Deletion or duplication on the distal portion of the long arm of chromosome 1 result in complex and highly variable clinical phenotype including.intellectual disability and autism.We report on a patient with intellectual disability and a 763.3 Kb duplication on 1q43 that includes only CHRM3, which was detected by next generation sequencing (NGS). The patient presented with intellectual disability, developmental delay, autistic behavior, limited or no speech, social withdrawal, self-injurious, feeding difficulties, strabismus, short stature, hand anomalie, and no seizures, anxiety, or mood swings, and clinodactyly.We propose that CHRM3 is the critical gene responsible for the common characteristics in the cases with 1q43 duplication and deletion. The clinical phenotype associated with chromosome 1q43-q44 deletion and duplication is characterized by substantial intellectual disability, limited or no speech, and craniofacial dysmorphology, hand and foot anomalies, congenital heart, genital malformation, and central nervous system (CNS) abnormalities, including microcephaly, seizures, and agenesis of the corpus callosum –11. AccoHere, we report the first case of a 763.3 kb duplication at chromosome 1q43 involving only CHRM3 which encodes for the M3-muscarinic receptor, who has intellectual disability, developmental delay, autistic behavior, short stature, strabismus, hand anomalies, limited or no speech, feeding difficulties, social withdrawal, self-injurious, and a tendency toward self-injurious stereotypic behavior. The clinical features of this patient with pure 1q43 duplication are different from a previously reported case of a ~ 3½ year-old boy with a pure 473 kb deletion at 1q43 comprising only CHRM3, who has no short stature and hand anomalies . We propThe patient, a boy, was born at term via uncomplicated spontaneous vaginal delivery to an 24-year-old gravida at 38 weeks of gestation. His birth weight was 3.5 kg. Prenatal course had no preeclampsia; neonatal history was benign. Both parents had no history of neurological disease and developmental delays. At ~ 12 months, his parents became concerned for delays in language skills. At 3 yrs., he was given a diagnosis of autism disorder by pediatric evaluation. His past medical history is significant for strabismus, short stature and hand anomalie , and was quantified by using Qubit dsDNA BR Assay Kit and Qubit 2.0 Fluorometer . KAPA HTP Library Preparation Kit Illumina® Platforms  was utilized for highthroughput library construction for Illumina sequencing, starting from fragmented, double-stranded DNA (dsDNA) according to manufacturer’s instructions. Reads of the sequencing run for patient, patient’s father and mother were 8.5 M, 9.2 M, 9.8 M, respectively. After quality control, coverage percentages of ROI were calculated using the filtered reads at 0.20X, 0.22X, 0.23X for patient, patient’s father and mother, respectively. Genome Analyzer Toolkit (GATK) was used for base quality score recalibration, and single-nucleotide variants (SNV) discovery across was performed for all three samples. The whole-exome enrichment and sequencing (WES) was determined based on the Agilent SureSelect Human All Exon platform (Agilent Technologies), which targets ~ 50 Mb of the human exonic regions. The SureSelect system uses ~ 120-base RNA probes to capture known coding DNA sequences (CDS) from the NCBI Consensus CDS Database as well as other major RNA coding sequence databases, such as Sanger miRBase.As a part of intellectual disability and autism workup, the patient undergoes genetic testing. NGS analysis showed a single duplication of 763.3 Kb at 1q43 region  Figs. .Fig. 2OvWe report the first case of a 763.3 kb duplication at chromosome 1q43 involving only CHRM3  which encodes for the M3-muscarinic acetylcholine receptor. M3-muscarinic acetylcholine receptor is the predominant muscarinic subtype mediating acetylcholine-induced airway smooth muscle contraction, one of 5 subtypes (M1-M5) of muscarinic receptors, found throughout the CNS and peripheral tissue. The clinical features of this patient with pure 1q43 duplication are different from a previously reported case of a ~ 3½ year-old boy with a pure 473 kb deletion at 1q43 comprising only CHRM3   which encoded by the genes CHRM1, CHRM2, CHRM3, CHRM4 and CHRM5. M1, M3 and M5 receptors tend to couple to G proteins of the Gq/11 family, whereas M2 and M4 receptors preferentially signal through the Gi/o family of G proteins . The M3 We report the first case of a 763.3 kb duplication at chromosome 1q43 involving only CHRM3. In view of these common characteristics in our patient and the case with 1q43 deletion, we propose CHRM3 as the critical gene responsible for intellectual disability, developmental delay, autistic behavior, limited or no speech, social withdrawal, self-injurious, feeding difficulties, strabismus, and other overlapping phenotype in our patient as well as other patients with 1q43 deletions and duplication including CHRM3.


Yak is always in a malnutrition condition under pure grazing in the cold season. Many studies have suggested that provision of supplementary feed to yak cannot only be an effective approach to avoid weight loss, but also improve productivity. Growth hormone (GH) and insulin-like growth factor 1 (IGF-1) could improve yak productivity. In current work, we evaluated the effects of different energy feedstuffs on the serum concentration of GH, IGF-1, and relative hepatic expression of their associated binding proteins and receptors, as well as growth performance of yak. The results indicated that average daily gain (ADG), body weight (BW), feed conversion ratio, serum IGF-1 concentration, and relative expression of IGF-1 and IGFBP-3 were increased with an increase in dietary energy level, while serum GH concentration and hepatic growth hormone (GHR) expression were decreased. Serum IGF-1 concentration, relative hepatic expression of IGF-1, and insulin-like growth factor binding protein 3 (IGFBP-3) showed positive correlation with ADG, but serum GH concentration and GHR expression were negatively correlated with ADG. Yak offered with medium energy diet (NEg: 6.2 MJ/kg) displayed better growth by gaining 0.883 kg/d and showing a superior feed conversion ratio during experiment.p < 0.05) throughout the experimental period. Serum GH concentration decreased (p < 0.05) with an increase in dietary energy level on d 30 and d 60. While IGF-1 concentration was higher (p < 0.05) in ME group, as compared to LE and HE groups on d 60. The expression level of growth hormone receptor (GHR) was decreased (p < 0.001) and IGF-1 was increased with the increase in the dietary energy level. The relative expression of insulin-like growth factor binding protein 3 (IGFBP-3) was higher (p < 0.001) in ME and HE groups, except the LE group. In conclusion, our findings provide a first insight into the combined effect of GH and IGF-1 in controlling the metabolism and productivity of yak. It also showed that medium energy level diet contributed to promote growth performance of yak during the cold season.The objective of this study was to investigate the effects of different dietary energy levels on serum concentrations of growth hormone (GH) and insulin-like growth factor 1 (IGF-1), as well as gene expression of their associated binding proteins and receptors in yak. Fifteen adult male yaks with BW of 276.1 ± 3.5 kg were allotted in three dietary groups and were fed with low (LE), medium (ME), and high energy (HE) level diet having different NEg of 5.5 MJ/kg, 6.2 MJ/kg, 6.9 MJ/kg, respectively. The effects of these treatments on ADG, BW, ADFI, and feed conversion ratio were significant ( Bos grunniens) is a multi-tasker grazing livestock species offering meat, milk, hides, transportation, and dung as fuel for the Tibetan nomads exclusively in the Hindu Kush-Himalayan region and the Qinghai-Tibetan Plateau [Yak , and insulin-like growth factor 1 (IGF-1) in cattle by reducing feed intake . GH and The use of animals was approved by Gansu Province Animal Care Committee , and experimental procedures in this study were in accordance with the Guide for the Care and Use of Laboratory Animals. The feeding trials were conducted between November 2015 and January 2016 at Hezuo  of Gannan Tibetan Autonomous Prefecture, Gansu Province, China. Hezuo is located in the South-Eastern of Qinghai-Tibetan Plateau with the average altitude around 2936 m, a representative plateau-climate and an annual average temperature around 1.7 °C. The average temperature and relative humidity in the barn during whole experimental period were 3 °C and 42%, respectively.2 per animal and floor was paved with dry wheat straw, which was replaced three times a week. Two feed troughs were fixed with beton for each animal. The barn was thoroughly sanitized before animals were housed in and was cleaned every morning. Diets were formulated by following the recommendations of Feeding Standard of Beef Cattle  were assigned randomly to three dietary treatments. All yaks were individually housed in tethered stalls in a good ventilated barn, with 6 mThe animals were adapted to each treatment for two weeks prior to a 60-day experimental period. The animals were fed ad libitum in two equal portions at 08:00–09:00 am and 17:00–18:00 pm on daily basis with 2.5 kg concentrates and 6.0 kg roughage. All the animals had free access to water. The amount of diet received by each animal was recorded to calculate average daily feed intake (ADFI), and ADFI was determined as the total feed intake/60. Feed troughs were cleaned every morning before feeding the animals, orts samples were collected once a week, weighted and stored at −20 °C. The samples were dried in the lab oven at 65 °C for 48 h and the oven-dried samples were grounded to pass 2-mm screen for analysis of DM (105 °C for 8 h), crude protein, crude fat, calcium and phosphorus with relevant AOAC methods . NeutralYaks were weighed on day 1, day 30, and at the end of the experiment in the morning before feeding and the records were used to determine average daily gain (ADG), while ADG was calculated as /60. The feed conversion ratio was calculated as total feed intake/total weight gain. All animals were transferred to slaughter house and humanely slaughtered by exsanguination after electrical stunning at the end of the trial.Blood samples (15 mL) were collected via jugular venipuncture in ethylenediaminetetraacetic acid dipotassium salt-containing tubes  of each animal without feeding between 08:00 am and 10:00 am on d 30 and d 60, then centrifuged at 3000 rpm for 15 min at 4 °C. Serum samples were stored at −20 °C for subsequent IGF-1 and GH concentrations analysis by using commercial ELISA kit . Liver samples were immediately collected in quadruplicate after slaughtering, washed with 0.9% NaCl solution, stored in 2 mL cryogenic vials  and snap frozen in liquid nitrogen. All liver tissues were stored at −80 °C in laboratory until total RNA extraction.Bos grunniens GHR, IGF-1, and IGFBP-3 are listed in The gene names, sequences, accession numbers, primer sequences for 2O at room temperature for 5 min. Then reverse transcription reaction was performed on a thermal cycler  with 10 μL gDNA purified sample, 1.0 μL PrimeScript RT Enzyme Mix I, 1 μL RT Primer Mix, 4 μL 5×PrimeScript Buffer 2  and 4 μL RNase Free dH2O by using following program: 37 °C for 15 min, 85 °C for 5 s and 4 °C for an indefinite time. RT-PCR products were stored at −20 °C until quantitative real-time PCR was performed.Total RNA from liver tissues was extracted by using RNAiso Plus kit  according to the manufacturer’s instructions. RNase free pipet tips, 1.5 mL microtubes and PCR tubes  were used. The extracted RNA was dissolved in 40 μL diethylpyrocarbonate (DEPC)-treated water  and concentration was measured by spectrophotometer  at 260/280 nm (OD260:OD280 = 1.8~2.0). To check purity and integrity of RNA, agarose gel electrophoresis having 1 pg/mL ethidium bromide was used to stain 28S and 18S bands. Total RNA was reversely transcribed in three replicates using a PrimeScript™ RT reagent kit  in 20-μL reaction volume. In brief, possible gDNA was removed through 2.0 μL 5×gDNA Eraser Buffer, 1.0 μL gDNA Eraser, 2 μL total RNA (200 ng/μL) and 5 μL RNase Free dH2O. Reactions were run on a fluorescence thermal cycler  and following program was used: 95 °C for 30 s, 40 cycles of 95 °C for 5 s, with respective annealing temperature  used as a reference gene. Quantitative real-time PCR was performed in triplicate to determine relative mRNA expression using SYBR® Premix Ex Taq™ II . Each 20-μL real-time reaction contained 10 μL SYBR Premix Ex Taq II (2×), 0.8 μL each of 10 μM primers, 1.0 μL cDNA and 7.4 μL RNase Free dHperature  for 30 si is the dietary treatments  as the fixed effect, Dj is experimental day  as the fixed effect, Bl is the blocks  as the fixed effect, TDij is the interaction between treatments and experimental day, Yik is yak  as the random effect and εijk is the random error associated with the observation of ijkl. The interaction between treatments and experimental day was found for BW and ADG, and then LSD procedure of PROC GLM model was used to conduct multiple comparisons of three treatments on same time or same experimental period using SAS 9.4. The model used for the analysis was as follows:i is the dietary treatments  as the fixed effect, Bj is the blocks  as the fixed effect, εij is the random error associated with the observation of ij.Data for BW and ADG were analyzed using the repeated measures procedure with the time taken as repeated measures by PROC MIXED model in SAS 9.4 . The model used for the analysis was as follows:p ≤ 0.05 and trends at 0.05 < p ≤ 0.10. ADFI, feed conversion ratio, serum concentrations of GH, and IGF-1 on day 30 and day 60, and gene relative expressions among three treatments were processed using one-way ANOVA analysis with LSD procedure for multiple comparisons of three treatments using SAS 9.4. Data were described as mean ± SEM (Standard Error of Mean). Significance of effect was declared at p < 0.05, p < 0.01 and p < 0.001, respectively.The Pearson correlation coefficient was also calculated by using the PROC CORR from SAS 9.4. The correlation analysis was based on individual values and was independent of the treatments. Fifteen observations for each group or index of the study were used for correlation analysis. *, ** and *** indicated p = 0.002), time (p < 0.001) and interaction between treatments (p = 0.009) on BW were significant BW on day 30 (p = 0.037) and day 60 (p = 0.012) were significantly increased with increasing dietary energy level. ADG was significantly affected by increasing dietary energy level during three treatments from day 1 to day 60, day 1 to day 30, and day 30 to day 60 (p < 0.001), and changing dietary NEg from 5.5 (LE) to 6.9 MJ/kg (HE) significantly increased ADG from 0.833 to 1.186 kg/d in yaks during day 1 to day 30 (p < 0.001). A slight decrease in ADG was observed in medium to high dietary energy groups , however ME group had a higher ADG than LE group from day 0 to day 60 and day 30 to day 60 . Additionally, the effects of experimental time on ADG in this study were noticeable showing lower value on d 30~60 than d 1~30 (p < 0.001). The interaction between dietary treatments and time on ADG was significant (p = 0.016). ADFI decreased (p = 0.01) when a higher energy diet was offered, but LE group displayed higher (p = 0.003) ADFI value than HE group. Feed conversion ratio was significantly affected (p < 0.001) by increasing dietary energy level and it was higher in LE group than that in ME and HE groups (p < 0.05).BW, ADG, ADFI, and feed conversion ratio on day 1, day 30, day 60, and from day 1~60 of Gannan yaks are presented in p < 0.05). The concentration of GH in LE dietary group was higher (p = 0.011) than HE dietary group on day 30 (p = 0.012 and p = 0.002) of GH as compared to ME (p = 0.012) and LE (p = 0.002) dietary groups. There was no significant effect of dietary energy level on IGF-1 concentration (p > 0.05) on day 30 (p = 0.010 and p = 0.044) was observed in the ME group but no significant difference (p > 0.05) was observed between LE and HE dietary groups on day 60 (The effect of different dietary energy levels on serum GH and IGF-1 concentrations in yak are presented in p < 0.001) with an increase in dietary energy levels, and yak fed with low energy diet showed high (p < 0.001) GHR expression than those offered with medium and high energy diets (p > 0.05) in both ME and HE dietary treatments, however significantly higher values (p < 0.001) were observed in both groups as compared to LE dietary group. The mRNA abundance of IGFBP-3 was greater in ME and HE dietary groups than LE group (p < 0.001), while no difference was observed between ME and HE dietary groups (p > 0.05).The relative hepatic expression of GHR significantly reduced (gy diets a. A linep = 0.044), as shown in p = 0.017) and day 60 (p = 0.006). The relationship between relative genes expression and growth performance of yak are shown in p = 0.042) with ADG, while the expression level of IGFBP-3 had significantly positive (p = 0.003) correlation with ADG.An inverse correlation between GH concentration and ADG was recorded on day 60 (Avena sativa), hay, highland barley, and concentrate as energy diet. The intention of supplementing concentrate in the feed was to increase dietary energy supply, organic matter (OM) digestibility, and BW gain. In the current study, ADG data indicated that this goal was achieved and days of experiment had great influence on ADG over the entire cold season. Similar results had been reported in Holstein steers, in which a significant increase in ADG was observed with an increase in dietary energy level [The yak evolved as a remarkable domesticated animal having compensatory growth potential to recover its body condition when enough nutrition was available  and our gy level . Howevergy level  reportedgy level , which wThe growth of animal is a complex process, in which fierce competition between anabolism and catabolism exists including a series of physiological and biochemical activities such as glucose and amino acid utilization, intra- and extracellular synthesis or dissociation of protein and fat, all of these reactions are regulated by many factors . In the The somatotrophic axis, including GH, IGF-1, GHR, and IGFBP-3, has a multifunctional role in the metabolism and physiology of mammals . InjectiGenerally, GH combines with GHR to stimulate target tissues to synthesize IGF-1 and its synthesis in liver is mainly modulated at the transcriptional level and stimulates the hepatic expression of IGFBP-3 ,40. It wThe current study assessed the effects of serum concentrations of GH, IGF-1, and hepatic expression of GHR, IGF-1, and IGFBP-3 on growth performance of yaks. The results indicated that ADG, feed conversion ratio, serum IGF-1 concentration and relative expression of IGF-1 and IGFBP-3 was increased with an increase in dietary energy level, while serum GH concentration and hepatic GHR expression decreased. Serum IGF-1 concentration, hepatic expressions of IGF-1 and IGFBP-3 showed positive correlation with ADG, but serum GH concentration and GHR expression were negatively correlated with ADG. Yaks offered with medium energy diet (NEg: 6.2 MJ/kg) showed superior feed conversion ratio by gaining 0.883 kg/d and displayed better growth performance during experiment, this study will provide the reference for improvement of supplementary feeding strategy for yak in the cold season.


The discovery of anticancer therapeutics effective in eliminating dormant cells is a significant challenge in cancer biology. Here, we describe new synthetic polymer-based anticancer agents that mimic the mode of action of anticancer peptides. These anticancer polymers developed here are designed to capture the cationic, amphiphilic traits of anticancer peptides. The anticancer polymers are designed to target anionic lipids exposed on the cancer cell surfaces and act by disrupting the cancer cell membranes. Because the polymer mechanism is not dependent on cell proliferation, we hypothesized that the polymers were active against dormant cancer cells. The polymers exhibited cytotoxicity to proliferating prostate cancer. Importantly, the polymer killed dormant prostate cancer cells that were resistant to docetaxel. This study demonstrates a new approach to discover novel anticancer therapeutics. The design of drugs which are able to target dormant cancer cells must meet at least two conditions: (i) the target mechanism/pathway and molecular target in cancer cells are not dependent on cell proliferation, and (ii) the molecular target must be distinctive from normal cells for selectivity. To that end, we hypothesize that cell membranes would serve as an anticancer target in dormant cancer cells to meet these requirements. Specifically, physically compromising the barrier function of cell membrane would be lethal, but not necessarily be dependent on the stages of cell cycle. For the second requirement, cancer cells overexpress phosphatidylserine (PS) on their cell membrane surfaces relative to normal cells, which could provide a cancer selective binding target4. Therefore, designer molecules capable of binding to PS lipids and disrupting cell membranes may effectively kill dormant cancer cells.The design and development of molecules effective in eliminating dormant cancer cells remain a significant challenge in drug discovery. Dormant cancer cells are not proliferating but are largely resistant to conventional chemotherapies which target only rapidly proliferating cells, leading to recurrence and metastasis7. This class of peptides, which we denote as anticancer peptides (ACPs), are relatively small  and adopt cationic amphiphilic α-helical structures. Cationic ACPs selectively bind to PS lipid-rich anionic cancer cell membranes preferentially to normal cells by electrostatic interactions. The bound ACPs insert the hydrophobic domain of their helix into the membranes, which causes membrane disruption, leakage of cellular components, and ultimately cancer cell death. The anticancer polymers used here are low molecular weight methacrylate copolymers with random-sequences of binary monomer compositions consisting of cationic and hydrophobic side chains, serving as a model platform to test our approach. We hypothesize that the ACP-mimetic polymers are selective to PCa cells and kill dormant PCa cells resistant to conventional anticancer drugs. Synthetic polymer platforms are more resistant to proteolytic degradation in physiological environments and are more chemically tunable for activity modulation than peptides. The anticancer activity of natural and synthetic cationic polymers has been previously examined and were shown to induce apoptosis in cancer cells9. However, the molecular mechanism is not clear, and thus it is difficult to control the activity by design or develop structure-activity relationships. In addition, many synthetic polymers used in anticancer therapeutics are simply drug delivery carriers in the forms of drug conjugates or drug-encapsulated particles where the anticancer activity originates primarily from the drugs, not the polymers12. Recently, Yang and Hedrick demonstrated cationic polymer assemblies with anticancer activity by lysing cancer cell membranes13. The membrane-targeting mechanism would be a new strategy to treat drug-resistant cancer cells.In this study, we report the design of new class of anticancer polymers that are effective in killing dormant prostate cancer (PCa) cells. The design is inspired by membrane-active host-defense peptides with anticancer activity5, and the molecular weight distributions of the polymers are relatively narrow (dispersity Đ = 1.1–1.3)  and bone metastasis (PC-3)14. The copolymers caused concentration-dependent cytotoxicity in these PCa cells and exhibited 100% killing in the culture at high polymer concentrations  against C4-2B and DU145 than PC-3 cells. The EC50 values decreased as the mole percentage of EMA monomer increased  compared to those with shorter cationic side chains (AEMA monomers). We have previously demonstrated that the elongation of cationic side chains of methacrylate random copolymers increases the depth of polymer insertion in anionic bacterial cell membranes (snorkeling effect), leading to higher antibacterial activity15. Similarly, the longer cationic side chains might enhance the polymer insertion into cancer cell membranes and disrupt the membranes more effectively. As a reference, the EC50 values of lytic peptide melittin were 2–6 µg/mL under the same assay conditions. While melittin exhibited higher activity than the polymers, it is highly toxic to normal cells, thus limiting its use as an anticancer agent17.To test our design approach, the copolymers were first examined for their ons Figs  and S2. sed Fig. , indicatP-5 for 24 hours was higher than that of similarly treated DU145 cells. Similar to DU145, the EC50 values for HDMECs also decreased as the mole percentage of EMA increased  was used as a measure of membrane-mediated acute cytotoxicity of copolymers. In general, the hemolytic activity of copolymers depended on their hydrophobicity. The percent hemolysis increased as the mole percentage of EMA increased, indicating that the copolymers with higher hydrophobicity are more hemolytic Table . The viased Fig. . These r50 values after 24 hour incubation with the selected copolymers were smaller than those against HDMEC and PCa cells  and human prostate epithelial cells (HPrECs). The ECls Table , indicatP-5 on the cell membranes. We selected P-5 as a model copolymer because it showed a potent activity against PCa cells and relative selectivity to PCa cells over HDMECs among the polymers tested in this study. We first observed the DU145 cells and HDMECs under a microscope after incubation with P-5 for 24 hours  from DU145 cells treated with P-5 for 24 hours was examined. P-5 caused significant LDH leakage at high polymer concentrations, suggesting that the cell membranes were permeabilized  after 72 hour incubation at high P-5 concentrations  with (DOPC/DOPS) or without PS lipids (DOPC). Lipid vesicles have been previously used to probe the mechanism of anticancer peptides21. It has been reported that the total amount of PS lipids in cells were largely different in cancer cell types, ranging from 2% to 10%22. In order to examine the effect of PS lipids on the copolymer activity evidently, we used GUVs with 10% PS lipids, which would be potentially the maximum amount of PS lipids exposed on the cancer cell surfaces. P-5 completely lysed all GUVs with PS lipids at high P-5 concentrations, although the GUVs without PS lipids remained intact  of copolymers and the level of Annexin binding /Cdt1(+)) made up 64% of the total cell population, while the actively proliferating cells (p27(-)/Cdt1(-)) were 19% , in which PS lipid exposure is no longer regulated and/or Annexin V freely binds to PS lipids in the inner leaflet of the cell membranes.The isolated dormant PC-3 cells were treated with docetaxel or P-5 were stained by both DAPI and Annexin V, suggesting significant membrane damage or necrotic cell death. At the P-5 concentrations of 15 µg/mL and 30 µg/ml, in which the cell viabilities of HDMECs were larger than 70% amino)butyl methacrylate (Boc-ABMA) , ethyl methacrylate (EMA) , 2-cyanoprop-2-yl-dithiobenzoate , and AIBN  were mixed in a flask. The oxygen of reaction mixture was removed by nitrogen gas bubbling for 5 minutes, and the reaction solution was stirred at 70 °C for 16 hours. The reaction was stopped by cooling the vial in a dry ice-acetone bath. The solvent was removed by evaporating under reduced pressure. The residue was dissolved in in dichloromethane, and the polymer was isolated by precipitation in excess hexanes twice. The mole percentage of EMA (MPethyl) was determined by comparing integrated peaks of butylene groups of Boc-ABMA and ethylene groups of EMA in the 1H NMR spectrum. The DP was calculated by comparing integrated peaks of phenyl group of chain transfer agent at the polymer ω-end and side chains. The number average molecular weight (Mn) was calculated using the DP, MPethyl, and the molecular weights of monomers and RAFT agent. Gel permeation chromatography ): Mn = 2,800 g/mol., Mw = 3,100 g/mol., dispersity (Đ) = 1.12. 1H NMR  δ: 7.90–7.26 , 5.25–4.70 , 4.20–3.83 , 3.25–2.92 , 2.15–1.77 , 1.70–1.45 , 1.42 , 1.32–1.17 , 1.16–0.80 . DP (1H NMR) = 15.0, MPethyl (1H NMR) = 30 mole %, Mn (1H NMR) = 3,400 g/mol.The copolymers were synthesized by RAFT polymerization according to the previous reportP-5 copolymer (1.00 g) was mixed with methyl 3-mercaptopropionate (MMP) (500 µL), followed by the addition of trifluoracetic acid (TFA) (5 mL). After stirring for 30 min, the TFA was removed by blowing with nitrogen gas. The residue was dissolved in methanol, and the deprotected P-5 was isolated by precipitating in excess diethyl ether. Subsequently, the precipitate was dissolved in distilled water and lyophilized to yield a light pink fluffy product . 1H NMR  δ: 7.90–7.40 , 4.20–3.90 , 3.10–2.90 , 2.20–1.80 , 1.80–1.65 , 1.50–1.10 , 1.20–0.80 . DP (1H NMR) = 15.9, MPethyl (1H NMR) = 31 mole %, Mn (1H NMR) = 2,500 g/mol.Boc-protected 2, and 100% humidity. Human dermal microvascular endothelial cells (HDMECs) were obtained from the PromoCell  and maintained in endothelial cell growth medium MV2  at 37 °C, 5% CO2, and 100% humidity. NHDF was obtained from PromoCell.The human prostate cancer cell lines, PC-3 (Cat #: CRL-1435), DU145 (Cat #: HTB-81) and HPrPC (Cat #: PCS-440-010) were obtained from the American Type Culture Collection. All prostate cancer cell lines were routinely grown in RPMI 1640  supplemented with 10% (v/v) fetal bovine serum (Invitrogen), 1% (v/v) penicillin-streptomycin (Invitrogen) and maintained at 37 °C, 5% COP-5, docetaxel, and vehicle  in a culture medium RPMI 1640 containing 0.5% FBS for 48 hours at 37 °C, 5% CO2, and 100% humidity. Apoptotic cells or necrotic cells were detected by flow cytometry using APC Annexin V Apoptosis Detection Kit .PC-3 cells expressing cell-cycle indicators were incubated with 4 cells) were seeded in ultra-low attachment 6-well cell culture plates (Corning #3471) or normal 6-well cell culture plates (Thermo Scientific #140675) in RPMI 1640 containing 10% (v/v) fetal bovine serum, and 48 hours later, Docetaxel or P-5 was added to the wells . Cell apoptosis/necrosis assay was performed 72 hours after the drug administration.PC-3 or DU145 cells (5 × 10Supplementary Information


APP), presenilin 1 (PSEN1), and presenilin 2 (PSEN2) are the frequent causes of AD. However, the clinical and genetic features of AD overlap with other neurodegenerative diseases. The present study aimed to identify the clinical and genetic characteristics in a Han Chinese AD cohort.Alzheimer's disease (AD) is the most common form of dementia characterized by memory loss at disease onset. The gene mutations in the amyloid precursor protein (APP), PSEN1, PSEN2, and microtubule‐associated protein tau (MAPT) genes were assessed in 83 sporadic AD patients by Sanger sequencing. A total of 25 probands from families with AD were subjected to next‐generation sequencing on 53 dementia‐associated genes to capture the target region, and Sanger sequencing was used to detect the variants in the DNA sequence.Detailed clinical assessment was applied to all the patients. We screened amyloid precursor protein (PSEN1 p.L226R was found in an early‐onset AD (EOAD) family characterized by language impairment at disease onset, a novel probably pathogenetic variant (p.D534H) was identified in a frontal‐temporal dementia gene, TANK‐binding kinase 1 (TBK1) with a typical AD phenotype in a late‐onset AD (LOAD) family, and a PSEN2p.H169N mutation and two benign MAPT (p.Q230R and p.V48L) mutations were detected in three EOAD patients.TBK1variant with a typical AD phenotype was detected. Also, the phenotypic characteristics of PSEN1 p.L226R, a PSEN2pathogenic mutation, and two likely benign MAPT variants were described. Hence, screening for mutations in other dementia genes could be further explored in clinically diagnosed AD patients.Thus, five variants were identified in a Han Chinese cohort. In the present study, a novel, probably damaging FTLD gene  Consequently, two known mutations, a PSEN1 p.L226R (chr14:73659480) mutation in an EOAD family and a PSEN2 p.H169N (chr1:227075798) in a LOAD family, a novel FTLD gene TANK‐binding kinase 1 (TBK1) p.D534H (chr12:64889341) variant with typical AD phenotype in a LOAD family, and two probably benign MAPTvariants p.Q230R and p.V48L (chr17:44060859 and chr17:44049233) in two sporadic EOAD patients, and some other insignificant variants were identified , which might easily lead to misdiagnosis . All patients were enrolled from the Department of Neurology of People's Hospital of Zhengzhou University, evaluated, and clinically diagnosed as AD according to the criteria of the National Institute of Neurological and Communicative Disorders and Stroke‐Alzheimer's Disease and Related Disorders Association , magnetic resonance angiography (MRA) and/or 18F‐fludeoxyglucose positron emission tomography (18F‐FDG‐PET), and computed tomographic angiography (CTA). The neuropsychological assessment such as clinical dementia rating (CDR), mini‐mental state examination (MMSE), and ADL was applied. All patients underwent laboratory evaluations such as blood cell count, the levels of thyroid hormone, erythrocyte sedimentation rate, C‐reactive protein, folic acid, and vitamin B12.2.3PSEN1, PSEN2, APP, and MAPT) in sporadic patients. Subsequently, these coding exons, splice sites, the immediately adjacent intron sequences, and high‐throughput sequencing data were analyzed in 53 genes associated with dementia in patients with a family history in order to identify the target region  under the following conditions: 94°C for 30 s, 55°C for 30 s, and 72°C for 1 min. Then, the PCR products of the proband and three other family members were purified and directly sequenced on an ABI3100 automated sequencer . The sequencing reads and lists were compared using Chromas software and NCBI blast. The single nucleotide polymorphisms were determined using the HGMD , NCBI dbSNP137 , Molgen , HapMap , 1,000 human genome data set , and ExAC Browser Beta . The pathogenicity of the novel single nucleotide mutation was predicted using PolyPhen‐2  and SIFT  along with the conservation score.Blood samples from all patients and 100 unrelated normal subjects were collected in 5 ml EDTA anticoagulant tubes after informed consent was obtained from each participant. Genomic DNA was extracted from peripheral blood leukocytes using standard procedures. The coding exons, splice sites, and the adjacent intron sequences were analyzed for four genes (33.1PSEN1 (c.677T>G p.L226R) mutation in an EOAD family  mutation in patient 2, with a LOAD family history  variation in a LOAD family  was a 70‐year‐old woman suffering with memory loss and disorientation for about 2 years. Her father had unclassified dementia at 70 years of age and died in his 80s, while her uncles died young , one novel heterozygous missense mutation in the FTLD gene with AD phenotype (TBK1 p.D534H), and two likely benign MAPT  variants in a Han Chinese AD cohort.Clinical and genetic crosstalk exists between AD and other neurodegenerative diseases, such as FTLD and PD . In the current study, the PSEN1 family demonstrated the clinical manifestation of language disability and altered personality at the beginning of the disease, which differed from the clinical manifestation of the initial memory loss in the AD. In addition, the PSEN1p.L226R mutation  variations, which might be benign polymorphisms. As described previously, MAPT gene consists of 16 exons and expresses six isoforms in the human brain by alternative mRNA splicing of exons 2, 3, 4A, 6, 8, and 10 (Goedert & Spillantini, MAPTwas detected in the family, and the patient's brother harboring the same mutation is currently 62 years old, with normal cognition, thereby suggesting that p.V48L is putatively benign.We found two rare TBK1 is a multifunctional kinase involved in the regulation of various cellular pathways, including immune response, inflammation, autophagy, cell proliferation, and insulin signaling (Freischmidt, Muller, Ludolph, Weishaupt, & Andersen, TBK1variants or functional deficits of TBK1 missense mutations cause a dominant form of ALS, FTD, ALS‐FTD (Freischmidt et al., TBK1p.D534H experienced memory loss and disorientation, which was in line with the diagnosis of AD. Thus, screening for mutations in TBK1might be advisable in clinically diagnosed AD patients (Van Mossevelde et al., TBK1p.D534H carrier is a LOAD patient with a late‐onset dementia family history, indicating the pathogenicity of the TBK1p.D534H variant. However, 75% of TBK1 mutation carriers developed the disease after the age of 62 years, while some remained unaffected at the age of >80 years (Van Mossevelde et al.,  TBK1p.D534H was pathogenic; however, the underlying mechanism is yet to be investigated.PSEN2and TBK1 mutations were not confirmed cosegregations in the families. Second, the study failed to demonstrate any changes in the biomarker in cerebrospinal fluid and during autopsy verification.Nevertheless, the present study had several limitations. First, the TBK1 variant with a typical AD phenotype was detected. Also, the phenotypic characteristics of PSEN1 p.L226R, which was not reported previously, a PSEN2 pathogenic mutation, and two likely benign MAPT variants were described. Hence, screening for mutations in other dementia genes might be advisable in clinically diagnosed AD patients.In summary, we identified five variants in a Han Chinese cohort. A novel, probably damaging FTLD gene The authors have no actual or potential conflicts of interest. Click here for additional data file. Click here for additional data file.


S)-protopanaxadiol (PPD) using a melting-solvent method with different polymers, in order to improve the solubility and dissolution performance of drugs with poor water solubility. The SDs were characterized via differential scanning calorimetry (DSC), powder X-ray diffraction (PXRD), Fourier transform infrared spectroscopy (FTIR), nuclear magnetic resonance (NMR), and molecular docking and dynamics study. DSC and PXRD results indicated that PPD crystallinity in SDs was significantly reduced, and that the majority of PPD is amorphous. No interaction was observed between PPD and polymers on FTIR and NMR spectra. Molecular docking and dynamic calculations indicated that the PPD molecule localized to the interpolated charged surface, rather than within the amorphous polymer chain network, which might help prevent PPD crystallization, consequently enhancing the PPD dispersion in polymers. An in vitro dissolution study revealed that the SDs considerably improved the PPD dissolution performance in distilled water containing 0.35% Tween-80 (T-80). Furthermore, among three PPD-SDs formulations, Poloxamer188 (F68) was the most effective in improving the PPD solubility and was even superior to the mixed polymers. Therefore, the SD prepared with F68 as a hydrophilic polymer carrier might be a promising strategy for improving solubility and in vitro dissolution performance. F68-based SD, containing PPD with a melting-solvent preparation method, can be used as a promising, nontoxic, quick-release, and effective intermediate for other pharmaceutical formulations, in order to achieve a more effective drug delivery.In this study, we prepared solid dispersions (SDs) of 20( Panax ginseng C. A. Meyer, has been used as a medicinal plant in China for thousands of years. In modern medicine, ginseng is generally used to treat inflammation, anxiety, depression, fatigue, diabetes, and other disorders [S)-protopanaxadiol    [14,15,1log P = 1.72). Researchers reported that this sapogenin exhibited low absolute bioavailability [However, PPD is a chemical biopharmaceutics classification system (BCS) Ⅱ drug with low solubility  , wherein one or more poorly soluble drugs are dispersed in hydrophilic polymers, is an effective method for improving the solubility and bioavailability of poorly soluble compounds ,31,32. HUp to now, few PPD formulations have been fully developed, among which only a few have improved water solubility, such as SD, emulsions, hydroxypropyl-β-cyclodextrin inclusion, and colloidal dispersions ,58,59,60p > 0.05, all against all SDs), respectively. The solubility of F1, F2, and F3 were 4.66 ± 0.93 μg/mL, 33.24 ± 2.04 μg/mL, and 14.33 ± 0.86 μg/mL, respectively. The solubility of the three formulations (F1–F3) significantly differed from each other (p < 0.05).The drug loading efficiency (DL) of the three SDs (F1–F3) were 8.17% ± 2.93%, 8.13% ± 2.56%, and 7.91% ± 0.74% (p < 0.05).The solubility measurement results exhibited different polymer effects on the drug dissolvability increase in water. The PPD water solubility in SDs clearly increased when compared with that of pure PPD at 37 °C (approximately 3 μg/mL ). F2 exhThe DSC thermograms of the drug, polymers, SDs, and Physical mixtures (PMs) are shown in In DSC analyses, PPD and polymer characteristic peaks were expected in the DSC thermograms of PMs. However, the DSC thermograms of PMs were quite similar to those of polymers, which can be explained by relating them to the SD formation process and the ratio of PPD and polymers in the formulation. As shown in DSC thermograms, polymer melting points (53 °C and 60 °C) were much lower than that of PPD (216 °C), during DSC analysis, wherein each sample was heated at 10 °C/min from 25 °C to 300 °C. Polymers were melted when the temperature reached 53 °C (F68) or 60 °C (PEG6000). This process was quite similar to SD formation, which indicated that melting PM actually existed in a state of SD. Moreover, large polymer amounts in PM (drug–polymer ratio = 1:10) had masked the existence of PPD DSC analysis, which might also lead to the disappearance of the endothermic peak of PPD in the PM thermograms ,62,63,64In PPD diffractograms, PPD exhibited high crystallinity, with characteristic crystalline peaks at 4.17°, 6.59°, 14.74°, and 15.68° A, which PXRD pattern results support the results of solubility measurements presented previously. Amorphous compounds have a less uniform molecular arrangement than crystalline compounds, and this disorder is the driving force for its greater solubility and faster dissolution rates ,66. Howe−1 (-C-O stretching). PEG6000 and F68 exhibited similar constitutional units (-CH2CH2O-); therefore, their FTIR spectra , PPD-F68 (F2), and PPD-PEG6000-F68 (F3) interactions at the molecular level. Optimized PPD and F68 conformations were initially produced from linear structures. F1 and F2 simulation results a,b revea−1, respectively. These negative values showed that PEG (or F68) could interact with PPD to prevent PPD crystal formation. This was consistent with the conclusions of the PXRD and DSC methods. The composite ratio of PPD and PEG6000 (or F68) can be considered as 2:1 according to the experimental data. Therefore, in the molecular docking protocol, two PPD and one PEG6000 (or F68) molecules were used. According to this composite ratio, the equilibrium constants of the PPD and F68 interaction should be greater than that between PPD and PEG6000. The computational result (docking-free energy of PPD-F68 in F2 was lower than that of PPD-PEG6000 in F1) was consistent with this estimation of their interaction. Therefore, we believe that an interaction between PEG6000 and F68 occurred . These interactions might lead to slight decrease in the PPD-PEG-F68 (F3) drug loading efficiency when compared with PPD-PEG6000 (F1) and PPD-F68 (F2), without any statistical difference (p > 0.05), as shown in the DL determination results. The docking-free energy of PPD-PEG6000 (F1) and PPD-F68 (F2) were −3.5 and −4.5 kcal·molThe molecular docking and dynamic study results suggested that the PPD molecule localized to the interpolated charged surface of PEG6000 (or F68), rather than within the amorphous polymer chain network. PEG6000 (or F68) might interact with PPD in order to prevent PPD crystal formation, which helped enhance the PPD dispersity in the polymers. The interaction between PEG6000 and F68 in F3 might lead to a lower drug loading efficiency. However, the effect was not obvious due to the low PEG6000 and F68 concentrations in F3.p < 0.05). Furthermore, the F2 dissolution rate significantly differed from that of F1 (p < 0.05).The in vitro dissolution behaviors of PPD, SDs, and PMs in the dissolution media under sink conditions are shown in The in vitro dissolution performance was measured to evaluate SD effects on promoting in vitro dissolution. The dissolution profile and statistical analyses revealed that F68 was a more suitable SD polymer than PEG6000 for the PPD-SDs formulations. SDs had a higher dissolution rate than PMs. The in vitro dissolution study results suggested that the F68 exerted superiorly favorable effects on the dissolution rate of PPD, whose effects might be related to the solubilization of these hydrophilic polymers. This might contribute to the development of new PPD agents and other poorly soluble drugs .S)-protopanaxadiol , PEG6000 , poloxamer 188 , and Tween®-80  were used in the study. The acetonitrile was of chromatographic grade and purchased from Merck Co. Ltd. Other solvents were of analytical grade.20(SD formulations were composed of PPD (purity > 98%), T-80, and polymers . The formulations are detailed in m/v/m). Subsequently, the solution was transferred into the melting matrix (polymer) in a 80 °C water bath and stirred for 15 min. The resultant was frozen at −20 °C for 24 h, porphyrized by an agate mortar and pestle, and stored in a desiccator at ambient temperature.SDs were prepared via a melting-solvent method at a mass ratio of 1:10 (drug:polymer). PPD was completely dissolved in the proper amount of absolute alcohol containing T-80  in a mortar until homogeneous. Resultant mixtures were sieved and then kept in a desiccator at room temperature until further study.PMs were prepared by the simple intensive mixing of PPD and PEG6000 and/or F68 at certain ratios  and eluted at a rate of 1.0 mL/min. The column temperature was maintained at 25 °C. The drug loading (DL) efficiency was measured as follows. After the prepared SD was completely dissolved in methanol, the supernatant was withdrawn and filtered through a membrane filter (0.45 μm). The amount of PPD in the resulting solution was quantitatively analyzed using HPLC. Each sample was measured in triplicate. Lastly, the DL was calculated using the following equation:The PPD content in the samples was determined through high-performance liquid chromatography (HPLC) using an Agilent 1200 system  equipped with a UV detector and it was detected at a wavelength of 203 nm. The separation was performed using a XB-C18 column . The mobile phase was composed of acetonitrile and deionized water , and each sample was heated at 10 °C/min from 25 °C to 300 °C.2θ) ranged from 3° to 40° at 1°/s.PXRD patterns were evaluated via PXRD (Bruker D8 Advance). Samples were exposed to Cu radiation under 40 mA and 40 kV. The scanning angle . Potassium bromide discs were prepared by mixing a small amount of the sample with potassium bromide and compressing the mixture. The frequency ranged from 4000 cm1H-NMR spectra [3) as the solvent.The  spectra  for SDs O-β-glucopyranosyl-20(S)-protopanaxadiol dehydrate obtained from Cambridge Structural Database (Refcode: JEPJEU) and reconstructed. PEG6000 and F68 molecular structures were constructed manually. The molecular dynamics program Amber 12  was used for energy minimization and molecular dynamics calculation. AmberTools in Amber 12 was used to prepare Amber starting stuffs. Partial atomic charges of PEG6000 or F68 units were calculated via Gaussian 09 package. The docking program AutoDock Vina 1.1.2  [PPD molecular structure was extracted from the single crystal structure of 20-CA, USA)  was usedFirstly, energy minimization protocol was executed to prepare PPD, PEG6000, and F68 models. Then, a 20 ns molecular dynamics simulation was run for PEG6000 and F68. Following this, the 10 highest scoring conformation with one or two PPD molecules separately docked to PEG6000 or F68 molecule were generated. Finally, one conformation with relatively low energy was used for the optimization of the successive structure.During the minimization protocol, all models were placed under vacuum. Optimized PEG6000 and F68 molecules were put into separated water boxes, which had a 10 Å width. Then, a 20 ns molecular dynamics simulation was performed. A non-bond cutoff distance of 18.5 Å, spline width of 1.0 Å, and buffer width of 0.5 Å were used. During the docking protocol, a Lamarckian Genetic Algorithm (LGA) combined with a grid-based energy evaluation method were used for pre-calculating grid maps according to the interatomic potentials of all of the atom types present in the host and guest molecules, including Lennard-Jones potentials for van der Waals interactions and Coulomb potentials for electrostatic interactions. A grid map of dimensions 80 Å × 80 Å × 80 Å, with a grid spacing of 0.375 Å, was used to cover the F68 or PEG6000 structure. Using AutoDockTools , the atomic partial charges were calculated using the Gasteiger-Marsili method . ParametThe water solubility was determined by a modified shake-flask method ,75. ExceThe in vitro dissolution studies of PPD, SDs, and PMs were performed using a Chinese Pharmacopoeia (2015) type III dissolution apparatus ,76,77. Tp < 0.05 was considered significant. Data analysis was performed with the SPSS software package .All data are presented as mean ± standard deviation or mean. The statistical significance of the differences was determined using ANOVA. In all tests, In this study, SDs were prepared with different polymers, and SD formations were characterized via different analytical methods. DSC and PXRD analyses results indicated that SD crystallinity reduced significantly, whereas the majority of PPD in SDs existed in an amorphous state. Furthermore, no interaction was observed between the PPD and polymers on FTIR and NMR spectra. Molecular docking and dynamic calculations indicated that the PPD molecule localized to the interpolated charged surface of PEG6000 (or F68) rather than within the amorphous polymer chain network, which might have helped prevent PPD crystal formation consequently enhancing the PPD dispersity in the polymers. An in vitro dissolution study revealed that, compared with pure PPD, SDs considerably improved the dissolution performance in distilled water (0.35% T-80). Furthermore, among three SD formulations, F68 was the most effective in improving the PPD solubility and was superior to F68 and PEG6000 (1:1).Consequently, this study revealed the potential of SDs to increase PPD solubility and dissolution performance. As a pharmaceutical intermediate, SDs can be further processed into tablets , capsule


HeLa cells were treated by different concentrations of cisplatin, bleomycin and ifosfamide alone and in various SMEDDS. The inhibitory effect on cell growth was analyzed by MTT cell viability assay. Inflammation is a driving force that accelerates cancer development. The inhibitory effect of these antitumor agents has also been tested on HeLa cells in the presence of inflammatory mediators  as an in vitro model of inflamed human cervix. Significant differences in the cytotoxicity of cytostatic drugs alone and in SMEDDS have been found in a concentration-dependent manner. The self-micro emulsifying system may potentiate the effectiveness of bleomycin, cisplatin and ifosfamide topically. The effect of SMEDDS containing antitumor agents was decreased significantly in the presence of inflammatory mediators. According to our experiments, the optimal SMEDDS formulation is 1:1:2:6:2 ratios of Isopropyl myristate, Capryol 90, Kolliphor RH 40, Cremophor RH40, Transcutol HP and Labrasol. It can be concluded that SMEDDS may increase the inhibitory effect of bleomycin, ifosfamide and cisplatin on human cervical cancer HeLa cells. Inflammation on HeLa cells hinders the effectiveness of SMEDDS containing antitumor agents. Our results might ensure useful data for development of optimal antitumor formulations.The aim of this study was to develop topical self-microemulsifying drug delivery systems (SMEDDS) containing antitumor agents  and to investigate their inhibitory potential in SMEDDS on human cervical cancer HeLa cells. The physicochemical properties of cytostatic drug loaded SMEDDS were characterized. The cytotoxicity of main components of SMEDDS was also investigated. Their IC HeLa cells are a reliable model of human cervix cancer cells [Uterine and cervix carcinoma is the first cause of gynecological cancer mortality in developing countries . Cervicaer cells .In the present study, our aim is to develop topical SMEDDS incorporating different antitumor agents  and to examine their inhibitory potential in SMEDDS in the presence of inflammatory mediators on human cervical cancer HeLa cells.Kolliphor RH 40 and Labrasol as surfactant, Capryol 90 or Lauroglycol FCC and Transcutol HP as co-surfactants were used in our experiments. Based on the results of the pseudoternary phase diagram, every composition formed microemulsion. The maximum microemulsion existing zones observed are shown in et al. method [Drug encapsulation efficiency was evaluated by Q. Li . method . The encn = 5.Evaluated droplet size of SMEDDS in water via DLS measurement. Evaluated average droplet sizes: composition 1: 137.81 ± 1.25 nm, composition 2: 68.44 ± 1.05 nm, composition 3:73.28 ± 0.95 nm, composition 4: 129.31 ± 1.95 nm, composition 5: 149.35 ± 1.00 nm, composition 6: 76.90 ± 1.00 nm. Every sample was found to be a monodisperse system. . The zet50: 0.23 ± 0.025 v/v %), Transcutol HP was less toxic (IC50: 3.42 ± 0.035 v/v %), and Kolliphor RH 40 was responsible for the lowest cell viability reduction (IC50: 5.12 ± 0.085 v/v %). However, there are significant differences in the cytotoxic properties of surfactants in a concentration-dependent manner, the IC50 values of each examined surfactant is much higher than the applied concentration range in SMEDDS compositions  dichloride), Bleomycin sulfate, Ifosfamide  tetrahydro-2H-1,3,2-oxazaphosphorin-2-amine-2-oxide) were obtained from FLUKA Analytical Ltd. . Labrasol, Capryol 90, Lauroglycol FCC and Transcutol HP were kind gifts from Gattefossé, Lyon, France. Kolliphor RH 40 were obtained from BASF, Ludwigshafen, Germany. 3--2,5-diphenyltetrazolium bromide (MTT), Dulbecco’s Modified Eagle’s Medium (DMEM), Hank’s Balanced Salt Solution (HBSS), phosphate buffered saline (PBS), Trypsin-EDTA, Heat-inactivated fetal bovine serum (FBS), l-glutamine, non-essential amino acids solution, penicillin-streptomycin were purchased from Sigma-Aldrich . 96 well cell plates and culturing flasks were obtained from Corning, New York . IL-1-β, TNF-α have been purchased from Sigma-Aldrich. Antitumor agents as Cisplatin . The cumulant Dynamic Light Scattering (DLS) method was used for determination of droplet size of formulated emulsions. To obtain the diffusion coefficient the intensity correlation function has been analyzed. The measurements have been performed by Brookhaven Photometer . During the operation temperature was 25 °C, the laser detection angle was adjusted to 90°, Lambda (λ) to 533 nm, index to 1.334 by Particle Sizing Program 3.1 . Diameters of dispersed droplets according to the diffusion coefficient have been evaluated automatically by the computer program . To eval3, 10% (v/v) heat-inactivated fetal bovine serum (FBS), 1% (v/v) non-essential amino acids solution, 1% (v/v) l-glutamine, 100 IU/mL penicillin, and 100 IU/mL streptomycin at 37 °C in an atmosphere of 5% CO2. The cells were routinely maintained by regular passaging. For cytotoxic and transport experiments, cells were used between passage numbers 20 and 40. The culture media was replaced with fresh media in every 72 h [HeLa  was obtained from the European Collection of Cell Cultures . Cells were grown in plastic cell culture flasks in Dulbecco’s Modified Eagle’s Medium , supplemented with 3.7 g/L NaHCOery 72 h .4 cells/well and allowed to grow in a CO2 incubator at 37 °C for 4 days. For these studies, the culture medium was removed, surfactant or SMEDDS solutions were added, and the cells were incubated for a further 30 min. After removing the samples, another 3-h-incubation in a medium containing MTT at the concentration of 0.5 mg/mL followed. The dark blue formazan crystals were dissolved in acidic isopropanol (isopropanol: 1.0 N hydrochloric acid = 25:1). The absorbance was measured at 570 nm against a 690 nm reference with FLUOstar OPTIMA Microplate Reader . Cell viability was expressed as the percentage of the untreated control [To exclude any toxic effect of the blank SMEDDS and their components on HeLa cells, MTT cell viability test was used . Cells w control .4 cells per well in 100 mL of medium, and incubated for 3–4 h. Cytostatic drugs alone in cell culture medium or incorporating in SMEDDS were prepared immediately before use and added in a volume of 50 μL and total volume of 200 μL (with 150 mL fresh medium supplement) per well at final concentrations of cytostatic drugs between 5 × 10−4 and 5 mg/mL. After 72 h the samples were removed and 100 mL of freshly diluted MTT solution at a concentration of 0.5 mg/mL, was pipetted into each well and the plate was incubated for 3 h at 37 °C in a humidified, 5% CO2 atmosphere. After a specific period, cell viability was evaluated by measurement of the absorbance at 520 nm, using a FLUOstar OPTIMA Microplate Reader (BMG LABTECH). All experiments were made in quadruplicate. Standard deviations were ≤10%.HeLa proliferation were also evaluated by using MTT cell viability assay. Cells were plated in 96-well sterile plates, at a density of 104 cells per well in 100 mL of medium, and incubated with 1.25 μL IL-1-β (0.1 μg/μL) and 3.75 μL TNF-α (0.1 μg/μL) for 3–4 h. After ignition, the previously described HeLa cell proliferation test was used.The inhibitory effect of different SMEDDS containing cytostatic drugs was evaluated in the presence of inflammatory mediators on human cervical cancer HeLa cells. HeLa cells were plated in 96-well sterile plates, at a density of 10p < 0.05. All experiments were carried out in triplicates and repeated at least three times.Data were analyzed using SigmaStat  and presented as means ± SD. Comparison of groups was performed by one-way ANOVA. This ANOVA was used to compare the differences of each values belong to certain concentrations in MTT. We marked the significant differences with asterisks in figures. After that, the results among the groups were presented by Tukey’s test. Differences were regarded as significant in case of It can be concluded that topically administered self-microemulsifying drug delivery systems containing different antitumor agents may provide an alternative treatment for cervical cancer. Inflammatory mediators can hinder the efficacy of therapy. Our results might provide useful data for development of optimal antitumor formulations.


Midazolam is known as a safe drug and is widely used as a sedative and an anesthetic adjuvant. Therefore, there is a lack of awareness that midazolam can cause anaphylaxis. Midazolam anaphylaxis is rare, and only a few cases have been reported, but such a risk is always present. In this study, we report a case of midazolam anaphylaxis by an intravenous injection, in the prone position, during general anesthesia.A 62-year-old woman was intravenously administered 1 mg midazolam during general anesthesia, and sudden severe hypotension, bronchospasm, decreased oxygen saturation, erythema, and diarrhea occurred.Midazolam anaphylaxis was presumptively diagnosed by clinical symptoms and was confirmed by an intradermal test after 9 weeks.The patient was treated with 100% oxygen, large volume of fluid, epinephrine, phenylephrine, ephedrine, dexamethasone and prednisolone, ranitidine, and flumazenil.Severe hypotension and decreased oxygen saturation were resolved within 20 minutes of the onset of anaphylaxis, and the patient was discharged after 3 days without any sequelae.Midazolam anaphylaxis is very rare, but it can happen always. Therefore, the possibility of anaphylaxis due to midazolam should be considered and always be prepared for treatment. Midazolam is known as a safe drug and is widely used as an anxiolytic, sedative, and adjuvant to general anesthetics in patients with drug allergy. Therefore, there is a lack of awareness that midazolam can cause anaphylaxis. Midazolam anaphylaxis is rare, and only a few cases have been reported.–9 But such a risk is always present. In this study, we report a case of anaphylactic shock caused by 1 mg midazolam that was intravenously administered as an adjuvant during general anesthesia.Anaphylaxis is an acute, potentially life threatening, systemic hypersensitivity reaction that occurs when a patient is re-exposed to a previously sensitized antigen.2A 62-year-old woman, weight 46 kg, height 153 cm, was scheduled for endoscopic discectomy due to the diagnosis of right L5S1 lateral stenosis.Her medical history showed that she underwent myomectomy 20 years ago and had taken alprazolam and diazepam per os for 2 months and underwent a colon polypectomy with midazolam 3 mg intravenous (IV) 2 months ago. She had no history of a drug or food allergy. The preoperative examination revealed no specific findings and the intradermal test for the antibiotic cefotetan injection was negative.2) 98%, Bispectral index 99, and body temperature 36.5 °C were recorded. Lidocaine 40 mg and 1% propofol 80 mg IV were administered to induce anesthesia, and tracheal intubation was performed after muscle relaxation with rocuronium 40 mg IV. Sevoflurane 1 to 2 vol%:O2:air was administered for maintenance of anesthesia. Artificial ventilation was maintained at a tidal volume of 400 mL, respiratory rate 11 breaths/min, and end tidal CO2 (ETCO2) 28 to 30 mmHg. Remifentanil 0.5 to 1 ng/mL was continuously infused using target-controlled infusion pump from the anesthetic induction. After the anesthetic induction, BP 145/65 mmHg and HR 80 beats/min were noted.On arrival to the operating room, electrocardiogram was normal and blood pressure (BP) 135/71 mmHg, heart rate (HR) 78 beats/min, peripheral oxygen saturation . Subsequently, we decided to inject midazolam 1 mg IV to prevent awareness of the patient without decrease of BP during general anesthesia.The patient was positioned in the prone position for surgery, and the vertebra level of the surgical site was checked with a C-arm x-ray. While simultaneously administering sevoflurane 2 vol% and remifentanil 0.5 ng/mL, BP 125/68 mmHg, HR 55 beats/min, and ETCO2 96%, and ETCO2 15 mmHg had declined and capnography showed an obstructive pattern. Wheezing sound in both lungs was noted on auscultation and some small erythema appeared on the back. Sevoflurane and remifentanil were discontinued and 100% O2 and ephedrine 10 mg IV were administered twice. However, BP 53/35 mmHg, HR 74 beats/min, and SpO2 91% were noted. SpO2 further reduced to 89% and BP was not detected. Ringer lactate solution at a high rate, 2 times of epinephrine 100 μg IV, and phenylephrine 100 μg IV were administered twice. BP 58/38 mmHg, HR 98 beats/min, and SpO2 92% were noted.One minute after administering midazolam 1 mg IV, Bispectral index decreased from 68 to 54. At the same time, BP 41/30 mmHg, HR 46 beats/min, SpO2 94%, and ETCO2 25 mmHg were noted, and phenylephrine 100 μg IV was administered. Vital signs improved to BP 86/61 mmHg, HR 110 beats/min, and SpO2 98% after 20 minutes of the anaphylactic shock. We then explained to the family the possibility of side effects due to midazolam and decided to postpone the operation. Glycopyrrolate 0.2 mg IV and pyridostigmine 10 mg IV were administered to reverse muscle relaxation. The wheezing sound disappeared and self-respiration of the patient gradually recovered. We performed tracheal extubation after 55 minutes of anesthesia induction. BP 121/60 mmHg, HR 110 beats/min, and SpO2 98% were noted at the time of extubation.The patient was changed to supine position, followed by Trendelenburg position. Based on the suspicion of anaphylaxis due to midazolam, we injected flumazenil 0.25 mg, dexamethasone 5 mg, methylprednisolone 125 mg, and ranitidine 100 mg intravenously. However, BP 73/48 mmHg, HR 104 beats/min, SpO2 99% were noted. The patient complained of diarrhea. After 15 minutes of arrival at the recovery room, BP and HR decreased again to 78/45 mmHg and 85 beats/min, respectively, and phenylephrine 100 μg IV was injected. On evaluation, BP was 88–92/48–50 mmHg and HR was 48 to 50 beats/min. Arterial blood gas analysis was performed, but there were no specific findings. After 80 minutes in the recovery room, BP, HR, and SpO2 were maintained at 92–100/51–52 mmHg, 51 beats/min, and 99%, respectively. The skin erythema disappeared, and the patient was transferred to the intensive care unit after 2 hours in the recovery room. The total amount of injected Ringer lactate solution was 1800 mL. Examinations for serum β-tryptase and serum immunoglobulin E were not performed.In the recovery room, oxygen 5 L/min was supplied and the patient's legs were elevated, and BP 90 to 100/53 to 55 mmHg, HR 85 to 100 beats/min, and SpOIn the intensive care unit, BP was maintained at 80/50 mmHg and HR at 60 beats/min. After 1 hour , the vital signs were recorded as BP 126/66 mmHg and HR 60 beats/min. The body temperature rose from 36.9 to 37.6 °C and was normalized after supportive care for 2 days. We examined Troponin-T, creatine kinase-MB isoenzyme, and echocardiogram to rule out the cardiovascular problem, which was normal.The day after the anaphylactic shock, the patient was transferred to the general ward. The main complaint, back pain, was treated with medication and physical therapy. After 2 days, the patient and her family were informed again about midazolam anaphylaxis and the patient was discharged without any sequelae. After 9 weeks of anaphylaxis, the patient showed a positive reaction for midazolam in the intradermal test.The study was approved by the Institutional Review Board of our University Hospital. Written informed consent was obtained from the patient for the publication of this case report.3 so proper diagnosis and treatment by the physician is very important.Anaphylaxis causes a very fast life-threatening condition and can involve multiple organs, In this case, the vena cava compression due to the prone position reduced the venous return and induced more severe hypotension. Therefore, it was recommended to change to a Trendelenburg position or supine position with leg elevation. The patient also developed fever on the day of anaphylaxis. This was due to the inflammatory reaction by newly formed proinflammatory phospholipid-derived mediators such as prostaglandin D2, leukotrienes, thromboxane A2, and platelet activating factor.Severe hypotension after the onset of anaphylaxis is due to vasodilation and increased vascular permeability by preformed mediators such as histamine, neutral protease , and proteoglycans (heparin) released from mast cells or basophils. Due to increased vascular permeability, 35% of the intravascular volume shifts to the interstitial space.2 agonist action, including bronchial dilatation, gastrointestinal smooth muscle relaxation, inhibition of further mediator release from mast cell and basophil, and inotropic/chronotropic effects.Treatments for anaphylactic reaction are administration of 100% oxygen, large volume of fluid, epinephrine, corticosteroid, and antihistamine. Epinephrine should be administered as early as possible and carefully. Epinephrine, in addition to vasoconstriction, has β2 decrease, abrupt drop in ETCO2, obstructive pattern on capnography, and erythema. Many drugs have been administered at various stages of anesthetic induction, but only midazolam was injected immediately before the anaphylactic response. We clinically suspected midazolam as the causative agent because anaphylactic signs appeared after 1 minute of IV injection of midazolam. An acute serum tryptase test within 2 hours after the onset of anaphylaxis was recommended. However, it is difficult to sample acute serum tryptase due to insufficient equipment or other practical limitations in urgent clinical situations. We also did not perform the test and it is a limitation of this case. Buka et al reported that, using a cutoff of 12.4 ng/mL (75th centile), sensitivity, specificity, positive predictive value, and negative predictive value of acute serum tryptase were 28%, 88%, 93%, and 17%, respectively. It means acute serum tryptase is a poor indicator for anaphylaxis. Nonetheless, acute serum tryptase is useful in clinical circumstances to distinguish from its mimics. In the cause analysis with the surgeon, they suspected deep anesthesia depth, heart disease, and other causes rather than midazolam anaphylaxis. We confirmed that there was a clear relationship between midazolam and anaphylaxis through skin test  performed after 9 weeks.The recognition of anaphylaxis during anesthesia is usually confusing because hypotension and bronchospasm can develop due to other reasons and because many drugs, including muscle relaxants and propofol, are administered during general anesthesia. In this case, the chief clinical factors of diagnosing anaphylaxis were sudden severe hypotension, bradycardia, SpOThis case clearly shows that midazolam is one of the causes of anaphylaxis.In conclusion, midazolam anaphylaxis is very rare, but it can happen always. Therefore, the possibility of anaphylaxis due to midazolam should be considered and always be prepared for treatment.Conceptualization: Yeon Su Jeon.Investigation: JinWoo Shim, Eun Hwa Jun, Seung Tae Choi.Writing – original draft: Yeon Su Jeon.Writing – review & editing: Hong Soo Jung.Yeon Su Jeon orcid: 0000-0002-2607-3107.JinWoo Shim: orcid: 0000-0002-9869-2812.Eun Hwa Jun orcid: 0000-0003-1342-0576.Seung Tae Choi orcid: 0000-0003-2041-5580.Hong Soo Jung orcid: 0000-0002-6812-1955.


Chitosan oligosaccharides (COS) display various biological activities. In this study, we aimed to explore the preventive effects of COS on glucolipid metabolism disorder using palmitic acid (PA)-induced HepG2 cells and high-fat diet (HFD)-fed C57BL/6J mice as experimental models in vitro and in vivo, respectively. The results showed that COS pretreatment for 12 h significantly ameliorated lipid accumulation in HepG2 cells exposed to PA for 24 h, accompanied by a reversing of the upregulated mRNA expression of proinflammatory cytokines  and glucolipid metabolism-related regulators . In addition, COS treatment alleviated glucolipid metabolism disorder in mice fed with HFD for five months, including reduction in body weight and fasting glucose, restoration of intraperitoneal glucose tolerance, and suppression of overexpression of proinflammatory cytokines and glucolipid metabolism-related regulators. Furthermore, our study found that COS pretreatment significantly reversed the downregulation of PPARγ at transcriptional and translational levels in both PA-induced HepG2 cells and liver tissues of HFD-fed mice. In summary, the study suggests that COS can improve glucolipid metabolism disorder by suppressing inflammation and upregulating PPARγ expression. This indicates a novel application of COS in preventing and treating glucolipid metabolism-related diseases. Data shows that over 30% of adults and 16.9% of children in the US are obese or overweight, and the figures are 14.3% and 12.8% in China, respectively . Thus, aFirmicutes and Lactobacillus spp. in the caecum and colon but increase the population of Bifidobacteria in caecum of pigs [N-acetylglucosamine, which have shorter chain length and lower molecular weights [Chitosan oligosaccharides (COS) are a kind of multifunctional oligosaccharides. COS are produced from chitosan, a polysaccharide constituent of crustaceans such as shrimps, crabs, lobsters, and prawns, by enzymatic degradation or acidic hydrolysis . COS are of pigs , indicat weights . Compare weights . Based oSo far, several functional polysaccharides have been proven to reverse glucose and lipid metabolism disorders by upregulating the activity of nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ) ,16,17. PThe purpose of this study was to determine whether COS administrated in advance could alleviate obesity-associated liver lipid metabolic disorder, both in vitro and in vivo, and if so, to determine the mechanism involved. Because of the critical role of PPARγ in the regulation of lipid metabolism, we specifically examined the effect of COS on PPARγ in palmitic acid (PA)-induced HepG2 cells and high-fat diet (HFD)-induced mice.p < 0.01, vs. the control group). On the other hand, the incubation of COS (100 μg/mL) for 12 h or pretreatment with 25, 50, and 100 μg/mL COS for 12 h and then exposure to 100 μM of PA for 24 h both caused no toxicity to HepG2 cells -2,5-diphenyltetrazolium bromide (MTT) assay. As shown in G2 cells B. TherefTo investigate whether COS could alleviate PA-induced lipid accumulation, HepG2 cells were pretreated with COS (25–100 μg/mL) for 12 h and then exposed to PA (100 μM) for 24 h. The results by oil red O staining showed that PA induced a considerable lipid accumulation in HepG2 cells, which was suppressed by COS pretreatment in a concentration-dependent manner .Considering that obesity is associated with low-grade chronic inflammation , we nextp < 0.01, vs. the control group) in HepG2 cells. In contrast, the PA-induced inflammation was remarkably suppressed by COS (100 μg/mL) pretreatment for 12 h .As shown in p < 0.01, vs. the control group) in HepG2 cells, which were evidently inhibited by COS (100 μg/mL) pretreatment for 12 h  for 24 h significantly increased the mRNA levels of fatty acid synthesis-related regulators  and glucogenesis-associated regulator PCK1-α . However, after COS pretreatment (100 μg/mL) for 12 h, the decreased expressions of PPARγ were statistically reversed in PA-induced HepG2 cells.To explore the potential molecular mechanism by which COS prevent PA-induced inflammation and glucolipid metabolism disorder, we measured the expression changes of PPARγ in PA-induced HepG2 cells, which plays a key role in the regulation of lipid metabolism. The results in p < 0.05, vs. the HFD group)  (p < 0.01), while COS treatment obviously reduced the AUC in HFD-fed mice, which was almost comparable to the CD group , HFD, CD plus COS (1 mg/mL in drinking water), or HFD plus COS for five months. The results showed that COS significantly lowered HFD-induced increase in body weight (D group) A and fasD group) B. In addD group) C. FurtheCD group D.To explore the effect of COS on hepatic steatosis, the lipid accumulation in all groups was measured using oil red O staining. As indicated in p < 0.05 or 0.01, vs. the CD group), which was reversed by COS treatment . Likewise, COS treatment led to evident transcriptional inhibition of glucolipid metabolism-related regulators  in liver tissues of HFD-fed mice (p < 0.01)  C.p < 0.01). However, COS treatment obviously reversed the reduction of PPARγ expression . COS were prepared in our laboratory . PA was 2. After reaching subconfluence, cells were incubated with 0, 25, 50, 100 μg/mL COS for 12 h and then exposed to 100 μM of PA diluted in culture medium for 24 h at 37 °C for further assay.HepG2 cells were maintained in MEM containing 10% FBS and 1% NEAA, 1% penicillin–streptomycin at 37 °C in humidified atmosphere with 5% CO4 cells/well in 96-well plates for 24 h. After treatment with PA at concentrations of 100 μM, 200 μM, and 400 μM or COS (100 μg/mL) for 24 h, the toxic effects of PA or COS alone on cell viability were determined. To assess the effect of COS plus PA on cell viability, the HepG2 cells were separately pretreated with 25, 50, and 100 μg/mL COS for 12 h and then exposed to 100 μM of PA for 24 h. After that, the cells were washed with PBS and incubated with MTT (5 mg/mL) in culture medium for 3 h at 37 °C. Next, the medium was discarded, and the formazan blue, which formed inside the cells, was dissolved using 100 μL dimethyl sulfoxide (DMSO). The optical density at 490 nm was determined with a Sunrise Remote Microplate Reader . The experiments were repeated 3 times independently, and 5 replicates were involved in each sample. The cell viability of each well was presented as the percentage of control level.HepG2 cells were plated at density of 1 × 10n = 5): CD group, HFD group, CD + COS  group, and HFD + COS group. The HFD was composed of 60% basic feed, 10% lard, 10% egg yolk powder, 2.5% cholesterol, 0.5% bile salts, 5% sucrose, 5% peanut, 5% milk powder, and 2% salt. Both diets used in this study were bought from Aoke Xieli Co., Ltd. . After the treatment for five months, the body weight and fasting glucose level of each mouse were detected. Then the mice were sacrificed, and liver tissues were collected. All samples were stored at −80 °C for further experiments.C57BL/6J wild-type male mice were obtained from Vital River Laboratory Animal Technology Co., Ltd. (Beijing China) and kept in a room with a 12 h light/dark cycle, a temperature of 22 ± 2 °C, and a relative humidity of 50 ± 5% during the whole experiment period. Mice were fed with a control diet for two weeks for adaptation. Then, all mice were randomly divided into four groups  and in accordance with the guidelines of the National Act on Use of Experimental Animals (China).n = 5) were fasted for 12 h and then given an intraperitoneal (i.p.) injection of glucose at a dose of 500 mg/kg body weight. Blood was collected from the tail vein at 0, 15, 30, 60, and 120 min, and the glucose levels were determined using a blood glucometer .An IGTT was conducted after the COS and HFD treatment for five months. Before the test, mice of each group (n = 5) were fixed overnight in 4% paraformaldehyde, embedded in paraffin, and made into 4 μm sections. After the deparaffinization and rehydration, the sections were stained with haematoxylin–eosin (HE) for 1 min and then with 0.5% oil red O solution for 30 min at room temperature. The visualized red oil droplets staining in the slides were observed using a Leica DMI4000 B light microscope .Liver tissues of each group  following the manufacturer’s instructions. Isolated RNA (1 µg) was reverse transcribed into cDNA using a HifiScript cDNA synthesis kit . For qRT-PCR, the reaction was performed using a 7500 Fast Real-Time PCR System  with the thermal cycle condition as follows: 95 °C for 2 min; 40 cycles of amplification . The experiments were repeated 3 times independently, and 3 replicates were involved in each sample in vitro. Five replicates were involved in each group in vivo. The primer sequences used in this study are listed in g for 15 min at 4 °C, and the supernatant was collected. The protein concentration was determined using a bicinchoninic acid . The lysate samples (40 µg/lane) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane. The membranes were blocked with 5% fat-free milk in Tris-buffered saline with 0.1% Tween-20 (TBST) buffer  for 1 h at room temperature and then incubated with primary antibodies against PPARγ overnight at 4 °C. Next, the membranes were incubated with HRP-conjugated secondary antibodies for 1 h. The protein bands were captured by enhanced chemiluminescence (ECL) , and the densitometry analysis was performed using an Image J2x software . The protein level of PPARγ of each sample was measured 3 times independently in vitro, and 5 samples expressing PPARγ protein of each group in vivo were assessed.HepG2 cells and liver tissues were homogenized using radio-immunoprecipitation assay (RIPA) buffer  supplemented with protease inhibitor cocktail . The sample homogenate was centrifuged at 15,000× p < 0.05 was regarded as statistically significant.Statistical analysis was carried out using SPSS 10.0 package . Data are presented as means ± SD. Differences between groups were assessed with one-way analysis variance (ANOVA), along with the Tukey–Kramer test.


Fgf8 morphogenetic signaling in establishing the molecular thalamic protomap, which was revealed by Igsf21, Pde10a and Btbd3 gene expression in the thalamic mantle layer. Fgf8 signaling activity was evidenced by pERK expression in radial glia cells and fibers, which may represent a scaffold that translates neuroepithelial positional information to the mantle layer. In this work, we describe the fact that Fgf8-hypomorphic mice did not express pERK in radial glia cells and fibers and presented disorganized thalamic regionalization, increasing neuronal death in the ventro-lateral thalamus and strong disruption of thalamocortical projections. In conclusion, Fgf8 encodes the positional information required for thalamic nuclear regionalization and the development of thalamocortical projections.Thalamic neurons are distributed between different nuclear groups of the thalamic multinuclear complex; they develop topologically ordered specific projections that convey information on voluntary motor programs and sensory modalities to functional areas in the cerebral cortex. Since thalamic neurons present a homogeneous morphology, their functional specificity is derived from their afferent and efferent connectivity. Adequate development of thalamic afferent and efferent connections depends on guide signals that bind receptors in nuclear neuropils and axonal growth cones, respectively. These are finally regulated by regionalization processes in the thalamic neurons, codifying topological information. In this work, we studied the role of The online version of this article (10.1007/s00429-018-1794-y) contains supplementary material, which is available to authorized users. Thalamic projection to specific areas of the cortex (Cx) is fundamental in the development of conscious sensory and motor functions, and thalamic neurons projecting to the Cx present homogeneous morphology Jones . NeverthNetrin1 (Ntn1), Slit 1, Robo, Sema3A and EphrinA5 in the ventral telencephalon (VTel) act as molecular signals for thalamic axon navigation, to innervate the Cx ; Phosphodiesterase 10A (Pde10a) and BTB (POZ) domain containing 3 (Btbd3). These genes exhibit a complementary expression pattern in the TML at E13.5 and E14.5, when the TML is still a structurally homogeneous mass of neurons, and TC pioneer axons are reaching the Cx. Next, we explored whether TML regionalization is regulated by signals that specify the neuroepithelial map. To this end, since the Fibroblast growth factor 8 (Fgf8) gene is expressed in the dorsal diencephalon  and the Fgf8 signal is involved in specifying thalamic progenitors (Kataoka and Shimogori Fgf8 hypomorphic mice (Fgf8null/neo), which express very low levels of Fgf8 in the Di , diencephalic neuroepithelial regions are specified by the expression of genes coding for morphogenetic signals and molecular regulators of cellular identities  and European Union laws on the protection of animals used in experimentation (Council Directive 86/609/EEC) and approved by the UMH-CEIE committee.neo/+ and Fgf8null/+ mice were bred to produce Fgf8null/neo mutants (C57BL/6 genetic background). PCR genotyping was performed as described by  at 4 °C.®. All images were taken using a Leica stereoscope (Leica MZ16FA) and digital cameras . The contrast and brightness of the photomicrographs were adjusted in Adobe PhotoShop, Macintosh or PC version, CS3 . Digoxigenin-11-UTP labelled RNA probes were prepared from full-length cDNA clones provided by Source BioScience imaGenes: Igsf21 (NM_198610.2) 32-1990 pb, Pde10a (NM_011866.2) 402-3324 pb; Btbd3 (NM_145534.2) 30-2439 pb. The Ntn1 probe was provided by O. Reiner, and Slit1 and Slit2 probes by O. Marín.The mouse brains were paraffin embedded  and sectioned in 7 µm-thick sagittal and transverse sections. The sections were washed with phosphate-buffered saline -0.1% Tween-20  and incubated in hybridization solution: deionized formamide 50%, standard SALT buffer 1X pH 7.5, Dextran sulfate 10X (Sigma-Aldrich), Denhart 1X (Sigma-Aldrich), yeast tRNA 2 mg/ml (Sigma-Aldrich) and water free of RNAase and DNAase (Sigma-Aldrich), for 1 h at 67 °C. Thereafter, the sections were hybridized overnight at 67 °C in hybridization solution containing 4 µl/ml riboprobe. Next, they were incubated overnight at 4 °C with alkaline phosphatase-coupled anti-digoxigenin antiserum ; followed by NBT/BCIP (nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate) solution as a chromogenic substrate to obtain blue labelling . The sections were dehydrated and covered with Eukitt2O2. For anti-dpERK immunohistochemistry , the brains were embedded in 4% agarose in PBS, and 70 µm sections were cut in coronal planes using a Leica vibratome (VT1000S). For these sections, the immunolabelling was revealed with 0.05% DAB, 0.025% ammonium nickel sulfate hexahydrate and 0.03% H2O2 in PBS.Paraffin sections were treated with 0.3% hydrogen peroxide in PBS + 0.3% Triton (PBT) for 15 min to inactivate endogenous peroxidase activity, blocked in PBT and 3% BSA and incubated with anti-DCC antibody . Then they were incubated with secondary biotinylated antiserum for 2 h at room temperature , washed with PBT and incubated in avidin–biotin complex  for 1 h at RT. The immunolabeling was revealed by 0.05% diaminobenzidine  in 0.05 M Tris buffer (pH 7.6), containing 0.03% HFluorescent immunohistochemistry: paraffin sections were incubated overnight with rabbit anti-phospho histone 3 , rabbit anti-Caspase 3 , rabbit anti-dpERK  and mouse anti-GFAP . After this, sections were incubated with anti-rabbit Alexa Fluor 488 (Molecular Probes #21206) and anti-mouse biotinylated antibody , followed by Cy3-streptavidin  and then counterstained with DAPI  a fluorescent nuclear dye, diluted in PBS at 0.001% and incubated for 10 min at RT.null/neo mutants paraffin sections were immunostained with anti-phospho histone 3 and anti-Caspase 3, as described above. Then, the number of positive cells for the corresponding antibody was quantified by counting positive cells per unit area (mitotic index). This unit area was a box of 10500 µm2 placed on the neuroepithelium from the epithalamus to the thalamus, following a previously described protocol , after which the medium was changed to Neurobasal/B-27 .E13.5 organotypic cultures of embryonic mouse brains .For axonal tracing, small DiI crystals  were inserted into the Th of hemi-dissected brains. These embryonic brains or cultured slices were incubated for 7 days, or 2 days, respectively, in 4% PFA at 37 °C. The brains were cut using a Leica vibratome (VT1000S) into 80–100 µm sections and counterstained with DAPI.http://www.eurexpress.org and http://www.brain-map.org). As a result, 90 genes were identified with expression in the TML at E13.5-14.5, but only 12 genes showed clear regional expression: BC055811, BC062109, Btbd3, EG628779, Elmo1, Igsf2, Pde10a, Rapgef3, Slc6a7, Slitrk6, Srgap2 and Trim9. From these, we selected three showing clear heterogeneous complementary expression in the TML: Igsf21, Pde10a and Btbd3  would develop , Igsf21 transcripts were reduced in Po and DL nuclei . Moreover, upregulation of this gene was detected in scattered cells of the lateral ventral nuclei, VPM/L  and downregulated in the VPM/L  was clearly smaller, there was no Igsf21 expression in the anterior thalamic pole  and mutant (n = 4) embryos at E17.5 was analyzed in transversal sections of the anterior telencephalon  than in later developmental stages  showed a significantly higher mitotic index (number of positive cells per unit area) in Th and Eth than mutants , respectively , both in the ventricular epithelium and TML fibers . These results suggest that Fgf8 morphogenetic activity in the ventricular epithelium is translated to the TML by the expression of pERK in radial glia fibers, which seems to be required for cell survival and appropriate nuclear patterning.To evidence a reduction in the ers Fig. k, l. To ers Fig. m–r. Moreers Fig. , we deciTML Fig. h–l. The Fgf8 signal regulates pERK expression in the thalamic radial glia, we performed “open book” E14.5 neuroepithelial explants of the WT , the pERK expression was strongly reduced in the thalamic and ZLI radial gland near the bead  implants. Increased cell death was induced on the experimental side around the DKK bead  significantly alter this tract  examined showed similar projection patterns. In contrast, DiI injections in the Fgf8null/neo embryos resulted in the derailment of thalamic axons in mutant mice (14/20). These axons followed rostrally towards the anterior forebrain through the PHy, but instead of making a dorsal turn, they continued on an ectopic rostral projection (ep) towards the anterior hypothalamus, into the preoptic area , some TC axons did turn dorsally and enter the pallium.The TC is also a marker for exploring thalamic regionalization, since mutations in thalamic genes . At E14.5 in the Di and anterior prosencephalon, Ntn1 expression was detected in the periventricular TML, with a gradient decreasing towards the lateral surface, both in control  and mutant animals . This may be the signal that induces axonal growth of the thalamic peduncle. However, the expression of Ntn1 in the POA and alar hypothalamic ventricular epithelium was downregulated in Fgf8null/neo mutants . DCC was detected in the PTh , with less intensity in the mutant mouse . At E18.5, DCC-positive axons were identified in the internal capsule ; however, in the Fgf8null/neo mouse, DCC-positive axons followed rostrally along an aberrant pathway into the lateral POA . In the control telencephalon, Ntn1 was also detected in the ventricular epithelium and mantle layer of the bed nucleus of stria terminalis (ST) and the pallidum  was also analyzed. These signaling molecules repel the axons expressing Robo1/Robo2 when they interact with Slit1/Slit2, deviating them from the hypothalamus and midline  .The expression of the el) Fig. q, s; hownts Fig. u, x. WhiSe; Fig. r, s in cSe; Fig. v, x. A cTel Fig. S, but noTel Fig. x. Slit2 LT; Fig. t, t′ in LT; Fig. y, y′. NoBtbd3 gene, differentiate into c-type neurons and project to layer III and IV of the primary sensorial Cx , suggesting that pERK may locally increase the Wnt signal. Conversely, ventro-medial and anterior thalamic nuclei are less sensitive to pERK/WNT3 signals and require other factors as SHH and GBX2  that act through a number of transduction pathways, like the highly conserved Ras-ERK mitogen-activated protein kinase (MAPK) , this may be related to the local specification and survival of ventro-lateral thalamic cells. We have shown that Wnt3 expression is induced by Fgf8 activity in thalamic neuroepithelium  that determines cyclic nucleotide signaling. PDE signaling regulates cAMP and cGMP concentrations in thalamic neurons in vitro  and neuronal death in VL Th.Fgf8 signal levels affect thalamic regionalization, acting on TML neurons. That molecular signaling contribute to the increase of TML complexity by projecting the 2D ventricular patterning onto a 3D organized structure following differences in signal intensity in radial glia fibers. This 3D pattern is exclusively coded by Fgf8 expression, since although Fgf15 is expressed in the TML, and regulated by Shh  did present partial distribution of the thalamic axons towards the Cx. Garel et al. . Our results strongly suggest that Ntn1 expression involved in the dorsal growth of TC to enter into the pallium requires normal pERK expression in the radial glia fibers. Previous work  in Fgf8l et al. . CongrueSlit1 is highly expressed in the ganglionic eminence (GE) at the stages in which TC fibers are elongating, probably repelling these fibers from the proliferative region Supplementary material 2 (TIF 3642 KB)


This assay reacted with the hepatopancreas (HP) of EHP-infected shrimps, and the highest copy numbers were found in HP and feces samples from Southeast Asian countries (106–108 copies mg−1), while HP samples from Latin America, Artemia, and EHP-contaminated water showed lower amounts (101–103 copies mg−1 or mL−1 of water). No false positive was found with the normal shrimp genome, live feeds, or other parasitic diseases. This tool will facilitate the management of EHP infection in shrimp farms.We developed a qPCR assay based on the  Enterocytozoon hepatopenaei (EHP), an intracellular spore-forming parasite, is a major threat to the commercial shrimp industry. The infection is specific to the tubular epithelial cells of the hepatopancreas (HP) of shrimps. Shrimps in an advanced stage of EHP infection show signs of lethargy, reduced feed intake, soft shells, and an empty midgut [Penaeus monodon [Enterocytozoon, in the family Enterocytozoonidae [P. vannamei cultured in various Asian countries, including China, Thailand, Indonesia, Malaysia, Vietnam, and India [The microsporidian y midgut . This pa monodon . It was zoonidae . Subseqund India ,5,6,7. Rnd India .Severe EHP infections have become common in ponds, and result in substantial economic losses to shrimp producers, since they are associated with inhibited growth in cultured shrimps . EHP infβ-tubulin gene sequence has sufficient variability for molecular diagnosis at the species level [β-tubulin gene.Several EHP diagnostic methods based on SSU rRNA are available, including PCR, in situ hybridization, and loop-mediated isothermal amplification assays ,2,3,4,9.es level . For morβ-tubulin gene sequence for EHP-specific quantification. This assay was then applied to examine shrimp and feces samples collected from various countries to quantify the level of EHP present. The method was also applicable for screening live shrimp feeds and water samples.In this study, we developed a qPCR assay based on the β-tubulin gene of the EHP genome (GenBank no. KY593130), using Primer Express v. 3.0 software (Life Technologies). The DNA fragment (84-bp) was amplified using the forward primer (EHP-bTub-F2: 5′-GATTTGAGAAAATTGGCGGTTAA-3′) and the reverse primer (EHP-bTub-R2: 5′-TTCTGAACACAGAGGCGCATA-3′), while the TaqMan probe (5′-TGATTCCATTTCCACGACTGCACTTTTTC-3′) was synthesized and labeled with 6-carboxyfluorescein (FAM) on the 5′ end and N,N,N′,N′-tetramethyl-6-carboxyrhododamine (TAMRA) on the 3′ end. The qPCR assay was performed by combining 1 µL extracted DNA and a qPCR mixture containing 2.5 μl TaqMan Faster Universal PCR Master Mix (Life Technologies), 0.3 μM of each primer, and 0.1 μM TaqMan probe for a final volume of 10 μl. Amplification was performed by thermal cycling for 20 s at 95 °C followed by 40 cycles of 3 s at 95 °C and 30 s at 60 °C. Amplification and data analysis were performed using a StepOnePlus PCR system (Life Technologies).The primers and TaqMan probe were designed based on the β-tubulin gene from the EHP isolate 16-505/HP (β-tubulin gene is a single copy gene in the EHP genome. The plasmid was designated pEHP-tubB-2 (3885-bp) and the concentration was determined by measuring OD260nm (2.38 × 108 copies per 1 ng of DNA). For a standard curve, the purified pEHP-tubB-2 plasmid was used, and the sensitivity was determined by preparing 10-fold dilutions. A standard curve presenting concentrations from 102–108 copies µL−1 is shown in Perezia sp. microsporidium [Paramoeba sp. amoeba [β-tubulin gene sequence are specific to EHP, showing no reaction to another shrimp parasites. In addition, DNA from different shrimp species and live shrimp feed were tested to ensure that the primers did not cause false positives  using a Microsep Advance Centrifugal Device (PALL Corporation). Approximately 30 mg of HP, feces, and artemia samples and 300 μL of water samples were used for DNA extraction using QIAamp tissue kit (Qiagen). After DNA extraction, the concentration of DNA was measured by a spectrophotometer . The EHP copy number was highest in the HP samples from Indonesia, with a range between 8.0 × 103–1.4 × 108 copies mg−1, followed by those from Thailand (2.5 × 102–9.1 × 107 copies mg−1) and Vietnam (1.9 × 103–4.8 × 107 copies mg−1). In contrast to the SE Asian samples, HP samples from Latin America presented a relatively low EHP quantity, with copy numbers between 2.1 × 101–1.3 × 102 copies mg−1. In the feces sample, 106–1.7 × 107 copies mg−1 were found, while Artemia samples contained EHP DNA at 4.3 × 103–2.8 × 105 copies mg−1. Finally, EHP was detected in the water samples at 1.7 × 103 copies per mL−1.To evaluate practical use, a total of 86 samples were collected from a population of EHP-affected vannamei . These sArtemia. This indicates good applicability of the qPCR assay regardless of the source of EHP and their host species. For the Artemia samples, substantial amounts of EHP (as DNA copies) were detected. Live shrimp feeds, including polychaetes, squids, and Artemia biomass, have been suspected to be carriers for EHP, so EHP quantification by qPCR in live shrimp feeds can be used as a tool to aid in disease prevention in broodstock populations. Using qPCR, we also confirmed that feces contained high numbers of EHP copies, indicating that this may act as an important source of infection in ponds. In penaeid shrimps, EHP multiplies rapidly in the HP tubule epithelium cells, and EHP spores are released into the digestive system, causing white feces in ponds [All samples tested in this study came from shrimps exhibiting slow growth in farms from SE Asia and Latin America, and from contaminated in ponds .β-tubulin gene, applicable in EHP management in shrimp farming.In conclusion, EHP infection is notorious in farmed shrimp ponds, and such EHP infections are likely to continue to be prevalent. The situation requires careful monitoring of EHP levels in shrimps and their feed using a highly specific diagnostic method. In addition, quantification of EHP in live feeds, pond environments, or feces becomes more important for EHP management. This study describes a new quantification method for EHP based on the