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Gene-knockout animal models with organ-deficient phenotypes used for blastocyst complementation are generally not viable. Animals need to be maintained as heterozygous mutants, and homozygous mutant embryos yield only one-fourth of all embryos. In this study, we generated organ-deficient embryos using the CRISPR-Cas9-sgRNA ms system that induces cell death with a single-guide RNA (sgRNA ms ) targeting multiple sites in the genome. The Cas9-sgRNA ms system interrupted cell proliferation and induced cell ablation in vitro. The mouse model had Cas9 driven by the Foxn1 promoter with a ubiquitous expression cassette of sgRNA ms at the Rosa26 locus (Foxn1 Cas9 ; Rosa26_ms). It showed an athymic phenotype similar to that of nude mice but was not hairless. Eventually, a rat cell-derived thymus in an interspecies chimera was generated by blastocyst complementation of Foxn1 Cas9 ; Rosa26_ms mouse embryos with rat embryonic stem cells. Theoretically, a half of the total embryos has the Cas9-sgRNA ms system because Rosa26_ms could be maintained as homozygous., (© 2024. The Author(s).)

Primary malignant melanoma of the esophagus (PMME) is an extremely rare but highly aggressive malignancy with a poor prognosis. Due to the scarcity of driver gene alterations, there is a need for more clinical data to comprehensively depict its molecular alterations. This study reviewed 26 PMME cases from three medical centers. Hybrid capture-based targeted sequencing of 295 and 1021 genes was performed in 14 and 12 cases, respectively. We found that PMME patients had a relatively low tumor mutation burden (median, 2.88 mutations per Mb) and were simultaneously accompanied by mutations in genes such as KIT (6/26, 23%), TP53 (6/26, 23%), SF3B1 (4/26, 15%), and NRAS (3/26, 12%). KIT, NRAS, and BRAF were mutually exclusive, and SF3B1 co-occurred with KIT mutation and amplification. The most common pathways affected were the mitogen-activated protein kinases and DNA damage response (DDR) pathways. Stage IV was a risk factor for both progression-free survival (hazard ratio [HR] = 5.14, 95% confidence interval [CI] = 1.32-19.91) and overall survival (OS), HR = 4.33, 95% CI = 1.22-15.30). Treatment with immune-checkpoint inhibitors (ICIs) was an independent factor for favorable OS (HR = 0.10, 95% CI = 0.01-0.91). Overall, PMME is a complex malignancy with diverse gene alterations, especially with harboring DDR alterations for potentially response from ICIs., (© 2023 The Authors. Pigment Cell & Melanoma Research published by John Wiley & Sons Ltd.)

Mutations in the human fasciculation and elongation protein zeta 1 ( FEZ1) gene are found in schizophrenia and Jacobsen syndrome patients. Here, using human cerebral organoids (hCOs), we show that FEZ1 expression is turned on early during brain development and is detectable in both neuroprogenitor subtypes and immature neurons. FEZ1 deletion disrupts expression of neuronal and synaptic development genes. Using single-cell RNA sequencing, we detected abnormal expansion of homeodomain-only protein homeobox (HOPX) - outer radial glia (oRG), concurrent with a reduction of HOPX + oRG, in FEZ1-null hCOs. HOPX - oRGs show higher cell mobility as compared to HOPX + oRGs. Ectopic localization of neuroprogenitors to the outer layer is seen in FEZ1-null hCOs. Anomalous encroachment of TBR2 + intermediate progenitors into CTIP2 + deep layer neurons further indicated abnormalities in cortical layer formation these hCOs. Collectively, our findings highlight the involvement of FEZ1 in early cortical brain development and how it contributes to neurodevelopmental disorders., Competing Interests: The authors declare no competing interests., (© 2023 The Authors.)

Fetal adenocarcinoma of the lung (FLAC) is a rare lung tumor classified into low-grade fetal adenocarcinoma of the lung (LG-FLAC) and high-grade fetal adenocarcinoma of the lung (HG-FLAC). It remains debatable whether HG-FLAC is a subset of FLAC or a distinct subtype of the conventional lung adenocarcinoma (CLA). In this study, samples of 4 LG-FLAC and 2 HG-FLAC cases were examined, and the clinicopathologic, immunohistochemical (IHC), and mutational differences between the 2 subtypes were analyzed using literature review. Morphologically, LG-FLACs had a pure pattern with complex glandular architecture composed of cells with subnuclear and supranuclear vacuoles, mimicking a developing fetal lung. In contrast, HG-FLACs contained both fetal lung-like (FLL) and CLA components. With regard to IHC markers, β-catenin exhibited a nuclear/cytoplasmic staining pattern in LG-FLACs but a membranous staining pattern in HG-FLACs. Furthermore, p53 was expressed diffusely and strongly in HG-FLACs, whereas in LG-FLACs, p53 staining was completely absent. Using next-generation sequencing targeting a 1021-gene panel, mutations of CTNNB1 and DICER1 were detected in all 4 LG-FLAC samples, and a novel mutation, MYCN P44L, was discovered in 2 LG-FLAC samples. DNA samples of the FLL and CLA components of HG-FLACs were separately extracted and sequenced. The FLL component harbored no CTNNB1, DICER1, or MYCN mutations; moreover, the FLL genetic profile largely overlapped with that of the CLA component. The morphologic, IHC, and genetic features of HG-FLAC indicate that it is a variant of CLA rather than a subset of FLAC. Thus, HG-FLAC should be treated differently from LG-FLAC., Competing Interests: Conflicts of Interest and Source of Funding: The authors have disclosed that they have no significant relationships with, or financial interest in, any commercial companies pertaining to this article., (Copyright © 2021 The Author(s). Published by Wolters Kluwer Health, Inc.)

The aim of this paper was to investigate the potential pharmacological effect of flavonoids in Sophora alopecuroides by network pharmacology. This study predicted the potential targets of 11 flavonoids of S. alopecuroides with help of reversed pharmacophore matching target recognition service platform (PharmMapper). The pathway information was acquired from DAVID and KEGG databases. Cytoscape software was used to construct the "ingredient-target-pathway" network of flavonoids active components of S. alopecuroides. The flavonoids active components of S. alopecuroides play anti-inflammatory, blood sugar regulating and other pharmacological effects by regulating 62 targets (such as INSR，KDR，MET) and intervening 44 pathways, such as B cell receptor signaling pathway, insulin signaling pathway, neurotrophin signaling pathway, and T cell receptor signaling pathway. In this study, the mechanism of "muti components-multitargets-multiple pathway" of flavonoids was studied. It reflects the multi-components, multi-targets and multiple pathway features of traditional Chinese medicine. Meanwhile, it provides a scientific basis for the elucidation the mechanism of S. alopecuroides as a medicine, and the development and utilization resources of S. alopecuroides., Competing Interests: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose., (Copyright© by the Chinese Pharmaceutical Association.)

It was aimed at exploring the potential pharmacological effects of alkaloids in Sophora alopecuroides by means of network pharmacology in this study. The main alkaloids in S. alopecuroides were collected for analysis of drug properties, prediction of potential targets and screening of signaling pathways. DAVID analysis tool combined with KEGG database was used to annotate and analyze the signaling pathway. The alkaloids-targets-signaling pathways network was built through Cytoscape software. Results showed that 17 alkaloids in S. alopecuroides involved 49 targets (170 times in all) and 22 important signaling pathways. Three nodes in model of network pharmacology were cross-linked, and the metabolic pathways were coordinated and regulated by each other. It indicated that alkaloids in S. alopecuroides may have therapeutic effect on diseases of cancer, metabolic disorder, endocrine system, digestive system, nervous system and so on., Competing Interests: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose., (Copyright© by the Chinese Pharmaceutical Association.)

To compare the appearances, tastes, contents of bioactive components and antioxidant activity of Lyceum ruthenicum under different drying methods, so as to direct its production practice. The folin-phenol colorimetric method, UV, extinction coefficient method and DPPH, as well as fluorescence recovery after photobleaching (FRAP) method to determine the contents of polyphenols, proanthocyanidins, total anthocyanin and antioxidant activity under different drying methods： vacuum freeze drying, low-temperature oven drying and air drying for L. ruthenicum. The results showed that the drying methods had certain effects on its appearances, tastes, contents of bioactive components and antioxidant activity. The appearances and tastes were best after the L. ruthenicum was dried by vacuum freeze drying, with significantly lower moisture than air drying method. The contents of total polyphenols, anthocyanin and proanthocyanidins were highest by air-drying but lowest by low temperature oven drying in L. ruthenicum. The scavenging ability to DPPH was strongest by freeze-drying and lowest by low temperature oven drying, while the antioxidant activity was strongest by air-drying in the FRAT method. In addition, the appearances and tastes were poor in air drying, with higher moisture but highest contents of the three bioactive components. Therefore, the drying methods for L. ruthenicum shall be comprehensively considered., Competing Interests: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose., (Copyright© by the Chinese Pharmaceutical Association.)

Panax ginseng has been studied for its chemo-preventive properties and pharmaceutical potential. Polyacetylenic compounds isolated from Panax ginseng root typically comprised of non-polar C 17 compound have been reported to exhibit bioactive properties. The objective of this project is to extract, isolate, and characterize bioactive polyacetylenes from Panax ginseng root using various extraction and separation methods Ginseng was extracted by reflux using methanol, ethanol, hexane, ethyl acetate, methanolic ultrasonication. The extracts were partitioned with hexane to obtain water-soluble portion and hexane-soluble portion. Hexane was subsequently removed under vacuum, and formed a crude polyacetylenes extract (crude PA). Silica gel chromatography and semi-preparative HPLC were utilized to prepare 5 fractions and the polyacetylenes were measure by HPLC and molecular weights confirm my APCI-MS and MNR. The bioactive effect was measured by MTT viability assay using murine 3T3-L1 cells. Extraction with methanol under reflux produced significantly larger amount of polyacetylenes (p<0.05). Liquid-liquid extraction and column chromatography were used to separate polyacetylenic compounds into five different fractions. Major polyacetylenes, panaxynol and panaxydol were found in fraction 1 and 2 respectively. Dose-response relationships were observed in 3T3-L1 cells and LC50 were 13.52±3.05μg/mL (fraction 1), 3.69±1.09μg/mL (fraction 2), 52.88±11.16μg/mL (fraction 3), 85.91±27.37μg/mL (fraction 4) and 135.52±32.91μg/mL (fraction 5). Fraction 2 containing panaxydol was found to have exhibited the greatest anti-proliferative effects on 3T3-L1 preadipocytes. Extraction with methanol under reflux produced significantly more polyacetylenes. Fractions that contain panaxydol was the most cytotoxic., (Copyright © 2017 Elsevier B.V. All rights reserved.)

This study is to construct a rapid and effective method for identification of wild and cultivated Glycyrrhiza uralensis (hereinafter referred to as Glycyrrhizae Radix et Rhizoma) from Ningxia by comparison of the difference in chromatography identification based on index components and near-infrared spectroscopy identification. HPLC and UV methods were used to determine the content of liquiritin, glycyrrhizate and total flavonoids for 9 wild Glycyrrhizae Radix et Rhizoma and 14 cultivated Glycyrrhizae Radix et Rhizoma samples,and the near-infrared spectroscopy was also,collected. The results illustrated that the chromatography identification based on index components could not identify wild and cultivated Glycyrrhizae Radix et Rhizoma from Ningxia, while near-infrared spectroscopy could quickly and effectively achieve it. It provides an effective method for the growth pattern identification and application of Glycyrrhizae Radix et Rhizoma., Competing Interests: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose., (Copyright© by the Chinese Pharmaceutical Association.)

The "master transcription factor" FOXP3 regulates the differentiation, homeostasis, and suppressor function of CD4 + regulatory T (Treg) cells, which are critical in maintaining immune tolerance. Epigenetic regulation of FOXP3 expression has been demonstrated to be important to Treg cell development, but the induction of human Treg cells through epigenetic modification has not been clearly described. We report that the combination of the DNA methyltransferase inhibitor 5-azacytidine (5-Aza) and suboptimal T cell receptor (TCR) stimulation promoted CD4 + CD25 h FOXP3 + T cell induction from human CD4 + CD25 - T cells. 5-Aza treatment enhanced the expression of Treg cell signature genes, such as CD25, FOXP3, CTLA-4, and GITR, in CD4 + CD25 h cells. Moreover, 5-Aza-treated CD4 + CD25 h T cells showed potent suppressive activity in a cell contact-dependent manner and reduced methylation in the Treg-specific demethylated region (TSDR) in the FOXP3 gene. The analysis of cytokine production revealed that CD4 + CD25 - T cells with 5-Aza treatment produced comparable levels of interferon (IFN)-γ and transforming growth factor (TGF)-β, but less IL-10 and more IL-2, when compared to cells without 5-Aza treatment. The increased IL-2 was indispensible to the enhanced FOXP3 expression in 5-Aza-treated CD4 + CD25 h cells. Finally, 5-Aza-treated CD4 + CD25 h T cells could be expanded with IL-2 supplementation alone and maintained FOXP3 expression and suppressor function through the expansion. Our findings demonstrate that DNA demethylation can enhance the induction of human Treg cells and promise to solve one of the challenges with using Treg cells in therapeutic approaches.

A denitrifying bacteria consortium was enriched from LiHe River's sediment, the dynamics of total nitrogen (TN), nitrate (NO3- -N), nitrite (NO2- -N), ammonium (NH4+ -N) and COD at different enrichment cultivation stages were studied, and the total volume, the releasing rates and the composition of gas released during the denitrification process were analyzed. The full-length 16S rDNA clone library was constructed, enclosing the diversity of the denitrifying bacteria consortium. The results showed, in the enrichment phase 4, under the load of TN 330 mg x L(-1), the best nitrogen removal effect was obtained, which the TN and NO3- -N removal rates reached 90.9% and 100% within 9 hours, respectively. The accumulation amounts of NO2- -N and NH4+ -N were merely 3.39 mg x L(-1) and 16.64 mg x L(-1). And the COD removal rate was 85%. The process released 260 mL of the compound gas, in which the main ingredient was N2 associated with a small quantity of CH4 and CO2. The denitrifying bacteria consortium consisted of the family Pseudomonadaceae and the family Rhodocyclaceae, belonging to Proteobacteria phylum, in which the OUT abundances were 57.8% and 31.6%, respectively. The family Pseudomonadaceae was the predominant group.

Purpose: To compare ingredients contained in top-selling brands of ocular nutritional supplements with the Age-Related Eye Disease Study (AREDS) and AREDS2 formulae and investigate the validity of claims made by manufacturers of leading brands of ocular nutritional supplements., Design: Descriptive., Participants: None., Methods: We examined the 5 top-selling brands of ocular nutritional supplements in the United States according to dollar sales tracked by SymphonyIRI (Waltham, MA) from June 2011 to June 2012. We reviewed the ingredients and manufacturer claims of 11 ocular nutritional supplements on the companies' consumer information websites; the ingredients were compared with those contained in the AREDS and AREDS2 formulae., Main Outcome Measures: Proportion of ocular nutritional supplements that contained the same ingredients, in the same doses, as the AREDS or AREDS2 formula; proportion of nutritional supplements with unsubstantiated claims made by the manufacturer., Results: All of the ocular nutritional supplements contained the ingredients from the AREDS or AREDS2 formula; 36% (4/11) of the supplements contained equivalent doses of AREDS or AREDS2 ingredients; 55% (6/11) included some information about the AREDS on their consumer information websites. Product descriptions from 4 of the 11 supplements (36%) stated that the supplements were important to maintain general eye health; none of these supplements duplicated the AREDS or AREDS2 formula. All the individual supplements claimed to "support," "protect," "help," or "promote" vision and eye health, but none specified that there is no proven benefit in using nutritional supplements for primary prevention of eye disease., Conclusions: The majority of top-selling ocular nutritional supplements did not contain the identical ingredient dosages of the AREDS or AREDS2 formula and had product description claims that lacked level 1 evidence, underscoring the importance of ophthalmologists educating their patients on the evidence-based role of nutritional supplements in the management of eye health., (Published by Elsevier Inc.)

Thiamine deficiency (TD) leads to Wernicke's encephalopathy (WE), in which focal histological lesions occur in periventricular areas of the brain. Recently, impaired neurogenesis has been reported in the hippocampus during the dietary form of TD, and in pyrithiamine-induced TD (PTD), a well-characterized model of WE. To further characterize the consequences of PTD on neural stem/progenitor cell (NSPC) activity, we have examined the effect of this treatment in the rat on both the subventricular zone (SVZ) of the rostral lateral ventricle and subgranular layer (SGL) of the hippocampus, and in the thalamus and inferior colliculus, two vulnerable brain regions in this disorder. In both the SVZ and SGL, PTD led to a decrease in the numbers of bromodeoxyuridine-stained cells, indicating that proliferation of NSPCs destined for neurogenesis in these areas was reduced. Doublecortin (DCX) immunostaining in the SGL was decreased, indicating a reduction in neuroblast formation, consistent with impaired NSPC activity. DCX labeling was not apparent in focal areas of vulnerability. In the thalamus, proliferation of cells was absent while in the inferior colliculus, numerous actively dividing cells were apparent, indicative of a differential response between these two brain regions. Exposure of cultured neurospheres to PTD resulted in decreased proliferation of NSPCs, consistent with our in vivo findings. Together, these results indicate that PTD considerably affects cell proliferation and neurogenesis activity in both neurogenic areas and parts of the brain known to display structural and functional vulnerability, confirming and extending recent findings on the effects of TD on neurogenesis. Future use of NSPCs in vitro may allow a closer and more detailed examination of the mechanism(s) underlying inhibition of these cells during TD.

Nanostructuring of Al₂O₃ is predominantly achieved by the anodization of aluminum film and is limited to obtaining porous anodized aluminum oxide (AAO). One of the main restrictions in developing approaches for direct fabrication of various types of Al₂O₃ patterns, such as lines, pillars, holes, etc, is the lack of a processable aluminum-containing resist. In this paper, we demonstrate a stable precursor prepared by reacting aluminum tri-sec-butoxide with 2-(methacryloyloxy)ethyl acetoacetate, a chelating monomer, which can be used for large area direct nanoimprint lithography of Al₂O₃. Chelation in the precursor makes it stable against hydrolysis whilst the presence of a reactive methacrylate group renders it polymerizable. The precursor was mixed with a cross-linker and their in situ thermal free-radical co-polymerization during nanoimprinting rigidly shaped the patterns, trapped the metal atoms, reduced the surface energy and strengthened the structures, thereby giving a ~100% yield after demolding. The imprinted structures were heat-treated, leading to the loss of organics and their subsequent shrinkage. Amorphous Al₂O₃ patterns with line-widths as small as 17 nm were obtained. Our process utilizes the advantages of sol-gel and methacrylate routes for imprinting and at the same time alleviates the disadvantages associated with both these methods. With these benefits, the chelating monomer route may be the harbinger of the universal scheme for direct nanoimprinting of metal oxides.

Our newly designed primers were evaluated for the molecular analysis of specific groups of the sqr gene encoding sulfide : quinone reductase (SQR) in sediment environments. Based on the phylogenetic analysis, we classified the sqr sequences into six groups. PCR primers specific for each group were developed. We successfully amplified sqr-like gene sequences related to groups 1, 2 and 4 from diverse sediments including a marine sediment (SW), a tidal flat (TS), a river sediment (RS) and a lake sediment (FW). We recovered a total of 82 unique phylotypes (based on a 95 % amino acid sequence similarity cutoff) from 243 individual sqr-like gene sequences. Phylotype richness varied widely among the groups of sqr-like gene sequences (group 1>group 2>group 4) and sediments (SW>TS>RS>FW). Most of the sqr-like gene sequences were affiliated with the Proteobacteria clade and were distantly related to the reference sqr gene sequences from cultivated strains (less than approximately 80 % amino acid sequence similarity). Unique sqr-like gene sequences were associated with individual sediment samples in groups 1 and 2. This molecular tool has also enabled us to detect sqr-like genes in a sulfur-oxidizing enrichment from marine sediments. Collectively, our results support the presence of previously unrecognized sqr gene-containing micro-organisms that play important roles in the global biogeochemical cycle of sulfur.

A taxonomic study was carried out on an isolate, strain NO2(T), from marine sediment collected from the East Sea, Korea. Comparative 16S rRNA gene sequence studies showed that this strain belonged to the Gammaproteobacteria and was most closely related to Glaciecola mesophila KMM 241(T) and Glaciecola polaris LMG 21857(T) (98.6 and 98.0 % 16S rRNA gene sequence similarity, respectively). The isolate was Gram-negative, aerobic and slightly halophilic and grew in 2-8 % NaCl and at 7-30 degrees C. Strain NO2(T) shared some physiological and biochemical properties with G. mesophila KMM 241(T) and G. polaris LMG 21857(T). The G+C content of the genomic DNA of strain NO2(T) was 45 mol%. Strain NO2(T) possessed C(16 : 0), summed feature 4 (C(16 : 1)omega7c and/or iso-C(15 : 0) 2-OH) and summed feature 7 (C(18 : 1)omega9c/omega12t/omega7c) as the major cellular fatty acids. DNA-DNA relatedness data indicated that strain NO2(T) represents a distinct species that is separate from G. mesophila and G. polaris. On the basis of polyphasic evidence, it is proposed that strain NO2(T) (=KCTC 12755(T)=LMG 23762(T)) represents the type strain of a novel species, Glaciecola agarilytica sp. nov.

Background/aims: Leptin has been recently implicated in the development of hepatic steatosis and fibrosis in chronic hepatitis C (CHC) infection. The serum levels of leptin are known to be strongly affected by anthropometric parameters such as body mass index (BMI) and total body fat, which show differences between races or ethnicities. In this study, we examined whether serum leptin levels are correlated with clinical, virological, and histological features, and with response to antiviral therapy in Korean CHC patients., Methodology: We evaluated correlations between serum leptin level and age, sex, BMI, fasting glucose, alanine aminotransferase, genotype, hepatitis C virus RNA titer, steatosis, fibrosis, and response to antiviral therapy after 24 weeks completing 24 weeks of interferon-alpha based therapy in 47 Korean CHC patients., Results: Of the variables examined, only female sex and a BMI > 25kg/m2 were identified as independent variables related to a higher leptin level by multivariate analysis. Baseline leptin levels and leptin changes before/after antiviral therapy were not correlated with response to therapy., Conclusions: In Korean CHC patients, serum leptin levels were found to be correlated with anthropometric parameters, but not with virological or histological features. In addition, serum leptin levels did not predict response to antiviral therapy.

Object: Individuals with unruptured intracranial aneurysms experience a higher rate of rupture if their history includes another aneurysm that has previously bled. The authors used systematic review and metaregression to estimate the annual rate of development of second de novo aneurysms after subarachnoid hemorrhage., Methods: This investigation included studies in which more than 300 patients with intracranial aneurysms were described, and in which the age of the patients and the proportion with multiple aneurysms were documented. Studies describing delayed follow-up angiography that was performed after treatment of aneurysms were also reviewed. Twenty studies were included in a between-study analysis. The univariate odds ratio (OR) for multiple intracranial aneurysms per year of age was 1.085 (95% confidence interval [CI] 1.015-1.165); this value was calculated using a hierarchical model for between-study heterogeneity. Five studies were included that provided age stratification. The estimated OR for multiple intracranial aneurysms per year was 1.011 (95% CI 1.005-1.018). Four follow-up studies were available., Conclusions: According to the three different approaches (study-level, patient-level, and follow-up analyses), the estimated annual rates of development of de novo aneurysms were 1.62% (95% CI 0.28-3.59%), 0.28% (95% CI 0.12-0.49%), and 0.92% (95% CI 0.64-1.25%), respectively. The estimated annual rate of development of second de novo aneurysms ranged from 0.28 to 1.62%.