Probing the Protein-Excipient Interaction in the Orally Delivered Protein by Solid-State Hydrogen-Deuterium Exchange Mass Spectrometry and Molecular Dynamics.

The oral administration of protein therapeutics in solid dosage form is gaining popularity due to its benefits, such as improved medication adherence, convenience, and ease of use for patients compared to traditional parental delivery. However, formulating oral biologics presents challenges related to pH barriers, enzymatic breakdown, and poor bioavailability. Therefore, understanding the interaction between excipients and protein therapeutics in the solid state is crucial for formulation development. In this Letter, we present a case study focused on investigating the role of excipients in protein aggregation during the production of a solid dosage form of a single variable domain on a heavy chain (VHH) protein. We employed solid-state hydrogen-deuterium exchange coupled with mass spectrometry (ssHDX-MS) at both intact protein and peptide levels to assess differences in protein-excipient interactions between two formulations. ssHDX-MS analysis revealed that one formulation effectively prevents protein aggregation during compaction by blocking ß-sheets across the VHH protein, thereby preventing ß-sheet-ß-sheet interactions. Spatial aggregation propensity (SAP) mapping and cosolvent simulation from molecular dynamics (MD) simulation further validated the protein-excipient interaction sites identified through ssHDX-MS. Additionally, the MD simulation demonstrated that the interaction between the VHH protein and excipients involves hydrophilic interactions and/or hydrogen bonding. This novel approach holds significant potential for understanding protein-excipient interactions in the solid state and can guide the formulation and process development of orally delivered protein dosage forms, ultimately enhancing their efficacy and stability.

In-Depth Characterization of Acidic Variants Induced by Metal-Catalyzed Oxidation in a Recombinant Monoclonal Antibody.

Characterization of antibody charge heterogeneity is an important task for antibody drug development. Recently, a correlation between acidic charge heterogeneity and metal-catalyzed oxidation has been observed for antibody drugs. However, to date, the acidic variants induced by metal-catalyzed oxidation have not been elucidated. In addition, it is challenging to satisfactorily explain the induced acidic charge heterogeneity, as the existing analytical workflows, which relied on either untargeted or targeted peptide mapping analysis, could lead to incomplete identification of the acidic variants. In this work, we present a new characterization workflow by combining untargeted and targeted analyses to thoroughly identify and characterize the induced acidic variants in a highly oxidized IgG1 antibody. As a part of this workflow, a tryptic peptide mapping method was also developed for accurate determination of the relative extent of site-specific carbonylation, where a new hydrazone reduction procedure was established to minimize under-quantitation artifacts caused by incomplete reduction of hydrazones during sample preparation. In summary, we identified 28 site-specific oxidation products, which are located on 26 residues and of 11 different modification types, as the sources of the induced acidic charge heterogeneity. Many of the oxidation products were reported for the first time in antibody drugs. More importantly, this study provides new insights to understanding acidic charge heterogeneity of antibody drugs in the biotechnology industry. Additionally, the characterization workflow presented in this study can be applied as a platform approach in the biotechnology industry to better address the need for in-depth characterization of antibody charge variants.

Translatability of In Vitro Stress for Predicting Deamidation and Oxidation Biotransformation on Biotherapeutics.

Biotransformation leading to single residue modifications (e.g., deamidation, oxidation) can contribute to decreased efficacy/potency, poor pharmacokinetics, and/or toxicity/immunogenicity for protein therapeutics. Identifying and characterizing such liabilities in vivo are emerging needs for biologics drug discovery. In vitro stress assays involving PBS for deamidation or AAPH for oxidation are commonly used for predicting liabilities in manufacturing and storage and are sometimes considered a predictive tool for in vivo liabilities. However, reports discussing their in vivo translatability are limited. Herein, we introduce a mass spectrometry workflow that characterizes in vivo oxidation and deamidation in pharmacokinetically relevant compartments for diverse protein therapeutic modalities. The workflow has low bias of <10% in quantitating degradation in the relevant pharmacokinetic concentration range for monkey and rabbit serum/plasma (1-100 µg/mL) and allows for high sequence coverage (∼85%) for discovery/monitoring of amino acid modifications. For oxidation and deamidation, the assay was precise, with percent coefficient of variation of <8% at 1-100 µg/mL and ≤6% method-induced artifacts. A high degree of in vitro and in vivo correlation was observed for deamidation on the six diverse protein therapeutics (seven liability sites) tested. In vivo translatability for oxidation liabilities were not observed for the 11 molecules tested using in vitro AAPH stress. One of the molecules dosed in eyes resulted in a false positive and a false negative prediction for in vivo oxidation following AAPH stress. Finally, peroxide stress was also tested but resulted in limited success (1 out of 4 molecules) in predicting oxidation liabilities.

Production of ginsenoside compound K by microbial cell factory using synthetic biology-based strategy: a review.

Ginsenoside compound K (CK) is a major intestinal bacterial metabolite of the protopanaxadiol-type ginsenoside family that can be absorbed in the systemic circulation. CK possesses diverse and important pharmacological properties. The low production and high cost of traditional manufacturing methods based on the extraction and biotransformation of total ginsenosides from ginseng have limited their medical application. However, considerable progress has been made in the area of de novo CK production via microbial cell factories using synthetic biology-based strategies. By introducing key enzymes responsible for CK biosynthesis into microbial cells, CK was produced via a series of in vivo enzymatic reactions that utilize the inherent precursors in microbial cells. After systematic optimization using various metabolic engineering strategies, the yield of CK increased significantly and exceeded the traditional plant extraction-biotransformation method, implying the commercial feasibility of this approach. This review summarizes recent novel advancements in the production of CK using microbial cell factories.

MIR99AHG inhibits EMT in pulmonary fibrosis via the miR-136-5p/USP4/ACE2 axis.

BACKGROUND: Long non-coding RNAs (lncRNAs) are closely related to the occurrence and development of cancer. Abnormally expressed lncRNA can be used as a diagnostic marker for cancer. In this study, we aim to investigate the clinical significance of MIR99AHG expression in lung adenocarcinoma (LUAD), and its biological roles in LUAD progression. METHODS: The relative expression of MIR99AHG in LUAD tissues and cell lines was analyzed using public databases and RT-qPCR. The biological functions of MIR99AHG were investigated using a loss-of-function approach. The effect of MIR99AHG on lung fibrosis was assessed by scratch assay, invasion assay and lung fibrosis rat model. FISH, luciferase reporter assay and immunofluorescence were performed to elucidate the underlying molecular mechanisms. RESULTS: LncRNA MIR99AHG expression level was downregulated in LUAD tissues and cell lines. Low MIR99AHG levels were associated with poorer patient overall survival. Functional analysis showed that MIR99AHG is associated with the LUAD malignant phenotype in vitro and in vivo. Further mechanistic studies showed that, MIR99AHG functions as a competitive endogenous RNA (ceRNA) to antagonize miR-136-5p-mediated ubiquitin specific protease 4 (USP4) degradation, thereby unregulated the expression of angiotensin-converting enzyme 2 (ACE2), a downstream target gene of USP4, which in turn affected alveolar type II epithelial cell fibrosis and epithelial-mesenchymal transition (EMT). In summary, the MIR99AHG/miR-136-5p/USP4/ACE2 signalling axis regulates lung fibrosis and EMT, thus inhibiting LUAD progression. CONCLUSION: This study showed that downregulated MIR99AHG leads to the development of pulmonary fibrosis. Therefore, overexpression of MIR99AHG may provide a new approach to preventing LUAD progression.

The m6A Methyltransferase METTL3-Mediated N6-Methyladenosine Modification of DEK mRNA to Promote Gastric Cancer Cell Growth and Metastasis.

Gastric cancer (GC) is the fifth most common cancer and the third deadliest cancer in the world, and the occurrence and development of GC are influenced by epigenetics. Methyltransferase-like 3 (METTL3) is a prominent RNA n6-adenosine methyltransferase (m6A) that plays an important role in tumor growth by controlling the work of RNA. This study aimed to reveal the biological function and molecular mechanism of METTL3 in GC. The expression level of METTL3 in GC tissues and cells was detected by qPCR, Western blot and immunohistochemistry, and the expression level and prognosis of METTL3 were predicted in public databases. CCK-8, colony formation, transwell and wound healing assays were used to study the effect of METTL3 on GC cell proliferation and migration. In addition, the enrichment effect of METTL3 on DEK mRNA was detected by the RIP experiment, the m6A modification effect of METTL3 on DEK was verified by the MeRIP experiment and the mRNA half-life of DEK when METTL3 was overexpressed was detected. The dot blot assay detects m6A modification at the mRNA level. The effect of METTL3 on cell migration ability in vivo was examined by tail vein injection of luciferase-labeled cells. The experimental results showed that METTL3 was highly expressed in GC tissues and cells, and the high expression of METTL3 was associated with a poor prognosis. In addition, the m6A modification level of mRNA was higher in GC tissues and GC cell lines. Overexpression of METTL3 in MGC80-3 cells and AGS promoted cell proliferation and migration, while the knockdown of METTL3 inhibited cell proliferation and migration. The results of in vitro rescue experiments showed that the knockdown of DEK reversed the promoting effects of METTL3 on cell proliferation and migration. In vivo experiments showed that the knockdown of DEK reversed the increase in lung metastases caused by the overexpression of METTL3 in mice. Mechanistically, the results of the RIP experiment showed that METTL3 could enrich DEK mRNA, and the results of the MePIP and RNA half-life experiments indicated that METTL3 binds to the 3'UTR of DEK, participates in the m6A modification of DEK and promotes the stability of DEK mRNA. Ultimately, we concluded that METTL3 promotes GC cell proliferation and migration by stabilizing DEK mRNA expression. Therefore, METTL3 is a potential biomarker for GC prognosis and a therapeutic target.

Interaction of MRPL9 and GGCT Promotes Cell Proliferation and Migration by Activating the MAPK/ERK Pathway in Papillary Thyroid Cancer.

Thyroid cancer remains the most common endocrine malignancy worldwide, and its incidence has steadily increased over the past four years. Papillary Thyroid Cancer (PTC) is the most common differentiated thyroid cancer, accounting for 80-85% of all thyroid cancers. Mitochondrial proteins (MRPs) are an important part of the structural and functional integrity of the mitochondrial ribosomal complex. It has been reported that MRPL9 is highly expressed in liver cancer and promotes cell proliferation and migration, but it has not been reported in PTC. In the present study we found that MRPL9 was highly expressed in PTC tissues and cell lines, and lentivirus-mediated overexpression of MRPL9 promoted the proliferation and migration ability of PTC cells, whereas knockdown of MRPL9 had the opposite effect. The interaction between MRPL9 and GGCT (γ-glutamylcyclotransferase) was found by immunofluorescence and co-immunoprecipitation experiments (Co-IP). In addition, GGCT is highly expressed in PTC tissues and cell lines, and knockdown of GGCT/MRPL9 in vivo inhibited the growth of subcutaneous xenografts in nude mice and inhibited the formation of lung metastases. Mechanistically, we found that knockdown of GGCT/MRPL9 inhibited the MAPK/ERK signaling pathway. In conclusion, our study found that the interaction of GGCT and MRPL9 modulates the MAPK/ERK pathway, affecting the proliferation and migration of PTC cells. Therefore, GGCT/MRPL9 may serve as a potential biomarker for PTC monitoring and PTC treatment.

Structural switch of lysyl-tRNA synthetase between translation and transcription.

Lysyl-tRNA synthetase (LysRS), a component of the translation apparatus, is released from the cytoplasmic multi-tRNA synthetase complex (MSC) to activate the transcription factor MITF in stimulated mast cells through undefined mechanisms. Here we show that Ser207 phosphorylation provokes a new conformer of LysRS that inactivates its translational function but activates its transcriptional function. The crystal structure of an MSC subcomplex established that LysRS is held in the MSC by binding to the N terminus of the scaffold protein p38/AIMP2. Phosphorylation-created steric clashes at the LysRS domain interface disrupt its binding grooves for p38/AIMP2, releasing LysRS and provoking its nuclear translocation. This alteration also exposes the C-terminal domain of LysRS to bind to MITF and triggers LysRS-directed production of the second messenger Ap(4)A that activates MITF. Thus our results establish that a single conformational change triggered by phosphorylation leads to multiple effects driving an exclusive switch of LysRS function from translation to transcription.

STAT3/miR-15a-5p/CX3CL1 Loop Regulates Proliferation and Migration of Vascular Endothelial Cells in Atherosclerosis.

Endothelial cell proliferation disorder caused by vascular injury seems to be one of the causes of atherosclerosis, which is the pathological basis of coronary heart disease. The role of STAT3 in the regulation of microRNAs and endothelial dysfunction in atherosclerosis is unclear. STAT3 can be activated by cytokine IL-6 and up regulate the expression of CX3CL1. In addition, microRNA-15a-5p (miR-15a-5p) inhibited the transcription of CX3CL1, the proliferation of vascular endothelial cells and the proliferation of STAT3 regulated vascular endothelial cells. STAT3 positively regulates the expression of CX3CL1, and then down-regulates the inhibition of CX3CL1 by over-expression of miR-15a-5p, thus forming an elimination feedback loop to control the proliferation of HUVECs and affect the progression of atherosclerosis. In conclusion, miR-15a-5p may be the therapeutic target of the pathological basis of coronary atherosclerosis.

A Surgical Management for Severe Rhinophyma With Five-Blade Scratcher.

ABSTRACT: Rhinophyma, the final stage of acne rosacea, severely influences the patient's appearance and can only be treated by surgical methods. This case reports a simple, safe, effective, and economical surgical method-five-blade scratcher. After the surgical treatment, the overall nasal contour of the patient, a male with severe rosacea, was restored without scar formation. Thus, this surgical method reported in this case is feasible and easy to operate, and worthy of clinical promotion.

Identification of a Novel ß-Thalassemia Mutation at Codon 130 (+T) (HBB: c.391insT) in Han Chinese.

ß-Thalassemia (ß-thal) is an inherited blood disorder characterized by reduced or absent synthesis of the ß chains of hemoglobin (Hb). Although more than 900 ß-globin gene mutations around the world have been identified, here we report a novel mutation detected in a Chinese subject of Han ethnicity. This allele develops by insertion of one nucleotide (+T) at codon 130 (HBB: c.391insT) in the third exon of the ß-globin gene. The mutation causes a frameshift that leads to a termination codon at codon 139. The identification of the novel mutation will facilitate future diagnosis of ß-thal and will also be useful the genetic counseling and prenatal diagnosis.

miR-133a-3p/FOXP3 axis regulates cell proliferation and autophagy in gastric cancer.

Although many methods and new therapeutic drugs have been developed, the overall survival rate and long-term survival rate of patients with gastric cancer (GC) are still not satisfactory. In this study, we investigated the effects of microRNA miR-133a-3p and transcription factor FOXP3 on proliferation and autophagy of GC cells and their interactions. Our results showed that knockdown of FOXP3 increased the proliferation and autophagy of GC cells. The relationship between FOXP3 and autophagy has not been reported previously. In addition, FOXP3 could directly bind the promoter region of TP53 and inhibit its expression. miR-133a-3p increased the proliferation and autophagy via decreasing the protein level of FOXP3 by targeting its 3'-UTR. Our research provides new insights into the development of GC and provides new ideas and theoretical basis for the clinical treatment of GC and the development of new drug targets.

Probing the Impact of the Knob-into-Hole Mutations on the Structure and Function of a Therapeutic Antibody.

Bispecific antibodies (BsAbs) have drawn increasing interest in the biopharmaceutical industry due to their advantage to bind two distinct antigens simultaneously. The knob-into-hole approach is an effective way to produce bispecific antibodies by driving heterodimerization with mutations in the CH3 domain of each half antibody. To better understand the conformational impact by the knob and hole mutations, we combined size-exclusion chromatography (SEC), differential scanning calorimetry (DSC), and hydrogen-deuterium exchange mass spectrometry (H/D exchange MS), to characterize the global and peptide-level conformational changes. We found no significant alteration in structure or conformational dynamics induced by the knob-into-hole framework, and the conformational stability is similar to the wild-type (WT) IgG4 molecules (except for some small difference in the CH3 domain) expressed in E. coli. Functional studies including antigen-binding and neonatal fragment crystallizable (Fc) receptor (FcRn) binding demonstrated no difference between the knob-into-hole and WT IgG4 molecules in E. coli.

Active Soil Nitrifying Communities Revealed by In Situ Transcriptomics and Microcosm-Based Stable-Isotope Probing.

Long-term nitrogen field fertilization often results in significant changes in nitrifying communities that catalyze a key step in the global N cycle. However, whether microcosm studies are able to inform the dynamic changes in communities of ammonia-oxidizing bacteria (AOB) and archaea (AOA) under field conditions remains poorly understood. This study aimed to evaluate the transcriptional activities of nitrifying communities under in situ conditions, and we found that they were largely similar to those of 13C-labeled nitrifying communities in the urea-amended microcosms of soils that had received different N fertilization regimens for 22 years. High-throughput sequencing of 16S rRNA genes and transcripts suggested that Nitrosospira cluster 3-like AOB and Nitrososphaera viennensis-like AOA were significantly stimulated in N-fertilized fresh soils. Real-time quantitative PCR demonstrated that the significant increase of AOA and AOB in fresh soils upon nitrogen fertilization could be preserved in the air-dried soils. DNA-based stable-isotope probing (SIP) further revealed the greatest labeling of Nitrosospira cluster 3-like AOB and Nitrosospira viennensis-like AOA, despite the strong advantage of AOB over AOA in the N-fertilized soils. Nitrobacter-like nitrite-oxidizing bacteria (NOB) played more important roles than Nitrospira-like NOB in urea-amended SIP microcosms, while the situation was the opposite under field conditions. Our results suggest that long-term fertilization selected for physiologically versatile AOB and AOA that could have been adapted to a wide range of substrate ammonium concentrations. It also provides compelling evidence that the dominant communities of transcriptionally active nitrifiers under field conditions were largely similar to those revealed in 13C-labeled microcosms.IMPORTANCE The role of manipulated microcosms in microbial ecology has been much debated, because they cannot entirely represent the in situ situation. We collected soil samples from 20 field plots, including 5 different treatments with and without nitrogen fertilizers for 22 years, in order to assess active nitrifying communities by in situ transcriptomics and microcosm-based stable-isotope probing. The results showed that chronic N enrichment led to competitive advantages of Nitrosospira cluster 3-like AOB over N. viennensis-like AOA in soils under field conditions. Microcosm labeling revealed similar results for active AOA and AOB, although an apparent discrepancy was observed for nitrite-oxidizing bacteria. This study suggests that the soil microbiome represents a relatively stable community resulting from complex evolutionary processes over a large time scale, and microcosms can serve as powerful tools to test the theory of environmental filtering on the key functional microbial guilds.

MKL-1 is a coactivator for STAT5b, the regulator of Treg cell development and function.

BACKGROUND: Foxp3+CD4+ regulatory T cells (Treg) constitutes a key event in autoimmune diseases. STAT5b is the critical link between the IL-2/15 and FOXP3, the master regulator of Treg cells. METHODS: The CD3+T cell and Foxp3+CD4+ regulatory T cells were overexpressioned or knockdown MKL-1 and STAT5a and tested for Treg cell development and function. Direct interaction of MKL-1 and STAT5a were analyzed by coimmunoprecipitation assays, Luciferase assay, Immunofluoresence Staining and Yeast two-hybrid screening. The effect of MKL-1 and STAT5a on the Treg genes expression was analyzed by qPCR and western blotting and Flow cytometry. RESULTS: However, the molecular mechanisms mediating STAT5b-dependent Treg genes expression and Treg cell phenotype and function in autoimmune diseases are not well defined. Here, we report that the MKL-1 is a coactivator for the major Treg genes transcription factor STAT5b, which is required for human Treg cell phenotype and function. The N terminus of STAT5b, which contains a basic coiled-coil protein-protein interaction domain, binds the C-terminal activation domain of MKL-1 and enhances MKL-1 mediated transcriptional activation of Treg-specific, CArG containing promoters, including the Treg-specific genes Foxp3. Suppression of endogenous STAT5b expression by specific small interfering RNA attenuates MKL-1 transcriptional activation in cultured human cells. The STAT5b-MKL-1 interaction identifies a role of Treg-specific gene regulation and regulated mouse Treg cell development and function and suggests a possible mechanism for the protective effects of autoimmune disease Idiopathic Thrombocytopenic Purpura (ITP). CONCLUSIONS: Our studies demonstrate for the first time that MKL-1 is a coactivator for STAT5b, the regulator of Treg cell development and function. Video abstract.

Premature Stroke Secondary to Severe Hypertension Results from Liddle Syndrome Caused by a Novel SCNN1B Mutation.

INTRODUCTION: Liddle syndrome (LS), an autosomal dominant and inherited monogenic hypertension syndrome caused by pathogenic mutations in the epithelial sodium channel (ENaC) genes SCNN1A, SCNN1B, and SCNN1G. OBJECTIVE: This study was designed to identify a novel SCNN1B missense mutation in a Chinese family with a history of stroke, and to confirm that the identified mutation is responsible for LS in this family. METHODS: DNA samples were collected from the proband and 11 additional relatives. Next-generation sequencing was performed in the proband to find candidate variants. In order to exclude genetic polymorphism, the candidate variantin SCNN1B was verified in other family members, 100 hypertensives, and 100 healthy controls by Sanger sequencing. RESULTS: Genetic testing revealeda novel and rare heterozygous variant in SCNN1B in the proband. This variant resulted in a substitution of threonine instead of proline at codon 617, altering the PY motif of ß-ENaC. The identified mutation was only verified in 5 relatives. In silico analyses indicated that this variant was highly pathogenic. In this family, phenotypic heterogeneity was present among 6 LS patients. Tailored medicine with amiloride was effective in controlling hypertension and improving the serum potassium concentration in patients with LS. CONCLUSIONS: We identified a novel SCNN1B mutation (c.1849C>A) in a family affected by LS. Patients with LS, especially those with severe hypertension, should be alert for the occurrence of premature stroke. Timely diagnosis using genetic testing and tailored treatment with amiloride can help LS patients to avoid severe complications.

Rapid Diagnosis of a Family Cluster of Pertussis Starting from a Child: a Case Report.

BACKGROUND: Pertussis, caused by Bordetella pertussis (B. pertussis), is a highly transmissible, acute respiratory disease that occurs in many countries. Diagnosis of pertussis continues to be a challenge using traditional tests due to their turn-around time and sensitivity. Herein, we rapidly and accurately screened a family cluster of pertussis from a child and her mother. METHODS: We used an automated nested multiplex PCR system which included B. pertussis, influenza A virus, and 19 other respiratory pathogens. RESULTS: We detected B. pertussis, influenza A virus H1-2009 (FluA-2009), adenovirus, and respiratory syncytial virus (RSV) in the child, and the mother of the child was positive for B. pertussis and FluA-2009. CONCLUSIONS: Active and timely screening for pertussis of adult family members should be considered. The detection of multiple respiratory pathogens may guide effective antibiotic therapies. This could be a novel test for the prevention of pertussis.

A Novel Spontaneous Mutation of the SOX10 Gene Associated with Waardenburg Syndrome Type II.

Waardenburg syndrome (WS), also known as auditory-pigmentary syndrome, is the most common cause of syndromic hearing loss. It is responsible for 2-5% of congenital deafness. WS is classified into four types depending on the clinical phenotypes. Currently, pathogenic mutation of PAX3, MITF, EDNRB, EDN3, SNAI2, or SOX10 can cause corresponding types of WS. Among them, SOX10 mutation is responsible for approximately 15% of type II WS or 50% of type IV WS. We report the case of a proband in a Chinese family who was diagnosed with WS type II. Whole exome sequencing (WES) of the proband detected a novel heterozygous spontaneous mutation: SOX10 c.246delC. According to analysis based on nucleic acid and amino acid sequences, this mutation may produce a truncated protein, with loss of the HMG structure domain. Therefore, this truncated protein may fail to activate the expression of the MITF gene, which regulates melanocytic development and plays a key role in WS. Our finding expands the database of SOX10 mutations associated with WS and provides more information regarding the molecular mechanism of WS.

Jervell and Lange-Nielsen Syndrome due to a Novel Compound Heterozygous KCNQ1 Mutation in a Chinese Family.

Jervell and Lange-Nielsen syndrome (JLNS) is a rare but severe autosomal recessive disease characterized by profound congenital deafness and a prolonged QTc interval (greater than 500 milliseconds) in the ECG waveforms. The prevalence of JLNS is about 1/1000000 to 1/200000 around the world. However, exceed 25% of JLNS patients suffered sudden cardiac death with kinds of triggers containing anesthesia. Approximately 90% of JLNS cases are caused by KCNQ1 gene mutations. Here, using next-generation sequencing (NGS), we identified a compound heterozygosity for two mutations c.1741A>T (novel) and c.477+5G>A (known) in KCNQ1 gene as the possible pathogenic cause of JLNS, which suggested a high risk of cardiac events in a deaf child. The hearing of this patient improved significantly with the help of cochlear implantation (CI). But life-threatening arrhythmias occurred with a trigger of anesthesia after the end of the CI surgery. Our findings extend the KCNQ1 gene mutation spectrum and contribute to the management of deaf children diagnosed with JLNS for otolaryngologists (especially cochlear implant teams).

Mutant breeding of Starmerella bombicola by atmospheric and room-temperature plasma (ARTP) for improved production of specific or total sophorolipids.

To enhance specific or total sophorolipids (SLs) production by Starmerella bombicola for specific application, mutant library consisting of 106 mutants from 7 batches was constructed via atmospheric and room-temperature plasma (ARTP). When compared to the wild strain, 11, 36 and 12 mutants performed increases over 30% in lactonic, acidic or total SLs production. Genetic stability investigation showed that 8, 7, and 4 mutants could maintain the improved SLs production capacity. Mutants of A6-9 and A2-8 were selected out for enhanced specific SLs and total SLs production in fed-batch cultivation in flask. Without optimization, A6-9 obtained the highest reported lactonic SLs production of 51.95 g/l and A2-8 performed comparable acidic and total SLs production of 68.75 g/l and 100.33 g/l with all the reported stains. The structural composition of the obtained SLs was analyzed by HPLC and LC/MS, and the results confirmed the enhancement of SLs and certain SL components. These mutants would be important in industrial applications because the production and purification costs of SLs could be greatly reduced. Besides, the acquisition of these mutants also provided materials for the investigation of regulation mechanism of SLs biosynthesis for further genetic engineering of S. bombicola. Furthermore, critical micelle concentration (CMC), minimum surface tension (STmin) and hydrophilic-lipophilic balance (HLB) of the SLs obtained from the wild and mutant strains were also examined and compared. These results demonstrated the feasibility of obtaining SLs with different properties from different strains and the high efficiency of mutation breeding of S. bombicola by ARTP.