A case report of using gauze packing to treat postoperative chest bleeding after left pneumonectomy for secondary rifampicin-resistant tuberculosis.

One patient with rifampin-resistant tuberculosis underwent emergency left pneumonectomy and thoracic gauze packing for hemoptysis due to recurrent hemoptysis after transcatheter arterial embolization. Vital signs were maintained by mechanical ventilation and medication. Tracheotomy and anti-tuberculosis treatment were performed. After half a year of follow-up, the patient's condition was stable.

Sialylation on vesicular integrin ß1 determined endocytic entry of small extracellular vesicles into recipient cells.

BACKGROUND: Small extracellular vesicles (sEV) are closely associated with the development and metastasis of many types of mammalian cancer. Glycoconjugates are highly expressed on sEV and play important roles in sEV biogenesis and their interaction with other cells. However, the study on vesicular glycoconjugates are far behind proteins and nucleic acids. Especially, the functions of sialic acids which are the terminal components of glycoconjugates, are poorly understood in sEV. METHODS: Sialic acid levels on sEV from plasma and bladder cancer cells were determined by ELISA and lectin blotting. Effects of sialylation on sEV uptake were determined by flow cytometry. Vesicular glycoproteins bearing sialic acids responsible for sEV uptake was identified by proteomics and density gradient centrifugation, and their site-specific sialylation functions were assayed by N-glycosylation site mutation. Effects of integrin ß1 bearing sialic acids on the pro-metastatic function of sEV in vivo were explored using Balb/c nu/nu mice. RESULTS: (1) Increased sialic acid levels were observed in sEV from malignant bladder cancer cells. (2) Elimination of sialic acids on sEV impaired sEV uptake by recipient cells. (3) Vesicular integrin ß1 bearing sialic acids was identified to play a key role in sEV uptake. (4) Desialylation of the hybrid domain of vesicular integrin ß1 inhibited its binding to matrix fibronectin, and reduced sEV entry into recipient cells. (5) Sialylation on integrin ß1 affected pro-metastatic function of sEV in Balb/c nu/nu mice. CONCLUSIONS: Taken together, our findings indicate important functional roles of sialic acids in sEV uptake and reprogramming plasticity of surrounding normal epithelial cells.

Highly efficient expression of Rasamsonia emersonii lipase in Pichia pastoris: characterization and gastrointestinal simulated digestion in vitro.

BACKGROUND: Acidic lipases with high catalytic activities under acidic conditions have important application values in the food, feed and pharmaceutical industries. However, the availability of acidic lipases is still the main obstacle to their industrial applications. Although a novel acidic lipase Rasamsonia emersonii (LIPR) was heterologously expressed in Escherichia coli, the expression level was unsatisfactory. RESULTS: To achieve the high-efficiency expression and secretion of LIPR in Pichia pastoris GS115, the combinatorial optimization strategy was adopted including gene codon preference, signal peptide, molecular chaperone co-expression and disruption of vacuolar sorting receptor VPS10. The activity of the combinatorial optimization engineered strain in a shake flask reached 1480 U mL-1 , which was 8.13 times greater than the P. pastoris GS115 parental strain. After high-density fermentation in a 5-L bioreactor, the highest enzyme activity reached as high as 11 820 U mL-1 . LIPR showed the highest activity at 40 °C and pH 4.0 in the presence of Ca2+ ion. LIPR exhibited strong tolerance to methanol, indicating its potential application in biodiesel biosynthesis. Moreover, the gastrointestinal digestion simulation results demonstrated that LIPR was tolerant to pepsin and trypsin, but its activity was inhibited by sodium taurodeoxycholate. CONCLUSION: This study provided an effective approach for the high expression of acidic lipase LIPR. LIPR was more appropriate for lipid digestion in the stomach than in intestine according to the gastrointestinal digestion simulation results. © 2024 Society of Chemical Industry.

Protein Signatures for Distinguishing Colorectal Cancer Liver Metastases from Primary Liver Cancer Using Tissue Slide Proteomics.

BACKGROUND: Colorectal cancer liver metastasis (CRLM) and hepatocellular carcinoma (HCC) are both high incidence tumors in China. In certain poorly differentiated cases they can exhibit comparable imaging and pathological characteristics, which impedes accurate clinical diagnosis. The use of protein-based techniques with tissue slides offers a more precise means to assess pathological changes and has the potential to assist with tumor diagnosis. METHODS: A simple in situ protein digestion protocol was established for protein fingerprint analysis of paraffin-embedded tissue slide samples. Additionally, machine learning techniques were employed to construct predictive models for CRLM and HCC. The accuracy of these models was validated using tissue slides and a clinical database. RESULTS: Analysis of differential protein expression between CRLM and HCC groups reliably identified 977 proteins. Among these, 53 were highly abundant in CRLM samples and 57 were highly abundant in HCC samples. A prediction model based on the expression of six proteins (CD9, GSTA1, KRT20, COL1A2, AKR1C3, and HIST2H2BD) had an area under curve (AUC) of 0.9667. This was further refined to three proteins (CD9, ALDH1A1, and GSTA1) with an AUC of 0.9333. CONCLUSIONS: Tissue slide proteomics can facilitate accurate differentiation between CRLM and HCC. This methodology holds great promise for improving clinical tumor diagnosis and for identifying novel markers for challenging pathological specimens.

Site- and Structure-Specific Glycosylation Signatures of Bovine, Caprine, Porcine, and Human Milk-Derived Extracellular Vesicles.

Extracellular vesicles (EVs) are membrane-bound vesicles released by living cells. As vesicles for macromolecule transmission and intercellular communication, EVs are broadly applied in clinical diagnosis and biomimetic drug delivery. Milk-derived EVs (MEVs) are an ideal choice for scale-up applications because they exhibit biocompatibility and are easily obtained. Herein, intact glycopeptides in MEVs from bovines, caprines, porcines, and humans were comprehensively analyzed by high-resolution mass spectrometry using the sceHCD, followed by the EThcD fragment method, revealing that protein glycosylation is abundant and heterogeneous in MEVs. The dominant glycans in all MEVs were sialic acid-modified N-linked glycans (over 50%). A couple of species-specific glycans were also characterized, which are potentially markers of different original EVs. Interestingly, the Neu5Gc-modified glycans were enriched in caprine milk-derived EVs (58 ± 2%). Heterogeneity of MEV protein glycosylation was observed for glycosites and glycan compositions, and the structural heterogeneity of protein glycosylation was also identified and validated. The glycosignatures of EV biogenesis- and endocytosis-related proteins (CD63 and MFGE8) were significantly different in these four species. Overall, we comprehensively characterized the glycosylation signature of MEVs from four different species and provided insight into protein glycosylation related to drug target delivery.

Sialylation: A Cloak for Tumors to Trick the Immune System in the Microenvironment.

The tumor microenvironment (TME), where the tumor cells incite the surrounding normal cells to create an immune suppressive environment, reduces the effectiveness of immune responses during cancer development. Sialylation, a type of glycosylation that occurs on cell surface proteins, lipids, and glycoRNAs, is known to accumulate in tumors and acts as a "cloak" to help tumor cells evade immunological surveillance. In the last few years, the role of sialylation in tumor proliferation and metastasis has become increasingly evident. With the advent of single-cell and spatial sequencing technologies, more research is being conducted to understand the effects of sialylation on immunity regulation. This review provides updated insights into recent research on the function of sialylation in tumor biology and summarizes the latest developments in sialylation-targeted tumor therapeutics, including antibody-mediated and metabolic-based sialylation inhibition, as well as interference with sialic acid-Siglec interaction.

Comprehensive proteome, phosphoproteome and kinome characterization of luminal A breast cancer.

Background: Breast cancer is one of the most frequently occurring malignant cancers worldwide. Invasive ductal carcinoma (IDC) and invasive lobular carcinoma (ILC) are the two most common histological subtypes of breast cancer. In this study, we aimed to deeply explore molecular characteristics and the relationship between IDC and ILC subtypes in luminal A subgroup of breast cancer using comprehensive proteomics and phosphoproteomics analysis. Methods: Cancer tissues and noncancerous adjacent tissues (NATs) with the luminal A subtype (ER- and PR-positive, HER2-negative) were obtained from paired IDC and ILC patients respectively. Label-free quantitative proteomics and phosphoproteomics methods were used to detect differential proteins and the phosphorylation status between 10 paired breast cancer and NATs. Then, the difference in protein expression and its phosphorylation between IDC and ILC subtypes were explored. Meanwhile, the activation of kinases and their substrates was also revealed by Kinase-Substrate Enrichment Analysis (KSEA). Results: In the luminal A breast cancer, a total of 5,044 high-confidence proteins and 3,808 phosphoproteins were identified from 10 paired tissues. The protein phosphorylation level in ILC tissues was higher than that in IDC tissues. Histone H1.10 was significantly increased in IDC but decreased in ILC, Conversely, complement C4-B and Crk-like protein were significantly decreased in IDC but increased in ILC. Moreover, the increased protein expression of Septin-2, Septin-9, Heterogeneous nuclear ribonucleoprotein A1 and Kinectin but reduce of their phosphorylation could clearly distinguish IDC from ILC. In addition, IDC was primarily related to energy metabolism and MAPK pathway, while ILC was more closely involved in the AMPK and p53/p21 pathways. Furthermore, the kinomes in IDC were primarily significantly activated in the CMGC groups. Conclusions: Our research provides insights into the molecular characterization of IDC and ILC and contributes to discovering novel targets for further drug development and targeted treatment.

Expression of a Siglec-Fc Protein and Its Characterization.

The emerging importance of the Siglec-sialic acid axis in human disease, especially cancer, has necessitated the identification of ligands for Siglecs. Recombinant Siglec-Fc fusion proteins have been widely used as ligand detectors, and also as sialic acid-targeted antibody-like proteins for cancer treatment. However, the heterogenetic properties of the Siglec-Fc fusion proteins prepared from various expression systems have not been fully elucidated. In this study, we selected HEK293 and CHO cells for producing Siglec9-Fc and further evaluated the properties of the products. The protein yield in CHO (8.23 mg/L) was slightly higher than that in HEK293 (7.46 mg/L). The Siglec9-Fc possesses five N-glycosylation sites and one of them is located in its Fc domain, which is important for the quality control of protein production and also the immunogenicity of Siglec-Fc. Our glycol-analysis confirmed that the recombinant protein from HEK293 received more fucosylation, while CHO showed more sialylation. Both products revealed a high dimerization ratio and sialic acid binding activity, which was confirmed by the staining of cancer cell lines and bladder cancer tissue. Finally, our Siglec9-Fc product was used to analyze the potential ligands on cancer cell lines.

Rare sugar L-sorbose exerts antitumor activity by impairing glucose metabolism.

Rare sugars are monosaccharides with low natural abundance. They are structural isomers of dietary sugars, but hardly be metabolized. Here, we report that rare sugar L-sorbose induces apoptosis in various cancer cells. As a C-3 epimer of D-fructose, L-sorbose is internalized via the transporter GLUT5 and phosphorylated by ketohexokinase (KHK) to produce L-sorbose-1-phosphate (S-1-P). Cellular S-1-P inactivates the glycolytic enzyme hexokinase resulting in attenuated glycolysis. Consequently, mitochondrial function is impaired and reactive oxygen species are produced. Moreover, L-sorbose downregulates the transcription of KHK-A, a splicing variant of KHK. Since KHK-A is a positive inducer of antioxidation genes, the antioxidant defense mechanism in cancer cells can be attenuated by L-sorbose-treatment. Thus, L-sorbose performs multiple anticancer activities to induce cell apoptosis. In mouse xenograft models, L-sorbose enhances the effect of tumor chemotherapy in combination with other anticancer drugs. These results demonstrate L-sorbose as an attractive therapeutic reagent for cancer treatment.

Accumulated precursors of specific GPI-anchored proteins upregulate GPI biosynthesis with ARV1.

We previously reported that glycosylphosphatidylinositol (GPI) biosynthesis is upregulated when endoplasmic reticulum-associated degradation (ERAD) is defective; however, the underlying mechanistic basis remains unclear. Based on a genome-wide CRISPR-Cas9 screen, we show that a widely expressed GPI-anchored protein CD55 precursor and ER-resident ARV1 are involved in upregulation of GPI biosynthesis under ERAD-deficient conditions. In cells defective in GPI transamidase, GPI-anchored protein precursors fail to obtain GPI, with the remaining uncleaved GPI-attachment signal at the C-termini. We show that ERAD deficiency causes accumulation of the CD55 precursor, which in turn upregulates GPI biosynthesis, where the GPI-attachment signal peptide is the active element. Among the 31 GPI-anchored proteins tested, only the GPI-attachment signal peptides of CD55, CD48, and PLET1 enhance GPI biosynthesis. ARV1 is prerequisite for the GPI upregulation by CD55 precursor. Our data indicate that GPI biosynthesis is balanced to need by ARV1 and precursors of specific GPI-anchored proteins.

Two modified density gradient centrifugation methods facilitate the isolation of mouse Leydig cells.

Preparation of sufficient mouse Leydig cells (LCs) with high purity is a prerequisite for investigations of the biological/pathological functions of LCs in mouse models. Density gradient centrifugation based on discontinuous Percoll gradients is an effective method (defined as regular method) for LC isolation. In this study, we developed two modified methods for LC isolation and compared their performance with that of the regular method. Modified method 1 integrated the crude LCs into the 50% Percoll solution before centrifugation. Modified method 2 sequentially used 50 and 60% Percoll solutions to isolate LCs. The purity of LCs was approximately 88.4, 91.3, and 79.7% derived from the regular, modified 1, and modified 2 methods, respectively. The yields of LCs in the same respective order were approximately 1.7 × 105, 3.9 × 105, and 11.9 × 105 cells per 108 interstitial cells input. Modified method 1 attained higher purity and yields than those of the regular method. Although the purity of LCs was relatively low for modified method 2, it could be used before further purification by, for example, fluorescence-activated or magnetic-activated cell sorting, owing to its simplicity and high yields. Therefore, our study provided alternative methods to facilitate LC isolation in mice.

Aloe Vera-Fermented Beverage Ameliorates Obesity and Gut Dysbiosis in High-Fat-Diet Mice.

Aloe vera has been proven to have various medicinal properties, including anti-inflammatory and anti-obesity functions. However, the effects of Aloe vera-fermented beverages (AFB) on obesity and its complications are still not clear. In this study, HepG2 cells in high-fat environment and high-fat diet (HFD) mice were used to investigate the potential obesity-preventing function of AFB. We found that AFB intervention decreased the amount of lipid droplets of HepG2 cells, suppressed the body weight gain and adipose accumulation, and reduced the serum contents of total cholesterol (TC), alanine aminotransferase (ALT), and interleukin 10 (IL-10) of HFD-mice. In addition, it also changed the composition of the gut microbiota. The ratio of Firmicutes/Bacteroidetes was decreased, while the relative abundance of Muribaculaceae, Alistipes and Rikenellaceae_RC9_gut_group was increased after the administration of AFB compared with HFD-mice. These results demonstrated that AFB can prevent diet-induced obesity (DIO) and provides a new option to modulate obesity-related gut dysbiosis.

miR-146-5p restrains calcification of vascular smooth muscle cells by suppressing TRAF6.

Vascular calcification is a prominent manifestation of advanced atherosclerosis. Tumor necrosis factor-receptor-associated factors (TRAFs) were reported to participate in atherosclerosis development. In this study, the role and mechanism of TRAF6 in vascular calcification were explored. To induce the vascular calcification, oxidized low-density lipoprotein (Ox-LDL) was applied to treat vascular smooth muscle cells (VSMCs). TRAF6 protein expression in VSMCs was assessed by western blotting. Osteogenic differentiation of VSMCs was assessed by alkaline phosphatase activity analysis. Mineral deposition in VSMCs was evaluated by von Kossa staining. VSMC proliferation, migration, apoptosis, inflammation, and reactive oxygen species (ROS) generation were detected using cell counting kit-8, Transwell, flow cytometry, reverse transcriptase quantitative polymerase chain reaction (RT-qPCR), and dichlorodihydrofluorescein diacetate staining, respectively. Luciferase reporter assay was utilized to identify the binding relationship between miR-146-5p and TRAF6 in VSMCs. We found that Ox-LDL administration induced the calcification of VSMCs and elevated the TRAF6 level. TRAF6 knockdown restrained VSMC calcification, proliferation, migration, inflammation, and ROS generation caused by Ox-LDL. Mechanically, TRAF6 was targeted by miR-146-5p in VSMCs. Furthermore, TRAF6 overexpression offset the inhibitory effects of miR-146-5p upregulation on vascular calcification in VSMCs under the Ox-LDL condition. Overall, miR-146-5p restrains the calcification of VSMCs by suppressing TRAF6.

Evaluation of strategies for identification of infants with pathogenic glucose-6-phosphate dehydrogenase variants in China.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency, which is caused by pathogenic variants of G6PD that result in decreased G6PD activity, is an X-linked inherited inborn error of metabolism that occurs worldwide. Individuals with G6PD deficiency and heterozygous females with normal G6PD activity (i.e., all individuals with pathogenic G6PD variants) are at risk of developing hemolytic anemia under increased oxidative challenge. However, this risk can be minimized by timely diagnosis. Currently, two assays are used to diagnose G6PD deficiency in China: evaluation of enzymatic activity and targeted genotyping. In terms of identification of all individuals with pathogenic G6PD variants, the performance and cost of different diagnostic strategies (isolated or combined evaluation of G6PD activity and G6PD genotyping) can vary, and these factors should be comprehensively evaluated. In this study, we examined 555 infants (437 males and 118 females) who were positive for the newborn screening of G6PD deficiency. We first evaluated the diagnostic performances of enzymatic testing and targeted genotyping. Both assays attained 100% specificities and positive predictive values for both male and female infants. In contrast, the sensitivities and negative predictive values (NPVs) of the diagnostic tests were different for male and female infants. For male infants, the sensitivities were 99.8 and 98.3%, and the NPVs were 94.1% and 69.6%, for enzymatic testing and targeted genotyping, respectively. For female infants, the sensitivities were 62.5% and 97.9%, and the NPVs were 37.9% and 91.7%, for enzymatic testing and targeted genotyping, respectively. We also evaluated the cost of the five different diagnostic strategies. The combination of G6PD activity testing of all infants, followed by genotyping of female infants with normal G6PD activity, attained high diagnostic sensitivity (99.8%) at a low cost (8.60 USD per diagnosed case). In the future, simultaneous examination of G6PD activity and whole-exon or whole-gene G6PD sequencing could become a standard clinical practice. Our data provide references for clinical practice on the standardization of current and future interventions for G6PD deficiency in China.

High strength chitosan hydrogels prepared from NaOH/urea aqueous solutions: the role of thermal gelling.

Upon heating and subsequent solvent displacement with pure water, the cold chitosan solution with aqueous alkaline/urea as solvent readily transforms into a hydrogel which is substantially stronger than traditional chitosan hydrogels regenerated from acidic solutions. In this work, we have systematically studied the effects of the treatment parameters in this two-step basic route and found that thermal gelation was the crucial step that dictated the structure and properties of the final gels. We hypothesized that the primary network formed in the thermal gelation step served as a template for the deposition of chitosan chains during solvent displacement, leading to a homogenous and compact structure. The primary network also provides crystalline seeds to facilitate the crystallization of the chitosan chains, leading to higher degree of crystallinity. This study provided a guideline for the preparation of chitosan hydrogels with high mechanical properties which is very meaningful to relevant research and applications.

Bioinspired, Ultra-Fast Polymerization of Dopamine under Mild Conditions.

Spontaneous oxidative polymerization of dopamine (DA) is widely exploited as a facile and versatile method for surface modification. However, the reaction is very slow and only occurs in alkaline solutions, which severely limit its applications. Herein it is reported that the reaction can be dramatically accelerated by using Fe2+ as catalyst. While it takes hours and days using conventional method, the Fe2+ -catalyzed reaction finishes almost immediately at pH 7.0. In addition, under the catalysis of Fe2+ , the reaction can occur at a pH down to 4.0. The fast Fe2+ -catalyzed polymerization of DA leads to fast deposition of polydopamine (PDA) coating, thus allowing fast surface modification and textile dyeing. The Fe2+ -catalyzed reaction also allows spatial control over the PDA deposition. The fast, simple, and mild surface modification method developed here will find applications in numerous fields.

Dysregulation and prometastatic function of glycosyltransferase C1GALT1 modulated by cHP1BP3/ miR-1-3p axis in bladder cancer.

BACKGROUND: Abnormal glycosylation in a variety of cancer types is involved in tumor progression and chemoresistance. Glycosyltransferase C1GALT1, the key enzyme in conversion of Tn antigen to T antigen, is involved in both physiological and pathological conditions. However, the mechanisms of C1GALT1 in enhancing oncogenic phenotypes and its regulatory effects via non-coding RNA are unclear. METHODS: Abnormal expression of C1GALT1 and its products T antigen in human bladder cancer (BLCA) were evaluated with BLCA tissue, plasma samples and cell lines. Effects of C1GALT1 on migratory ability and proliferation were assessed in YTS-1 cells by transwell, CCK8 and colony formation assay in vitro and by mouse subcutaneous xenograft and trans-splenic metastasis models in vivo. Dysregulated circular RNAs (circRNAs) and microRNAs (miRNAs) were profiled in 3 pairs of bladder cancer tissues by RNA-seq. Effects of miR-1-3p and cHP1BP3 (circRNA derived from HP1BP3) on modulating C1GALT1 expression were investigated by target prediction program, correlation analysis and luciferase reporter assay. Functional roles of miR-1-3p and cHP1BP3 on migratory ability and proliferation in BLCA were also investigated by in vitro and in vivo experiments. Additionally, glycoproteomic analysis was employed to identify the target glycoproteins of C1GALT1. RESULTS: In this study, we demonstrated upregulation of C1GALT1 and its product T antigen in BLCA. C1GALT1 silencing suppressed migratory ability and proliferation of BLCA YTS-1 cells in vitro and in vivo. Subsets of circRNAs and miRNAs were dysregulated in BLCA tissues. miR-1-3p, which is reduced in BLCA tissues, inhibited transcription of C1GALT1 by binding directly to its 3'-untranslated region (3'-UTR). miR-1-3p overexpression resulted in decreased migratory ability and proliferation of YTS-1 cells. cHP1BP3 was upregulated in BLCA tissues, and served as an miR-1-3p "sponge". cHP1BP3 was shown to modulate migratory ability, proliferation, and colony formation of YTS-1 cells, and displayed tumor-suppressing activity in BLCA. Target glycoproteins of C1GALT1, including integrins and MUC16, were identified. CONCLUSIONS: This study reveals the pro-metastatic and proliferative function of upregulated glycosyltransferase C1GLAT1, and provides preliminary data on mechanisms underlying dysregulation of C1GALT1 via miR-1-3p / cHP1BP3 axis in BLCA.

Polymerase chain reaction-based assays facilitate the breeding and study of mouse models of Klinefelter syndrome.

Klinefelter syndrome (KS) is one of the most frequent genetic abnormalities and the leading genetic cause of nonobstructive azoospermia. The breeding and study of KS mouse models are essential to advancing our knowledge of the underlying pathological mechanism. Karyotyping and fluorescence in situ hybridization are reliable methods for identifying chromosomal contents. However, technical issues associated with these methods can decrease the efficiency of breeding KS mouse models and limit studies that require rapid identification of target mice. To overcome these limitations, we developed three polymerase chain reaction-based assays to measure specific genetic information, including presence or absence of the sex determining region of chromosome Y (Sry), copy number of amelogenin, X-linked (Amelx), and inactive X specific transcripts (Xist) levels. Through a combined analysis of the assay results, we can infer the karyotype of target mice. We confirmed the utility of our assays with the successful generation of KS mouse models. Our assays are rapid, inexpensive, high capacity, easy to perform, and only require small sample amounts. Therefore, they facilitate the breeding and study of KS mouse models and help advance our knowledge of the pathological mechanism underlying KS.

Newborn Screening for G6PD Deficiency in Xiamen, China: Prevalence, Variant Spectrum, and Genotype-Phenotype Correlations.

Background: Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common inherited enzymatic defect. The purpose of this study was to evaluate the profile of G6PD deficiency and investigate the factors associated with the accuracy of newborn screening (NBS) in Xiamen, China. Methods: A total of 99,546 newborns were screened by modified fluorescent spot test at the Women and Children's Hospital, Xiamen University. High-risk neonates were recalled for diagnosis by either a measurement of G6PD activity or genetic testing for the presence of pathogenic G6PD variants using a quantitative G6PD enzymatic assay or the MeltPro® G6PD assay, respectively. Results: In the first-tier screening, 1,256 newborns were categorized as high risk. Of these, 1,051 were diagnosed with G6PD deficiency, indicating a prevalence of 1.39% in Xiamen, China. Among the 1,013 neonates who underwent genotyping, 851 carried hemizygous, heterozygous, homozygous, or compound heterozygous variants, for a positive predictive value (PPV) of 84.01%. In total, 12 variants and 32 genotypes were identified, and the six most common variants were c.1376G>T, c.1388G>A, c.95A>G, c.1024C>T, c.871G>A, and c.392G>T, which accounted for approximately 94% of the identified alleles. Different variants showed characteristic enzymatic activities, although high phenotypic heterogeneity was observed for each variant. The use of cold-chain transportation significantly improved the PPV of NBS. Conclusions: We determined the profile of G6PD deficiency in Xiamen, including the prevalence, variant spectrum, and genotype-phenotype correlations and confirmed that maintaining a low temperature during sample transport is essential to ensure the high screening accuracy of NBS. Our data provides epidemiological, genotypic, phenotypic, and clinical practice references to standardize future interventions for G6PD deficiency.