Sensitive Screening Method Research Articles

A simple, cost-effective, and efficient method for screening CRISPR/Cas9 mutants in plants

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated genome editing system is widely used for targeted mutagenesis in a growing number of plant species. To streamline the screening process for mutants, especially those generated from low-efficiency editing events, there is a need for a rapid, cost-effective, and efficient method. Although several screening methods have been developed to process initial samples, these methods often tend to be time-consuming, expensive, or inefficient when dealing with larger sample sizes. Here we describe a simple, rapid, low-cost, and sensitive screening method for screening CRISPR/Cas9 mutants called PCR-Bsl I-associated analysis (PCR-BAA). This method requires only standard PCR and Bsl I restriction enzyme digestion, as well as agarose gel electrophoresis analysis. This method is particularly well suited for the efficient screening of mutants from larger populations of transformants. The simplicity, low cost, and high sensitivity of the PCR-BAA method make it particularly suitable for rapid screening of CRISPR/Cas9-induced mutants, especially those from low-efficiency editing events.

Rapid screening and identification strategy of lactic acid bacteria and yeasts based on Ramanomes technology and its application in fermented food

There is an urgent need for quick, sensitive, thorough, and low-cost preliminary screening and rapid identification method for probiotic resource development. Here, we constructed a culture-free, accurate and sensitive “separation-enrichment-detection” rapid evaluation and identification method of probiotics in fermented food based on Ramanomes technology, and established a Ramanome reference of lactic acid bacteria and yeasts by AgNPs nanostructure array (AgNPs NSA) chips with uniform “hot spots” and high signal enhancement ability as SERS substrates. Systematic cluster analysis could clearly distinguish among different genera of lactic acid bacteria and yeast, and could indicate the affinity and difference between lactic acid bacteria of the same genus and yeast of the same genus. The recognition accuracy of convolutive neural networks was 100 %, and the recognition sensitivity was less than 10 CFU/mL. We constructed a probiotic isolation and enrichment method by velocity gradient centrifugation and density gradient differential centrifugation, and the average recoveries of lactobacilli and yeasts were greater than 98 % in the practical application, and the accuracy of the verified classification was 100 %. In conclusion, this study has established a preliminary screening strategy of “screening before cultivating” for lactic acid bacteria and yeasts, which breaks through the principle limitation of the current traditional paradigm of “cultivating before screening”. It can greatly improve the preliminary screening rate of probiotics in fermented foods, and provide a good technical support for the mining of probiotic resources from environmental or complex matrix samples.

Maximizing Breast Cancer Detection Through Screening: A Comparative Analysis of Imaging-Based Approaches

Introduction/BackgroundThis study estimates the percentage of detectable breast cancers in the screening population that could be found with primary and supplemental screening, and provides cost estimates for population wide supplemental screening in the U.S. Materials and MethodsPublished estimates on cancer detection rates of 2D mammography, tomosynthesis (DBT), whole breast ultrasound (US), molecular breast imaging (MBI), contrast-enhanced mammography (CEM), and MRI, the number of mammograms conducted in the United States in 2023, and the proportion of dense breast tissue, were utilized. The maximum number of detectable cancers was projected from incremental cancer detection rates of the most sensitive supplemental screening method. The proportion of cancers detectable for each modality was calculated. In 2023, Medicare reimbursement rates were used to estimate supplemental screening costs. ResultsOut of 469,437 detectable cancers, 2D mammography could detect 190,531 (41%), leaving 278,906 undetected. Adding supplemental screening could detect a combined 220,165 cancers (47%) with DBT, 237,596 (51%) with US, 331,727 (71%) with MBI, 377,049 (80%) with CEM and 469,437 (100%) with MRI. The imaging cost in US dollars to provide supplemental screening to all individuals with dense breasts in 2023 was $933M for tomosynthesis, $1.84B for US, $3.87B for CEM, $4.16B for MBI, and $6.36B for MRI. ConclusionThe study highlights potential benefits from supplemental breast cancer screening, suggesting the combination of mammography and breast MRI offers the most effective detection method though at the highest imaging cost. These findings provide valuable insights to guide future research and inform decision-making in supplemental breast cancer screening strategies.

Rapid Identification of the Spruce Bark Beetle <i>Ips typographus</i> (Linnaeus) Basing on a New Amplification and Analysis Platform

Insects, one of the major disturbance agents, are regarded as a big challenge to forests. Bark beetles (Coleoptera: Curculionidae: Scolytinae) are among the most destructive pests around the world. The European spruce bark beetle <i>I. typographus </i>(Linnaeus) is considered the most dangerous species to mature spruce forests throughout Eurasia. In order to improve efficiency, accuracy, and operability of identification, a rapid, simple, highly sensitive and specific screening method is in urgent need. In this study, a rapid classification approach for <i>I. typographus</i> was established based on the enzyme-mediated duplex exponential amplification (EmDEA) amplification and analysis platform. The method development process consists of target gene selection, primer design, primer screening, and method validation. Parameter analysis demonstrated that this new method has a detection limit of 1.96×103 copies/μL, which is comparable to conventional molecular tools such as PCR. Stable repeatability and high specificity were confirmed by testing 5 samples of <i>I. typographus</i> and 4 related beetles. Besides, this screening protocol was easy to use, and could be completed in 30 min. With the advantage of isothermal amplification, this method could be further applied in non-laboratory scenarios such as port rapid screening and wild survey. This rapid screening method for <i>I. typographus</i> is believed to assist precise prediction and efficient prevention of exotic insect species.

HGG-26. MLH1, MSH2, MSH6, AND PMS2 IMMUNOHISTOCHEMISTRY REPRESENTS A HIGHLY SENSITIVE SCREENING METHOD FOR MISMATCH REPAIR DEFICIENCY SYNDROMES IN PEDIATRIC HIGH-GRADE GLIOMA

Abstract BACKGROUND Pediatric central nervous system (CNS) tumors can occur as first manifestations of cancer predisposition syndromes (CPS) resulting from pathogenic variants in DNA mismatch repair (MMR) genes MLH1, MSH2, MSH6, and PMS2. Early detection of MMR deficiencies (MMRD) may warrant the therapeutic use of checkpoint inhibitors and enable genetic consulting for the patient’s families. METHODS We prospectively screened 128 pediatric high-grade gliomas (pedHGG) for MMR protein expression by immunohistochemistry (IHC) as part of the HIT-HGG-2013 clinical trial. We compared IHC results to independently collected patient information on CPS including MMRD to test the method’s efficiency in screening for Lynch syndrome and Constitutional MMRD syndrome CMMRD. RESULTS From 128 successfully tested tumors, ten cases (10/128, 7.8%), showed loss of expression of at least one of the MMR proteins. In seven of the ten affected patients (7/128, 5.5%), genetic testing uncovered heterozygous (6/10) or homozygous (1/10) pathogenic germline variants in MMR genes (MLH1, MSH2 or MSH6) confirming the diagnosis of Lynch syndrome respectively CMMRD. In three cases (3/128 2.3%), MMR alterations were restricted to tumor tissue. The group of diffuse midline gliomas, H3 K27-altered, (67/128, 52.3%) did not show MMR alterations, while either somatic or germline MMR mutations were detected in two of eight patients with diffuse hemispheric glioma, H3 G34-mutant. In 88 patients (88/128, 68.8%) either clinical or molecular-genetic information on CPS could be provided. All ten patients with signs of MMRD were successfully detected by IHC from tumor tissue. CONCLUSIONS IHC for MMR proteins represents a highly sensitive screening method for MMRD in pedHGG. Considering the occurrence of at least 11.5% (7/61) of MMRD in pediatric patients with diffuse high-grade glioma excluding DMG, MMRD IHC should be part of routine diagnostics as cost-effective, broadly available and robust screening method.

Screening of malaria infections in human blood samples with varying parasite densities and anaemic conditions using AI-Powered mid-infrared spectroscopy

BackgroundEffective testing for malaria, including the detection of infections at very low densities, is vital for the successful elimination of the disease. Unfortunately, existing methods are either inexpensive but poorly sensitive or sensitive but costly. Recent studies have shown that mid-infrared spectroscopy coupled with machine learning (MIRs-ML) has potential for rapidly detecting malaria infections but requires further evaluation on diverse samples representative of natural infections in endemic areas. The aim of this study was, therefore, to demonstrate a simple AI-powered, reagent-free, and user-friendly approach that uses mid-infrared spectra from dried blood spots to accurately detect malaria infections across varying parasite densities and anaemic conditions.MethodsPlasmodium falciparum strains NF54 and FCR3 were cultured and mixed with blood from 70 malaria-free individuals to create various malaria parasitaemia and anaemic conditions. Blood dilutions produced three haematocrit ratios (50%, 25%, 12.5%) and five parasitaemia levels (6%, 0.1%, 0.002%, 0.00003%, 0%). Dried blood spots were prepared on Whatman™ filter papers and scanned using attenuated total reflection-Fourier Transform Infrared (ATR-FTIR) for machine-learning analysis. Three classifiers were trained on an 80%/20% split of 4655 spectra: (I) high contrast (6% parasitaemia vs. negative), (II) low contrast (0.00003% vs. negative) and (III) all concentrations (all positive levels vs. negative). The classifiers were validated with unseen datasets to detect malaria at various parasitaemia levels and anaemic conditions. Additionally, these classifiers were tested on samples from a population survey in malaria-endemic villages of southeastern Tanzania.ResultsThe AI classifiers attained over 90% accuracy in detecting malaria infections as low as one parasite per microlitre of blood, a sensitivity unattainable by conventional RDTs and microscopy. These laboratory-developed classifiers seamlessly transitioned to field applicability, achieving over 80% accuracy in predicting natural P. falciparum infections in blood samples collected during the field survey. Crucially, the performance remained unaffected by various levels of anaemia, a common complication in malaria patients.ConclusionThese findings suggest that the AI-driven mid-infrared spectroscopy approach holds promise as a simplified, sensitive and cost-effective method for malaria screening, consistently performing well despite variations in parasite densities and anaemic conditions. The technique simply involves scanning dried blood spots with a desktop mid-infrared scanner and analysing the spectra using pre-trained AI classifiers, making it readily adaptable to field conditions in low-resource settings. In this study, the approach was successfully adapted to field use, effectively predicting natural malaria infections in blood samples from a population-level survey in Tanzania. With additional field trials and validation, this technique could significantly enhance malaria surveillance and contribute to accelerating malaria elimination efforts.

Isolation of aptamers with excellent cross-reactivity and specificity to sulfonamides towards a ratiometric fluorescent aptasensor for the detection of nine sulfonamides in seafood

Sulfonamides (SAs) is a class of antibiotics that extensively used for treating infectious diseases in livestock industries and aquaculture. Thus, it is urgent need to obtain the bio-receptor, which has excellent cross-reactivity and specificity to SAs, for developing high-throughput methods for the determination of multiple SAs even all commonly-used SAs, to realize the quick screening/detection of total SAs in animal-derived foods. We herein isolated several SAs-specific cross-reactive aptamers by using a library-immobilized SELEX with multi-SAs parallel selection strategy. Two of the isolated aptamers (Sul-01 and Sul-04) can specifically recognize and bind seven SAs respectively with higher binding affinity and no interference of non-sulfonamide antibiotics, and thus can be applied as bio-receptors for developing high-throughput aptasensors for the quick screening/detection of multiple SAs. By using the mixture of Sul-01 and Sul-04 as bio-receptor, a ratiometric fluorescent aptasensor was created for the quick detection of nine SAs including sulfamethoxydiazine (SMD), sulfapyridine (SPD), sulfaquinoxaline (SQ), sulfathiazole (ST), sulfamonomethoxine (SMM), sulfamerazine (SMR), sulfaguanidine (SG), sulfamethazine (SMZ) and sulfadiazine (SD) with a detection limit (LOD) of 0.10–0.50 μM, or total of above nine SAs with a LOD of 0.20 μM. The fluorescent aptasensor was successfully applied to detect each or total of SMD, SPD, SQ, ST, SMM, SMR, SG, SMZ and SD in fish samples with a recovery of 83 %–92 % and a relative standard deviation (RSD, n = 5) < 5 %. This study not only provided several promising bio-receptors for the development of diverse high-throughput aptasensors to achieve the quick screening of multiple SAs residues, but also provided a simple, stable and sensitive method for the quick screening of SMD, SPD, SQ, ST, SMM, SMR, SG, SMZ and SD in seafood.

Detecting Mosaicism of Monosomy X Using FISH in Prenatal Samples: Post High Risk NIPT

AbstractNoninvasive prenatal testing (NIPT) is a highly specific and sensitive aneuploidy screening method with low false positive results. Sex chromosome aneuploidy (SCA) is not picked up in prenatal ultrasounds, as they may not have antenatally identifiable features, except for hydrops in monosomy X cases. Women with high risk NIPT results for SCAs are recommended to go for invasive prenatal diagnosis for confirmation by diagnostic tests like chromosome microarray, karyotyping, and/or fluorescence in situ hybridization (FISH). We present two cases that showed a high risk for monosomy X on NIPT. Chromosomal microarray was negative for SCA. Further, FISH was done to confirm the results and confirm the presence of low level mosaicism for monosomy X. FISH proves to be the test of choice to detect low level mosaicism in high risk NIPT cases with high positive predictive values.

Label-free voltammetric screening of human blood serum

The current study presents a comprehensive voltammetric investigation into the direct analysis of untreated human blood serum in a phosphate buffer at an unmodified, graphite electrode by means of voltammetry. By employing advanced square-wave voltammetry at an edge plane pyrolytic graphite electrode (EPPGE), the basic principles were established for developing a sensitive, fast, simple, and label-free method for the simultaneous screening of uric acid, bilirubin, and albumin analytes that are present in human blood serum and are quite essential for rapid medical diagnostics. The electrochemical protocol utilizes the specific structural patterns of the EPPGE, the inherent redox and adsorption properties of the analysed analytes, and the sensitivity and rapidity of the employed advanced voltammetric technique. The methodology has been successfully applied for quantification of the considered analytes in a series of samples of human blood serum and was compared with the standard methods used in a clinical biochemical laboratory. This novel method represents a significant advancement towards the development of point-of-care devices aimed at swiftly and simultaneously quantifying uric acid, bilirubin, and albumin levels in human serum.

Development of a multiplex real-time quantitative reverse-transcription polymerase chain reaction for the detection of four bee viruses

Viruses in the families Dicistroviridae and Iflaviridae are among the main threats to western honey bees (Apis mellifera) and native bee species. Polymerase chain reaction (PCR) is the gold standard for pathogen detection in bees. However, high throughput screening for bee virus infections in singleplex PCR reactions is cumbersome and limited by the high quantities of sample RNA required. Thus, the development of a sensitive and specific multiplex PCR detection method for screening for multiple viruses simultaneously is necessary. Here, we report the development of a one-step multiplex reverse-transcription quantitative polymerase chain reaction (RT-qPCR) assay to detect four viruses commonly encountered in pollinator species. The optimized multiplex RT-qPCR protocol described in this study allows simultaneous detection of two dicistroviruses (Israeli acute paralysis virus and Black queen cell virus) and two iflaviruses (Sacbrood virus and Deformed wing virus) with high efficiency and specificity comparable to singleplex detection assays. This assay provides a broad range of detection and quantification, and the results of virus quantification in this study are similar to those performed in other studies using singleplex detection assays. This method will be particularly useful for data generation from small-bodied insect species that yield low amounts of RNA.

Quantitative Phase Imaging as Sensitive Screening Method for Nanoparticle-Induced Cytotoxicity Assessment

Quantitative Phase Imaging as Sensitive Screening Method for Nanoparticle-Induced Cytotoxicity Assessment

Screening anabolic androgenic steroids in human urine: an application of the state-of-the-art gas chromatography-Orbitrap high-resolution mass spectrometry.

Over the past few decades, anabolic androgenic steroids (AASs) have been abused in and out of competition for their performance-enhancing and muscle-building properties. Traditionally, AASs were commonly detected using gas chromatography-mass spectrometry in the initial testing procedure for doping control purposes. Gas chromatography-Orbitrap high-resolution mass spectrometry (GC-Orbitrap-HRMS) is a new technology that has many advantages in comparison with GC-MS (e.g., a maximum resolving power of 240,000 (FWHM at m/z 200), excellent sub-ppm mass accuracy, and retrospective data analysis after data acquisition). Anti-doping practitioners are encouraged to take full advantage of the updated techniques of chromatography-mass spectrometry to develop sensitive, specific, and rapid screening methods for AASs. A new method for screening a wide range of AASs in human urine using GC-Orbitrap-HRMS was developed and validated. The method can qualitatively determine 70 anabolic androgenic steroids according to the minimum required performance limit of the World Anti-Doping Agency. Moreover, the validated method was successfully applied to detect six metabolites in urine after the oral administration of metandienone, and their excretion curves in vivo were studied. Metandienone M6 (17β-hydroxymethyl-17α-methyl-18-nor-androst-1,4,13-trien-3-one) has been identified as a long-term urinary metabolite which can be detected up to 7 weeks, thus providing a longer detection window compared with previous studies. This study provides a rationale for GC-Orbitrap-HRMS in drug metabolism and non-targeted screening.

Primary sclerosing cholangitis-Diagnosis and treatment 2024

The etiology of primary sclerosing cholangitis (PSC) remains unclear, which explains in part the lack of acausal treatment. The differential diagnostic distinction from the even rarer immunoglobulin 4 (IgG4)-associated cholangitis (IAC) is becoming increasingly more successful. Advances in the understanding of different clinical courses, improvements in noninvasive diagnostics through modern magnetic resonance imaging (MRI) and the introduction of liver elastography have led to the development of improved prognostic models. The evidence for recommendations on medicinal (e.g., ursodeoxycholic acid) or endoscopic treatment (e.g., balloon dilatation and/or stent insertion) for PSC is still low. In contrast, the long-term results of liver transplantation in PSC patients are constantly improving. Due to the lack of highly sensitive and specific screening methods the early recognition of cholangiocellular carcinoma (CCC) as the most important complication is rarely successful. The continuous improvement of endoscopic retrograde cholangiopancreatography (ERCP) and direct cholangioscopy in combination with molecular biological and fluorescence in situ hybridization (FISH) analyses of bile duct tissue samples are promising for refined diagnostics. Due to the significantly increased risk of colorectal cancer, an annual colonoscopy is recommended in the presence of inflammatory bowel disease. Improvement of the early diagnostics of PSC and successful testing of new treatment strategies raise hope for acontinuous improvement in the medical support of these complex patients.

Assessing long-term, vestibulotoxic side effects after gentamicin therapy in neonatal sepsis or infection using video head impulse test.

Newborn infection and sepsis remain serious problems. Guideline-compliant therapy includes, among other therapeutics, calculated intravenous antibiosis with gentamicin. One of the known side effects of gentamicin is severe vestibulotoxicity, which can be detected using the video head impulse test (VHIT), which is a sensitive examination method for the detection of vestibular hypofunction in children and adults. Previous studies on the vestibulotoxicity of gentamicin in newborns were carried out using caloric testing, rotary testing, and electronystagmography. Nevertheless, there are currently no data available on VHIT examinations in children who have been treated with neonatal gentamicin therapy. A single-center, prospective cross-sectional study, was conducted at a tertial referral center. VHIT was performed on 23 children aged 3-7 years who had received intravenous gentamicin therapy for at least five days as part of the treatment of newborn sepsis between 2012 and 2016. Main outcome was median gain and occurrence of refixational saccades as measured with VHIT. In addition, the children's parents received questionnaires to detect possible risk factors and vestibular and cochlear abnormalities. Out of 23 children with a mean age of four years and seven months (ranging from 3 to 7 years), 11 (47.8%) indicated abnormal results in VHIT. The VHIT results were unilaterally abnormal in six children (26.1%) and bilaterally abnormal in five others (21.7%). Additionally, five of the children with an abnormal HIT had abnormalities, as found in the questionnaire results. and Relevance: Almost half of the children observed after having undergone gentamicin therapy as newborns showed abnormalities in VHIT, although they did not show any clinical signs of disbalance or vestibular hypofunction. VHIT can serve as a sensitive investigation method for the early screening of post-therapeutic vestibulotoxic side effects after gentamicin therapy in children. Additionally, VHIT can enable early intervention in these children.

Dual-FRET aptasensor for rapid screening of Ochratoxin A in food samples

Simple, low cost, and sensitive OTA screening method is important for food safety control. The discovery of the OTA aptamer enabled the development of various aptasensors, but the structural change caused by target binding is not appreciable enough, thus limiting the sensitivity. The introduction of various nanomateirals could amplify the structure change, but such aptasensors are difficult to be standardized, thus is not suitable for screening analysis. Herein, we proposed a full DNA-based dual-FRET aptasensor, in which the aptamer was rigidified to amplify the OTA-induced structure change. Meanwhile, a dual FRET strategy was employed to increase the OTA detection sensitivity. Upon both experimental and theoretical selection, aptamer OBA33 was selected, leading to a detection limit of 2.1 nM that is lower than the limit set by Chinese National Food Safety Standard (12 nM), thus is appealing for screening of potential OTA-positive cases in a large number of rice samples. To facilitate rapid screening analysis, a mini 3D-printed sensor device was constructed by integrating LED excitation, cellphone camera image taking, and RGB extraction. Due to the technological maturity of DNA production, the proposed aptasensor could be easily standardized for fast OTA screening analysis (15 min).

Comparison of effectiveness and cost of different HCV testing strategies in high-risk populations in China.

High-risk populations are the predominant populations affected by hepatitis C virus(HCV) infection, and there is an urgent need for efficient and cost-effective HCV testing strategies for high-risk populations to identify potential undiagnosed HCV-infectedindividuals. This study compared several commonly used testing strategies and conducted effectiveness and cost analysis to select the appropriate testing strategy for diagnosing HCV infection in high-risk populations. Among the 2093 samples from high-risk populations in this study, 1716 were HCV negative, 237 were current HCV infection, 137 were past HCV infection, and three were acute early HCV infection. It was found that out of 237 patients with HCV current infection, Strategy A could detect 225 cases, with a missed detection rate of 5.06%, and the total cost was 33 299 RMB. In addition, Strategy B could detect 237 cases of current HCV infection, and the HCV missed detection rate was 0.00%, and the total cost was 147 221 RMB. While 137 cases of past HCV infection could be distinguished by strategy C, but 14 cases with current HCV infection were missed, with an HCV-positivemissed detection rate of 5.91%, and the total cost for Strategy C was 43 059 RMB. In conclusion, in high-risk populations, the HCV positivity rate is typically higher. If feasible, the preferred approach is to directly conduct HCV RNA testing, which effectively minimizes the risk of missing cases. However, in situations with limited resources, it is advisable to initially choose a highly sensitive method for anti-HCV screening, followed by HCV RNA testing on reactive samples.

Saliva Based Diagnostic Prediction of Oral Squamous Cell Carcinoma using FTIR Spectroscopy.

Oral cancer ranks as the sixth most prevalent form of cancer worldwide, presenting a significant public health concern. According to the World Health Organization (WHO), within a 5-year period following diagnosis, the mortality rate among oral cancer patients of all stages stands at 45%. In this study, a total of 60 patients divided into 2 groups were recruited. Group A included 30 histo-pathologically confirmed OSCC patients and Group B included 30 healthy controls. A standardized procedure was followed to collect saliva samples. FTIR spectroscopy was done for all the saliva samples collected from both Group A and B. An IR Prestige-21 (Shimadzu Corp, Japan) spectrometer was used to record IR spectra in the 40-4000cm-1 range SVM classifier was applied in the classification of disease state from normal subjects using FTIR data. The peaks were identified at wave no 1180cm-1, 1230cm-1, 1340cm-1, 1360cm-1, 1420cm-1, 1460cm-1, 1500cm-1, 1540cm-1, 1560cm-1, and 1637cm-1. The observed results of SVM demonstrated the accuracy of 91.66% in the classification of Cancer tissues from the normal subjects with sensitivity of 83.33% while specificity and precision of 100.0%. The development of oral cancer leads to noticeable alterations in the secondary structure of proteins. These findings emphasize the promising use of ATR-FTIR platforms in conjunction with machine learning as a reliable, non-invasive, reagent-free, and highly sensitive method for screening and monitoring individuals with oral cancer.

Adapting lipidomic sample processing methods for boars housed in commercial settings.

Multiple reaction monitoring (MRM) profiling is a sensitive method of lipid screening that has the capability to distinguish between different fertility phenotypes in gilts. However, MRM profiling has not yet been utilized to evaluate fertility phenotypes in boars. Markers indicative of fertility status in boars would be valuable as inclusion of subfertile boars in breeding programs results in a loss of efficiency and negative economic consequences. In addition, semen samples for lipidomic analysis are transported in liquid nitrogen or on dry ice to suspend metabolic activity within the sperm cells, however, these cryopreservation techniques are not commonly available at commercial boar studs. Therefore, the objective of this study was to develop a method of sample processing for MRM profiling that suspends metabolic activity within semen without freezing the sample. Five, sexually mature boars of similar genetics enrolled in a commercial breeding program were collected for the study. Following collection, ejaculates were aliquoted into methanol to suspend metabolic activity and shipped to Purdue University overnight for lipid extraction. Lipids were extracted using the Bligh and Dyer method and MRM profiling was used for lipid screening. A total of 329 ion transitions (MRMs) related to lipids were detected with most lipids being characterized as plasma membrane lipids (74%) which were comprised of phosphatidylcholines (40%), ceramides (16%), phosphatidylethanolamines (11%), and phosphatidylserines (7%). acylcarnitines (AC) represented approximately 8% of the ejaculate lipidome. Hierarchical cluster and principal component analysis revealed that boars have a distinct ejaculate lipidome profile based on major plasma membrane lipid classes. In addition, we observed that one boar was unique in his abundance of AC which are related to progressive motility and sperm cell metabolism. These results indicate that this method of sample processing for MRM profiling is suitable to be used to evaluate the lipidome of ejaculates from commercial boars and has the potential for broader applications across different livestock species in commercial environments.

Utility of Abdominal Fat Pad Aspiration in the Early Diagnosis of Systemic Amyloidosis: A Cross-Sectional Study

Introduction: Abdominal Subcutaneous Fat Pad aspiration (AFPA) is a quick, sensitive, and specific screening and diagnostic method for detecting amyloid deposits. This procedure can be performed in an outpatient setting. The present study aimed to demonstrate amyloid deposits in subcutaneous fat pad aspirates on Congo Red stained slides using polarised microscopy. Aim: To evaluate the diagnostic accuracy of abdominal fat pad aspiration cytology as a screening procedure for systemic amyloidosis. Materials and Methods: A cross-sectional study was conducted over a three-year period (January 2019 to July 2021) at SDM College of Medical Sciences and Hospital, Sattur, Dharwad. Fat pad aspirate slides stained with Congo Red stain were independently reviewed by two cytopathologists. Slides were examined under a polarising microscope and interpreted as either positive or negative for amyloid deposits based on the presence or absence of apple green birefringence. The data were evaluated using Microsoft Excel and Statistical Package for the Social Sciences (SPSS) software version 20.0. Results: The present study included 54 abdominal fat pad aspirates. The ages ranged between 29 and 80 years, with a mean age of presentation of 59 years. Among all AFPA samples, 35 (64.8%) were positive for amyloid, 16 (29.6%) were negative, and three (5.5%) were inconclusive for interpretation. Follow- up deep/visceral biopsies, such as kidney and bone marrow biopsies, were performed in 31 (57.4%) cases, and approximately half of these cases showed amyloid deposition on deep biopsy. AFPA demonstrated a sensitivity, specificity, positive predictive value, and diagnostic accuracy of 94%, 92%, 94%, and 93.3%, respectively, considering visceral biopsies including bone marrow and/or kidney as the gold standard. Conclusion: Abdominal fat pad aspiration is a quick, minimally invasive, outpatient-based procedure that helps in the early diagnosis of systemic amyloidosis before patients clinically present with complications.

Device comparison study to measure nasal nitric oxide in relation to primary ciliary dyskinesia

Primary ciliary dyskinesia (PCD) is a genetic respiratory disease characterized by chronic cough, recurrent respiratory infections, and rhinosinusitis. The measurement of nasal nitric oxide (nNO) against resistance has been suggested as a sensitive screening method. However, current recommendations argue for the use of expensive, chemiluminescence devices to measure nNO. This study aimed to compare nNO measurement using three different devices in distinguishing PCD patients from healthy controls and cystic fibrosis (CF) patients and to evaluate their diagnostic precision. The study included 16 controls, 16 PCD patients, and 12 CF patients matched for age and sex. nNO measurements were performed using a chemiluminescence device (Eco Medics CLD 88sp), and two devices based on electrochemical sensors (Medisoft FeNO+ and NIOX Vero) following standardized guidelines. Correlation estimation, Bland–Altman, ROC curve, and one-way ANOVA were used to assess device differences and diagnostic performance. Significantly lower nNO output values were observed in PCD and CF patients compared to controls during exhalation against resistance. The correlation analysis showed high agreement among the three devices. ROC curve analysis demonstrated 100% sensitivity and specificity at different cut-off values for all devices in distinguishing PCD patients from controls (optimal cut-offs: EcoMedics 73, Medisoft 92 and NIOX 87 (nl min−1 )). Higher nNO output values were obtained with the Medisoft and NIOX devices as compared to the EcoMedics device, with a bias of−19 nl min−1 (95% CI: −73–35) and −21 nl min−1 (−73–31) accordingly. These findings indicate that all three tested devices can potentially serve as diagnostic tools for PCD if device specific cut-off values are used. This last-mentioned aspect warrants further studies and consideration in defining optimal cut-offs for individual device.