Genetic background and intraoperative haemodynamic instability in patients with pheochromocytoma and paraganglioma: a multicenter retrospective study.

BACKGROUND: Perioperative management to maintain intraoperative haemodynamic stability is crucial during surgical treatment of pheochromocytomas and paragangliomas (PPGLs). Although approximately 70% of PPGLs carry pathogenic variants (PVs) in susceptibility genes, whether intraoperative haemodynamic instability (IHI) is associated with genetic background remains unclear. This study aimed to analyse IHI in patients with PPGL due to PVs in different genes. MATERIALS AND METHODS: This retrospective study recruited 756 patients with abdominal PPGL from two tertiary care centres. Clinical information including sex, age, catecholamine-associated signs and symptoms (CAS), tumour location and size, biochemistry, and perioperative characteristics were collected. Genetic mutations were investigated using next-generation sequencing. RESULTS: Among the 671 patients included in the analysis, 61.8% (415/671) had IHI. IHI was significantly associated with genetic background in patients with PPGL. Most (80.9%, 89/110) patients with PPGL due to PVs in HRAS suffered IHI. In contrast, only half (31/62) of patients with PPGL due to PVs in VHL had IHI. In the multivariate regression analysis, compared to those with negative genetic testing results, patients with PPGL due to PVs in HRAS (OR 3.82, 95% CI 2.187-6.679, P<0.001), the other cluster 2 genes (OR 1.95, 95% CI 1.287-2. 569, P< 0.05), and cluster 1 genes other than VHL (OR 2.35, 95% CI 1.338-4.111, P<0.05) were independent risk factors for IHI, while PVs in VHL was not independent risk factor (OR 1.09, 95% CI 0.605-1.953, P>=0.05). In addition, age at diagnosis of primary tumour, presenting of CAS, and tumour size were identified as independent factors for IHI. The nomogram illustrated that genetic background as sharing the largest contribution to IHI, followed by tumour size, age, and presenting of CAS. CONCLUSION: IHI is associated with the genetic background in patients with PPGL. The perioperative management of patients with PPGL can be personalized according to their genetic backgrounds, tumour size, age, and presenting of CAS.

Meshing theory and contact analysis of double enveloping hourglass worm drive with planar generatrix.

The meshing and limit equations of worm drive usually have strong nonlinearities such as multiple solutions, solution nonexistence and equation singularity. Meanwhile, the tooth surfaces of worm drive are in nonconforming line contact, which often requires mesh refinement of contact region for the loaded contact analysis. These two challenges cause that the modeling of worm drive heavily relies on manual adjustment and the loaded contact analysis of worm drive is still rare especially when edge contact and assembly error are concerned. Focusing on the double enveloping hourglass worm (DEHW) drive with planar generatrix, this work presents procedures to solve meshing and limit equations with global convergence. The instantaneous contact line, meshing limit line, curvature interference limit line and tooth surface grid discretization are adaptively generated, without manual trial or adjustment. On the basis of adaptive mesh refinement of tooth surface, the mortar virtual element method is adopted for loaded contact analysis of DEHW drive with edge contact and center distance error. Under sliding friction, the discrete system of governing equalities and inequalities is solved by semi-smooth Newton algorithm after constraint condensation. Numerical results for meshing theory and loaded contact analysis of DEHW drive with planar generatrix are discussed.

Differently Distributed Iron Deposition in Mild and Moderate Stroke with Small Artery Occlusion: A Pilot Voxel-Based Susceptibility Mapping Study.

BACKGROUND: Small artery occlusion (SAO) is a common ischemic stroke subtype. However, its clinical outcome can be more severe than commonly understood. The severity of SAO can vary, ranging from mild to moderate. Iron deposition has been associated with the development and progression of stroke. However, its specific distribution and relationship with stroke severity in SAO remain unclear. The study's purpose is to investigate the differences in iron deposition between mild stroke with SAO (SAO-MiS) and moderate stroke with SAO (SAO-MoS) through quantitative susceptibility mapping (QSM) and its association with neurological deficits. METHODS: Sixty-eight SAO participants within 24 hours of first onset were enrolled and separated into SAO-MiS and SAO-MoS according to the National Institutes of Health Stroke Scale (NIHSS) scores. QSM helped calculate the susceptibility maps, reflecting the iron content within the brain. The susceptibility maps were analyzed using voxel-wise statistical analysis to compare the iron deposition between SAO-MiS and SAO-MoS. Then, differentially distributed iron deposition helped differentiate between mild and moderate stroke using support vector machine (SVM) methods. RESULTS: Compared with SAO-MiS, SAO-MoS depicted elevated iron deposition in the left pallidum, parahippocampal gyrus, and superior frontal gyrus medial region, and is lower in the right superior/middle frontal gyrus and bilateral supplementary motor area. Based on iron deposition, the SVM classifier's analysis revealed a high power to discriminate SAO-MoS from SAO-MiS. In addition, fibrinogen, triglyceride (TG), and total cholesterol (TC) were linked with QSM values in specific brain regions. CONCLUSIONS: Our study first revealed the brain iron distribution after SAO and differently distributed iron deposition in SAO-MiS and SAO-MoS. The results indicate that iron deposition could play a role in the pathophysiology of SAO and its correlation with stroke severity.

High-Efficiency Perovskite Solar Cells with Improved Interfacial Charge Extraction by Bridging Molecules.

The interface between the perovskite layer and electron transporting layer is a critical determinate for the performance and stability of perovskite solar cells (PSCs). The heterogeneity of the interface critically affects the carrier dynamics at the buried interface. To address this, a bridging molecule, (2-aminoethyl)phosphonic acid (AEP), is introduced for the modification of SnO2/perovskite buried interface in n-i-p structure PSCs. The phosphonic acid group strongly bonds to the SnO2 surface, effectively suppressing the surface carrier traps and leakage current, and uniforming the surface potential. Meanwhile, the amino group influences the growth of perovskite film, resulting in higher crystallinity, phase purity, and fewer defects. Furthermore, the bridging molecules facilitate the charge extraction at the interface, as indicated by the femtosecond transient reflection (fs-TR) spectroscopy, leading to champion power conversion efficiency (PCE) of 26.40% (certified 25.98%) for PSCs. Additionally, the strengthened interface enables improved operational durability of ≈1400 h for the unencapsulated PSCs under ISOS-L-1I protocol.

CRISPR/Cas9-Editing K562 Cell Line as a Potential Tool in Transfusion Applications: Knockout of Vel Antigen Gene.

Introduction: The Vel- phenotype is a rare blood group, and it is challenging for identifying this phenotype due to limited available reagents. Moreover, there are relatively few studies on genomic editing of erythroid antigens and generation of knockout (KO) cell lines at present. Methods: To identify the high-efficiency small-guiding RNA (sgRNA) sequence, candidate sgRNAs were transfected into HEK 293T cells and analyzed using Sanger sequencing. Following this, the high-efficiency sgRNA was transfected into K562 cells using lentivirus transduction to generate KO Vel blood group gene cells. The expression of the Vel protein was detected using Western blot on single-cell clones. Additionally, flow cytometry was used to detect the erythroid markers CD235a and CD71. Hemoglobin quantification and Giemsa staining were also performed to evaluate the erythroid differentiation of KO clones induced by hemin. Results: The high-efficiency sgRNA was successfully obtained and used for CRISPR-Cas9 editing in K562 cells. After limiting dilution and screening, two KO clones had either deleted 2 or 4 bases and showed no expression of the Vel protein. In the hemin-induced KO clone, there was a significant difference in erythroid marker and hemoglobin quantification compared to untreated cells. The morphological changes were also observed for the hemin-induced KO clone. Conclusion: In this study, a highly efficient sgRNA was screened out and used to generate Vel erythroid antigen KO single-cell clones in K562 cells. The edited cells could then be induced to undergo erythroid differentiation with the use of hemin.

Spatial heterogeneity, sediment-water exchange, and diffusion mechanisms of organophosphate esters from river to coastal aquatic system in a typical economic circle of northern China.

Organophosphate esters (OPEs) have proven to be pervasive in aquatic environments globally. However, understanding their partitioning behavior and mechanisms at the sediment-water interface remains limited. This study elucidated the spatial heterogeneity, interfacial exchange, and diffusion mechanisms of 14 OPEs (∑14OPEs) from river to coastal aquatic system. The transport tendencies of OPEs at the sediment-water interface were quantitatively assessed using fugacity methods. The total ∑14OPEs concentrations in water and sediments ranged from 154 ng/L to 528 ng/L and 2.41 ng/g dry weight (dw) to 230 ng/g dw, respectively. This result indicated a descending spatial tendency with moderate variability. OPE distribution was primarily influenced by temperature, pH, and dissolved oxygen levels. As the carbon atom number increased, alkyl and chlorinated OPEs transitioned from diffusion towards the aqueous phase to equilibrium. In contrast, aryl OPEs and triphenylphosphine oxide, which had equivalent carbon atom counts, maintained equilibrium throughout. Diffusion trends of individual OPE congener at the sediment-water interface varied at the same total organic carbon contents (foc). As the foc increased, the fugacity fraction values for all OPE homologs showed a declining trend. The distinct molecular structure of each OPE monomer might lead to unique diffusive behaviors at the sediment-water interface. Higher soot carbon content had a more pronounced effect on the distribution patterns of OPEs. The sediment-water distribution of OPEs was primarily influenced by total organic carbon, sediment particle size, dry density, and moisture content. OPEs displayed the highest sensitivity to fluctuations in ammonium and dissolved organic carbon. This study holds significant scientific and theoretical implications for elucidating the interfacial transport and driving forces of OPEs and comprehending their fate and endogenous release in aquatic ecosystems.

Spiral-Pipe Gas Anaerobic Digester.

The reduction of carbon dioxide to methane using hydrogen is an important process in biogas production. However, designing gas anaerobic digesters (GADs) based on this reaction presents several challenges. In this study, we developed an innovative spiral-pipe gas anaerobic digester (SGAD) to increase the displacement distance between the bubbles, thus prolonging the gas retention time and facilitating the reduction of CO2 to CH4 via H2. The process was successfully demonstrated by using a CO2/H2 ratio of 1:3 and a gas-feeding rate of 3.9 L Lr -1 d-1. During the experiment, more than 98% of the CO2 and 96% of the H2 were consumed, resulting in biogas containing ca. 86-96% CH4. Additionally, we applied our proposed evaluation methodology for assessing GAD performance to evaluate the performance of the SGAD. This methodology serves as a reference for evaluating and designing GAD systems. The innovative design of the SGAD and the corresponding evaluation methodology offer new insights into the design of reactors.

Identification of TonB-dependent siderophore receptor inhibitors against Flavobacterium columnare using a structure-based high-throughput virtual screening method.

TonB-dependent siderophore receptors play a critical transport role for Flavobacterium columnare virulence formation and growth, and have become valuable targets for the development of novel antimicrobial agents. Traditional Chinese medicine has demonstrated notable efficacy in the treatment of fish diseases and includes potential antibacterial agents. Herein, we performed molecular docking-based virtual screening to discover novel TonB-dependent siderophore receptor inhibitors from traditional Chinese medicine and provide information for developing novel antibacterial agents. Firstly, we efficiently obtained 11 potential inhibitors with desirable drug-like characteristics from thousands of compounds in the TCM library based on virtual screening and property prediction. The antibacterial activity of Enoxolone, along with its interaction characteristics, were determined via an MIC assay and molecular dynamic simulation. Transcriptional profiling, along with validation experiments, subsequently revealed that an insufficient uptake of iron ions by bacteria upon binding to the TonB-dependent siderophore receptors is the antibacterial mechanism of Enoxolone. Finally, Enoxolone's acceptable toxicity was illustrated through immersion experiments. In summary, we have used virtual screening techniques for the first time in the development of antimicrobial agents in aquaculture. Through this process, we have identified Enoxolone as a promising compound targeting the TonB-dependent siderophore receptor of F. columnare. In addition, our findings will provide new ideas for the advancement of innovative antimicrobial medications in aquaculture.

Performance evaluation of ML models for preoperative prediction of HER2-low BC based on CE-CBBCT radiomic features: A prospective study.

To explore the value of machine learning (ML) models based on contrast-enhanced cone-beam breast computed tomography (CE-CBBCT) radiomics features for the preoperative prediction of human epidermal growth factor receptor 2 (HER2)-low expression breast cancer (BC). Fifty-six patients with HER2-negative invasive BC who underwent preoperative CE-CBBCT were prospectively analyzed. Patients were randomly divided into training and validation cohorts at approximately 7:3. A total of 1046 quantitative radiomic features were extracted from CE-CBBCT images and normalized using z-scores. The Pearson correlation coefficient and recursive feature elimination were used to identify the optimal features. Six ML models were constructed based on the selected features: linear discriminant analysis (LDA), random forest (RF), support vector machine (SVM), logistic regression (LR), AdaBoost (AB), and decision tree (DT). To evaluate the performance of these models, receiver operating characteristic curves and area under the curve (AUC) were used. Seven features were selected as the optimal features for constructing the ML models. In the training cohort, the AUC values for SVM, LDA, RF, LR, AB, and DT were 0.984, 0.981, 1.000, 0.970, 1.000, and 1.000, respectively. In the validation cohort, the AUC values for the SVM, LDA, RF, LR, AB, and DT were 0.859, 0.880, 0.781, 0.880, 0.750, and 0.713, respectively. Among all ML models, the LDA and LR models demonstrated the best performance. The DeLong test showed that there were no significant differences among the receiver operating characteristic curves in all ML models in the training cohort (P > .05); however, in the validation cohort, the DeLong test showed that the differences between the AUCs of LDA and RF, AB, and DT were statistically significant (P = .037, .003, .046). The AUCs of LR and RF, AB, and DT were statistically significant (P = .023, .005, .030). Nevertheless, no statistically significant differences were observed when compared to the other ML models. ML models based on CE-CBBCT radiomics features achieved excellent performance in the preoperative prediction of HER2-low BC and could potentially serve as an effective tool to assist in precise and personalized targeted therapy.

Modulating confinement space in metal-organic frameworks enables highly selective indole C3-formylation.

Here, Selective C3-formylation of indole was achieved under mild conditions using a metal-organic framework (MOF) catalyst. The confined reaction space within the MOF pores effectively suppressed undesired side reactions and promoted the formation of the targeted product by controlling the reaction pathway. Density functional theory (DFT) calculations corroborated the experimental observations.

Sustainable 3D printing by reversible salting-out effects with aqueous salt solutions.

Achieving a simple yet sustainable printing technique with minimal instruments and energy remains challenging. Here, a facile and sustainable 3D printing technique is developed by utilizing a reversible salting-out effect. The salting-out effect induced by aqueous salt solutions lowers the phase transition temperature of poly(N-isopropylacrylamide) (PNIPAM) solutions to below 10 °C. It enables the spontaneous and instant formation of physical crosslinks within PNIPAM chains at room temperature, thus allowing the PNIPAM solution to solidify upon contact with a salt solution. The PNIPAM solutions are extrudable through needles and can immediately solidify by salt ions, preserving printed structures, without rheological modifiers, chemical crosslinkers, and additional post-processing steps/equipment. The reversible physical crosslinking and de-crosslinking of the polymer through the salting-out effect demonstrate the recyclability of the polymeric ink. This printing approach extends to various PNIPAM-based composite solutions incorporating functional materials or other polymers, which offers great potential for developing water-soluble disposable electronic circuits, carriers for delivering small materials, and smart actuators.

Systemic immune-inflammation index as a novel predictor of major adverse cardiovascular events in patients undergoing percutaneous coronary intervention: a meta-analysis of cohort studies.

BACKGROUND: The Systemic Immune-Inflammation Index (SII), a novel marker of inflammation based on neutrophil, platelet, and lymphocyte counts, has demonstrated potential prognostic value in patients undergoing percutaneous coronary intervention (PCI). Our aim was to assess the correlation between the SII and major adverse cardiovascular events following percutaneous coronary intervention. METHODS: We searched PubMed, Web of Science, Embase, and The Cochrane Library from inception to November 20, 2023, for cohort studies investigating the association between SII and the occurrence of MACEs after PCI. Statistical analysis was performed using Revman 5.3, with risk ratios (RRs) and 95% confidence intervals (CIs) as relevant parameters. RESULTS: In our analysis, we incorporated a total of 8 studies involving 11,117 participants. Our findings revealed that a high SII is independently linked to a increased risk of MACEs in PCI patients (RR: 2.08,95%CI: 1.87-2.32, I2 = 42%, p < 0.00001). Additionally, we demonstrated the prognostic value of SII in all-cause mortality, heart failure, and non-fatal myocardial infarction. CONCLUSIONS: Elevated SII may serve as a potential predictor for subsequent occurrence of MACEs in patients undergoing PCI. TRIAL REGISTRATION: Our protocol was registered in PROSPERO (registration number: CRD42024499676).

Diagnosis and management of urinary bladder paragangliomas: A Sino-American-European retrospective observational study.

OBJECTIVE: Paragangliomas of the urinary bladder (UBPGLs) are rare neuroendocrine tumours and pose a diagnostic and surgical challenge. It remains unclear what factors contribute to a timely presurgical diagnosis. The purpose of this study is to identify factors contributing to missing the diagnosis of UBPGLs before surgery. DESIGN, PATIENTS AND MEASUREMENTS: A total of 73 patients from 11 centres in China, and 51 patients from 6 centres in Europe and 1 center in the United States were included. Clinical, surgical and genetic data were collected and compared in patients diagnosed before versus after surgery. Logistic regression analysis was used to identify clinical factors associated with initiation of presurgical biochemical testing. RESULTS: Among all patients, only 47.6% were diagnosed before surgery. These patients were younger (34.0 vs. 54.0 years, p < .001), had larger tumours (2.9 vs. 1.8 cm, p < .001), and more had a SDHB pathogenic variant (54.7% vs. 11.9%, p < .001) than those diagnosed after surgery. Patients with presurgical diagnosis presented with more micturition spells (39.7% vs. 15.9%, p = .003), hypertension (50.0% vs. 31.7%, p = .041) and catecholamine-related symptoms (37.9% vs. 17.5%, p = .012). Multivariable logistic analysis revealed that presence of younger age (<35 years, odds ratio [OR] = 6.47, p = .013), micturition spells (OR = 6.79, p = .007), hypertension (OR = 3.98, p = .011), and sweating (OR = 41.72, p = .013) increased the probability of initiating presurgical biochemical testing. CONCLUSIONS: Most patients with UBPGL are diagnosed after surgery. Young age, hypertension, micturition spells and sweating are clues in assisting to initiate early biochemical testing and thus may establish a timely presurgical diagnosis.

Response of antioxidation and immunity to combined influences of pH and ammonia nitrogen in the spotted babylon (Babylonia areolata).

Spotted babylon were exposed to three different pH levels (7.0, 8.0 and 9.0) and four different concentrations of ammonia nitrogen (0.02, 1.02, 5.10 and 10.20 mg/L) in seawater to determine their acute toxicity and physiological responses to environmental fluctuation. The study evaluated four antioxidant enzymes: catalase (CAT), alkaline, superoxide dismutase (SOD), peroxidase (POD) and glutathione peroxidase (GSH-PX), and two immunoenzymes: acid phosphatase (ACP) and phosphatase (AKP). Over time, the immunoenzyme activity was significantly affected by pH and ammonia nitrogen concentration. After being exposed to pH and ammonia nitrogen, the spotted babylon showed signs of unresponsiveness to external stimuli, reduced vitality, slow movement, and an inability to maintain an upright position. Over time, the spotted babylon exhibited a trend of increasing and then decreasing GSH-PX, CAT, and SOD activities to adapt to the changing environment and enhance its immunity. On the contrary, the POD and ACP activities exhibited a decreasing trend initially, followed by an increasing trend over time and the AKP activity showed a gradual increase with time. The combined effect of pH and ammonia was found to be stronger than the effect of either factor alone. The interaction between pH and ammonia increased the activity of the spotted babylon antioxidant enzymes, induced oxidative stress, and reduced the ability of the spotted babylon's non-specific immune system to reverse it. Thus, the reverse-back of the spotted babylon was higher when pH and ammonia stress were dual than when pH or ammonia were single-factor stresses. The study results will establish a theoretical basis for analyzing the risk of multiple factors to the spotted babylon, and also enrich the basic information about the shellfish immune system.

Xinnaotongluo liquid protects H9c2 cells from H/R-induced damage by regulating MDM2/STEAP3.

Xinnaotongluo liquid has been used to improve the clinical symptoms of patients with myocardial infarction. However, the molecular mechanism of Xinnaotongluo liquid is not completely understood. H9c2 cells exposed to hypoxia/reoxygenation (H/R) was used to simulate damage to cardiomyocytes in myocardial infarction in vitro. The biological indicators of H9c2 cells were measured by cell counting kit-8, enzyme linked immunoabsorbent assay, and western blot assay. In H/R-induced H9c2 cells, a markedly reduced murine double minute 2 (MDM2) was observed. However, the addition of Xinnaotongluo liquid increased MDM2 expression in H/R-induced H9c2 cells. And MDM2 overexpression strengthened the beneficial effects of Xinnaotongluo liquid on H9c2 cells from the perspective of alleviating oxidative damage, cellular inflammation, apoptosis and ferroptosis of H/R-induced H9c2 cells. Moreover, MDM2 overexpression reduced the protein expression of p53 and Six-Transmembrane Epithelial Antigen of Prostate 3 (STEAP3). Whereas, STEAP3 overexpression hindered the function of MDM2-overexpression in H/R-induced H9c2 cells. Our results insinuated that Xinnaotongluo liquid could protect H9c2 cells from H/R-induced damage by regulating MDM2/STEAP3, which provide a potential theoretical basis for further explaining the working mechanism of Xinnaotongluo liquid.

The latest emerging drugs for the treatment of diabetic cardiomyopathy.

INTRODUCTION: Diabetic cardiomyopathy (DCM) is a serious complication of diabetes mellitus involving multiple pathophysiologic mechanisms. In addition to hypoglycemic agents commonly used in diabetes, metabolism-related drugs, natural plant extracts, melatonin, exosomes, and rennin-angiotensin-aldosterone system are cardioprotective in DCM. However, there is a lack of systematic summarization of drugs for DCM. AREAS COVERED: In this review, the authors systematically summarize the most recent drugs used for the treatment of DCM and discusses them from the perspective of DCM pathophysiological mechanisms. EXPERT OPINION: We discuss DCM drugs from the perspective of the pathophysiological mechanisms of DCM, mainly including inflammation and metabolism. As a disease with multiple pathophysiological mechanisms, the combination of drugs may be more advantageous, and we have discussed some of the current studies on the combination of drugs.

Realizing Stable Perovskite Solar Cells with Efficiency Exceeding 25.6% Through Crystallization Kinetics and Spatial Orientation Regulation.

Organic-inorganic hybrid perovskites have emerged as highly promising candidates for photovoltaic applications, owing to the exceptional optoelectronic properties and low cost. Nonetheless, the performance and stability of solar cells suffer from the defect states of perovskite films aroused by non-optically active phases and non-centralized crystal orientation. Herein, a versatile organic molecule, Hydantoin, to modulate the crystallization of perovskite, is developed. Benefiting from the diverse functional groups, more spatially oriented perovskite films with high crystallinity are formed. This enhancement is accompanied by a conspicuous reduction in defect density, yielding efficiency of 25.66% (certified 25.15%), with superb environmental stability. Notably, under the standard measurement conditions (ISOS-L-1I), the maximum power point (MPP) output maintains 96.8% of the initial efficiency for 1600 h and exhibits excellent ion migration suppression. The synergistic regulation of crystallization and spatial orientation offers novel avenues for propelling perovskite solar cell (PSC) development.

Integrated analyses reveal unexpected complex inversion and recombination in RH genes.

ABSTRACT: Phenotype D-- is associated with severe hemolytic transfusion reactions and hemolytic disease of the fetus and newborn. It is typically caused by defective RHCE genes. In this study, we identified a D-- phenotype proband and verified Rh phenotypes of other 6 family members. However, inconsistent results between the phenotypic analysis and Sanger sequencing revealed intact RHCE exons with no mutations in the D-- proband, but the protein was not expressed. Subsequent whole-genome sequencing by Oxford Nanopore Technologies of the proband revealed an inversion with ambiguous breakpoints in intron 2 and intron 7 and copy number variation loss in the RHCE gene region. Given that the RHCE gene is highly homologous to the RHD gene, we conducted a comprehensive analysis using Pacific Biosciences long-read target sequencing, Bionano optical genome mapping, and targeted next-generation sequencing. Our findings revealed that the proband had 2 novel recombinant RHCE haplotypes, RHCE∗Ce(1-2)-D(3-10) and RHCE∗Ce(1-2)-D(3-10)-Ce(10-8)-Ce(3-10), with clear-cut breakpoints identified. Furthermore, the RH haplotypes of the family members were identified and verified. In summary, we made, to our knowledge, a novel discovery of hereditary large inversion and recombination events occurring between the RHD and RHCE genes, leading to a lack of RhCE expression. This highlights the advantages of using integrated genetic analyses and also provides new insights into RH genotyping.

Targeted next-generation sequencing and long-read HiFi sequencing provide novel insights into clinically significant KLF1 variants.

BACKGROUND: Krüppel-like factor 1 (KLF1), a crucial erythroid transcription factor, plays a significant role in various erythroid changes and haemolytic diseases. The rare erythrocyte Lutheran inhibitor (In(Lu)) blood group phenotype serves as an effective model for identifying KLF1 hypomorphic and loss-of-function variants. In this study, we aimed to analyse the genetic background of the In(Lu) phenotype in a population-based sample group by high-throughput technologies to find potentially clinically significant KLF1 variants. RESULTS: We included 62 samples with In(Lu) phenotype, screened from over 300,000 Chinese blood donors. Among them, 36 samples were sequenced using targeted Next Generation Sequencing (NGS), whereas 19 samples were sequenced using High Fidelity (HiFi) technology. In addition, seven samples were simply sequenced using Sanger sequencing. A total of 29 hypomorphic or loss-of-function variants of KLF1 were identified, 21 of which were newly discovered. All new variants discovered by targeted NGS or HiFi sequencing were validated through Sanger sequencing, and the obtained results were found to be consistent. The KLF1 haplotypes of all new variants were further confirmed using clone sequencing or HiFi sequencing. The lack of functional KLF1 variants detected in the four samples indicates the presence of additional regulatory mechanisms. In addition, some samples exhibited BCAM polymorphisms, which encodes antigens of the Lutheran (LU) blood group system. However, no BCAM mutations which leads to the absence of LU proteins were detected. CONCLUSIONS: High-throughput sequencing methods, particularly HiFi sequencing, were introduced for the first time into genetic analysis of the In(Lu) phenotype. Targeted NGS and HiFi sequencing demonstrated the accuracy of the results, providing additional advantages such as simultaneous analysis of other blood group genes and clarification of haplotypes. Using the In(Lu) phenotype, a powerful model for identifying hypomorphic or loss-of-function KLF1 variants, numerous novel variants have been detected, which have contributed to the comprehensive understanding of KLF1. These clinically significant KLF1 mutations can serve as a valuable reference for the diagnosis of related blood cell diseases.

Restoration of virulence in the attenuated Candid#1 vaccine virus requires reversion at both positions 168 and 427 in the envelope glycoprotein GPC.

Live-attenuated virus vaccines provide long-lived protection against viral disease but carry inherent risks of residual pathogenicity and genetic reversion. The live-attenuated Candid#1 vaccine was developed to protect Argentines against lethal infection by the Argentine hemorrhagic fever arenavirus, Junín virus. Despite its safety and efficacy in Phase III clinical study, the vaccine is not licensed in the US, in part due to concerns regarding the genetic stability of attenuation. Previous studies had identified a single F427I mutation in the transmembrane domain of the Candid#1 envelope glycoprotein GPC as the key determinant of attenuation, as well as the propensity of this mutation to revert upon passage in cell culture and neonatal mice. To ascertain the consequences of this reversion event, we introduced the I427F mutation into recombinant Candid#1 (I427F rCan) and investigated the effects in two validated small-animal models: in mice expressing the essential virus receptor (human transferrin receptor 1; huTfR1) and in the conventional guinea pig model. We report that I427F rCan displays only modest virulence in huTfR1 mice and appears attenuated in guinea pigs. Reversion at another attenuating locus in Candid#1 GPC (T168A) was also examined, and a similar pattern was observed. By contrast, virus bearing both revertant mutations (A168T+I427F rCan) approached the lethal virulence of the pathogenic Romero strain in huTfR1 mice. Virulence was less extreme in guinea pigs. Our findings suggest that genetic stabilization at both positions is required to minimize the likelihood of reversion to virulence in a second-generation Candid#1 vaccine.IMPORTANCELive-attenuated virus vaccines, such as measles/mumps/rubella and oral poliovirus, provide robust protection against disease but carry with them the risk of genetic reversion to the virulent form. Here, we analyze the genetics of reversion in the live-attenuated Candid#1 vaccine that is used to protect against Argentine hemorrhagic fever, an often-lethal disease caused by the Junín arenavirus. In two validated small-animal models, we find that restoration of virulence in recombinant Candid#1 viruses requires back-mutation at two positions specific to the Candid#1 envelope glycoprotein GPC, at positions 168 and 427. Viruses bearing only a single change showed only modest virulence. We discuss strategies to genetically harden Candid#1 GPC against these two reversion events in order to develop a safer second-generation Candid#1 vaccine virus.