Combination of untargeted and targeted proteomics for secretome analysis of L-WRN cells.

Organoid culture is a promising biomedical technology that requires specialized growth factors. Recently, a recombinant L-WRN cell line has been extensively used to generate conditioned medium (L-CM) for organoid culture. Nevertheless, methods for evaluating the stability of the L-WRN cells have been limited. In this study, a novel proteomics-based approach was developed to analyze the secretome of the cells. Serum-free L-CM was lyophilized, precipitated by trichloroacetic acid, and desalted prior to analysis by liquid chromatography-tandem mass spectrometry. Data-dependent acquisition (DDA) was conducted for the untargeted secretome profiling of the cells, and parallel reaction monitoring (PRM) was applied for the targeted quantification of the Wnt3A, R-spondin3, and noggin proteins (WRNs). This study also compared the performance of two types of PRM methods, namely MS1-independent PRM and MS1-dependent PRM, that can be executed on an Orbitrap instrument. The results showed that the growth of mouse intestinal organoids was closely related to the use of L-CM. The composition of L-CM could be markedly affected by the medium collection scheme. A total of 1725, 2302, and 2681 proteins were identified from the L-CM collected on day 5, day 9, and day 13, respectively. The MS1-independent PRM outperformed the MS1-dependent PRM and effectively quantified the WRNs with high repeatability and specificity. In conclusion, by integrating untargeted and targeted proteomics, this study develops a mass spectrometry-based method for the secretome analysis and quality control of the L-WRN cells. The methodology and findings of the present work will benefit future studies on organoids and secretomes.

Aqueous Supramolecular Co-Assembly of Anionic and Cationic Photoresponsive Stiff-Stilbene Amphiphiles.

Fabrication of macroscopic soft functional materials, such as macroscopic photoresponsive soft materials and artificial muscles, can be commonly prepared by charge screening of supramolecular assemblies with inorganic salt solutions using a shear-flow method. However, some of the charged end-groups of photoresponsive molecular amphiphiles cannot be stabilized with inorganic salt solutions to fabricate macroscopic soft materials. Stiff stilbene amphiphiles (SAs) functionalized with anionic phosphite and cationic quaternary ammonium end groups are designed and synthesized and their photochemical and supramolecular assembly properties are determined. Supramolecular co-assembly of anionic and cationic nanotubes of SAs allows to transform into nanoribbons, confirmed by transmission electron microscopy, critical aggregation concentration, and Zeta potential measurements. Nanoribbons of anionic and cationic SAs can be prepared into macroscopic soft materials with inorganic salt solutions and surprisingly also with deionized water. The macroscopic soft material of anionic and cationic SAs can be stabilized at low concentration ≈5 mm. Meanwhile, the photoresponsiveness of the macroscopic soft materials is retained to provide macroscopic morphological change upon photoirradiation. These results exhibit the feasibility in fabrication of macroscopic functional soft materials from supramolecular assembly across multiple length-scale without help of inorganic salts and offer ample opportunity in developing future soft supramolecular robotic systems.

Esophageal Granular Cell Tumor: A Case and Review of the Literature.

Granular cell tumors (GCTs) are rare and benign tumors that can occur at any anatomical site. GCTs are thought to originate from nerve cells, particularly Schwann cells. Their name derives from the fact that an accumulation of cytoplasmic lysosomes imparts the tumor with a granular appearance. They are most commonly observed in the oral cavity, skin and subcutaneous tissue, breast, and respiratory tract. GCTs rarely affect the gastrointestinal tract. We report a 56-year-old female with a medical history of human immunodeficiency virus, hepatitis C, and cholelithiasis, who presented with abdominal pain. Upper endoscopy revealed a 1 - 2 cm solitary yellowish appearing nodule just distal to the GE junction. Biopsy of the nodule followed by histopathology was positive for S100, but negative for pancytokeratin immunostains. PAS staining highlighted cytoplasmic granules, further supporting the diagnosis of gastrointestinal GCT.

Systems and synthetic biology of the vessel wall.

Atherosclerosis is intimately coupled to blood flow by the presence of predilection sites. The coupling is through mechanotransduction of endothelial cells and approximately 2000 gene are associated with this process. This paper describes a new platform to study and identify new signalling pathways in endothelial cells covering an atherosclerotic plaque. The identified networks are synthesized in primary cells to study their reaction to flow. This synthetic approach might lead to new insights and drug targets.

Single-dose bioequivalence assessment of two formulations of polysaccharide iron complex capsules in healthy adult male Chinese volunteers: A sequence-randomized, double-blind, two-way crossover study.

BACKGROUND: Iron deficiency anemia (IDA) is a common nutritional disease worldwide. Iron supplementation is an efficient method for treating patients with IDA. Polysaccharide iron complex is an oral iron supplement that is associated with generally good tolerability and good bioavailability. OBJECTIVE: The aim of this study was to evaluate the bioequivalence of 2 branded formulations of polysaccharide iron complex in healthy adult male Chinese volunteers by determining the pharmacokinetic parameters after single-dose oral admi ni strati on. METHODS: This sequence-randomized, double-blind, 2-way crossover study was carried out in the Affiliated Hospital, Institute of Medical Sciences of Qingdao University, Qingdao, China. Healthy adult male Chinese volunteers were enrolled and evenly randomized to receive 1 of 2 formulations on day 1. Subjects received an oral dose of 150 mg (1 capsule) of polysaccharide iron complex with 150 mL of warm water in the morning. Capsules were of similar size, shape, and color to ensure blinding. Four hours after administration, the subjects were given standardized meals. After a 1-week washout period, the subjects were crossed over to receive the other formulation in a similar manner. The serum iron concentration 12 hours after study drug administration was determined using atomic-absorption spectrometry. The pharmacokinetic parameters Cmax, Tmax, AUC0-t, and AUC0-∞ were obtained and analyzed using the Schuir mann 2 one-sided t test. The 2 formulations were considered bioequi valent if the test/reference ratios of Cmax, AUC0-t, and their 90% CIs were within the range of 70% to 143% for Cmax and within 80% to 125% for AUC0-t. Tolerability was monitored by inquiring whether the subjects had experienced adverse events (AEs), with a focus on gastrointestinal AEs, during the clinic visits during the 24-hour period after drag administration and subsequently via telephone throughout the study. RESULTS: Thirty adult male Chinese volunteers were assessed for inclusion. Twenty healthy male volunteers (10 in each group) (mean [SD] age, 21.5 [2.9] years [range, 19-23 years]; weight, 66.2 [5-8] kg [range, 56-80 kg]; height, 172.5 [5.1] cm [range, 162-180 cm]) were enrolled and completed the study. The pharmacokinetic parameters of the test and reference formulations were as follows: AUCO-t, 6.58 (2.09) and 6.58 (1.91) µg/mL · h(-1); Cmax, 1.10 (0.28) and 1.07 (0.25) µg/mL; Tmax, 3.93 (0.37) and 3-93 (0.37) hours; t½, 8.33 (0.36) and 8.38 (0.41) hours; and AUC0-∞, 6.93 (2.23) and 6.95 (2.13) µg/mL · h(-1), respectively. There were no statistically significant differences in AUC0-∞ or Tmax by formulation, period, or subject between the test and reference formulations. Similarly, there were no statistically significant differences in Cmax by period; however, a significant difference was found in Cmax by formulation (P = 0.012). No clinically significant AEs were reported with either formulation. CONCLUSIONS: In these healthy adult male Chinese volunteers, the test formulation of polysaccharide iron complex was found to be bioequivalent to the reference formulation according to the Chinese regulatory definition. A significant difference by formulation was found in Cmax. The sample size was smaller than that recommended by the US Food and Drug Administration for a bioequivalence study, and additional studies with larger sample sizes are needed.

Rna interference of annexin II gene in PC3 cells by using small interference RNA synthesized with in vitro transcription.

OBJECTIVE: To silence annexin II gene expression by using small interference RNA (siRNA) in prostate cancer cell line PC3. METHODS: For in vitro transcription, four sequences of 29-nucleotide DNA template oligonucleotides were designed, and one pair of the sequences were complementary to annexin II gene. The other pair was negative control. The 8 nucleotides at the 3' end of each oligonucleotide were complementary to the T7 Promoter Primer. The sense and antisense siRNA templates were transcribed by T7 RNA polymerase and the resulting RNA transcripts were hybridized to create dsRNA. The siRNA was transfected into prostate cancer cell PC3. For assaying the efficiency of siRNA, confocal microscopy, Northern blotting, and Western blotting were employed to examine the expression of annexin II protein and its mRNA. 3H thymidine was used to measure DNA synthesis. RESULTS: The siRNA sequence specific to annexin II gene was capable of inhibiting the expression of annexin II protein and its mRNA. And cellular DNA synthesis was significantly reduced in siRNA transfected cells. CONCLUSIONS: The protocol for the synthesis of siRNA by T7 RNA polymerase is feasible. Annexin II might be involved in DNA synthesis.

Cross-sensitivity in a child with anticonvulsant hypersensitivity syndrome.

We describe a fulminant picture of anticonvulsant hypersensitivity syndrome (AHS) and the possible role of nitrazepam. A 5-month-old boy developed fever and rash after the use of phenobarbitone. Allergy to phenobarbitone was suspected. Nitrazepam was substituted for seizure control. Over the next few days he progressively collapsed with fever, facial oedema and multi-organ involvement. The diagnosis of AHS was delayed because nitrazepam has not been implicated in the development of cross-sensitivity. AHS is a severe multi-organ reaction to aromatic anti-epileptic drugs. It has been thought to occur as a consequence of pre-existing pharmacogenetic and immunologic abnormalities. Careful selection of anti-epileptic drugs is essential as cross-sensitivity is common. Intermittent benzodiazepines have been recommended in managing breakthrough seizures in AHS. However, the structure of benzodiazepines contains aromatic rings and potential cross-reactivity cannot be totally ignored. Although we do not have direct proof, we believe that nitrazepam prolonged the clinical course.