High Specificity Research Articles

Design and Performance Evaluation of Network Intrusion Detection System Based on Deep Learning

Today’s internets are made up of nearly half million various networks. In any network connection, detecting attacks by their kinds is challenging task as various attacks may have several connections, their number vary from few to hundreds of network connections. In this paper, Design and Performance Evaluation of Network Intrusion Detection System Based on Deep Learning (NID-SPGAN-STO) is proposed. Initially, input data are collected by NSL-KDD dataset. Afterward, data are fed to preprocessing. In preprocessing, Distributed Set-Membership Fusion Filtering is used to remove redundant and biased records from input data. The pre-processed output is given to feature selection for selecting optimal features utilizing Piranha foraging Optimization Algorithm. Finally the selected features are transferred into the Semantic-Preserved Generative Adversarial Network (SPGAN) for detecting Network Intrusion, like DoS, Probe, R2L, U2R and Normal. Generally SPGAN doesn’t reveal some adoption of optimization techniques for computing optimal parameters for promising precise network intrusion detection. Hence Siberian Tiger Optimisation (STO) is used to enhance weight parameters of SPGAN. The proposed NID-SPGAN-STO method is implemented using Python. To detect network intrusion detection, performance metrics likes precision, sensitivity, FI-score, specificity, accuracy, RoC, computational time are considered. The NID-SPGAN-STO method attains 30.58%, 28.73% and 25.62%, higher precision, 20.48%, 24.73%, 29.32% higher specificity and 30.98%, 26.66% and 21.32% higher F-score, 26.78%, 34.47%, and 22.86% higher recall analysed, with existing techniques likes improved binary gray wolf optimizer with SVM for intrusion detection system in WSNs (NID-SVM-IDS), network intrusion detection system utilizing deep learning (NID-DNN), design with improvement of efficient network intrusion detection system utilizing ML methods (NID-IDS-ANN) respectively.

Interspecific variation in Rubisco CO2/O2 specificity along the leaf economic spectrum across 23 woody angiosperm plants in the Pacific islands.

The coordinated interspecific variation in leaf traits and leaf lifespan is known as the leaf economic spectrum (LES). The limitation of CO2 diffusion to chloroplasts within the lamina is significant in C3 photosynthesis, resulting in a shortage of CO2 for Rubisco. Although Rubisco CO2/O2 specificity (SC/O) should be adaptively adjusted in response to the interspecific variation in CO2 concentrations [CO2] associated with Rubisco, SC/O variations across species along the LES remain unknown. We investigated the coordination among leaf traits, including SC/O, CO2 conductance, leaf protein content, and leaf mass area, across 23 woody C3 species coexisting on an oceanic island through phylogenetic correlation analyses. A high SC/O indicates a high CO2 specificity of Rubisco. SC/O was negatively correlated with [CO2] at Rubisco and total CO2 conductance within lamina, while it was positively correlated with leaf protein across species, regardless of phylogenetic constraint. A simulation analysis shows that the optimal SC/O for maximizing photosynthesis depends on both [CO2] at Rubisco sites and leaf protein per unit leaf area. SC/O is a key parameter along the LES axis and is crucial for maximizing photosynthesis across species and the adaptation of woody plants.

AL–AMYLOIDOSIS RELATED ADVANCED HEART FAILURE DIAGNOSED THROUGH F–18–FLUTEMETANOL PET SCAN

Abstract Background Systemic amyloidosis (SA) is a group of rare diseases in which abnormal misfolded proteins, amyloid fibrils, accumulate in tissue. The gold standard for diagnosis is tissue biopsy of the affected organ, but it is invasive and needs specific proteomic or immunohistochemistry analyses to characterize the involved protein. We report a case of a complex diagnosis of SA in an advanced heart failure (HF) patient. Methods and Results A 68–year–old woman with history of recurrent uveitis started to complain dyspnea in 2022, after Covid19. Since her symptoms worsened, in June 2023 she was diagnosed in another center with restrictive cardiomyopathy with preserved left ventricular (LV) ejection fraction. She underwent coronary angiography (unremarkable), cardiac MRI (aspecific fibrosis), and a FDG–PET scan which showed non–homogeneous LV fixation. The patient was treated with steroids for the suspicion of an immunologic–inflammatory disease, without benefit. Fourteen months after the initial presentation, she was admitted to our centre for advanced inotrope–dependent HF. She had mildly increased LV hypertrophy and severe systolic dysfunction with restrictive physiology and, notably, peri–orbital subcutaneous hemorrhages (Figure 1). Hematological screening for amyloidosis showed elevated serum free lambda light chains. Repeated cardiac MRI scan was consistent with infiltrative disease. A total–body PET scan with dual tracer was performed. The control session with F–18–FDG showed mild aspecific cardiac uptake in the basal septum and lateral wall (Figure 2), while off–label use of F–18–Flutemetanol, an Alzheimer‘s disease approved experimental tracer that binds to amyloid fibrils beta sheets with very high sensitivity and specificity, showed intense cardiac, hepatic, pulmonary and muscular uptake, with wash–out–timing diagnostic for AL–SA (Figure 3). Periumbilical fat biopsy confirmed the clinical suspicion of SA. Hematological investigations led to the diagnosis of stage I–A lambda micromolecular multiple myeloma, with systemic amyloidosis and a IIIb cardiac prognostic score. Conclusions Endomyocardial biopsy is an invasive procedure and periumbilical fat biopsy discriminates the amyloid protein type only by proteomic analysis, which is available in very few Italian centres. The off–label use of Alzheimers’ PET tracers could reduce the delay of cardiac AL–SA diagnosis, allowing early disease identification and prompting timely hematological therapy.

Identification of four mitochondria-related genes in sepsis based on RNA sequencing technology

ObjectivesThe purpose of this study was to identify and analyze the mitochondrial genes associated with sepsis patients in order to elucidate the underlying mechanism of sepsis immunity and provide new ideas for the clinical treatment of sepsis.MethodsThe hospitalized cases of sepsis (n = 20) and systemic inflammatory response syndrome (SIRS) (n = 12) admitted to the Emergency Intensive Care Unit (EICU) of the Affiliated Hospital of Southwest Medical University from January 2019 to December 2019 were collected consecutively. RNA-seq was used to sequence the RNA (mRNA) of peripheral blood cells. Bioinformatics techniques were used to screen and identify differentially expressed RNAs, with an absolute value of fold change (FC) greater than or equal to 1.2 and a false discovery rate (FDR) less than 0.05. At the same time, mitochondrial genes were obtained from the MitoCarta 3.0 database. Differential genes were then intersected with mitochondrial genes. The resulting crossover genes were subjected to GO, KEGG, and PPI analysis. Subsequently, the GSE65682 dataset was downloaded from the GEO database for survival analysis to assess the prognostic value of core genes, and GSE67652 was downloaded for ROC curve analysis to validate the diagnostic value of core genes. Finally, the localization of core genes was clarified through 10X single-cell sequencing.ResultsThe crossing of 314 sepsis differential genes and 1136 mitochondrial genes yielded 28 genes. GO and KEGG analysis showed that the crossover genes were mainly involved in the mitochondrion, mitochondrial matrix, and mitochondrial inner membrane. Survival analysis screened four genes that were significantly negatively associated with the prognosis of sepsis, namely FIS1, FKBP8, GLRX5, and GUK1. A comparison of peripheral blood RNA-seq results between the sepsis group and the SIRS group showed that the expression levels of these four genes were significantly decreased in the sepsis group compared to the SIRS group. ROC curve analysis based on GSE67652 indicates these four genes’ high sensitivity and specificity for sepsis detection. Additionally, single-cell RNA sequencing found that the core genes were mainly expressed in macrophages, T cells, and B cells.ConclusionsMitochondria-related genes (FIS1, FKBP8, GLRX5, GUK1) were underexpressed in the sepsis group, negatively correlated with survival, and mainly distributed in immune cells. This finding may guide studying the immune-related mechanisms of sepsis. This study protocol was reviewed by the Ethics Committee of the Affiliated Hospital of Southwest Medical University (ethics number: KY2018029), the clinical trial registration number is ChiCTR1900021261, and the registration date is February 4, 2019.

Potential Advantage of Magnetic Resonance Imaging in Detecting Thoracic Wall Infiltration in Pleural Mesothelioma. A Retrospective Single Center Analysis.

Abstract Background Thoracic wall infiltration in pleural mesothelioma (PM) can be an important prognostic factor when determining the extent of resection. Currently, standardized imaging for restaging after neoadjuvant systemic therapy comprises contrast enhanced Computed Tomography (CT) or Positron Emission Tomography (PET/CT) scan. Aims However, thoracic Magnetic Resonance Imaging (MRI) could be a better discriminator of chest wall infiltration prior to surgery. To increase the preoperative staging accuracy, we introduced a MRI protocol at our center. Methods A retrospective analysis of this prospective new imaging protocol was performed from 07/2018 to 08/2023, including descriptive analysis for patient`s sex, age, nicotine consumption, asbestos exposure, histological subtype, TNM-stage, RECIST criteria and number of neoadjuvant therapy cycles. Preoperative restaging included routine imaging and MRI. After histological diagnosis of PM, neoadjuvant therapy was conducted, followed by partial pleurectomy or extended pleurectomy/decortication, with intraoperative biopsies of suspicious chest wall lesions. The CT/MRI results were compared to the intraoperative biopsies. Sensitivity and specificity of both modalities were analyzed. Results Twenty-five patients (mean age 65.4, 12% female) with possible chest wall infiltration were included out of the 35 patients with PM treated during the observation period. Of the 10 patients with actual chest wall infiltration, 9 (90%) had a T-Stage of 3 or higher, 9 (90%) had PM of epithelioid histologic subtype and 4 (40%) a R2-Resection. The mean overall survival of all patients was 18.88 months (8-58). In our study, thoracic MRI showed a high sensitivity (90%) and specificity (100%) for the detection of chest wall infiltration, especially when compared to the CT scan (sensitivity of 10%). Conclusion With the adjunctive use of thoracic MRI we demonstrated a higher sensitivity for detection of chest wall infiltration compared to conventional imaging prior to surgery. This may facilitate the preoperative assessment of the extent of resection. Nevertheless, larger studies are required to confirm these results.

Multiplexed detection of eight respiratory viruses based on nanozyme colorimetric microfluidic immunoassay

Pandemics caused by respiratory viruses, such as the SARS-CoV-1/2, influenza virus, and respiratory syncytial virus, have resulted in serious consequences to humans and a large number of deaths. The detection of such respiratory viruses in the early stages of infection can help control diseases by preventing the spread of viruses. However, the diversity of respiratory virus species and subtypes, their rapid antigenic mutations, and the limited viral release during the early stages of infection pose challenges to their detection. This work reports a multiplexed microfluidic immunoassay chip for simultaneous detection of eight respiratory viruses with noticeable infection population, namely, influenza A virus, influenza B virus, respiratory syncytial virus, SARS-CoV-2, human bocavirus, human metapneumovirus, adenovirus, and human parainfluenza viruses. The nanomaterial of the nanozyme (Au@Pt nanoparticles) was optimized to improve labeling efficiency and enhance the detection sensitivity significantly. Nanozyme-binding antibodies were used to detect viral proteins with a limit of detection of 0.1 pg/mL with the naked eye and a microplate reader within 40 min. Furthermore, specific antibodies were screened against the conserved proteins of each virus in the immunoassay, and the clinical sample detection showed high specificity without cross reactivity among the eight pathogens. In addition, the microfluidic chip immunoassay showed high accuracy, as compared with the RT-PCR assay for clinical sample detection, with 97.2%/94.3% positive/negative coincidence rates. This proposed approach thus provides a convenient, rapid, and sensitive method for simultaneous detection of eight respiratory viruses, which is meaningful for the early diagnosis of viral infections. Significantly, it can be widely used to detect pathogens and biomarkers by replacing only the antigen-specific antibodies.

THE APEX TALKS…CARDIAC METASTASIS MIMICKING ST SEGMENT ELEVATION MYOCARDIAL INFARCTION

Abstract A 78–year–old man with a recent diagnosis of lung squamous cell carcinoma was referred to our cardiology department for marked ECG abnormalities (ST segment elevation in V2–V4 and T waves inversion in the antero–lateral leads, Fig.1) in the absence of ischemic symptoms and for the detection of an intramyocardial mass at the apical segments on the CT scan. He had no significant previous cardiovascular history. A transthoracic echocardiogram (TTE) was performed and showed an isoechoic mass at the LV apical segments measuring 3 x 4 cm apparently incorporating the myocardial wall without a clear cleavage plane (Fig.2a). The EF was normal. No pericardial effusion present. The contrast echocardiography with Sonovue revealed a late uptake of the contrast (Fig.2b). Cardiac magnetic resonance (CMR) showed an ovular image in the para–apical area of 40 x 23 cm consistent with a secondary lesion (Fig.3). Unfortunately, the exam was interrupted early due to a patient’s claustrophobic crisis, without having acquired the post–contrast images. Due to infiltrative appearance of the mass and the oncology history, this formation was highly suspicious for metastasis. No complicated ventricular arrhythmias were detected on ECG monitoring. No symptoms of heart failure were reported. Due to the marked EKG repolarization abnormalities the patient underwent coronary angiography which excluded significant stenosis. FDG PET/CT showed areas of increased metabolism also at liver and adrenal left gland. Finally, the patient was transferred to the oncology department for specific management. Cardiac metastases are more common than primary cardiac malignancy and are more frequently related to primary lung cancer, followed by breast cancer and hematologic malignancies. Typical presentation includes arrhythmias or conduction disturbances. The imaging findings of cardiac metastases are non–specific but mostly infiltrative, heterogeneous, and multiple masses may be present. Echocardiography is the initial imaging test for the detection of cardiac metastasis, although CMR, CT and PET/CT may be helpful. Herein we described a case of cardiac metastasis with EKG mimicking acute coronary syndrome. This finding is not uncommon among these patients. EKG findings of myocardial ischemia or injury, particularly localized and prolonged ST elevation, in the absence of ischemic symptoms have been reported in previous studies as high specificity for cardiac metastasis in patients with malignancy.

Rapid and sensitive diagnosis of live Mycobacterium tuberculosis using clustered regularly interspaced short palindromic repeat‐Cas13a point‐of‐care RNA testing

AbstractMycobacterium tuberculosis (MTB) is the causal pathogen of tuberculosis (TB). Rapid and accurate detection of live MTB is important for transmission control and patient treatment. Here, we described a clustered regularly interspaced short palindromic repeat (CRISPR)‐Cas13a‐based molecular diagnosis approach for rapid and specific detection of live MTB. This detection method, which we termed CRISPR‐Live‐MTB, contained two consecutive reactions including nuclear acid sequence‐based amplification (NASBA) and CRISPR‐Cas13a collateral cleavage reaction. CRISPR‐Live‐MTB could efficiently detect MTB single‐stranded RNA (ssRNA) in 2 hours with high specificity over double‐stranded DNA (dsDNA). Importantly, CRISPR‐Live‐MTB exhibited a limit of detection of 2.4 copies for MTB ssRNA, which was 1000 times lower than that of the clinically used NASBA method. Moreover, lateral flow was integrated into the CRISPR‐Live‐MTB method to enable point‐of‐care testing application with a sensitivity of 95% and a specificity of 100%. Overall, our study demonstrated the feasibility of CRISPR‐Live‐MTB as a rapid, sensitive, and specific approach for live MTB detection.

The molecular interaction of six single-stranded DNA aptamers to cardiac troponin I revealed by docking and molecular dynamics simulation.

Cardiac troponin I (cTnI) is a cardiac biomarker for diagnosing ischemic heart disease and acute myocardial infarction. Current biochemical assays use antibodies (Abs) due to their high specificity and sensitivity. However, there are some limitations, such as the high-cost production of Abs due to complex instruments, reagents, and steps; the variability of Abs quality from batch to batch; the low stability at high temperatures; and the difficulty of chemical modification. Aptamer overcomes the limitations of antibodies, such as relatively lower cost, high reproducibility, high stability, and ease of being chemically modified. Aptamers are three-dimensional architectures of single-stranded RNA or DNA that bind to targets such as proteins. Six aptamers (Tro1-Tro6) with higher binding affinity than an antibody have been identified, but the molecular interaction has not been studied. In this study, six DNA aptamers were modeled and docked to cTnI protein. Molecular docking revealed that the interaction between all aptamer and cTnI happened in the similar cTnI region. The interaction between aptamer and cTnI involved hydrophobic interaction, hydrogen bonds, π-cation interactions, π-stack interactions, and salt-bridge formation. The calculated binding energy of all complexes was negative, which means that the complex formation was thermodynamically favorable. The electrostatic energy term was the main driving force of the interaction between all aptamer and cTnI. This study could be used to predict the behavior of further modified aptamer to improve aptamer performance.

Focused ultrasound as a treatment modality for gliomas

Ultrasound waves were initially used as a diagnostic tool that provided critical insights into several pathological conditions (e.g., gallstones, ascites, pneumothorax, etc.) at the bedside. Over the past decade, advancements in technology have led to the use of ultrasound waves in treating many neurological conditions, such as essential tremor and Parkinson’s disease, with high specificity. The convergence of ultrasound waves at a specific region of interest/target while avoiding surrounding tissue has led to the coined term “focused ultrasound (FUS).” In tumor research, ultrasound technology was initially used as an intraoperative guidance tool for tumor resection. However, in recent years, there has been growing interest in utilizing FUS as a therapeutic tool in the management of brain tumors such as gliomas. This mini-review highlights the current knowledge surrounding using FUS as a treatment modality for gliomas. Furthermore, we discuss the utility of FUS in enhanced drug delivery to the central nervous system (CNS) and highlight promising clinical trials that utilize FUS as a treatment modality for gliomas.

Three-dimensional telomere profiling predicts risk of progression in smoldering multiple myeloma.

Smoldering multiple myeloma (SMM) is a precursor stage that precedes multiple myeloma (MM). SMM is heterogenous with nearly 40% of patients progressing to MM in the first 5 years. The high rate of progression of SMM patients highlights the need for early intervention, which underscores the importance of identifying SMM patients with the highest risk of progression. Several risk stratification models showed utility in identifying high-risk SMM patients; however, these systems showed limited sensitivity. To date, identifying high-risk SMM patients remains an important clinical need. In this study, we present the 3-dimensional telomere profiling as a structural biomarker capable of stratifying SMM patients as a function of genomic instability. Quantifying telomere dysfunction using the TeloView technology showed utility in risk stratification of cancer patients, particularly hematological malignancies. In this study, we analyzed 168 SMM patients. We report an AUC in ROC analysis of 0.8 using a subset of the patients as a training dataset. We then conducted a blind validation on a different cohort and demonstrated a positive predictive value of 85% and negative predictive value of 73%, with sensitivity and specificity of 83% and 76%, respectively. We examined the correlation between the TeloView prediction and the 20-2-20 scoring system, and cytogenetic abnormalities. We report a correlation of 53% with the 20-2-20 scores and over 60% correlation with cytogenetic abnormalities. The result of this study presents the telomere profiling as an effective biomarker able to stratify SMM patients to their respective risk groups with high sensitivity and specificity.

Test characteristics of glycated albumin in the diagnosis of gestational diabetes mellitus

This study determined the test characteristics of glycated albumin in the diagnosis of gestational diabetes mellitus. The gold standard for diagnosing gestational diabetes mellitus is the oral glucose tolerance test which requires patient preparation, drinking of glucose solution, and multiple sample collections. A possible alternative biomarker for the diagnosis of gestational diabetes is glycated albumin does not require patient preparation and only one sample is collected. Glycated albumin levels are higher among Black Americans than in Caucasians.The study involved 200 pregnant women attending the antenatal clinic at the University of Port Harcourt Teaching Hospital. The diagnosis of gestational diabetes mellitus was made using the World Health Organization 2013 diagnostic criteria. The test characteristics of glycated albumin were determined using the area under the curve of the receiver operator characteristic curve, sensitivity, specificity, positive predictive value, and negative predictive value. The prevalence of gestational diabetes mellitus was 9.0%. The area under the receiver operator characteristic curve for glycated albumin was 0.8 (95% CI 0.7-0.9; p=0.0001). The sensitivity and specificity of glycated albumin were 83.3% and 86.8% respectively. The positive predictive value was 38.5% and the negative predictive value was 98.1%. Glycated albumin has high sensitivity, specificity, and negative predictive values and therefore, can be used as a preliminary test for gestational diabetes mellitus.

Direct investigations of interactions between nucleolins and aptamers on pancreatic cancer and normal cells by atomic force microscopy

Nucleolin is overexpressed on the surface of pancreatic cancer cells and are regarded as the remarkable therapeutic target. Aptamers are capable of binding the external domain of nucleolin on the cell surface with high affinity and specificity. But nucleolin has not been localized on pancreatic cancer cells at very high spatial resolution, and the interactions between nucleolin and aptamers have not been investigated at very high force resolution level. In this work, nucleolin was localized on pancreatic cancer and normal cells by aptamers (9FU-AS1411-NH2, AS1411-NH2 and CRONH2) in Single Molecule Recognition Imaging mode of Atomic Force Microscopy. There are plenty of nucleolin on the surfaces of pancreatic cancer cells (area percentage about 5 %), while there are little nucleolin on the surfaces of normal cells. The interactions between three types of aptamers and nucleolins on the surfaces of pancreatic cancer cells were investigated by Single Molecule Force Spectroscopy. The unbinding forces of nucleolins-(9FU-AS1411-NH2) are larger than nucleolins-(AS1411-NH2). The dissociation activation energy on nucleolin-(9FU-AS1411-NH2) is higher than nucleolin-(AS1411-NH2), which indicates that the former complex is more stable and harder to dissociate than the later complex. There are no unbinding forces between nucleolin and CRONH2. All these demonstrate that nucleolin was localized on pancreatic cancer and normal cells at single molecule level quantitatively, and the interactions (unbinding forces and kinetics) between nucleolin and aptamers were studied at picoNewton level. The approaches and results of this work will pave new ways in the investigations of nucleolin and aptamers, and will also be useful in the studies on other proteins and their corresponding aptamers.

Choriocapillaris reduction accurately discriminates against early-onset Alzheimer's disease.

This study addresses the urgent need for non-invasive early-onset Alzheimer's disease (EOAD) prediction. Using optical coherence tomography angiography (OCTA), we present a choriocapillaris model sensitive to EOAD, correlating with serum biomarkers. Eighty-four EOAD patients and 73 controls were assigned to swept-source OCTA (SS-OCTA) or the spectral domain OCTA (SD-OCTA) cohorts. Our hypothesis on choriocapillaris predictive potential in EOAD was tested and validated in these two cohorts. Both cohorts revealed diminished choriocapillaris signals, demonstrating the highest discriminatory capability (area under the receiver operating characteristic curve: SS-OCTA 0.913, SD-OCTA 0.991; P<0.001). A sparser SS-OCTA choriocapillaris correlated with increased serum amyloid beta (Aβ)42, Aβ42/40, and phosphorylated tau (p-tau)181 levels (all P<0.05). Apolipoprotein E status did not affect choriocapillaris measurement. The choriocapillaris, observed in both cohorts, proves sensitive to EOAD diagnosis, and correlates with serum Aβ and p-tau181 levels, suggesting its potential as a diagnostic tool for identifying and tracking microvascular changes in EOAD. Optical coherence tomography angiography may be applied for non-invasive screening of Alzheimer's disease (AD). Choriocapillaris demonstrates high sensitivity and specificity for early-onset AD diagnosis. Microvascular dynamics abnormalities are associated with AD.

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.

Engineering of a Baeyer-Villiger monooxygenase to improve substrate scope, stereoselectivity and regioselectivity.

Baeyer-Villiger monooxygenases belong to a family of flavin-binding proteins that catalyze the Baeyer-Villiger (BV) oxidation of ketones to produce lactones or esters, which are important intermediates in pharmaceuticals or sustainable materials. Phenylacetone monooxygenase (PAMO) from Thermobifida fusca with moderate thermostability catalyzes the oxidation of aryl ketone substrates, but is limited by high specificity and narrow substrate scope. In the present study, we applied loop optimization by loop swapping followed by focused saturation mutagenesis in order to evolve PAMO mutants capable of catalyzing the regioselective BV oxidation of cyclohexanone and cyclobutanone derivatives with formation of either normal or abnormal esters or lactones. We further modulated PAMO to increase enantioselectivity. Crystal structure studies indicate that rotation occurs in the NADP-binding domain and that the high B-factor region is predominantly distributed in the catalytic pocket residues. Computational analyses further revealed dynamic character in the catalytic pocket and reshaped hydrogen bond interaction networks, which is more favorable for substrate binding. Our study provides useful insights for studying enzyme-substrate adaptations.

Changes of serum cortisol during pregnancy and labor initiation: an onsite cross-sectional study

BackgroundIncreased maternal cortisol secretion has been observed during pregnancy and labor. However, due to the limitations in diagnostic methods, the dynamic change of cortisol during the short period between threatened labor and labor is unknown. In this study, we aim to evaluate the changes in serum cortisol during late pregnancy and full-term labor initiation, verifying if cortisol could serve as a biomarker for the diagnosis of labor initiation from threatened labor.MethodsThis cross-sectional onsite study involved 564 participants of 6 different gestational stages (C: Control; T1: Trimester 1; T3: Trimester 3; E: expectant; TL: threatened labor; L: labor), all patients in the E, TL, and L groups were at full term. The serum cortisol concentration was quantified with a point-of-care test (POCT), and the gestation, age, parity, and BMI of participants were documented. Morning serum cortisol was collected between 8:00 and 10:00 a.m., except for the TL and L group women who were tested upon arrival or during latent labor. With cortisol levels or all five variables, L was distinguished from TL using machine learning algorithms.ResultsSignificant elevation of cortisol concentration was observed between T1 and T3, or TL and L group (P&amp;lt; 0.001). Women belonging to the E and TL group showed similar gestation week and cortisol levels. Diagnosis of labor initiation using cortisol levels (cutoff = 21.46 μg/dL) yielded sensitivity, specificity, and AUC of 86.50%, 88.60%, and 0.934. With additional variables, a higher specificity (89.29%) was achieved. The diagnostic accuracy of all methods ranged from 85.93% to 87.90%.ConclusionSerum cortisol could serve as a potential biomarker for diagnosis of L form TL. The rapid onsite detection of serum cortisol with POCT could facilitate medical decision-making for admission and special treatments, either as an additional parameter or when other technical platforms are not available.

Assessment of the phenotypic severity of hemophilia A: using rotational thromboelastometry (ROTEM) and APTT-clot waveform analysis

BackgroundHemophilia A (HA) is an X-linked inherited bleeding disorder caused by reduced factor VIII (FVIII) levels. Approximately 10–15% of patients with severe HA (SHA) do not present with the anticipated bleeding pattern. Here, we assessed the phenotypic severity of hemophilia A using rotational thromboelastometry (ROTEM) and activated partial thromboplastin time-clot waveform analysis (APTT-CWA).MethodsPatients diagnosed with hemophilia A were enrolled. Clinical phenotype assignment was performed according to the published literature, and patients were classified into four phenotypic subgroups. The whole blood sample was first run on ROTEM in INTEM mode using platelet-poor plasma, APTT was run, and the APTT-CWA graph was simultaneously recorded.ResultsA total of 66 patients were recruited for this study. Statistically significant differences were observed between the four phenotypically categorized groups using ROTEM and APTT-CWA. On comparing patients with mild/moderate-to-severe phenotypes (Group II) with SHA without inhibitors (Group IV), no significant difference was found for all parameters of ROTEM or APTT-CWA. The MCF, MA30, MAXV, and Alpha angle values using ROTEM were found to be the lowest in patients with SHA with inhibitors, which helped differentiate them from those with SHA without inhibitors. However, these two groups could not be differentiated using the APTT-CWA parameters.ConclusionROTEM can be used to distinguish patients with SHA with inhibitors from those with SHA without inhibitors using a combination of parameters with high sensitivity and specificity. However, APTT-CWA cannot be used to differentiate these patient groups.

Opportunities and challenges in accurate early diagnosis and treatment of lung cancer/pulmonary nodules

Lung cancer remains the most prevalent and lethal malignancy in our country. Early diagnosis and treatment are crucial for improving patient prognosis in lung cancer/pulmonary nodules. Recent advancements in non-invasive/minimally invasive liquid biopsy, multi-omics, and artificial intelligence technologies have significantly enhanced the accuracy of early lung cancer/pulmonary nodule diagnosis. However, an early diagnostic method with both high sensitivity and specificity is yet to be established. Furthermore, addressing the methods and extent of early precision surgery, local precision therapy, perioperative combined treatment, and postoperative recurrence and metastasis monitoring are urgent challenges in the early management of lung cancer/pulmonary nodules. Integrating the advantages of various treatment strategies and formulating personalized and precise treatment plans is key to further improving patient survival. In the future, while exploring new therapeutic strategies, it is necessary to continuously search for biomarkers to identify the population that will benefit from the treatment effectively. Additionally, large-sample randomized controlled clinical studies should be conducted to investigate the benefits of long-term patient survival under a diverse range of treatment strategies.

Structural and mechanistic insights into Quinolone Synthase to address its functional promiscuity

Quinolone synthase from Aegle marmelos (AmQNS) is a type III polyketide synthase that yields therapeutically effective quinolone and acridone compounds. Addressing the structural and molecular underpinnings of AmQNS and its substrate interaction in terms of its high selectivity and specificity can aid in the development of numerous novel compounds. This paper presents a high-resolution AmQNS crystal structure and explains its mechanistic role in synthetic selectivity. Additionally, we provide a model framework to comprehend structural constraints on ketide insertion and postulate that AmQNS’s steric and electrostatic selectivity plays a role in its ability to bind to various core substrates, resulting in its synthetic diversity. AmQNS prefers quinolone synthesis and can accommodate large substrates because of its wide active site entrance. However, our research suggests that acridone is exclusively synthesized in the presence of high malonyl-CoA concentrations. Potential implications of functionally relevant residue mutations were also investigated, which will assist in harnessing the benefits of mutations for targeted polyketide production. The pharmaceutical industry stands to gain from these findings as they expand the pool of potential drug candidates, and these methodologies can also be applied to additional promising enzymes.