[Expert consensus over genetic counseling for carrier screening of Spinal muscular atrophy].

Spinal muscular atrophy (SMA), an autosomal recessive neuromuscular disease with a carrier frequency of 1/60 ~ 1/40, is characterized by severe clinical symptoms, high mortality rate, and expensive treatment costs. Carrier screening is of paramount importance to detect high-risk couples, and therefore to reduce the occurrence of SMA. In China, SMA carrier screening has become widespread, though there is still a lack of genetic counseling expertise. This article has focused on the current challenges for SMA carrier screening, including the screening methods, target population, screening procedures, and pre-/post-testing counseling. The aim is to standardize its application and counseling in the clinical practice.

[Expert consensus on the genetic counseling for Dystrophinopathies].

Dystrophinopathies caused by variants of DMD gene are a group of muscular diseases including Duchenne muscular dystrophy, Becker muscular dystrophy, and DMD-associated dilated cardiomyopathy. With the advancement of genetic testing techniques and wider implementation of genetic screening, especially the expanded carrier screening, more and more individuals carrying DMD gene variants have been identified, whereas the genetic counseling capacity is relatively insufficient. Currently there is still a lack of professional norms for genetic counseling on dystrophinopathies. In this consensus, the main points to be covered in the pre- and post-test consultation have been discussed, with an aim to provide genetic counseling guidance for the disease diagnosis, treatment, and family reproduction.

Molecular epidemiology and clinical characterization of human rhinoviruses circulating in Shanghai, 2012-2020.

Human rhinoviruses (HRVs) cause acute upper and lower respiratory tract infections and aggravation of asthma and chronic obstructive pulmonary disease. The 5' untranslated region (5' UTR) and the VP4/VP2 region are widely used for genotyping of HRVs. Members of the species Rhinovirus A and Rhinovirus C have been reported to be more frequently associated with severe disease than members of the species Rhinovirus B. We report the clinical and molecular epidemiological characteristics of HRVs circulating from 2012 to 2020 in Shanghai. A total of 5832 nasopharyngeal swabs from patients with acute respiratory infections were collected. A real-time reverse transcription polymerase chain reaction assay was used for virus detection. The 5' untranslated region and VP4/VP2 region were amplified and sequenced for genotyping and phylogenetic analysis. The overall rate of rhinovirus detection was 2.74% (160/5832), with members of species A, B, and C accounting for 68.13% (109/160), 20.00% (32/160), and 11.88% (19/160) of the total, respectively. A peak of HRV infection was observed in autumn (5.34%, 58/1087). Patients in the 3- to 14-year-old age group were the most susceptible to HRV infection (χ2 = 23.88, P = 0.017). Influenza virus and Streptococcus pneumoniae were detected more frequently than other pathogens in cases of coinfection. Recombination events were identified in 10 strains, which were successfully genotyped by phylogenetic analysis based on the 5' UTR-VP4/VP2 region but not the 5' UTR region alone. We observed a high degree of variability in the relative distribution of HRV genotypes and the prevalence of HRV infection in Shanghai and found evidence of recombination events in the portion of the genome containing the 5' UTR and the VP4/VP2 region between HRV-C strains and HRV-A-like strains. This study is important for surveillance of the spread of HRVs and the emergence of new variants.

Simultaneous detection of fetal aneuploidy, de novo FGFR3 mutations and paternally derived ß-thalassemia by a novel method of noninvasive prenatal testing.

OBJECTIVE: The aim is to develop a novel noninvasive prenatal testing (NIPT) method that simultaneously performs fetal aneuploidy screening and the detection of de novo and paternally derived mutations. METHODS: A total of 68 pregnancies, including 26 normal pregnancies, 7 cases with fetal aneuploidies, 7 cases with fetal achondroplasia or thanatophoric dysplasia, 18 cases with fetal skeletal abnormalities, and 10 cases with ß-thalassemia high risk were recruited. Plasma cell-free DNA was amplified by Targeted And Genome-wide simultaneous sequencing (TAGs-seq) to generate around 99% of total reads covering the whole-genome region and around 1%  covering the target genes. The reads on the whole-genome region were analyzed for fetal aneuploidy using a binary hypothesis T-score and the reads on target genes were analyzed for point mutations by calculating the minor allelic frequency of loci on FGFR3 and HBB. TAGs-seq results were compared with conventional NIPT and diagnostic results. RESULTS: In each sample, TAGs-seq generated 44.7-54 million sequencing reads covering the whole-genome region of 0.1-3× and the target genes of >1000×depth. All cases of fetal aneuploidy and de novo mutations of achondroplasia/thanatophoric dysplasia were identified with high sensitivities and specificities except for one false-negative paternal mutation of ß-thalassemia. CONCLUSIONS: TAGs-seq is a novel NIPT method that combines the fetal aneuploidy screening and the detection of de novo FGFR3 mutations and paternal HBB mutations.

Combined exome sequencing and deep phenotyping in highly selected fetuses with skeletal dysplasia during the first and second trimesters improves diagnostic yield.

OBJECTIVE: To investigate the genetic etiology of skeletal dysplasia in highly selected fetuses during the first and second trimesters using deep phenotyping and exome sequencing (ES). METHOD: Fetuses with short femurs were identified using the established prenatal diagnostic approach. A multidisciplinary team reviewed fetal phenotypic information (prenatal ultrasound findings, fetal postmortem, and radiographs) in a cohort of highly selected fetuses with skeletal dysplasia during the first and second trimesters. The affected families underwent multiplatform genetic tests. RESULTS: Of the 27 affected fetuses, 21 (77.8%) had pathogenic or potential pathogenic variations in the following genes: COL1A1, FGFR3, COL2A1, COL1A2, FLNB, DYNC2LI1, and TRIP11. Two fetuses had compound heterozygous mutations in DYNC2LI1 and TRIP11, respectively, and the other 19 carried de novo autosomal dominant variants. Novel variants were identified in COL1A1, COL2A1, COL1A2, DYNC2LI1, and TRIP11 in 11 fetuses. We also included the first description of the phenotype of odontochondrodysplasia in a prenatal setting. CONCLUSIONS: ES or panel sequencing offers a high diagnostic yield for fetal skeletal dysplasia during the first and second trimesters. Comprehensive and complete phenotypic information is indispensable for genetic analysis and the expansion of genotype-phenotype correlations in fetal skeletal abnormalities.

iRGD and TGN co-modified PAMAM for multi-targeted delivery of ATO to gliomas.

A poly(amidoamine) dendrimer (PAMAM, G5) based drug delivery system was developed for the treatment of glioma. PAMAM was modified with polyethylene glycol (PEG) to improve its in vivo stability and reduce immunogenicity. Further, the internalized RGD (iRGD) recognition ligand of the integrin αvß3 receptor and the blood-brain barrier (BBB)-targeting group TGN were introduced. Arsenic trioxide (ATO) was loaded into the internal cavity through electrostatic interactions to form iRGD/TGN-PEG-PAMAM-ATO. The drug delivery system of iRGD/TGN dual-modified PAMAM, which entrapped ATO, had a high entrapment efficiency of approximately 71.92% ± 1.17% and displayed sustainable acid-dependent drug release. Assessment of antiglioma effects revealed that survival rate was significantly higher in the iRGD/TGN comodified group than in the other groups. Overall, iRGD/TGN-based dual targeting by combining nanocarriers and targeting technology increased the amount of drug that crossed BBB, thus achieving targeted enrichment and activation of the drug in tumor tissue. This activation ultimately increased therapeutic effects and reduced side effects of ATO. This strategy using a multistep-targeted delivery system shows great promise for targeted glioma therapy.

Two novel TCTN2 mutations cause Meckel-Gruber syndrome.

Meckel-Gruber syndrome (MKS) is a clinically and genetically heterogeneous ciliopathy characterized by a triad of occipital encephalocele, polycystic kidneys, and postaxial polydactyly. Pathogenesis of MKS is related to dysfunction of primary cilia. However, reports on MKS caused by Tectonic2 (TCTN2) mutations are scanty whilst. There is no direct evidence of ciliogenesis in such MKS patients. Here, we identified two novel nonsense variants of TCTN2 (c.343G > T, p.E115*; c.1540C > T, p.Q514*) in a Chinese MKS fetus. Compared to reported TCTN2-causing MKS patients, our case represented an endocardial pad defect, which was not reported previously. We also found primary cilia protruded normally from the surface of epithelial cells in the affected fetal kidney tubules compared to controls, indicating TCTN2 is not necessary for ciliogenesis in the kidney. To our knowledge, this is the first case of MKS fetus caused by TCTN2 mutations from China.

A modular pathway engineering strategy for the high-level production of ß-ionone in Yarrowia lipolytica.

BACKGROUND: The GRAS and oleaginous yeast Yarrowia lipolytica (Y. lipolytica) is an attractive cell factory for the production of chemicals and biofuels. The production of many natural products of commercial interest have been investigated in this cell factory by introducing heterologous biosynthetic pathways and by modifying the endogenous pathways. However, since natural products anabolism involves long pathways and complex regulation, re-channelling carbon into the product of target compounds is still a cumbersome work, and often resulting in low production performance. RESULTS: In this work, the carotenogenic genes contained carB and bi-functional carRP from Mucor circinelloides and carotenoid cleavage dioxygenase 1 (CCD1) from Petunia hybrida were introduced to Y. lipolytica and led to the low production of ß-ionone of 3.5 mg/L. To further improve the ß-ionone synthesis, we implemented a modular engineering strategy for the construction and optimization of a biosynthetic pathway for the overproduction of ß-ionone in Y. lipolytica. The strategy involved the enhancement of the cytosolic acetyl-CoA supply and the increase of MVA pathway flux, yielding a ß-ionone titer of 358 mg/L in shake-flask fermentation and approximately 1 g/L (~ 280-fold higher than the baseline strain) in fed-batch fermentation. CONCLUSIONS: An efficient ß-ionone producing GRAS Y. lipolytica platform was constructed by combining integrated overexpressed of heterologous and native genes. A modular engineering strategy involved the optimization pathway and fermentation condition was investigated in the engineered strain and the highest ß-ionone titer reported to date by a cell factory was achieved. This effective strategy can be adapted to enhance the biosynthesis of other terpenoids in Y. lipolytica.

Novel Strategy of Gene Delivery System Based on Dendrimer Loaded Recombinant Hirudine Plasmid for Thrombus Targeting Therapy.

This study proposed a new nonviral gene delivery system for thrombus targeting therapy based on PEGlyation polyamides dendrimer (PAMAM) modified with RGDyC to condense the pDNA with recombinant hirudine (rHV) gene (RGDyC-rHV-EGFP). The RGDyC-mPEG-PAMAM was synthesized and characterized by 1H NMR, PAMAM/pDNA was characterized by particle size, zeta potential, cellular uptake, and gel retraction assay. The transfection was carried out between lipofectamine 2000 and PAMAM/pDNA on HUVEC cells at various N/P ratios. The antithrombotic effect in vivo was evaluated by venous thrombosis model on Wistar rats. It showed that the drug delivery system of RGDyC modified PAMMA, which entrapped pDNA could significantly improve the transfection efficiency. It was about 7.56-times higher than that of lipofectamine 2000. In addition, the expression level of hirudine fusion protein was the highest at N/P ratio of 0.5. The results of antithrombotic effect showed that the weight of thrombus was reduced in RGDyC modified group; compared with heparin group, there was no significant difference ( P > 0.05). Overall, we take the advantage of the unique advantages of hirudine, combining the genetic engineering, nanocarriers, and targeting technology, to achieve the targeted enrichment and activation the hirudine fusion protein in the thrombus site, to improve the concentration of drugs in the thrombus site, finally increasing the curative effect and reduce the risk of bleeding. The strategy of gene delivery system holds unique properties as a gene delivery system and has great promises in thrombus targeting therapy.

Noninvasive prenatal test for FGFR3-related skeletal dysplasia based on next-generation sequencing and plasma cell-free DNA: Test performance analysis and feasibility exploration.

OBJECTIVE: To explore the feasibility and accuracy of a noninvasive prenatal test for fibroblast growth factor receptor 3 (FGFR3)-related skeletal dysplasia based on next-generation sequencing (NGS) of plasma cell-free DNA. METHOD: Fragmented genome DNA (gDNA) of fetuses with achondroplasia (ACH) and thanatophoric dysplasia type I (TD I) was mixed with postdelivery maternal plasma cell-free DNA to generate spiked samples of different modeled fetal fractions. Multiplex polymerase chain reaction was used to amplify the 19 FGFR3 loci, and the amplification products were then sequenced by NGS to detect the fetal mutant alleles. Then, maternal plasma samples of pregnant women carrying ACH (n = 4) and TD I fetuses (n = 2), as well as healthy controls (n = 15), were tested by NGS, and the test performance was evaluated. RESULTS: Fetal FGFR3 mutations were detected in all artificial mixtures with fetal gDNA concentrations above 3%. In clinical validation, our method identified all fetal FGFR3 mutant alleles from maternal plasma, with no false positive results. The sensitivity and specificity of our method were 100% (95% CI, 54.1%-100%) and 100% (78.2%-100%), respectively. CONCLUSION: Our method had a favorable performance for noninvasively detecting fetal FGFR3 mutations in maternal plasma, highlighting its promising value in developing a noninvasive prenatal test for de novo and paternally inherited disorders.

[Preparation and in vitro evaluation of arsenic trioxide glioma targeting drug delivery system loaded by PAMAM dendrimers co-modified with RGDyC and PEG].

Arsenic trioxide (ATO) is an effective component of traditional Chinese medicine arsenic. The existing studies have shown its good inhibition and apoptosis ability on a variety of tumours. However, its toxicity and difficulties in the permeability into the blood brain barrier (BBB) has the limitation in the application of glioma treatment. Polyamide-amine dendrimer (PAMAM) is a synthetic polymer with many advantages, such as a good permeability, stability and biocompatibility. Additionally, the 5th generation of PAMAM is an ideal drug carrier due to its three-dimensional structure. In this study, the 5th generation of PAMAM co-modified with RGDyC and PEG, then confirmed by ¹H-NMR. The average particle size of nanoparticles was about 20 nm according to the nanoparticle size-potential analyser and transmission electron microscopy. in vitro release showed that the nanocarrier not only has the sustained release effect, but also some pH-sensitive properties. The cell results showed that PAMAM co-modified with RGDyC and PEGAM has a lower cytotoxicity than the non-modified group in vitro. Accordingly, the drug delivery system has a better anti-tumour effect across the blood brain barrier (BBB) in vitro, which further proves the tumour targeting of RGDyC.

A quantitative cSMART assay for noninvasive prenatal screening of autosomal recessive nonsyndromic hearing loss caused by GJB2 and SLC26A4 mutations.

PurposeThe aim of this study was to assess the performance of a noninvasive prenatal screening (NIPS) assay for accurate fetal genotyping of pregnancies at genetic risk for autosomal recessive nonsyndromic hearing loss (ARNSHL).MethodsA total of 80 pregnant couples carrying known mutations in either the GJB2 or SLC26A4 genes associated with a risk for ARNSHL were recruited to the study. Fetal amniocyte samples were genotyped by invasive prenatal screening (IPS), whereas the cell-free fetal DNA present in maternal plasma samples was genotyped using a novel NIPS method based on circulating single-molecule amplification and resequencing technology (cSMART).ResultsIPS of the 80 at-risk pregnancies identified 20 normal homozygote, 42 heterozygote, 5 affected homozygote, and 13 affected compound heterozygote fetuses. Benchmarking against IPS, 73 of 80 fetuses (91.3%) were correctly genotyped by the cSMART NIPS assay. A low fetal DNA fraction (<6%) was identified as the main contributing factor in five of seven discordant NIPS results. At fetal DNA fractions >6%, the sensitivity and specificity of the cSMART assay for correctly diagnosing ARNSHL were 100 and 96.5%, respectively.ConclusionBased on key performance indicators, the cSMART NIPS assay has clinical potential as an alternative to traditional IPS of ARNSHL.

Expressions of programmed death (PD)-1 and PD-1 ligand (PD-L1) in cervical intraepithelial neoplasia and cervical squamous cell carcinomas are of prognostic value and associated with human papillomavirus status.

AIM: The programmed death 1/programmed death 1 ligand (PD-1/PD-L1) pathway can decrease the immune clearance effects of antigen-presenting cells and T lymphocytes to promote immune evasion of cervical cancer cells. However, the effects of this pathway on cervical intraepithelial neoplasia (CIN) progression and squamous cell carcinoma (SCC) metastasis are not clear. We herein investigated whether human papillomavirus infection could affect PD-1 and PD-L1 expression in CIN, and whether their expression is associated with CIN progression and SCC metastasis. METHODS: We collected paraffin-embedded samples from two cohorts of patients: (i) CIN samples from cohort I (40 women who tested positive or negative for high-risk human papillomavirus [HR-HPV] with grades 0, I, and II-III CIN); and (ii) paired primary and metastatic tumor samples from cohort II (20 SCC patients with or without metastasis). Immunohistochemistry was used to detect expressions of PD-L1 in tumor cells and PD-1 in tumor-associated macrophages and tumor-infiltrating lymphocytes. We also measured P16INK4a expression and interferon-γ levels in the cervical tissues. RESULTS: The most common HPV type seen in both cohorts of patients was HPV16, followed by HPV18. Increase in PD-L1 and PD-1 expression was positively correlated with HPV-positivity, increase in CIN grade, and tumor metastasis. Furthermore, upregulation of the PD-1/PD-L1 pathway was associated with decreased expression of the pro-inflammatory cytokine, interferon-γ and increased expression of P16INK4a . CONCLUSION: Expression of PD-L1 and PD-1 could be used as clinical prognostic biomarkers for evaluating CIN and cervical cancer because of its positive correlation with CIN progression and tumor metastasis.

[Analysis of the gene mutation in an inherited FVII deficiency patient].

OBJECTIVE: The identify the gene defect of an inherited FVII deficiency patient. METHODS: The promoter, all the exons and exon-intron boundaries and 3' UTR of F7 gene of the proband were analyzed by direct sequencing. The defected mutations were confirmed by sequencing the complementary strand. The mutations would be screened in the related database and 150 healthy donors to identify the SNP. By splice site prediction, we analyzed the pathogenesis of defected mutations. RESULTS: Genetic analysis revealed G to A transition at 15975 in the intron 6 of F7 gene (IVS6-1G>A) and A to G transition at 16813 in the intron 7 of F7 gene (IVS7+7 A>G). According to the fruitfly, the acceptor site could not be recognized when a G to A substitution took place. The closest candidate splice site was located 132 bp downstream. The distance was probably too far to allow the use of the cryptic splice site and resulted in the skipping of exon6. A to G transition at 16813 in the intron 7 of F7 gene could not change the splice site, but modify a different molecular interaction that is important for the splice process. CONCLUSION: The heterozygous mutation of IVS6-1G>A combined with polymorphism of IVS7+7 A>G in F7 gene relates to the FVII deficiency.

[Correlation between primary cilium and Wnt signaling pathway].

Primary cilium is a microtubule-based organelle,which develops from the mother centriole of the centrosome. It is an antenna-like structure that anchors at the cell membrance, protruding from the cell surface. Primary cilium acts as a sensory organelle that receives different kinds of signals from the environment and transmits signals to cells to elicit cellular responses. Recent studies have revealed that primary cilium play an important role in transmitting Wnt signaling, which is critical for embryonic development. Dysfunction of primary cilium deregulates Wnt signaling, causing a series of pathological changes in different organs of the embryo, resulting in ciliopathies. In this review, we summarize correlation among primary cilium,Wnt/ß-catenin signaling,Wnt/PCP signaling and ciliopathies. Current therapies in ciliopathies are also discussed. Highlights on these researches will encourage the development of Wnt-associated diagnostic tools and therapy for ciliopathies.

Functional mutation of SMAC/DIABLO, encoding a mitochondrial proapoptotic protein, causes human progressive hearing loss DFNA64.

SMAC/DIABLO is a mitochondrial proapoptotic protein that is released from mitochondria during apoptosis and counters the inhibitory activities of inhibitor of apoptosis proteins, IAPs. By linkage analysis and candidate screening, we identified a heterozygous SMAC/DIABLO mutation, c.377C>T (p.Ser126Leu, refers to p.Ser71Leu in the mature protein) in a six-generation Chinese kindred characterized by dominant progressive nonsyndromic hearing loss, designated as DFNA64. SMAC/DIABLO is highly expressed in human embryonic ears and is enriched in the developing mouse inner-ear hair cells, suggesting it has a role in the development and homeostasis of hair cells. We used a functional study to demonstrate that the SMAC/DIABLO(S71L) mutant, while retaining the proapoptotic function, triggers significant degradation of both wild-type and mutant SMAC/DIABLO and renders host mitochondria susceptible to calcium-induced loss of the membrane potential. Our work identifies DFNA64 as the human genetic disorder associated with SMAC/DIABLO malfunction and suggests that mutant SMAC/DIABLO(S71L) might cause mitochondrial dysfunction.

Adsorption of PNIPAm(x)-PEO20-PPO70-PEO20-PNIPAm(x) pentablock terpolymer on gold surfaces: effects of concentration, temperature, block length, and surface properties.

The effects of concentration, relative block length and environmental temperature as well as the surface chemical and wetting properties of solid substrates on the adsorption behaviors and mechanisms of a series of pentablock terpolymer poly(N-isopropylacrylamide)x-poly(ethylene oxide)20-poly(propylene oxide)70-poly(ethylene oxide)20-poly(N-isopropylacrylamide)x (PNIPAmx-PEO20-PPO70-PEO20-PNIPAmx or PNIPAmx-P123-PNIPAmx) with x of 10, 63 and 97 on gold were studied by using a quartz crystal microbalance (QCM) technique. It was found that increasing the solution concentration did not alter the adsorption mechanism of thickness growth mode but increase the adsorption amount of PNIPAm97-P123-PNIPAm97 on a bare gold substrate at 20 °C. Increasing the length x of PNIPAm block decreased the adsorption rate constant and shifted the adsorption mechanism from the densification adsorption process for PNIPAm10-P123-PNIPAm10 to the thickness growth mode for PNIPAm63-P123-PNIPAm63 and PNIPAm97-P123-PNIPAm97 on bare (unmodified) gold substrate at 20 °C. The adsorption mechanisms of PNIPAm97-P123-PNIPAm97 at 20 °C on the hydrophobic and hydrophilic gold surfaces were the thickness growth mode and densification adsorption process, respectively. A complex adsorption behavior with large adsorption amounts was observed at the lower critical solution temperature (LCST) of PNIPAm block, i.e. 34.7 °C, for the adsorption of PNIPAm97-P123-PNIPAm97 not only on hydrophobic gold substrates but also on hydrophilic gold substrates. The adsorption mechanism of PNIPAm97-P123-PNIPAm97 micelles at 45 °C was the densification adsorption process regardless of the surface wetting and chemical properties of gold substrate. Overall, the adsorption behavior and mechanism of PNIPAmx-P123-PNIPAmx pentablock terpolymers were mainly determined by the interactions of the pentablock terpolymers with different chain conformations in dilute aqueous solutions at various temperatures and the gold substrates with surface wetting and chemical properties.

[Sensitivity and specificity of noninvasive prenatal fetal RhD genotesting: a meta-analysis].

OBJECTIVE: To explore the sensitivity, specificity and clinical validity of fetal Rh genotyping from maternal blood. METHODS: A comprehensive literature search of PubMed, Embase and Web of Science was performed for describing fetal RhD determination from maternal blood. The inclusion criteria were established based on the validity criteria for diagnostic research. And the eligible entries were collected and analyzed with MetaDisc4.0. RESULTS: This meta-analysis included 55 studies with a total of 17 138 samples. The random-effect model was used for analysis because of heterogeneity. The pooled sensitivity and specificity were 98.5% and 97.3% respectively. The SROC curve was plotted and the area under the curve (AUC) calculated (AUC = 0.994). The subgroup and sensitivity analyses were performed. The sensitivity of 25 studies with samples<100 (94.6%) was significantly lower than those of 19 studies with samples 100-300 (98.5%) and 11 studies with samples>300 (99.0%) (χ² = 36.800, 106.062, P < 0.05). The sensitivity of 19 studies with samples 100-300 (98.5%) was not different from that of 11 studies with samples >300 (99.0%)( χ² = 3.068, P > 0.05). CONCLUSIONS: Noninvasive prenatal diagnosis of fetal RhD status from maternal blood represents a significant achievement in the application of research with high sensitivity and specificity. It may be applied for screening testing of all RhD⁻ negative pregnant women.

The Regulatory Effect of Huangshui Polysaccharides on Intestinal Microbiota and Metabolites during In Vitro Fermentation.

Huangshui polysaccharides (HSPs) have attracted extensive attention recently for their biological activity and physicochemical property. This research investigated the extraction, structural characterization, and prebiotic activity of three different HSPs (HSP40-0, HSP60-0, and HSP80-0) in vitro to reveal the scientific support for the high-value utilization of Huangshui. HSPs were heteropolysaccharide with diverse structures and surface morphologies. Comprehensive analysis was conducted through 16S rRNA gene sequencing and metabolite profiling techniques, and results showed that HSPs had different potentials to regulate the gut microbiota due to their different structures; for instance, both HSP40-0 and HSP80-0 could notably increase the relative abundance of Bacteroidota, whereas HSP60-0 could increase the relative abundance of Phascolarctobacterium. In addition, HSPs upregulated beneficial differential metabolites, especially short-chain fatty acids (SCFAs). Fermentation products containing these metabolites exhibited anti-inflammatory effects on LPS-treated Caco-2 cells. This study will provide reference for exploring the relationship between the natural polysaccharide structure and the prebiotic activity and widen the application of Huangshui.

Solution behaviors and microstructures of PNIPAm-P123-PNIPAm pentablock terpolymers in dilute and concentrated aqueous solutions.

The solution behaviors and microstructures of poly(N-isopropylacrylamide)x-poly(ethylene oxide)20-poly(propylene oxide)70-poly(ethylene oxide)20-poly(N-isopropylacrylamide)x (PNIPAmx-PEO20-PPO70-PEO20-PNIPAmx or PNIPAmx-P123-PNIPAmx) pentablock terpolymers with various PNIPAm block lengths in dilute and concentrated aqueous solutions were investigated by micro-differential scanning calorimetry (micro-DSC), static and dynamic light scattering (SLS & DLS), and synchrotron small angle X-ray scattering (SAXS). Two lower critical solution temperatures (LCSTs) were observed for PNIPAmx-P123-PNIPAmx pentablock terpolymers in dilute solutions, which corresponded to LCSTs of PPO and PNIPAm blocks, respectively. The LCST of PPO block shifted from 24.4 °C to 29 °C when the length x of PNIPAm block increased from 10 to 97. The LCST of PNIPAm is around 34.5 °C-35.3 °C and less dependent on the block length x. The PNIPAmx-P123-PNIPAmx pentablock terpolymers formed "associate" structures and micelles with hydrophobic PNIPAm and PPO blocks as cores and soluble PEO blocks as coronas in dilute aqueous solutions at 20 °C and 40 °C, respectively, regardless of the relative lengths of PNIPAm, PPO and PEO blocks. The size of "associate" structures of PNIPAmx-P123-PNIPAmx pentablock terpolymers at 20 °C increased with increasing the length of PNIPAm block. The microstructures of PNIPAmx-P123-PNIPAmx hydrogels formed in concentrated aqueous solutions (40 wt%) were strongly dependent on the environmental temperatures and relative lengths of PNIPAm, PPO and PEO blocks as revealed by SAXS. Increasing the length of PNIPAm block weakened the order structures of PNIPAmx-P123-PNIPAmx hydrogels. The microstructures of PNIPAmx-P123-PNIPAmx hydrogels changed from mixed fcc and hex structures for PNIPAm10-P123-PNIPAm10 to isotropic structure for PNIPAm97-P123-PNIPAm97. Increasing temperature led to the transition from mixed hex and fcc structure to pure hex structure for PNIPAm10-P123-PNIPAm10 hydrogel at temperature above the LCSTs.