[Clinical phenotype and genetic analysis of a fetus with Cardiac valvular dysplasia type 1].

OBJECTIVE: To explore the genetic basis for a fetus with Cardiac valvular dysplasia type 1 (CVDP1). METHODS: A CVDP1 fetus identified at the Ningbo Women and Children's Hospital on July 7, 2022 was selected as the study subject. Clinical data of the fetus was collected. The fetus and its parents were subjected to trio-whole exome sequencing (trio-WES), and candidate variants were verified by Sanger sequencing. RESULTS: The fetus had exhibited generalized edema, complex cardiac malformation, abdominal effusion, and enhanced intestinal and renal parenchymal echoes. Trio-WES revealed that it has harbored compound heterozygous variants of the PLD1 gene, namely c.2977C>T (p.R993*) and c.1460G>A (p.W487*), which were respectively inherited from its father and mother. Neither variant was reported previously. Based on the guidelines from the American College of Medical Genetics and Genomics (ACMG), the c.2977C>T (p.R993*) variant was evaluated to be likely pathogenic (PVS1_Moderate+PM2_Supporting+PM3+PP4), whilst the c.1460G>A (p.W487*) variant was evaluated to be pathogenic (PVS1+PM2_Supporting+PP4). CONCLUSION: The c.2977C>T (p.R993*) and c.1460G>A (p.W487*) compound heterozygous variants of the PLD1 gene probably underlay the CVDP1 in the fetus. Above discovery has enriched the mutational spectrum of the PLD1 gene and provided a guidance for genetic counseling and prenatal diagnosis in this family.

Improved Principal Component Analysis (IPCA): A Novel Method for Quantitative Calibration Transfer between Different Near-Infrared Spectrometers.

Given the labor-consuming nature of model establishment, model transfer has become a considerable topic in the study of near-infrared (NIR) spectroscopy. Recently, many new algorithms have been proposed for the model transfer of spectra collected by the same types of instruments under different situations. However, in a practical scenario, we need to deal with model transfer between different types of instruments. To expand model applicability, we must develop a method that could transfer spectra acquired from different types of NIR spectrometers with different wavenumbers or absorbance. Therefore, in our study, we propose a new methodology based on improved principal component analysis (IPCA) for calibration transfer between different types of spectrometers. We adopted three datasets for method evaluation, including public pharmaceutical tablets (dataset 1), corn data (dataset 2), and the spectra of eight batches of samples acquired from the plasma ethanol precipitation process collected by FT-NIR and MicroNIR spectrometers (dataset 3). In the calibration transfer for public datasets, IPCA displayed comparable results with the classical calibration transfer method using piecewise direct standardization (PDS), indicating its obvious ability to transfer spectra collected from the same types of instruments. However, in the calibration transfer for dataset 3, our proposed IPCA method achieved a successful bi-transfer between the spectra acquired from the benchtop and micro-instruments with/without wavelength region selection. Furthermore, our proposed method enabled improvements in prediction ability rather than the degradation of the models built with original micro spectra. Therefore, our proposed method has no limitations on the spectrum for model transfer between different types of NIR instruments, thus allowing a wide application range, which could provide a supporting technology for the practical application of NIR spectroscopy.

The significance of machine learning in neonatal screening for inherited metabolic diseases.

Background: Neonatal screening for inherited metabolic diseases (IMDs) has been revolutionized by tandem mass spectrometry (MS/MS). This study aimed to enhance neonatal screening for IMDs using machine learning (ML) techniques. Methods: The study involved the analysis of a comprehensive dataset comprising 309,102 neonatal screening records collected in the Ningbo region, China. An advanced ML system model, encompassing nine distinct algorithms, was employed for the purpose of predicting the presence of 31 different IMDs. The model was compared with traditional cutoff schemes to assess its diagnostic efficacy. Additionally, 180 suspected positive cases underwent further evaluation. Results: The ML system exhibited a significantly reduced positive rate, from 1.17% to 0.33%, compared to cutoff schemes in the initial screening, minimizing unnecessary recalls and associated stress. In suspected positive cases, the ML system identified 142 true positives with high sensitivity (93.42%) and improved specificity (78.57%) compared to the cutoff scheme. While false negatives emerged, particularly in heterozygous carriers, our study revealed the potential of the ML system to detect asymptomatic cases. Conclusion: This research provides valuable insights into the potential of ML in pediatric medicine for IMD diagnosis through neonatal screening, emphasizing the need for accurate carrier detection and further research in this domain.

Review of the Application of Raman Spectroscopy in Qualitative and Quantitative Analysis of Drug Polymorphism.

Drug polymorphism is an important factor affecting the drugs quality and clinical efficacy. Therefore, great attention should be paid to the crystal analysis of drugs with their researching and evaluating part. With the booming development of Raman spectroscopy in recent years, more and more crystal analysis investigations were based on vibrational spectroscopy. This review mainly discussed the qualitative and quantitative analysis of active pharmaceutical ingredients (API) and pharmaceutical preparation with Raman spectroscopy. On basis of the determination of the vibration mode of drug molecules and the analysis of their chemical structure, this method had the advantages of universal, non-destructive, fast determination, low samples and cost, etc. This review provides theoretical and technical support for crystal structure, which are worth popularizing. It is expected that it will be helpful to relevant government management institutions, pharmaceutical scientific research institutions and pharmaceutical manufacturers.

Real-time in-line prediction of drug loading and release rate in the coating process of diclofenac sodium spheres based on near infrared spectroscopy.

The preparation of diclofenac sodium spheres by fluidized bed is a common production mode for the pharmaceutical preparations at present, but the critical material attributes in the production process is mostly analyzed off-line, which is time-consuming and laborious, and the analysis results lag behind. In this paper, the real-time in-line prediction of drug loading of diclofenac sodium and the release rate during the coating process was realized by using near infrared spectroscopy. For the best near infrared spectroscopy (NIRS) model of drug loading, R2cv, R2p, RMSECV, RMSEP were 0.9874, 0.9973, 0.002549 mg/g, 0.001515 mg/g respectively. For the best NIRS model of three release time points, the R2cv, R2p, RMSECV and RMSEP were 0.9755, 0.9823, 3.233%, 4.500%; 0.9358, 0.9965, 2.598%, 0.7939% and 0.9867, 0.9927, 0.4085%, 0.4726% respectively. And the analytical ability of these model was verified. The organic combination of these two parts of work constituted an important basis for ensuring the safety and effectiveness of diclofenac sodium spheres from the perspective of production process.

An Integrated Strategy to Identify and Quantify the Quality Markers of Xinkeshu Tablets Based on Spectrum-Effect Relationship, Network Pharmacology, Plasma Pharmacochemistry, and Pharmacodynamics of Zebrafish.

Xinkeshu tablets (XKST), a traditional Chinese patent medicine (CPM), have served in the clinical treatment of cardiovascular diseases (CVDs) for decades. However, its pharmacodyamic material basis was still unclear, and the holistic quality control has not been well established due to the lack of systematic research on the quality markers. In this experiment, the heart rate recovery rate of a zebrafish larva was used to evaluate the traditional pharmacological effect of XKST i.e., antiarrhythmic effect. The HPLC fingerprints of 16 batches of XKST samples were obtained, and antiarrhythmic components of XKST were identified by establishing the spectrum-effect relationship between HPLC fingerprints and heart rate recovery rate of zebrafish larva with orthogonal signal correction and partial least squares regression (OSC-PLSR) analysis. The anticardiovascular disease components of XKST were identified by mapping the targets related to CVDs in network pharmacology. The compounds of XKST absorbed and exposed in vivo were identified by ultra-high performance liquid chromatography Q-Exactive high-resolution mass spectrometry (UHPLC-Q-Exactive HRMS). Based on the earlier studies, combined with five principles for identifying quality markers and verified by a zebrafish arrhythmia model, danshensu, salvianolic acid A, salvianolic acid B, daidzein, and puerarin were identified as quality markers of XKST. In total, 16 batches of XKST samples were further quantified with the method established in this study. Our study laid the foundation for the quality control of XKST. The integrated strategy used in the study of XKST could be applied for the identification and quantification of quality markers of other CPMs as well.

Investigation of protective effect of ethanol on the natural structure of protein with infrared spectroscopy.

The stability of biological drugs with protein as an active substance depends heavily on the retention of natural protein structure during freeze-drying. Stabilizers have become important substances in the process of protein freeze-drying. In order to further understand the mechanism of the interaction between protein and stabilizers, human serum albumin (HSA) and simple hydroxyl compound ethanol were used as models. Infrared (IR) spectroscopy combined with chemometrics was implemented to investigate the changes of secondary structure and hydration of HSA when different concentrations of ethanol were considered as interference. Through the analysis of the protein secondary structure and hydrated layer, we found that the addition of ethanol-d6 increased the α-helix of HSA and reduced the disordered structure. The hydrogen bond structure around HSA was enhanced and intermolecular aggregation was reduced through the action of the water molecules. The hypothesis was verified by circular dichroism (CD) and transmission electron microscopy (TEM) observation by adding different concentrations of ethanol-d6. It was found that a small amount of ethanol could protect the native conformation of HSA. In conclusion, this study revealed the mechanism of ethanol as a protein protector, provided a new idea for protein purification process and a theoretical basis for biomolecular interaction.

Random forest classifier improving phenylketonuria screening performance in two Chinese populations.

Phenylketonuria (PKU) is a genetic disorder with amino acid metabolic defect, which does great harms to the development of newborns and children. Early diagnosis and treatment can effectively prevent the disease progression. Here we developed a PKU screening model using random forest classifier (RFC) to improve PKU screening performance with excellent sensitivity, false positive rate (FPR) and positive predictive value (PPV) in all the validation dataset and two testing Chinese populations. RFC represented outstanding advantages comparing several different classification models based on machine learning and the traditional logistic regression model. RFC is promising to be applied to neonatal PKU screening.

Newborn Screening and Genetic Analysis Identify Six Novel Genetic Variants for Primary Carnitine Deficiency in Ningbo Area, China.

Primary carnitine deficiency (PCD) is an autosomal recessive disorder that could result in sudden death. It is caused by a defect in the carnitine transporter encoded by SLC22A5 (Solute Carrier Family 22 Member 5, MIM:603377). Currently, a number of variants in SLC22A5 have been identified, however, the PCD prevalence and its variants in Ningbo area are unclear. In this study, we screened 265,524 newborns by using tandem mass spectrometry. Variants in SLC22A5 were further detected by next-generation sequencing in individuals with abnormal free carnitine levels (C0). We identified 53 newborns with abnormal C0 levels and 26 with variants in SLC22A5. Among them, 16 with compound heterozygous or homozygous variants in SLC22A5 were diagnosed with PCD, suggesting the PCD birth prevalence in Ningbo city was 1/16,595. Moreover, the C0 level was significantly (P = 0.013) higher in PCD patients than in those with one variant. Besides, the c.1400C > G (p. S467C) and c.51C > G (p. F17L) variants were the most frequent and six novel variants are all predicted to be damaging. This study reports the largest PCD patients in Ningbo area by newborn screening and expands the variant spectrum of SLC22A5. Our findings demonstrate the clinical value of combining NBS program results with DNA analysis for the diagnosis of PCD.

Abnormal Intrinsic Functional Interactions Within Pain Network in Cervical Discogenic Pain.

Cervical discogenic pain (CDP) is mainly induced by cervical disc degeneration. However, how CDP modulates the functional interactions within the pain network remains unclear. In the current study, we studied the changed resting-state functional connectivities of pain network with 40 CDP patients and 40 age-, gender-matched healthy controls. We first defined the pain network with the seeds of the posterior insula (PI). Then, whole brain and seed-to-target functional connectivity analyses were performed to identify the differences in functional connectivity between CDP and healthy controls. Finally, correlation analyses were applied to reveal the associations between functional connectivities and clinical measures. Whole-brain functional connectivity analyses of PI identified increased functional connectivity between PI and thalamus (THA) and decreased functional connectivity between PI and middle cingulate cortex (MCC) in CDP patients. Functional connectivity analyses within the pain network further revealed increased functional connectivities between bilateral PI and bilateral THA, and decreased functional connectivities between left PI and MCC, between left postcentral gyrus (PoCG) and MCC in CDP patients. Moreover, we found that the functional connectivities between right PI and left THA, between left PoCG and MCC were negatively and positively correlated with the visual analog scale, respectively. Our findings provide direct evidence of how CDP modulates the pain network, which may facilitate understanding of the neural basis of CDP.

A Novel Missense Variant in the Gene PPP2R5D Causes a Rare Neurodevelopmental Disorder with Increased Phenotype.

PPP2R5D-related neurodevelopmental disorder, which is mainly caused by de novo missense variants in the PPP2R5D gene, is a rare autosomal dominant genetic disorder with about 100 patients and a total of thirteen pathogenic variants known to exist globally so far. Here, we present a 24-month-old Chinese boy with developmental delay and other common clinical characteristics of PPP2R5D-related neurodevelopmental disorder including hypotonia, macrocephaly, intellectual disability, speech impairment, and behavioral abnormality. Trio-whole exome sequencing (WES) and Sanger sequencing were performed to identify the causal gene variant. The pathogenicity of the variant was evaluated using bioinformatics tools. We identified a novel pathogenic variant in the PPP2R5D gene (c.620G>T, p.Trp207Leu). The variant is located in the variant hotspot region of this gene and is predicted to cause PPP2R5D protein dysfunction due to an increase in local hydrophobicity and unstable three-dimensional structure. We report a novel pathogenic variant of PPP2R5D associated with PPP2R5D-related neurodevelopmental disorder from a Chinese family. Our findings expanded the phenotypic and mutational spectrum of PPP2R5D-related neurodevelopmental disorder.

Identification of Six Novel Variants of ACAD8 in Isobutyryl-CoA Dehydrogenase Deficiency With Increased C4 Carnitine Using Tandem Mass Spectrometry and NGS Sequencing.

Isobutyryl-CoA dehydrogenase deficiency (IBDHD, MIM: #611283) is a rare autosomal recessive hereditary disease, which is caused by genetic mutations of acyl-CoA dehydrogenase (ACAD) 8 and associated with valine catabolism. Here, tandem mass spectrometry (MS/MS) was applied to screen 302,993 neonates for inherited metabolic diseases (IMD) in Ningbo of China from 2017 to 2020. The results suggest that 198 newborns (0.7‰) were initially screened positive for IBDHD with C4-Carnitine, and 27 cases (0.1‰) were re-screened positive. Genetic diagnosis was performed on 21 of the 27 cases. Seven compound heterozygous variations, three biallelic variations, and one heterozygous variation of ACAD8 were found with a pathogenicity rate of 33.3% (7/21). In addition, seven biallelic variations, one heterozygous variation of acyl-CoA dehydrogenase short chain (ACADS), and one biallelic variation of acyl-CoA dehydrogenase short/branched chain (ACADSB) was detected. Further research showed that ACAD8 mutations of 11 IBDHD cases distributed in six different exons with total 14 mutation sites. Five of which were known suspected pathogenic sites (c.286G > A, c.553C > T, c.1000C > T, c.409G > A, c.500del) and six were novel mutation sites: c.911A > T, c.904C > T, c.826G > A, c.995T > C, c.1166G > A, c.1165C > T. This finding enriched the mutation spectrum of ACAD8 in IBDHD.

Static and Dynamic Changes of Amplitude of Low-Frequency Fluctuations in Cervical Discogenic Pain.

Cervical discogenic pain (CDP) is a clinically common pain syndrome caused by cervical disk degeneration. A large number of studies have reported that CDP results in brain functional impairments. However, the detailed dynamic brain functional abnormalities in CDP are still unclear. In this study, using resting-state functional magnetic resonance imaging, we explored the neural basis of CDP with 40 CDP patients and 40 age-, gender-matched healthy controls to delineate the changes of the voxel-level static and dynamic amplitude of low frequency fluctuations (ALFF). We found increased static ALFF in left insula (INS) and posterior precuneus (PCu), and decreased static ALFF in left precentral/postcentral gyrus (PreCG/PoCG), thalamus (THA), and subgenual anterior cingulate cortex in CPD patients compared to healthy controls. We also found decreased dynamic ALFF in left PreCG/PoCG, right posterior middle temporal gyrus, and bilateral THA. Moreover, we found that static ALFF in left PreCG/PoCG and dynamic ALFF in THA were significantly negatively correlated with visual analog scale and disease duration, respectively. Our findings provide the neurophysiological basis for CDP and facilitate understanding the neuropathology of CDP.

High specificity of spot urinary free metanephrines in diagnosis and prognosis of pheochromocytomas and paragangliomas by HPLC with electrochemical detection.

BACKGROUND: The metanephrines (MNs) in plasma and urine were proposed as biomarkers for the diagnosis of pheochromocytomas and paragangliomas (PPGLs). However, plasma free MNs and 24h urinary fractionated MNs were not satisfactory enough in specificity for the diagnosis of PPGLs. Moreover, the collection of 24h urine was inconvenient. This work examined the diagnostic and prognostic efficiency of free MNs in spot urine for PPGLs. METHODS: We measured free MNs concentration in spot urine and plasma of 28 PPGLs patients and 155 control subjects by HPLC with electrochemical detection. Postoperative free MNs levels in spot urine and plasma of 14 PPGLs patients were also determined. Creatinine (Cr) concentration was used for the correction of urine volume. RESULTS: The specificity of spot urinary free MNs/Cr in the diagnosis of PPGLs was significantly higher than that of plasma free MNs [normetanephrine (NMN), 98.7% (95.4%-99.8%) vs 93.0% (87.4%-96.6%); metanephrine (MN), 93.6% (88.5%-96.9%) vs 84.5% (77.5%-90.0%)]. Meanwhile, the positive likelihood ratios for spot urinary free NMN/Cr and MN/Cr were 69.21 and 13.29, compared with 12.68 and 5.30 for plasma free NMN and MN, respectively. For the PPGLs patients underwent surgery, the plasma free MNs level appeared an abnormal elevation and yielded false-positive results for some patients. Our findings were validated in an independent cohort, resulting in the specificity of 100% for both urinary free NMN/Cr and MN/Cr, and 97.3% and 83.8% for plasma free NMN and MN, respectively. CONCLUSIONS: Spot urinary free MNs/Cr, superior to plasma free MNs, presented a promising biomarker for the diagnosis and prognosis of PPGLs.

Identification of potential microRNA-target pairs associated with osteopetrosis by deep sequencing, iTRAQ proteomics and bioinformatics.

MicroRNAs aberrantly express in many human diseases including some metabolic bone disorders. They have been found to be associated with osteoclast differentiation and function, which makes them attractive candidates for the therapy of bone. However, the potential clinical application of microRNAs in therapeutics rests heavily upon our in-depth understanding of microRNAs and their targets. To identify potential microRNA-target pairs associated with osteopetrosis, we performed a system approach including deep sequencing, iTRAQ quantitative proteomics, and bioinformatics in the peripheral blood mononuclear cells (PBMCs) taken from patients with osteopetrosis and health donors. Notably, 123 differently expressed microRNAs, 173 differently expressed proteins, and 117 computationally predicted microRNA-target pairs with reciprocally expressed level in PBMCs were found in the two sample groups. Functional annotation identified that the microRNA-target pairs were involved in cell growth, differentiation, cellular signaling network, and the network highlighted the microRNA-target pair of has-miR-320a and ADP ribosylation factor 1 (Arf1) potentially associated with CLCN7 mutations in osteopetrosis. The pair of has-miR-320a and Arf1 was further verified by real-time PCR, western blot, and the interaction between has-miR-320a and its targeted sequence on the Arf1 mRNAs was confirmed by luciferase assay. Collectively, the present study established a new system approach for the investigation of microRNAs, and the microRNA-target pairs, particular has-miR-320a and Arf1, may have important roles in osteopetrosis.

[Simultaneous determination of tryptophan and its metabolites in plasma by high performance liquid chromatography with on-column derivatization].

A high performance liquid chromatography-ultraviolet/fluorescence detection (HPLC-UV/FLD) with on-column derivatization was established to simultaneously determine tryptophan (Trp), kynurenine (Kyn), 5-hydroxyindole acetic acid (5-Hiaa) and kynurenic acid (Kyna). A Hypersil C-18 column (250 mm x 4.0 mm, 5 microm) was used for the analysis at 30 degrees C. The separation was carried out with the mobile phase consisting of 250 mmol/L zinc acetate (pH 5.5) and acetonitrile (95: 5, v/v) at a flow rate of 0.8 mL/min using 3-nitrotyrosine as internal standard (IS). The excitation (Ex) and emission (Em) wavelengths were set at 278 nm (lambda(ex))/343 nm (lambda(em)) for 5-Hiaa and 244 nm (lambda(ex))/400 nm (lambda(em)) for Kyna, while the wavelengths of ultraviolet detection were set at 360 nm for Kyn and IS, 302 nm for Trp. The recoveries were in the range of 91.62% to 114.17%. The linearities were from 2.50 micromol/L to 320.00 micromol/L for Trp, 0.32 micromol/L to 15.36 micromol/L for Kyn, 3.27 nmol/L to 104.60 nmol/L for 5-Hiaa, and 14.00 nmol/L to 464.80 nmol/L for Kyna. The detection limits were 0.078 micromol/L, 0.056 micromol/L, 0.690 nmol/L and 1.290 nmol/L for Trp, Kyn, 5-Hiaa, and Kyna, respectively. Thirty plasma samples of normal pregnant women and 28 plasma samples of healthy controls were tested, and the results exhibited that the concentrations of Trp, Kyn and Kyna in the plasma of the normal pregnant women were significantly different from those of the control group (all P < 0.01). The method is simple and sensitive with good reproducibility, and it is suitable for clinical measurements.

[Combination surface shaded display with multiplanar reconstruction in the evaluation of acetabular morphologies in patients with developmental dysplasia of the hip before total hip arthroplasty].

OBJECTIVE: To explore the methods and application value of surface shaded display (SSD) and multiplanar reconstruction (MPR) in the evaluation of acetabular morphology in patients with developmental dysplasia of the hip (DDH) before total hip arthroplasty (THA). METHODS: From October 2003 to November 2006, 17 patients (3 males and 14 females, aging from 35 years to 61 years) with osteoarthritis secondary to DDH were scanned with spiral CT preoperatively. According to the Crowe standard, 19 dysplasia hips were classified as type I in 4 hips, type II in 9 hips, type III in 6 hips. The obtained hip CT data were developed with SSD and MPR to observe spatial position and bone stock of the acetabula. RESULTS: The dislocated extent was 25%-89% in these dysplasia hips according to the Crowe method and their sharp angles all exceeded 45 degrees. Bone defect occurred to each of the acetabula, among which it was located in anterosuperior acetabulum in 5 hips, in superolateral acetabulum in 11 hips and in posterosuperior acetabulum in 3 hips. The hip images made with MPR showed that the minimum thickness of the medial wall of acetabula ranged from 2.0 mm to 10.9 mm. Among 15 unilateral dysplasia patients, the opening difference and depth difference between the dysplasia acetabulum and the contralateral one ranged from 2.7 mm to 19.1 mm and from 2.3 mm to 13.1 mm, respectively. CONCLUSION: SSD and MPR of spiral CT are effective methods in evaluating acetabular morphology preoperation and contribute to intraoperative acetabular reconstruction in patients with DDH performed THA.