demonstrated poor fit or discrimination capability. The final RUTISS comprised a 3-item symptom frequency section, a 1-item global rating of change scale and an 11-item general 'rUTI symptom and pain severity' subscale with four sub-factor domains measuring 'urinary symptoms', 'urinary presentation', 'UTI pain and discomfort' and 'bodily sensations'. The bifactor model fit indices were excellent (root mean square error of approximation [RMSEA] =0.041, comparative fit index [CFI] =0.995, standardised root mean square residual [SRMSR] =0.047), and the mean-square fit statistics indicated that all items were productive for measurement (mean square fit indices [MNSQ] =0.64 - 1.29). Eighty-one per cent of the common model variance was accounted for by the general factor and sub-factors collectively, and all factor loadings were greater than 0.30 and communalities greater than 0.60. Items indicated high discrimination capability (slope parameters >1.35). The 15-item RUTISS is a patient-generated, psychometrically robust questionnaire that dynamically assesses the patient experience of recurrent UTI symptoms and pain. This brief tool offers the unique opportunity to enhance patient-centred care by supporting shared decision-making and patient monitoring.

B-052 Establishment of New Antibodies and ELISA System to Detect the Potato Alkaloids α-Solanine and α-Chaconine

Abstract Background Food poisoning by potato alkaloids, α-solanine (SO) and α-chaconine (CHA), currently occurs worldwide, however laboratory methods have not yet been established to detect these toxins in biological samples. In this study, we established new antibodies to detect SO and CHA and evaluated the practicality of enzyme-linked immunosorbent assays (ELISAs) using these antibodies in laboratory medicine. Methods Solanidine, a chemical compound found in both SO and CHA, was conjugated with bovine albumin, and this complex was repeatedly injected into two rabbits for two months. Two types of polyclonal solanidine antibodies were then purified from rabbit serum using affinity column chromatography. Using these antibodies, two forms of direct ELISAs (Sold ELISA-1, 2) were constructed. The basic detection performance of the ELISAs was confirmed by measuring the levels of SO and CHA diluted in phosphate-buffered saline (PBS). To simulate biological samples from patients with potato food poisoning, SO and CHA powders were mixed with commercially available human serum and urine, and SO and CHA levels were then measured using ELISA. Lastly, SO and CHA levels were detected in extracts from rotten potato (Irish Cobbler) sprouts, peels, and tubers. Results In samples of SO diluted in PBS, the limit of quantification (LoQ) was determined as 2.56 ng/mL and 9.55 ng/mL for Sold ELISA-1 and -2, respectively. In CHA diluted in PBS, the LoQs of Sold ELISA-1 and -2 were 1.36 ng/mL and 4.8 ng/mL, respectively. The LoQs of SO in the serum were calculated as 14.25 ng/mL and 19.41 ng/mL using Sold ELISA-1 and -2, respectively. In contrast, the LoQs of CHA in the serum were 12.48 ng/mL and 16.92 ng/mL using Sold ELISA-1 and -2, respectively. The LoQs of SO in the urine were determined as 30.28 ng/mL and 45.16 ng/mL using Sold ELISA-1 and -2, respectively. Conversely, the LoQs of CHA in the urine were 22.92 ng/mL and 38.15 ng/mL using Sold ELISA-1 and -2, respectively. Using Sold ELISA-1, both SO and CHA in the extracts from rotten potato tubers and peels were detected as approximately 1.7- and 2.3-fold higher, respectively, in each part than those in the extracts from fresh potatoes. When both SO and CHA were measured in the same samples using Sold ELISA-2, the levels were approximately 1.6- and 2.4-fold higher, respectively, in the tubers and peels than those in fresh potato samples. Furthermore, the potato sprout extract contained approximately 80-fold more SO and CHA than the tuber and 8-fold more than the peel. Discussion and Conclusion Based on our results, Sold ELISA-1 presented greater performance in detecting SO and CHA than Sold ELISA-2. The non-specific reactions in the serum samples and influence of urea in the urine samples may underlie the reduced detection performance of SO and CHA in ELISA, respectively. Although the sensitivity depends on the sample type, these ELISAs may be effective as future clinical and food testing methods.

Application of 3D motion analysis to quantify a clinical test method assessing wrist spasticity

Application of 3D motion analysis to quantify a clinical test method assessing wrist spasticity

FM latency, FM ratio, F-wave latency versus F-wave index in the diagnosis of diabetic neuropathy

BackgroundDiabetes is one of the most common causes for peripheral neuropathy which warrants early diagnosis and treatment. The conventional nerve conduction studies appear to be normal if few of the nerve roots are escaping the lesion. In view of identifying a better clinical testing method, a late response index comprising most of the parameter of F wave was constructed and compared with the diagnosing ability of other parameters.MethodsThis hospital-based cross-sectional study was conducted among male diabetics aged 30 to 50 years in a tertiary care hospital. Maintaining an ambient temperature of 28 °C and following a supramaximal stimulation of the median nerve of both upper limbs, using a digitalized nerve conduction/electromyography/EP machine, the F-wave tracing was obtained.ResultsThe F-wave index detected a greater number of subjects with diabetic peripheral neuropathy when compared to F-wave mean latency, F M latency and F–M ratio.ConclusionF-wave index can be used as a surrogate electrophysiological study to detect peripheral neuropathy at an early stage and aids in better prognosis.

Use of genome sequencing to hunt for cryptic second-hit variants: analysis of 31 cases recruited to the 100 000 Genomes Project

BackgroundCurrent clinical testing methods used to uncover the genetic basis of rare disease have inherent limitations, which can lead to causative pathogenic variants being missed. Within the rare disease arm...

Diagnosing dry-eye: Which tests are most accurate?

PurposeTo demonstrate how the likelihood of making a correct diagnosis of dry eye disease varies according to the clinical test methods used. MethodsThe probability of a person having dry eye, given that they return a positive test, was calculated for a range of standard tests, using the Bayes-Price rule. Global specificity and sensitivity values for each test were estimated by employing the Beta distribution to combine all relevant data obtained from a literature review. ResultsAt an assumed prevalence of 11.6%, the single test with the highest probability of a correct diagnosis was corneal staining (probability = 0.28) and the lowest was the ocular surface disease index - OSDI (0.14). The best combination of symptoms with a single test of tear film homeostasis was the 5-item dry eye questionnaire (DEQ-5) + corneal staining (0.42) while OSDI + tear film break up time (TBUT) was the worst (0.23). The simultaneous observation of conjunctival and corneal staining was associated with a probability of 0.49. The probability of a correct diagnosis increased with the number of positive tests, up to a maximum of 0.90 when all of DEQ-5, conjunctival and corneal staining, osmolarity and TBUT were positive. ConclusionA significant risk of misdiagnosis is associated with using any single test for dry eye disease, or the minimum TFOS DEWS II criterion of symptoms plus any single test of tear film homeostasis. To minimize this risk, the maximum number of tests available should be performed and the results used to inform diagnosis. The simultaneous occurrence of conjunctival and corneal staining should be considered a key outcome and be specified in future guidelines.

Autoimmune Dysphagia Related to Rheumatologic Disorders: A Focused Review on Diagnosis and Treatment.

Autoimmune dysphagia is defined as dysphagia caused by autoimmune processes affecting various components of the swallowing processsuch as muscle, neuromuscular junction, nerves, roots, brainstem, or cortex. These autoimmune causes can be classified into gastroenterological, dermatological, rheumatologic, and neurologic. Rheumatological disorders, such as scleroderma, Sjogren's syndrome, systemic lupus erythematosus, rheumatoid arthritis, sarcoidosis, Behcet's disease, anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis, or granulomatosis with polyangiitis, have been associated with dysphagia. Autoimmune dysphagia in the context of rheumatological disorders is particularly significant because it can occur as a sole manifestation or as part of a symptom complex associated with the underlying disorder and often responds to immunosuppressive therapies. However, diagnosing autoimmune dysphagia can be challenging as it requires the exclusion of structural and primary motility disorders through procedures such as endoscopy and manometry. Early diagnosis is important to improve the quality of life and prevent significant mortality and morbidity. Management focuses on treating the underlying disease activity, and a multidisciplinary approach involving various medical specialties may be necessary to achieve success. This article aims to review the autoimmune rheumatological conditions that can lead to dysphagia and discuss the associated pathophysiological mechanisms. We also outline the clinical clues and laboratory testing methods that facilitate early diagnosis, with the goal of improving patient outcomes through timely intervention and appropriate management.

Several potential serum proteomic biomarkers for diagnosis of osteoarticular tuberculosis based on mass spectrometry

BackgroundOsteoarticular tuberculosis is one of the extrapulmonary tuberculosis (EPTB) diseases, which is mainly caused by infection of Mycobacterium tuberculosis (MTB) in bone and joints. The limitation of current clinical test methods is leading to a high misdiagnosis rate and affecting the treatment and prognosis. This study aims to search serum biomarkers that can assist in the diagnosis of osteoarticular tuberculosis. MethodsProteomics can serve as an important method in the discovery of disease biomarkers. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to analyze proteins in 90 serum samples, which were collected from June 2020 to December 2021, then evaluated by statistical analysis to screen potential biomarkers. After that, potential biomarkers were validated by enzyme-linked immunosorbent assay (ELISA) and diagnostic models were also established for observation of multi-index diagnostic efficacy. Results118 differential expressed proteins (DEPs) were obtained in serum after statistical analysis. After the diagnostic efficacy evaluation and clinical verification, inter-alpha-trypsin inhibitor heavy chain H2 (ITIH2), complement factor H-related protein 2 (CFHR2), complement factor H-related protein 3 (CFHR3), and complement factor H-related protein 5 (CFHR5) were found as potential biomarkers, with 0.7167 (95 %CI: 0.5846–0.8487), 0.8600 (95 %CI: 0.7701–0.9499), 0.8150 (95 %CI: 0.6998–0.9302), and 0.9978 (95 %CI: 0.9918–1.0040) AUC value, respectively. The remaining DEPs except CFHR5 were constructed as diagnostic models, the diagnostic model contained CFHR2 and CFHR3 had good diagnostic efficacy with 0.942 (95 %CI: 0.872–0.980) AUC value compared to other models. ConclusionThis study provides a reference for the discovery of serum protein markers for osteoarticular tuberculosis diagnosis, and the screened DEPs can also provide directions for subsequent pathogenesis research.

Detection of multiple types of cancer driver mutations using targeted RNA sequencing in non-small cell lung cancer.

DNA-based next-generation sequencing has been widely used in the selection of target therapies for patients with nonsmall cell lung cancer (NSCLC). RNA-based next-generation sequencing has been proven to be valuable in detecting fusion and exon-skipping mutations and is recommended by National Comprehensive Cancer Network guidelines for these mutation types. The authors developed an RNA-based hybridization panel targeting actionable driver oncogenes in solid tumors. Experimental and bioinformatics pipelines were optimized for the detection of fusions, single-nucleotide variants (SNVs), and insertion/deletion (indels). In total, 1253 formalin-fixed, paraffin-embedded samples from patients with NSCLC were analyzed by DNA and RNA panel sequencing in parallel to assess the performance of the RNA panel in detecting multiple types of mutations. In analytical validation, the RNA panel achieved a limit of detection of 1.45-3.15 copies per nanogram for SNVs and 0.21-6.48 copies per nanogram for fusions. In 1253 formalin-fixed, paraffin-embedded NSCLC samples, the RNA panel identified a total of 124 fusion events and 26 MET exon 14-skipping events, in which 14 fusions and six MET exon 14-skipping mutations were missed by DNA panel sequencing. By using the DNA panel as the reference, the positive percent agreement and the positive predictive value of the RNA panel were 98.08% and 98.62%, respectively, for detecting targetable SNVs and 98.15% and 99.38%, respectively, for detecting targetable indels. Parallel DNA and RNA sequencing analyses demonstrated the accuracy and robustness of the RNA sequencing panel in detecting multiple types of clinically actionable mutations. The simplified experimental workflow and low sample consumption will make RNA panel sequencing a potentially effective method in clinical testing.

Development of New Antibodies and an ELISA System to Detect the Potato Alkaloids α-Solanine and α-Chaconine.

Food poisoning can be caused by the potato alkaloids α-solanine (SO) and α-chaconine (CHA). Therefore, this study aimed to establish new enzyme-linked immunosorbent assays (ELISAs) for detecting these two toxins in biological samples and potato extracts. Two antibodies that bind to solanidine, a chemical compound found in both SO and CHA, were newly developed, and two types of ELISAs (Sold1 ELISA and Sold2 ELISA) were constructed. We measured SO and CHA diluted in phosphate-buffered saline (PBS), serum, and urine. The detection performance of the two ELISAs for SO and CHA in PBS was higher than in serum and urine, and the sensitivity of Sold2 ELISA was lower than that of Sold1 ELISA. Thus, we used these ELISAs to measure SO and CHA in potato part extracts and found that potato sprouts contained approximately 80-fold more SO and CHA than tubers and 8-fold more SO and CHA than peels. Although the detection sensitivity of SO and CHA depends on the sample types, these ELISAs may be effective as future clinical and food testing methods after further improvements.

MUC5B Alters Oral Mucosa of Oral Lichen Planus Patients with Xerostomia

Oral Lichen planus（OLP）is a chronic autoimmune cell-mediated inflammatory disease. Most OLP patients complain of xerostomia/dry mouth due to changes in the oral mucosal protective barrier. Mucin 5B（MUC5B）is the predominate gel-forming mucin in the oral cavity that protects the underlying tissue from mechanical damage, chemical insult, and pathogenic microbes. This study aims to assess MUC5B in OLP subjects and correlate its role in etiopathogenesis. The study included 20 OLP subjects and 10 healthy controls. OLP subjects were divided further into 10 keratotic（reticular）and 10 non-keratotic（atrophic/erythematous）groups based on the clinical criteria. Dryness of mouth was confirmed by the subjective（Xerostomia Inventory, XI; Bother Index, BI）and clinical testing（Clinical Oral Dryness Score, CODS）method. Stimulated, unstimulated saliva and blood samples were collected from subjects. Samples were centrifuged and analysed for MUC5B level using an ELISA kit. The subjective and clinical testing methods which clinically define xerostomia are correlated with the age and sex of the OLP subjects. The stimulated and unstimulated salivary flow rate of OLP subjects was lower（p<0.05）than controls and was associated with the XI, BI, and CODS（p<0.01）. In contrast, the MUC5B level was increased in the serum of OLP subjects compared to controls（p<0.05）. OLP subjects with xerostomia, displayed lower salivary flow, decrease MUC5B in saliva and increase MUC5B in serum. Alteration of MUC5B in the saliva causes oral mucosal alterations affecting the protection of the oral cavity and increase xerostomia in OLP.