Purification, Characterization, and Anti-Inflammatory Potential of Free and Bound Polyphenols Extracted from Rosa roxburghii Tratt Pomace.

Rosa roxburghii Tratt pomace (RRTP), an underutilized byproduct, is rich in polyphenol compounds. This study aimed to further explore the purification, characterization, anti-inflammatory activities, and underlying molecular mechanisms of free polyphenols (RRTP-FP) and bound polyphenols (RRTP-BP) from RRTP. The results indicated that AB-8 macroporous resin emerged as the preferred choice for subsequent separation and purification. The purities of purified RRTP-FP (P-RRTP-FP) and purified RRTP-BP (P-RRTP-BP) increased by 103.34% and 66.01%, respectively. Quantitative analysis identified epigallocatechin, epicatechin, and ellagic acid as the main phenolic compounds in P-RRTP-FP. In P-RRTP-BP, the primary phenolic compounds were ellagic acid, epicatechin, and gallic acid. In vitro antioxidant assays demonstrated the superior DPPH and ABTS radical scavenging activities of P-RRTP-FP and P-RRTP-BP compared to vitamin C. Treatment with P-RRTP-FP and P-RRTP-BP reduced nitric oxide (NO) and reactive oxygen species (ROS) production, mitigated the decline in cellular membrane potential, and significantly downregulated the mRNA expression of pro-inflammatory cytokines and inducible nitric oxide synthase (iNOS) in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. Additionally, P-RRTP-FP and P-RRTP-BP inhibited the phosphorylation of pertinent proteins in the nuclear factor kappa-B (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways. This finding suggests potential utility of RRTP-derived polyphenols as anti-inflammatory agents for managing severe inflammatory conditions.

Structural changes and degradation mechanism of type 3 resistant starch during in vitro fecal fermentation.

The colonic fermentation metabolites of resistant starch (RS) are recognized to have various health benefits. However, the relationship between the structural variation of RS and the colonic fermentation properties, remains inadequately studied, especially for type 3 resistant starch. The in vitro fecal fermentation properties with multi-structure evolution of A- and B-type polymorphic resistant starch spherulites (RSS) were investigated. Both polymorphic types of RSS showed similar fermentation rate and total short-chain fatty acid profiles, while the butyrate concentration of the A-type RSS subjected to 24 h of fermentation was significantly higher compared to B-type RSS. In the case of recrystallized starch spherulites, irrespective of the polymorphic type, gut bacteria preferentially degraded the intermediate chains and crystalline regions, as the local molecule-ordered area potentially serves as suitable attachment sites or surfaces for microbial enzymes.

Influence of Three Modification Methods on the Structure, Physicochemical, and Functional Properties of Insoluble Dietary Fiber from Rosa roxburghii Tratt Pomace.

Rosa roxburghii Tratt pomace is rich in insoluble dietary fiber (IDF). This study aimed to investigate the influence of three modification methods on Rosa roxburghii Tratt pomace insoluble dietary fiber (RIDF). The three modified RIDFs, named U-RIDF, C-RIDF, and UC-RIDF, were prepared using ultrasound, cellulase, and a combination of ultrasound and cellulase methods, respectively. The structure, physicochemical characteristics, and functional properties of the raw RIDF and modified RIDF were comparatively analyzed. The results showed that all three modification methods, especially the ultrasound-cellulase combination treatment, increased the soluble dietary fiber (SDF) content of RIDF, while also causing a transition in surface morphology from smooth and dense to wrinkled and loose structures. Compared with the raw RIDF, the modified RIDF, particularly UC-RIDF, displayed significantly improved water-holding capacity (WHC), oil-binding capacity (OHC), and swelling capacity (SC), with increases of 12.0%, 84.7%, and 91.3%, respectively. Additionally, UC-RIDF demonstrated the highest nitrite ion adsorption capacity (NIAC), cholesterol adsorption capacity (CAC), and bile salt adsorption capacity (BSAC). In summary, the combination of ultrasound and cellulase treatment proved to be an efficient approach for modifying IDF from RRTP, with the potential for developing a functional food ingredient.

Enhanced in vitro bioavailability of resveratrol-loaded emulsion stabilized by ß-lactoglobulin-catechin with excellent antioxidant activity.

The study aimed to fabricate ß-Lactoglobulin-catechin (ß-La-Ca) conjugates as a natural designed antioxidant emulsifier to improve the physicochemical stability of resveratrol emulsion delivery system. Fourier transform infrared (FT-IR) and fluorescence spectroscopy analysis confirmed the formation of conjugates using free radical grafting. The antioxidant ability of emulsion was evaluated by DPPH scavenging activities and ORAC experiments. The emulsion stabilized by ß-La-Ca conjugates exhibited strong antioxidant activity with ORAC value of 2541.39 ± 29.58 µmol TE/g, which was significantly higher than that by ß-Lactoglobulin alone with 387.96 ± 23.45 µmol TE/g or their mixture with 948.23 ± 32.77 µmol TE/g. During the whole simulated gastrointestinal digestion, emulsion stabilized by ß-La-Ca conjugates exhibited excellent oxidative stability that the lipid was mainly digested in the small intestine. This behavior attributed to the greater stability of resveratrol to chemical transformation leading to a higher overall bioavailability in vivo. These results suggested that the ß-La-Ca conjugates could be used to fabricate the emulsion-based delivery system to improve the oxidative stability and bioavailability of chemically labile hydrophobic bioactive compounds.

Interfacial Behavior and Long-Term Stability of the Emulsions Stabilized by Sugar Beet Pectin-Ca2+ Complexes with Different Cross-Linking Degrees.

Recent studies showed that sugar beet pectin exhibited more excellent emulsifying properties than traditional citrus peel pectin and apple pectin ascribed to the higher content of neutral sugar, protein, ferulic acid, and acetyl groups. It is precisely because of the extremely complex molecular structure of pectin that the emulsifying properties of the pectin-Ca2+ complex are still unclear. In this study, SBP-Ca2+ complexes with different cross-linking degrees were prepared. Subsequently, their interfacial adsorption kinetics, the resistance of interfacial films to external perturbances, and the long-term stability of the emulsions formed by these SBP-Ca2+ complexes were measured. The results indicated that the highly cross-linked SBP-Ca2+ complex exhibited slower interfacial adsorption kinetics than SBP alone. Moreover, compared with SBP alone, the oil-water interfacial film loaded by the highly cross-linked SBP-Ca2+ complex exhibited a lower elasticity and a poorer resistance to external perturbances. This resulted in a larger droplet size, a lower ζ-potential value, a larger continuous viscosity, and a worse long-term stability of the emulsion formed by the highly cross-linked SBP-Ca2+ complex. This study has very important guiding significance for deeply understanding the emulsification mechanism of the pectin-Ca2+ complex.

Gastro-Intestinal Digested Bovine Milk Osteopontin Modulates Gut Barrier Biomarkers In Vitro.

SCOPE: Osteopontin (OPN) is a multifunctional protein naturally present in mammals' milk, associated with immune homeostasis and intestinal maturation. This study aims to investigate the protein digestion pattern and the cellular bioactivity of bovine milk OPN digesta in vitro. METHODS AND RESULTS: A modified INFOGEST static in vitro infant digestion protocol and a Caco-2/HT-29 co-culture cell model are employed to evaluate the digestion properties and the anti-inflammatory effects of OPN. OPN is resistant to gastric hydrolysis but degraded into large peptides during intestinal digestion. Its 10 kDa digesta permeate with predicted extensive bioactivities protects the co-culture cell model from the inflammation-induced dysfunction by dose-dependently recovering the expression of occludin, claudin-3, and ZO-1. Low dosage of OPN significantly decreases the production of IL-8 and IL-6, and downregulates the mRNA and protein expression of MyD88, NF-κB p65, and IκB-α, whereas a high dose evokes a mild pro-inflammatory response. Interestingly, anti-inflammatory effect of OPN digesta is stronger than lactoferrin and whey protein concentrate counterparts. CONCLUSION: The findings demonstrate that the bioactive peptides released from in vitro infant gastrointestinal digestion of bovine milk OPN alleviates intestinal epithelial cell inflammation by inhibiting NF-κB pathway activation and potentiates the barrier function of the intestinal epithelium.

Stabilization and release of thymol in pre-formed V-type starch: A comparative study with traditional method.

Recently, pre-formed V-type starch has become popular as a versatile carrier in encapsulation systems of containing starch-guest inclusion complexes (ICs). However, the differences in stabilizing and dissociating guests between ICs prepared by either the traditional method or the pre-formed "empty" helix method have not yet been elucidated. Here, starch-thymol ICs were prepared using the traditional high temperature-water method and the pre-formed method, covering different complexation temperatures and solvents, to compare the loading capacity, crystalline structure, thermal stability, and release properties. The highest content of thymol in ICs prepared by the pre-formed and the traditional method was 74.2 and 65.3 mg/g, respectively. Different from ICs prepared by the traditional method (V7-type crystal), ICs prepared by the pre-formed method mostly exhibited a V6a structure with larger crystallinities and a better short-range ordered structure. ICs prepared at 90 °C were type II complexes and efficiently protected thymol from rapid heat loss. A slow release was observed in both cases: about 45 % and 75 % of thymol were released from ICs prepared by the pre-formed and traditional methods, respectively, after two weeks of storage at 25 °C.

Characterization and emulsifying ability evaluation of whey protein-pectin conjugates formed by glycosylation.

Glycosylation is a method that enhances the functional properties of proteins by covalently attaching sugars to them. This study aimed at preparing three conjugates (WP-HG, WP-SBP, and WP-RGI) by dry heating method to research the influence of different pectin structures on the functional properties of WP and characterize properties and structures of these conjugates. The research results manifested that the degree of glycosylation (DG) of HG, SBP and RGI were 13.13 % ± 0.07 %, 23.27 % ± 0.3 % and 36.39 % ± 0.3 % respectively, suggesting that the increase of the number of branch chains promoted the glycosylation reaction. The formation of the conjugate was identified by the FT-IR spectroscopy technique. And SEM showed that WP could covalently bind to pectin, resulting in a smoother and denser surface of the conjugates. The circular dichroism analysis exhibited that the glycosylation reaction altered the secondary structure of WP and decreased the α-Helix content. This structural change in the protein spatial conformation led to a decrease in the hydrophobicity of protein surface. But the addition of pectin further regulated the hydrophilic-hydrophobic ratio on the surface of the protein, thus improving the emulsification properties of WP. In addition, the glycosylation could improve the stability of the emulsion, giving it a smaller droplet size, higher Zeta-potential and more stable properties. In a word, this study pointed out the direction for the application of different pectin structures in the development of functional properties of glycosylation products in food ingredients.

Modulation Effects of Sargassum pallidum Extract on Hyperglycemia and Hyperlipidemia in Type 2 Diabetic Mice.

The aim of this study was to investigate the antidiabetic effect of the extract from Sargassum pallidum (SPPE) on type 2 diabetes mellitus (T2DM) mice. SPPE treatment alleviated hyperglycemia, insulin resistance (IR), liver and pancreatic tissue damage, hyperlipidemia and hepatic oxidative stress resulting from T2DM. SPPE reversed phosphoenolpyruvate carboxylase (PEPCK) and hexokinase (HK) activities to improve gluconeogenesis and glycogen storage in the liver. Furthermore, SPPE modulated glucose metabolism by regulating the levels of mRNA expression involving the PI3K/Akt/FOXO1/G6pase/GLUT2 pathway and could inhibit fatty acid synthesis by reducing the gene expression levels of fatty acid synthase (FAS) and acetyl-CoA carboxylase-1 (ACC-1). A 16 sRNA analysis indicated that SPPE treatment also reversed gut dysbiosis by increasing the abundance of beneficial bacteria (Bacteroides and Lactobacillus) and suppressing the proliferation of harmful bacteria (Enterococcus and Helicobacter). Untargeted metabolomics results indicated that histidine metabolism, nicotinate and nicotinamide metabolism and fatty acid biosynthesis were significantly influenced by SPPE. Thus, SPPE may be applied as an effective dietary supplement or drug in the management of T2DM.

Clinical application of transcatheter arterial methylene blue angiography in the localization of lower gastrointestinal arterial bleeding / 介入放射学杂志

Objective To evaluate the clinical application value of transcatheter arterial methylene blue angiography in the localization of lower gastrointestinal arterial bleeding.Methods Ten patients with lower gastrointestinal arterial bleeding received interventional celiac artery angiography.After the bleeding responsible arteries were identified,a microcatheter was super-selectively placed in the bleeding responsible artery.During surgical procedure,the methylene blue solution was injected through the microcatheter to display the bleeding segment of the intestinal tract,providing precise localization of the bleeding intestinal segment for surgical resection.Results Transcatheter arterial methylene blue angiography could clearly display the bleeding segment of the intestinal tract.The bleeding segments of the intestinal tract in the 10 patients were quickly and accurately removed.After surgery,the gastrointestinal bleeding stopped,and no surgery-related complications occurred.Conclusion Transcatheter arterial methylene blue angiography can accurately detect the arterial bleeding segment of the lower gastrointestinal tract,which provides precise localization for quickly removing the bleeding segment of intestinal tract,therefor,this technique is worthy of widespread clinical application.(J Intervent Radiol,2023,32:1230-1232)

Double heterozygous pathogenic mutations in KIF3C and ZNF513 cause hereditary gingival fibromatosis / 国际口腔科学杂志·英文版

Hereditary gingival fibromatosis (HGF) is a rare inherited condition with fibromatoid hyperplasia of the gingival tissue that exhibits great genetic heterogeneity. Five distinct loci related to non-syndromic HGF have been identified; however, only two disease-causing genes, SOS1 and REST, inducing HGF have been identified at two loci, GINGF1 and GINGF5, respectively. Here, based on a family pedigree with 26 members, including nine patients with HGF, we identified double heterozygous pathogenic mutations in the ZNF513 (c.C748T, p.R250W) and KIF3C (c.G1229A, p.R410H) genes within the GINGF3 locus related to HGF. Functional studies demonstrated that the ZNF513 p.R250W and KIF3C p.R410H variants significantly increased the expression of ZNF513 and KIF3C in vitro and in vivo. ZNF513, a transcription factor, binds to KIF3C exon 1 and participates in the positive regulation of KIF3C expression in gingival fibroblasts. Furthermore, a knock-in mouse model confirmed that heterozygous or homozygous mutations within Zfp513 (p.R250W) or Kif3c (p.R412H) alone do not led to clear phenotypes with gingival fibromatosis, whereas the double mutations led to gingival hyperplasia phenotypes. In addition, we found that ZNF513 binds to the SOS1 promoter and plays an important positive role in regulating the expression of SOS1. Moreover, the KIF3C p.R410H mutation could activate the PI3K and KCNQ1 potassium channels. ZNF513 combined with KIF3C regulates gingival fibroblast proliferation, migration, and fibrosis response via the PI3K/AKT/mTOR and Ras/Raf/MEK/ERK pathways. In summary, these results demonstrate ZNF513 + KIF3C as an important genetic combination in HGF manifestation and suggest that ZNF513 mutation may be a major risk factor for HGF.

Clinical characteristics of intestinal stricture after neonatal necrotizing enterocolitis / 国际儿科学杂志

Neonatal necrotizing enterocolitis(NEC)is a common acute abdomen in newborns, while intestinal stricture is one of the frequent complications of NEC.Post-NEC stricture often occurs in the colon, and has clinical features such as vomiting, abdominal distension and bloody stools.This complication has a high incidence, high risk of death, and is also affected by multiple factors such as disease severity, treatment method and recovery time of enteral nutrition.Early prediction, diagnosis and intervention can reduce the adverse effects of the disease on the growth and development of children.This article reviews the clinical characteristics, influencing factors and prediction of the post-NEC stricture.

VPS4B mutation impairs the osteogenic differentiation of dental follicle cells derived from a patient with dentin dysplasia type I / 国际口腔科学杂志·英文版

A splicing mutation in VPS4B can cause dentin dysplasia type I (DD-I), a hereditary autosomal-dominant disorder characterized by rootless teeth, the etiology of which is genetically heterogeneous. In our study, dental follicle cells (DFCs) were isolated and cultured from a patient with DD-I and compared with those from an age-matched, healthy control. In a previous study, this DD-I patient was confirmed to have a loss-of-function splicing mutation in VPS4B (IVS7 + 46C > G). The results from this study showed that the isolated DFCs were vimentin-positive and CK14-negative, indicating that the isolated cells were derived from the mesenchyme. DFCs harboring the VPS4B mutation had a significantly higher proliferation rate from day 3 to day 8 than control DFCs, indicating that VPS4B is involved in cell proliferation. The cells were then replenished with osteogenic medium to investigate how the VPS4B mutation affected osteogenic differentiation. Induction of osteogenesis, detected by alizarin red and alkaline phosphatase staining in vitro, was decreased in the DFCs from the DD-I patient compared to the control DFCs. Furthermore, we also found that the VPS4B mutation in the DD-I patient downregulated the expression of osteoblast-related genes, such as ALP, BSP, OCN, RUNX2, and their encoded proteins. These outcomes confirmed that the DD-I-associated VPS4B mutation could decrease the capacity of DFCs to differentiate during the mineralization process and may also impair physiological root formation and bone remodeling. This might provide valuable insights and implications for exploring the pathological mechanisms underlying DD-I root development.

Structural characteristics of the deciduous teeth of Tibetan miniature pigs / 南方医科大学学报

OBJECTIVE@#To explore the histological structure of the deciduous teeth and the tooth germs of Tibetan miniature pigs for studies of dental tissue diseases and tooth regeneration.@*METHODS@#The structure of the deciduous teeth of Tibetan miniature pigs was observed by X-ray. The ultrastructure of the enamel and dentin of deciduous teeth was characterized by scanning electron microscopy. The jaws and teeth were three-dimensionally reconstructed using Mimics software based on Micro-CT scanning of the deciduous teeth. Image J software was used to calculate the gray value and the mineralization density of the deciduous teeth. Hisotological structure of the tooth germ and the pulp tissue of Tibetan miniature pigs was observed using HE staining.@*RESULTS@#The deciduous teeth of Tibetan miniature pigs were composed of enamel, dentin and medullary pulp tissue. The permanent tooth germ were formed during the deciduous dentition. The enamel and dentin ultrastructure of deciduous teeth were consistent with that of human deciduous teeth. The enamel and dentin mineralization densities were 2.47±0.09 g/cm and 1.72±0.07 g/cm, respectively. The pathological structures of tooth germ and pulp tissue were similar to those of human teeth, and the pulp tissue of the deciduous teeth was in an undifferentiated state.@*CONCLUSIONS@#The deciduous teeth of Tibetan miniature pig have similar anatomy, ultrastructure and histopathological structure to human teeth and can serve as a good animal model for studying human dental tissue diseases and the mechanisms of tooth regeneration.

Progress of cholestasis liver diseases with low serum γ-glutamyl transpeptidase level / 中华实用儿科临床杂志

γ-glutamyl transpeptidase(GGT) is an important index of cholestasis.Most patients with cholestasis liver diseases have high GGT level, but some of the patients are characterized by low GGT level.This paper summarizes the progress of cholestasis liver diseases with low GGT level in order to improve the understanding of these diseases.

Progress of cholestasis liver diseases with low serum γ-glutamyl transpeptidase level / 中华实用儿科临床杂志

γ-glutamyl transpeptidase (GGT) is an important index of cholestasis.Most patients with cholestasis liver diseases have high GGT level,but some of the patients are characterized by low GGT level.This paper summarizes the progress of cholestasis liver diseases with low GGT level in order to improve the understanding of these diseases.

Whole exome sequencing identifies an AMBN missense mutation causing severe autosomal-dominant amelogenesis imperfecta and dentin disorders / 国际口腔科学杂志·英文版

Tooth development is a complex process that involves precise and time-dependent orchestration of multiple genetic, molecular, and cellular interactions. Ameloblastin (AMBN, also named "amelin" or "sheathlin") is the second most abundant enamel matrix protein known to have a key role in amelogenesis. Amelogenesis imperfecta (AI [MIM: 104500]) refers to a genetically and phenotypically heterogeneous group of conditions characterized by inherited developmental enamel defects. The hereditary dentin disorders comprise a variety of autosomal-dominant genetic symptoms characterized by abnormal dentin structure affecting either the primary or both the primary and secondary teeth. The vital role of Ambn in amelogenesis has been confirmed experimentally using mouse models. Only two cases have been reported of mutations of AMBN associated with non-syndromic human AI. However, no AMBN missense mutations have been reported to be associated with both human AI and dentin disorders. We recruited one kindred with autosomal-dominant amelogenesis imperfecta (ADAI) and dentinogenesis imperfecta/dysplasia characterized by generalized severe enamel and dentin defects. Whole exome sequencing of the proband identified a novel heterozygous C-T point mutation at nucleotide position 1069 of the AMBN gene, causing a Pro to Ser mutation at the conserved amino acid position 357 of the protein. Exfoliated third molar teeth from the affected family members were found to have enamel and dentin of lower mineral density than control teeth, with thinner and easily fractured enamel, short and thick roots, and pulp obliteration. This study demonstrates, for the first time, that an AMBN missense mutation causes non-syndromic human AI and dentin disorders.

Analysis of hematological characteristics of patient with different genotypes in lightβ-thalassemia / 实用医学杂志

Objective To investigate the hematological characteristics of patients with light β- thalassae-mia and rapidly identify different mutational genotypes. Methods RBC、Hb、MCV、MCH、MCHC、RDW-CV and HbA2 were studied in the 646 patients,the differences between β0/βN and β +/βN mutations were also compared. Results Most of them were microcytic hypochromic anemia. The most common genotype were β654/βN(33%)、β41-42/βN(32.5%)、β17/βN(14.4%)、β - 28/βN(10%)respectively,β0/βN were relatively higher. The differences in RBC、MCV、MCH、RDW-CV and HbA2 were significant between β0/βN and β +/βN. Compared with β +/βN patients,the MCV and MCH of β0/βN were significantly reduced,RDW-CV and HbA2 were significantly higher. Conclusion Light β- thalassaemia with different genotypes has its own unique hematological features and can be quickly and ef-fectively identified. Clinical efficiency can be improved through hematological analysis.

Analysis of DSPP gene mutation in a Chinese pedigree affected with hereditary dentinogenesis imperfecta / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To analyze the clinical phenotype of a Chinese pedigree affected with hereditary dentinogenesis imperfecta and mutation of dentin sialophosphoprotein (DSPP) gene.</p><p><b>METHODS</b>Affected members underwent intraoral photography, dental film and panoramic radiography. Genomic DNA was extracted from peripheral venous blood samples. Coding regions of the DSPP gene were subjected to PCR amplification and Sanger sequencing. Functional effect of the mutation was predicted with SIFT and PolyPhen-2. The tertiary structure of wild type and mutant proteins were predicted by Swiss-Port.</p><p><b>RESULTS</b>A heterozygous c.50C to T (p.P17L) mutation was identified in exon 2 of the DSPP gene in the proband and her father. The same mutation was not found among 200 unrelated healthy controls. The Pro-17 residues and its surrounding positions in DSPP are highly conserved across various species. The mutation was predicted to be damaging to the structure of DSPP protein.</p><p><b>CONCLUSION</b>The c.50C to T (p.P17L) mutation of the DSPP gene probably underlies the disease in this pedigree. Above finding has expanded the spectrum of DSPP gene mutations and provided a basis for genetic counseling and prenatal diagnosis for this family.</p>

Screening of CTSC gene mutations in a Chinese pedigree affected with Papillon-Lefevre syndrome / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To analyze the clinical phenotype of a Chinese pedigree affected with Papillon-Lefevre syndrome(PLS) and detect mutation of CTSC gene.</p><p><b>METHODS</b>Clinical phenotypes were noted, and oral examination for the proband was carried out for the clinical diagnosis of PLS. PCR and Sanger sequencing were used to identify potential mutation of the CTSC gene. Functional effect of the mutation was predicted with SIFT and PolyPhen-2. Swiss-Port was used to predict the tertiary structure of wild type and mutant proteins. The mRNA and protein expression were analyzed by real-time PCR and Western blotting.</p><p><b>RESULTS</b>A homozygous mutation c.901G>A (p.G301S) in exon 7 of CTSC gene was identified in the patient. Both parents of the patient had carried a heterozygous c.901G>A mutation. The mutation was located in the conserved region of CTSC enzyme and was predicted to be damaging by changing the structure of the protein, which could affect the activity of Cathepsin C. However, no significant difference was found in the expression of p.G301S variant at the mRNA and protein levels compared with that of the wild type CTSC gene.</p><p><b>CONCLUSION</b>The c.901G>A mutation of the CTSC gene was first reported in China, which has expanded its mutation spectrum.</p>