Whether body mass index (BMI) is a significant risk factor for recurrence of primary spontaneous pneumothorax (PSP) remains controversial. The purpose of this study was to examine whether BMI and other factors are linked to risk of PSP recurrence.n = 81) and those who did not (n = 192), as well as into those who had low BMI (n = 75) and those who had normal or elevated BMI (n = 198). The two pairs of groups were compared in terms of baseline data, and Cox proportional hazards modeling was used to identify predictors of PSP recurrence.A consecutive cohort of 273 patients was retrospectively evaluated. Patients were divided into those who experienced recurrence (P < 0.001).Rates of recurrence among all 273 patients were 20.9% at 1 year, 23.8% at 2 years, and 28.7% at 5 years. Univariate analysis identified the following significant predictors of PSP recurrence: height, weight, BMI, size of pneumothorax, and treatment modality. Multivariate analyses identified several risk factors for PSP recurrence: low BMI, pneumothorax size ≥50%, and non-surgical treatment. Kaplan–Meier survival analysis indicated that patients with low BMI showed significantly lower recurrence-free survival than patients with normal or elevated BMI (Low BMI, pneumothorax size ≥50%, and non-surgical treatment were risk factors for PSP recurrence in our cohort. Low BMI may be a clinically useful predictor of PSP recurrence. Pneumothorax is defined as the presence of air or gases between the parietal and visceral pleural space. Pneumothorax can be clinically classified as either traumatic or spontaneous, and the spontaneous type can be further categorized as primary or secondary based on etiology . PrimaryEven following optimal treatment to fully expand collapsed lungs, 20–60% of PSP patients suffer relapse, and this rate may be increasing . StudiesThe possible association between BMI and risk of PSP recurrence remains controversial, with some studies suggesting a significant relationship , 12 but The study protocol was approved by the Institutional Review Board of the First Affiliated Hospital of Guangxi Medical University , and it was conducted in accordance with the Declaration of Helsinki. Medical records were retrospectively reviewed for patients with PSP admitted to the thoracic surgery or emergency departments of the First Affiliated Hospital of Guangxi Medical University between October 2010 and September 2014. Patients were enrolled in the study if (1) they fulfilled the diagnostic criteria of PSP based on chest radiography or computed tomography (CT), (2) they had no history of PSP, and (3) they were between 12 and 30 years old when PSP occurred. Patients were excluded from the study if they had secondary, traumatic, or iatrogenic pneumothorax.Data were collected on age, sex, height, weight, BMI, location and size of pneumothorax, and treatments. Pneumothorax size was quantified in terms of percentages as described : pneumot2. This cutoff corresponds to the World Health Organization (WHO) threshold for classifying people as underweight or as normal/overweight/obese [2), overweight (BMI ≥25 and <30 kg/m2), or obese (BMI ≥30 kg/m2).Patients were also assigned to groups depending on whether their BMI was <18.5 or ≥18 kg/mht/obese . Since wTreatments given to the patients in our cohort for the first PSP episode included oxygen therapy, needle aspiration, chest tube drainage, and video-assisted thoracoscopic surgery (VAST). The clinicians and techniques used to administer these treatments remained the same throughout the study period. Asymptomatic patients with small pneumothorax were treated by oxygen inhalation therapy; symptomatic patients with small pneumothorax were treated by chest tube drainage, as were symptomatic or asymptomatic patients with any other size of pneumothorax. VAST was administered to patients who experienced air leakage lasting 4–5 days, tension pneumothorax, hemopneumothorax, or occupational demand. Surgery was also used to treat patients with bullae visible on chest X-rays or CT scans, depending on the wishes of patients or their relatives.All patients were followed up after discharge until recurrence or October 2015. In principle, patients were followed up every one month during the first two years after discharge, and every 3–6 months thereafter. Long-term data on PSP recurrence were obtained through telephone interviews.t test or Mann–Whitney U test. Categorical data were expressed as number (%), and intergroup differences were assessed using the Chi-squared or Fisher’s exact tests (2-tailed) as appropriate. Variables with a P value less than 0.05 in univariate analysis were entered in multivariate analysis. At the same time, height and weight that are closely related to BMI cannot be entered in multivariate Cox proportional hazards. BMI Kaplan–Meier survival curves were estimated and then compared using the log-rank test. The threshold of significance in all analyses was P < 0.05. All analyses were performed using SPSS 19.0 .Continuous data were expressed as mean ± standard deviation (SD), and intergroup differences were assessed for significance using the During the study period, 305 patients who experienced first PSP were admitted to our hospital and treated by conservative or surgical techniques. Excluded from the present series were 16 patients (5.2%) who had history of PSP, 7 patients (2.3%) with secondary pneumothorax, or 9 patients (3.0%) older than 30 years with PSP. Therefore, 273 patients (89.5%) were included in the analysis. Most patients (81.0%) were male, with a mean age of 19.8 years (SD 3.5). Average height was 173.6 ± 4.7 cm, and average weight was 59.2 ± 7.3 kg. The distribution of BMI was as follows: underweight, 27.5%; normal, 70.3%; overweight, 2.2%; and obese, 0%. Only 7.0% of patients had a smoking history. Location of pneumothorax was right in 53.1% of patients, left in 46.2%, and bilateral in 0.7%. Two patients who experienced bilateral pneumothorax underwent VAST, one of them had a relapse, and the other did not  and non-surgical in the remaining 97 (35.5%). Among all 273 patients, 81 (29.7%) experienced recurrence and 192 did not. Rates of PSP recurrence were 20.9% at 1 year, 23.8% at 2 years, and 28.7% at 5 years (Table n = 75) or not (198). While the underweight group was slightly younger on average (19.3 ± 4.5 vs. 20.2 ± 4.8 years), the two groups did not differ significantly in gender, smoking history, location or size of pneumothorax, or treatment modality. The two groups did, however, differ significantly in height, weight, and PSP recurrence rates. Recurrence rates at 1, 2, and 5 years were 40.0, 48.0, and 58.7% in the underweight group, compared to 13.6, 15.2, and 18.7% in the normal/overweight group . During follow-up, 44 patients in the underweight group (58.7%) relapsed, compared to 37 (18.5%) in the normal/overweight group. Rates of recurrence-free survival at 1, 2, and 5 years were 60, 52, and 41.3%, respectively, in the underweight group, which were significantly lower 1 than the rates of 86.4, 84.8, and 81.3%, respectively, in the normal/overweight group (–60. DuriThis retrospective cohort study associated low BMI (underweight) with risk of PSP recurrence and a lower rate of recurrence-free survival. These results suggest that BMI may be a helpful clinical indicator of PSP recurrence risk in patients experiencing their first pneumothorax.Males predominated over females in our cohort by a ratio of 4:1, which is consistent, albeit slightly lower, than ratios of 9:1 reported in two Asian cohorts , 16. OurPrevious studies have reported conflicting results about whether BMI is associated with risk of PSP recurrence , 22, 23,1-antitrypsin. This leads, in turn, to major apical emphysema-like changes [1-antitrypsin was closely associated with bilateral bronchial abnormalities, which may be responsible for bilateral recurrences of PSP [This association has several possible explanations. One is that most PSP patients with low BMI show unbalanced physical development. This significantly increases chest negative pressure, especially at the cupula of pleura, where it increases risk of bleb or bulla formation and pneumothorax . A secon changes . Deficies of PSP . A thirds of PSP , increasP < 0.001). The main reason may be that surgical excision for bleb or bulla indeed decreases the risk of recurrence of PSP. Consistent with this, our multivariate analysis identified pneumothorax size ≥50% and non-surgical treatment as independent predictors of PSP recurrence, in addition to low BMI. The hazard ratio associated with low BMI in our cohort was 3.668 , greater than the corresponding ratios associated with pneumothorax size ≥50% and non-surgical treatment. This suggests that BMI may be a clinically more important risk factor for PSP recurrence.Risk of PSP recurrence may depend on several factors . No mattP < 0.001). This strengthens our finding that BMI may be a clinically useful indicator of recurrence risk. At the same time, our study has several limitations. First, it retrospectively analyzed patients from a single medical center, increasing risk of selection bias, which needed multicenter clinical trials to avoid such bias and verify the results. Second, most patients were high school and university students from the Guangxi Autonomous Region in western China, who are generally slightly shorter and thinner than Chinese from other parts of the country. Thus, our results may not be generalizable to other parts of China or to other countries. Third, we have failed to compare BMI at a later stage after initial treatment, the change of which may be of more sense to explain association between BMI and recurrence of PSP. Fourth, although we did examine several baseline characteristics as candidate risk factors for PSP recurrence, we may have failed to analyze other factors that are playing a role. Future studies should examine a more exhaustive set of possible risk factors.Kaplan–Meier survival curves were used to estimate PSP recurrence in patients stratified by BMI, and the result showed that low BMI showed significantly lower recurrence-free survival than patients with normal or elevated BMI (Despite these limitations, our study provides evidence that low BMI is associated with higher risk of PSP recurrence and shorter recurrence-free survival. This work justifies larger clinical trials and mechanistic studies to elucidate how low BMI may influence PSP recurrence.


Staphylococcus aureus and its drug-resistant strains, which threaten public health and food safety, are in need of effective control by biopreservatives. A novel bacteriocin, pentocin JL-1, produced by Lactobacillus pentosus that was isolated from the intestinal tract of Chiloscyllium punctatum, was purified by a four-step chromatographic process. Mass spectrometry based on MALDI-TOF indicated that pentocin JL-1 has a molecular mass of 2987.23 Da. Only six of the twenty-five amino acids could be identified by Edman degradation. This bacteriocin is thermostable and tolerates a pH range of 5–7. Also, it is sensitive to proteinase K, trypsin, pepsin, and alkaline protease. This bacteriocin has a broad inhibitory spectrum against both Gram-positive and Gram-negative strains and in particular is effective against multidrug-resistant S. aureus. Additionally, we showed that the cell membrane is the target of pentocin JL-1 against methicillin-resistant S. aureus (MRSA), causing a loss of proton motive force. Furthermore, pentocin JL-1 has a drastic impact on the structure and integrity of MRSA cells. These results suggest that pentocin JL-1 has potential as a biopreservative in the food industry. Staphylococcus aureus belongs to the Gram-positive Micrococcaceae family and is one of the most serious bacterial pathogens globally [ S. aureus (MRSA) was first found among S. aureus clinical isolates [ S. aureus have been detected in the past few years [ S. aureus strains are a potential risk to humans as they could transfer the resistance to other pathogenic bacteria of humans through the food chain, the genetic pool of bacteria, bacteriophages, or DNA fragments [ S. aureus and its drug-resistant strains for either food preservation or prevention and control of bacterial infectious diseases [globally . It can globally –3. In 19isolates  and carrisolates . In addiew years –8. Drug-ragments . There idiseases .β-carbon of other amino acid residues; class II, small (<10 kDa), linear peptides without posttranslationally modified amino acids; class III, proteins (>10 kDa); class IV, small (<10 kDa), circular peptides without posttranslationally modified amino acids and with an amide bond between the N- and C-termini; class V, small (<5 kDa), linear or circular peptides containing extensively posttranslationally modified amino acids with thioether bridges formed between α-carbon of other amino acid residues and the thiol groups of Cys residues. However, there is no international standard of classification, and other schemes have been proposed along with information regarding their characteristics [Bacteriocins are prokaryotic proteins or peptides, which exhibit inhibitory activity against other prokaryotes , 11. Pareristics , 16. Lactococcus lactis, pediocin produced by Pediococcus acidilactici, and a combination of three bacteriocins , all produced by Carnobacterium maltaromaticum UAL307, which has been commercialized in the USA and Canada, with the name of Micocin®, are used as food preservatives commercially [In recent years, many useful LAB bacteriocins have been identified and studied, such as lactococcin A , pentociercially , 23. Othercially . S. aureus growth in food. Many researchers have shown that LAB bacteriocins have anti-MRSA ability [ S. aureus such as anticiprofloxacin, anticefoxitin, and antigentamicin [ Lactobacillus pentosus isolated from the intestinal tract of Chiloscyllium punctatum, exhibiting a broad inhibitory spectrum. This bacteriocin can inhibit not only MRSA but also other multidrug-resistant S. aureus strains. In addition, the mode of action by which pentocin JL-1 causes cell membrane damage in MRSA was characterized.Treatment with LAB bacteriocins is an effective and safe way to inhibit ability , 24, 25.ntamicin . In our  C. punctatum (grey carpet shark) were dissected and homogenized in 20 mL saline solution under sterile conditions and were then plated in serial dilutions in deMan, Rogosa, and Sharpe (MRS) medium. The plates were incubated aerobically at 30°C for 24 h. Several colonies were picked at random and incubated again in MRS broth. For screening for bacteriocin-producing strains, the agar-well diffusion test was used to detect antimicrobial activity in cell-free supernatants (filtered through a 0.22 μm Millipore filter) obtained by centrifugation  after 18, 36, 60, and 72 h incubation [ Escherichia coli O157:H7 GIM 1.707 were used as indicator strains. Before the experiments, the indicator strains were grown to 106 CFU/mL. Then, 1 mL of the culture was mixed with 100 mL soft agar medium and poured onto individual Petri dishes. Subsequently, 8 mm diameter wells were punched onto the plates, and each well was filled with 100 μL of the cell-free supernatants under sterile conditions. The plates were incubated overnight at their respective optimum temperatures, and the clear zones of inhibition were measured in diameter and indicated the presence of antimicrobial activity. In our study, all bacteria culture media and chemical reagents were supplied by Sigma-Aldrich (USA).The intestinal tracts of E. coli O157:H7 GIM 1.707 was selected and named JL-1. It was stored at −80°C in MRS broth with 25% (v/v) glycerol. The strain JL-1 was then identified by 16S rRNA gene sequencing with the forward primer 5′-AGAGTTTGATCCTGGCTCAG-3′ and the reverse primer 5′-CTACGGCTACCTTGTTACGA-3′. Subsequently, sequence homologies were analyzed by comparing the sequence with those in the NCBI database and phylogenetic analysis was carried out using MEGA 5.0.The strain with the highest antibacterial activity against MRSA GIM 1.771 andg, 30 min, 4°C), and the cell-free supernatant was absorbed with the macroporous resin D4020 . The column was eluted with 20% (v/w) ethanol and active fractions were collected. The antimicrobial activity of each fraction was determined by measuring the diameter of the inhibition zone around the wells compared with nisin and expressed as international units (IU) per mL [18 column  using the LC-6AD semipreparative High Performance Liquid Chromatography (HPLC) system  at 2.5 mL/min with a gradient elution of 100% buffer A  to 100% buffer B (100% acetonitrile and 0.1% trifluoroacetic acid). The active fraction was collected and repurified using an analytical C18 column  with an elution with 40% acetonitrile. The active fraction was collected and lyophilized for mass spectrum (MS) detection. Purified pentocin JL-1 was lyophilized using a freeze-dryer . MRSA GIM 1.77 was used as the indicator strain for the activity test.The strain JL-1 was grown in MRS medium at 30°C for 72 h. The ferments were centrifuged  per mL . The actThe molecular mass of the purified pentocin JL-1 was detected by MALDI-TOF-MS , which was analyzed by GL Biochem . The N-terminal amino acid sequence of the purified pentocin JL-1 was detected by PPSQ33A automatic sequencing system , which was analyzed by Shanghai Sangon Biotech Company, China.μg/mL, pH 5.5) [The agar-well diffusion test as described previously was used to detect the antimicrobial activity of the purified pentocin JL-1 (15  pH 5.5) . The ind6 CFU/mL was collected and 50 μL of each was grown in 96 well-microtiter plates  with different concentrations of pentocin JL-1, ranging from 50 ng/mL to 15 μg/mL, at 37°C for 24 h. Each concentration was done in triplicate. The MIC represents the bacteriocin concentration at which 100% of growth is inhibited measured by the absorbance at 540 nm [Additionally, the minimal inhibitory concentration (MIC) of pentocin JL-1 against MRSA GIM 1.771 was tested. Overnight culture of MRSA GIM 1.771 with the concentration of around 2 × 10t 540 nm .w/v) acetic acid at 15 μg/mL and was adjusted with 1.0 M HCl or 1.0 M NaOH to different pH values of 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, and 10.0. Then the above samples were incubated at 37°C for 1 h and were adjusted to pH 5.5 with 1.0 M HCl or 1.0 M NaOH.To determine pH stability, lyophilized purified pentocin JL-1 was dissolved in 0.05% (w/v) acetic acid at 15 μg/mL was evaluated at different temperatures  for 1 h and also at the autoclaved condition (121°C at 15 psi for 15 min).To determine thermal sensitivity, lyophilized purified pentocin JL-1 dissolved in 0.05% (w/v) acetic acid at 15 μg/mL was treated with the following enzymes at 75 μg/mL under their respective optimum pH and temperatures: proteinase K , trypsin , pepsin , and alkaline protease  . After incubation for 1 h, the bacteriocin-enzyme mixture was boiled for 5 min to inactivate the enzymes.To determine enzymatic sensitivity, lyophilized purified pentocin JL-1 dissolved in 0.05% (w/v) acetic acid at 15 μg/mL with pH 5.5 was used as a control in all assays.The residual antibacterial activities of the above samples were calculated using the agar-well diffusion test with MRSA GIM 1.771 as the indicator strain. The area of inhibition was calculated from the diameter of the inhibition zones, and the decrease ratio was displayed as a percentage. Lyophilized pentocin JL-1 dissolved in 0.05% (w/v) acetic acid was added to the cultures at a final concentration of 1x MIC and the same volume of 0.05% (w/v) acetic acid was added to the aforementioned media as a control. Samples were incubated at 37°C and bacterial suspensions were taken each hour for 24 h, and the absorbance was measured at OD600. In addition, the viable cell counts on TSB agar medium after the addition of 1x MIC, 2x MIC, and 3x MIC of pentocin JL-1 were also quantified every 10 min for 1 h.The bacteriostatic or bactericidal mode of action of pentocin JL-1 was tested as described previously by Zhu et al. with some modifications . MRSA GI2(5) . MRSA GIM 1.771 cells were grown to the exponential phase in 50 mL TSB culture medium, harvested, and washed twice with 50 mL buffer A  at 4°C, resuspended in 5 mL of the same buffer and stored on ice for the fluorescence measurements. Then, the cells were added to a fluorescence cuvette together with 0.5 μM DisC2(5). The fluorescence emission was monitored at room temperature using an F-4600 spectrofluorometer  with an excitation wavelength (Ex) of 647 nm and emission wavelength (Em) of 680 nm for 400 s. When the reduction of fluorescence was stable, final concentrations of 1x, 2x, and 3x MIC of pentocin JL-1 were added to the cuvette, respectively, and 0.05% (w/v) acetic acid was added as a negative control. Full dissipation of the membrane potential was indicated by addition of 1% Triton X-100. To the control, an equivalent volume of 3x MIC of pentocin JL-1 was added.To test the effect of pentocin JL-1 on membrane integrity, the cell PMF was assayed. PMF includes the transmembrane electrical potential (ΔΨ) and the transmembrane pH gradient (ΔpH) . ΔΨ was μM BCECF AM pH probe was added and the solution was incubated at 37°C for 1 h in the dark. Then 1 mL of the incubated solution was added to a fluorescence cuvette together with a final concentration of 1x, 2x, and 3x MIC of pentocin JL-1, respectively. Acetic acid  was used as a negative control and 1% (w/v) Triton X-100 was used as a positive control. To the control, an equivalent volume of 3x MIC of pentocin JL-1 was added. The fluorescence intensity was monitored at 50 s intervals for 400 s immediately after mixing at Ex 488 nm and Em 535 nm using an F-4600 spectrofluorometer .ΔpH was monitored by the fluorescent pH probe 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester (BCECF AM) . MRSA GIM 1.771 cells were grown to the exponential phase in 50 mL TSB culture medium, harvested, washed twice with 50 mL 5 mM HEPES buffer at 4°C, and resuspended in 5 mL of the same buffer for incubation on ice for 1 h. Subsequently, the 1 g for 5 min, and washed gently with 500 μL phosphate buffer saline  twice. Subsequently, the cells were fixed in 2.5% glutaraldehyde at 4°C for 16 h and washed gently with 500 μL PBS twice. Then the cells were dehydrated with gradient ethanol solutions  at 4°C and centrifuged at 6000g for 15 min. The cells were then freeze-dried using a freeze-dryer , coated with gold, and imaged using an SU8010 scanning electron microscope .MRSA GIM 1.771 cells in exponential phase were supplemented with 1x MIC of pentocin JL-1 and incubated at 37°C for 10 min. Cells without pentocin JL-1 were used as controls. Cells were collected by centrifugation at 4°C, 6000t-test and p < 0.05 was considered statistically significant.All related experiments were done in triplicate and the results are expressed as mean ± standard deviation. Data analysis was performed with SPSS 19.0 and Origin 8.0. Comparison of data on stability of pentocin JL-1 against pH, temperatures, and enzymes were performed using independent sample  E. coli O157:H7 GIM 1.707 with a diameter of the inhibition zones of 24.8 ± 0.5 mm and 23.9 ± 0.2 mm, respectively. It is a Gram-positive and catalase-negative Bacillus. The 16S rRNA gene sequence was amplified by PCR, and a 1485 bp gene fragment was sequenced after cloning and aligned using the NCBI database. We found that the 16S rRNA sequence of strain JL-1 had 99% similarity with that of L. pentosus KC422317.1. Additionally, a phylogenetic tree was constructed using MEGA 5.0 , with a specific activity reaching up to 432 IU/mg (p < 0.05). F3Aa was repurified by analytical HPLC (data not shown), and the active fraction was collected and lyophilized for MS detection. The purification process and antibacterial activity are listed in  L. pentosus SJ65 was purified 52-fold at a yield of 8% [ L. lactis KU24 was purified 24.58-fold [ L. plantarum ZJ5 was purified 139.5-fold at a yield of 1.7% [The bacteriocin, pentocin JL-1, is one of the secondary metabolites of32 IU/mg . The actld of 8% , bacteri.58-fold , and pla L. gasseri EV146 after the addition of the supernatant from a previous bacteriocin-producing EV1461 culture (autoinduction) or through cocultivation with several other Gram-positive strains (inducing bacteria) [ L. plantarum, L. lactis, L. sakei, S. thermophilus, and various other LAB have been used as hosts for heterologous expression [The yield of pentocin JL-1 can be enhanced by optimizing the production conditions. In addition to incubation temperatures, initial pH values, inoculum density, loading volume, and culture medium optimization, coculture with other strains and autoinduction by a signal peptide produced by the strain itself are effective ways to improve the yield of bacteriocins. For example, at low cell densities, gassericin E was produced byacteria) . In addipression , 35, 36.18 reverse-phase HPLC (C18 RP-HPLC). Many bacteriocins such as bacteriocin KU24 [In addition, from the purification process, we observed that pentocin JL-1 is a cationic and hydrophobic peptide, which was shown by cation-exchange, hydrophobic-interactions, and Ccin KU24 , bactericin KU24 , plantarcin KU24 , and oth L. pentosus reported previously, including the bacteriocin from L. pentosus RL2e of around 20 kDa [ L. pentosus of about 5 kDa [https://blast.ncbi.nlm.nih.gov/Blast.cgi). Thus, pentocin JL-1 may be a novel LAB bacteriocin. To obtain more detailed information, further chemical and mass spectrometry techniques needed to be performed. In addition, the complete genome sequencing of our L. pentosus will be performed to gain insights into the genetic elements involved in bacteriocin production in the future.Pentocin JL-1 was identified by MALDI-TOF-MS. The results indicated that the purified pentocin JL-1 has a molecular mass of 2987.23 Da , which wut 5 kDa , pentociut 5 kDa , and penut 5 kDa . In addiut 5 kDa  and ZJ00ut 5 kDa . Howeverp > 0.05). However, the antimicrobial activity decreased significantly when the pH decreased from 4 to 2 and increased from 8 to 10 (p < 0.05). Some other bacteriocins have also been reported to have a similar pH stability [The effects of pH, temperatures, and enzymes on the antibacterial activity of the bacteriocin were determined . No signtability , 40. It tability . Howeverp > 0.05). An antibacterial activity of 84.10% remained even after autoclavation . This thermostable characteristic makes pentocin JL-1 suitable for use in a sterilization process.At different temperatures, pentocin JL-1 was stable, and no significant differences were detected from −20°C to 100°C (p < 0.05). However, the inhibitory action was completely abolished by treatment with pepsin and alkaline protease. Thus, pentocin JL-1 has a proteinaceous nature like most other bacteriocins [When purified pentocin JL-1 was treated with different hydrolytic enzymes, its inhibitory action was significantly reduced by treatment with proteinase K and trypsin  (w/v) acetic acid was added, did not indicate any increase or decrease during the 400 s experiment.As many bacteriocins are assumed to kill the target microorganism via permeabilization of the cell membrane , the eff2(5)  and the 2(5) . As show2(5)  caused a2(5)  was subsOnce BCECF AM is absorbed into the cell membrane, it is cleaved by an esterase into BCECF, which is a fluorescent probe that indicates a transmembrane pH gradient. As shown in In general, cationic bacteriocins initially interact with the anionic cell membrane through electrostatic attraction . Then, bΔΨ and ΔpH dissipations were nearly complete within 100 s, which shows that the membrane permeabilization caused by pentocin JL-1 is a relatively rapid process. This is in accordance with the well-known antibiotic peptide clavanin , which iSEM was used to further demonstrate the membrane damage of MRSA GIM 1.771 caused by pentocin JL-1. Morphological changes of MRSA GIM 1.771 after 10 min exposure to 1x MIC of pentocin JL-1 are presented in  L. pentosus isolated from the intestinal tract of C. punctatum, was purified and found to have a molecular mass of 2987.23 Da. It is sensitive to proteinase K, trypsin, pepsin, and alkaline protease, indicating that it has a proteinaceous nature. Also, this bacteriocin has a broad inhibitory spectrum, is thermostable, and stable over a pH range of 5–7. Hence, pentocin JL-1 appears to have promising potential as a biopreservative in the food industry, especially for controlling multidrug-resistant S. aureus. Additionally, our results show that the cell membrane is the target of pentocin JL-1 against MRSA GIM 1.771, causing a loss of PMF in only a few minutes, and that it has a drastic impact on the structure and integrity of the MRSA GIM 1.771 cell that finally leads to cell death, which was indicated by the growth curve and time-killing kinetics. In further studies, more detailed information on the mode of action, the exact amino acid sequence, and the structure of pentocin JL-1 will be addressed.In the present study, the bacteriocin pentocin JL-1, produced byL. pentosus JL-1 by SP-Sepharose Fast Flow chromatography.Figure S1: Purification of the bacteriocin produced by L. pentosus JL-1 by Ultrahydrogel TM 250 gel filtration chromatography.Figure S2: Purification of the bacteriocin produced by


Acupuncture (Acup) and moxibustion (Moxi) are commonly used interventions in clinical practice. However, the difference between Acup and moxibustion mechanisms is unclear. In current study, blood perfusion responses resulted from Acup or Moxi at Weishu acupoint (BL21) and control points were explored, respectively. The time series of blood flux signals at BL21 and control points were transformed with Morlet wavelet, and the differences in each frequency interval were observed. The results suggested that acupoint response to different stimulation is a comprehensive process which related to all components of blood perfusion signals. Whereas the different response at control points was not observed, there has been significant difference coherence value between Acup and Moxi stimulation. The results suggested the influence of Acup and Moxi not only on the level of blood perfusion at local area; the intrinsic relevance after stimulation which can be evaluated by coherence analysis is also an appropriate index to distinguish different stimulations. Acup has been widely used to treat diseases at least for 2500 years . As a tyPrevious studies have shown that acupoint areas are enriched in nerve endings, blood vessels, and mast cells, and these components constitute the basic structure of axon reflex –8. When A total of 10 healthy subjects were recruited to the study. The general characters are presented in This study was approved by the Institutional Research Ethics Boards of Acup & Moxi, China Academy of Chinese Medical Sciences. In accordance with the Declaration of Helsinki, each subject provided informed consent and had an adequate understanding of the procedure and purpose of this study.Some studies suggested that the meridian system may contain a continuous channel  to facilWeishu acupoints (BL21), upper point, and down point were marked by senior Acup doctor. BL21 is in the upper back region, at the same level as the inferior border of spinous process of the 12th thoracic vertebra (T12), 1.5 B-cun lateral to the posterior median line [In the present study, a commercially available FLPI system  was used to measure blood flux at BL21. Before and after stimulation, respectively, as shown in Blood perfusion signals of upper and down point were recorded with PeriFlux System 5000  at 32 Hz sample rate and 0.2 s time constant. An optical fiber probe connected with the PeriFlux 5000 was used to illuminate and collect the scattered light from the skin tissue. The probe was attached to the surface of interest by a two-sided adhesive tape . In this study, upper point was recorded by the first channel and the down point was recorded by the second channel. In the current study, all records were performed at the same time.All measurements were carried out in a quiet, temperature controlled (24–26°C) laboratory. On arrival to the laboratory, subjects were asked to empty their bladders. The participant was asked to assume a comfortable supine position and the skin of BL21 was sterilized. Following a period for cardiovascular stability (40 min), a baseline recording of blood flux was made for 20 min . After bFor Acup, after baseline recording, a small Acup needle  was gently inserted in a depth of 15 mm in the BL21 acupoint. Before stopping Acup intervention, the needle was slowly rotated for 5 min to maintain the soreness and numbness sensation of De-Qi. For Moxi intervention, after baseline recording, the ignited moxa roll was held approximately 2-3 cm above the BL21, which produced a mild warm and comfortable sensation. Moxi at BL21 lasted for 5 minutes.The recording file of upper and down point was opened in the software of PeriSoft for Windows . The detailed data were exported as txt format and saved. The recording file of BL21 was opened in the software of moorFLPI full-field laser perfusion imager review  and the detailed data was also exported as txt format. Then the data were imported to the Matlab software and analyzed. For each recording point, the mean of blood flux for 20 min was calculated.Previous studies indicated that blood flux oscillations at frequencies from 0.0095 to 1.6 Hz might reflect different physiological rhythms , which cCxy(f)) can be used to examine the relation between two signals, and the analysis method can be referenced as in our previous study [Cxy(f) is between 0 and 1 and indicates how well x corresponds to y at each frequency. In present study, x is the upper point blood flux signal, while y is the down point blood flux signal. To calculate the coherence between upper point and down point, the coherence value was estimated by the following equation [Coherence value (us study . Cxy(f) equation , 20. TheP < 0.05. Statistical analysis was performed by paired t-test with SPSS software . All reported P values are two-sided.Data are expressed as mean ± SE. The level of significance was defined as t = 1.076, P = 0.310, paired t-test), while after stimulation, the mean blood perfusion of BL21 has significant difference .The recording points  and bloot = −4.435, P = 0.002, paired t-test), respectively. In frequency interval II (0.02–0.06 Hz), the blood perfusion was 167.62 ± 18.71 PU versus 258.28 ± 26.48 PU , respectively. In frequency interval III (0.06–0.15 Hz), the blood perfusion was 107.16 ± 12.12 PU versus 156.54 ± 11.70 PU , respectively. In frequency interval IV (0.15–0.4 Hz), the blood perfusion was 124.98 ± 7.73 PU versus 173.83 ± 18.69 PU , respectively, and in frequency interval V (0.4–1.6 Hz), the blood perfusion was 59.96 ± 6.02 PU versus 84.27 ± 8.75 PU , respectively.To examine the underlying mechanisms after stimulation, blood flux signals were transformed by Morlet mother wavelet. There was significant difference between Acup and Moxi in the different frequency interval . In freqt = −0.480, P = 0.643, paired t-test), respectively. In frequency interval II (0.02–0.06 Hz), the blood perfusion was 15.05 ± 2.79 PU versus 14.96 ± 2.70 PU , respectively. In frequency interval III (0.06–0.15 Hz), the blood perfusion was 10.09 ± 2.00 PU versus 8.89 ± 1.32 PU , respectively. In frequency interval IV (0.15–0.4 Hz), the blood perfusion was 3.92 ± 0.76 PU versus 3.54 ± 0.53 PU , respectively, and in frequency interval V (0.4–1.6 Hz), the blood perfusion was 1.83 ± 0.23 PU versus 1.52 ± 0.18 PU , respectively.There were no significant differences at the upper point Figures  resultint = 1.022, P = 0.334, paired t-test), respectively. In frequency interval II (0.02–0.06 Hz), the blood perfusion was 15.56 ± 4.62 PU versus 14.39 ± 2.51 PU , respectively. In frequency interval III (0.06–0.15 Hz), the blood perfusion was 7.73 ± 2.14 PU versus 7.07 ± 1.09 PU , respectively. In frequency interval IV (0.15–0.4 Hz), the blood perfusion was 5.51 ± 2.59 PU versus 3.49 ± 0.53 PU , respectively, and in frequency interval V (0.4–1.6 Hz), the blood perfusion was 3.11 ± 1.25 PU versus 1.96 ± 0.36 PU , respectively.There were also no significant differences at down point Figures  resultint = 3.710, P = 0.0048, paired t-test).The coherence-frequency responses to stimuli are shown in The main finding of this study is that there have been different responses of blood perfusion at BL21 acupoint after Acup or Moxi stimulation. To the best of our knowledge, the current pilot study is the first to compare vascular response after Acup-related stimulations by Morlet wavelet analysis. Whereas other studies have detected changes in local vascular response , this stPrevious studies indicated that blood flux oscillations at frequencies from 0.0095 to 1.6 Hz might reflect different physiological rhythms , which cIn Acup theory, the Acup effect is based on the integrity function of the meridian . Some stCoherence analysis is a method to estimate the correlation of the signals on different frequencies, which can be used for analysis correlation of two blood flux signals in frequency domain –26. In tThere have been different responses of blood perfusion at BL21 acupoint after Acup or Moxi stimulation. Coherence analysis along BL meridian can also as be an appropriate index to distinguish different stimulations.


Prenylated flavonols are known as phytoestrogen and have good bioactivties. However, their abundances in nature are pretty low. It is required to find an efficient synthesis technique. Icariin is a prenylated flavonol glycoside with low cost. It can be used to synthesize different prenylated flavonols. A combination of cellulase and trifluoacetic acid hydrolysis could effectively remove rhamnose and glucose from icariin. Icaritin, anhydroicaritin and wushanicaritin were the leading prenylated flavonol products. Their affinities to estrogen receptors α and β were predicted by docking study. The weak affinity of wushanicaritin indicated that prenyl hydroxylation impaired its affinity to estrogen receptor β. The prenyl cyclization led to a loss of affinity to both receptors. The interactions between icaritin and ligand binding cavity of estrogen receptor β were simulated. π-π stacking and hydrophobic forces were predicted to be the dominant interactions positioning icaritin, which induced the helix (H12) forming an activated conformation. Even though flavonoids are widely present at a high level in nature2, the levels of prenylated flavonoids are usually much lower. Prenylated flavonoids have been documented to be more bioactive than their flavonoid precursors, as prenylation increases the lipophilicity of flavonoids, leading to a higher permeability and bioavailability in vivo4. For instance, 8-prenyl quercetin shows a higher cellular uptake and a lower efflux than quercetin in Caco-2 and C2C12 myotube cells. 8-Prenyl substitution also enhances the bioavailability of quercetin in different tissues in vivo5. Prenylated flavonoids possess a wide variety of bioactivities, such as estrogenic activity, immunomodulatory activity, anticancer activity and antioxidant activity7.Prenylated flavonoids are an important sub-class of flavonoids, which combine a flavonoid skeleton with a lipophilic prenyl side chain9. It can reduce the incidence of steroid-associated oesteonecrosis in rabbit with inhibition of both intravascular thrombosis and extravascular lipid deposition for maintaining the integrity of intraosseous vasculature10. The anti-breast cancer activity of icaritin remains confused. Icaritin at 5 μM can inhibit the proliferation of breast cancer stem/progenitor cells even better than tamoxifen11. However, a proliferation stimulatory effect has been observed for icaritin and desmethylicaritin in MCF-7 cells12. Moreover, icaritin shows anti-inflammatory activity and inhibitory activities against other cancer cells13.Icaritin is a monoprenylated flavonol with 4′-methoxyl. It has been documented to have osteoblastic and neuroprotective activities14. Estrogen receptor β expression is positively correlated with an improved clinical response to therapy and its loss is considered as an important step of cancer progression15. Therefore, it is of interest to find a selective estrogen receptor modulatory agent. As prenylated flavonols are potent phytoestrogens, it is worthy to find a selective estrogen receptor modulatory agents from this class of chemicals. However, due to their limited abundances in nature, it is required to develop an efficient synthesis technique. Though Epimedium genus is a good source of icaritin derivatives, the icaritin level is very low when determined by HPLC-MS16. As icariin is abundant in plants of Epimedium genus, using it as initial substrate to synthesize icaritin and its derivatives can be a possible way.There are two classes of estrogen receptors, α and β. They are expressed in different tissues. Estrogen receptor α regulates the proliferation and differentiation of breast cancer cells. However, estrogen receptor β antagonizes the activities of estrogen receptor α, inhibiting carcinogenesis and tumor progressionIcariin, β-dextranase, β-glucosidase were purchased from Solarbio . Cellulase was obtained from Yakult pharmaceutical Industry Co., LTD . HPLC grade acetonitrile was purchased from ANPEL Laboratory Technologies Inc. .Icariin (100 mg) were added into 25 ml of water. Five milligrams of enzymes were added to start the hydrolysis reaction at 37 °C for 2.5 h. The reaction products were extracted by acetyl acetate. The extract was dried by a vacuum rotary evaporator and redissolved in methanol. The products were qualified and quantified by ultra-performance liquid chromatography.The effects of temperature, time and enzyme type were investigated. Three enzymes, including β-dextranase, β-glucosidase and cellulase, were tested to compare their hydrolysis efficiencies. When analysing the effect of temperature, 20, 37, 60 and 80 °C were used. The time were set as 1.5, 2.5, 3.5 and 4.5 h, respectively.Icariin (100 mg) were suspended in 25 ml of water. Trifluoacetic acid (TFA) was added to a final concentation of 2 M. The hydrolysis was conducted at 60 °C for 1 h. The reaction products were extracted by acetyl acetate. The extract was dried by vacuum rotary evaporator and redissolved in methanol. The products were qualified and quantified by ultra-performance liquid chromatography.Hydrolysis by cellulase first and then TFA: Icariin (100 mg) were added into 25 ml of water. Five milligrams of cellulase were added to start the hydrolysis reaction at 37 °C for 2.5 h. Then TFA was added to a final concentation of 2 M. The hydrolysis was conducted at 60 °C for 1 h. The reaction products were extracted by acetyl acetate. The extract was dried by vacuum rotary evaporator and redissolved in methanol. The products were qualified and quantified by ultra-performance liquid chromatography.Hydrolysis by TFA first and then cellulose: Icariin (100 mg) were added into 25 ml of water. TFA was added to a final concentation of 2 M. The hydrolysis was conducted at 60 °C for 1 h. The hydrolysates were dried by vacuum rotary evaporator. Deionized water was added and dried again. This step was repeated for four times to completely remove acid. The dry products were redissolved in 25 ml of water. Five milligrams of cellulase were added to start the hydrolysis at 37 °C for 2.5 h. The reaction products were extracted by acetyl acetate. The extract was dried by vacuum rotary evaporator and redissolved in methanol. The products were qualified and quantified by ultra-performance liquid chromatography.d4 or acetone-d6. 1D and 2D NMR spectra were recorded.The enzymatic products were purified by semi-preparative reversed-phase liquid chromatography. 1.5 g of reaction products were loaded into a column with 15 × 460 mm. Methanol and water were used for elution. The column was equilibrated at 10% methanol for 12 min; 12–52 min, 75% methanol; 52–92 min, 80% methanol; 92–132 min, 85% methanol; 132–172 min, 90% methanol; 172–212 min, 100% methanol. The flow rate is 10 ml/min. The structures of the products were characterized by nuclear magnetic resonnance (NMR) spectroscopy and mass spectrometry (MS) . The purified products were vacuum evaporated to dryness, dissolved in methanol-17. The analyses were performed on an Agilent ZORBAX SB-C18 column . The flow rate was 0.3 ml/min, the column temperature was set at 40 °C, and the injection volume was 10 μl. The chromatogram was monitored at 280 nm. The mobile phases comprised solvents A (ultrapure water) and B (methanol). The elution program was as follows: 0–30 min, 5–100% B; 30–40 min, 100% B18.The analyses of reaction products and determination of conversion rate were performed on an Agilent 1260 Infinity UPLC system m/z 50–1000. The daughter ions were monitored at a collision voltage range of 28–42 eV19.UPLC-MS/MS was analysed on a maXis LC-ESI-QTOF-MS system  equipped with an Agilent ZORBAX SB-C18 column . The elution program was the same as UPLC analysis. Ionization of the analytes was achieved by using electron spray ionization interface in negative mode. The collision voltage was 10 eV. Mass scan was set in the range of 20. Autodocktools (version 1.5.6rc3) was used to dock estrogen receptor α/β and the tested chemicals21. The protein was checked for any misssing atoms, removed water and added hydrogen. The ligand was drawed and saved as a mol2 file. It was opened in autodocktools and saved as PDBQT file. A grid was generated for protein with the ligand centered. Genetics algorithm was used for docking22. The binding affinity was recorded to evaluate the potential to be estrogen receptor agonist.The X-ray structure of estrogen receptor α and β in complex with agonist -(4-hydroxyphenyl)-6-hydroxy-cyclopentyl[c]3,4-dihydro-2H-1- benzopyran (PDB id: 2i0j and 2i0g) were used as the templates1H and 13C chemical shifts of this chemical are listed in Table 1, Fig. 23. It confirmed that these enzymes could not degrade this unit. The above information confirmed the presence of baohuoside I (2).Three enzymes, including cellulase, β-glucosidase and β-dextranase, were used to hydrolyse icariin, respectively. There was only one product generated after enzyme hydrolysis Fig. . It was − at m/z 513.1761 was observed. m/z 367.1112 indicated the loss of rhamnosyl. Two peaks at m/z 323.0921 and 311.0573 suggested the cleavage of prenyl and loss of C3H6 and C4H8 moieties, respectively24.UPLC-QTOF-MS is used to determine the precise molecular weight and fragmentation pattern of baohuoside I in negative mode Table . A paren25, its effectiveness was much lower than cellulase in this work  were not detected. It indicated the removal of rhamnosyl from icariin. By comparison with NMR signals of icariin, 3 was identified as icariside I.Icariin was hydrolysed by TFA and four products were detected Fig. . They we4 was analysed to have a parent ion [M-H]− at m/z 547.1892. It was 18 larger than icariside I (3), which implicated the presence of H2O. In NMR spectra, a quaternary carbon signal C-3″ at 71.7 ppm and C-2″ signal at 18.8 ppm further indicated the hydration of prenyl. Through comparing with NMR signals of icariside I and literatures27, chemical 4 was identified as maohuoside A.Chemical 5 was detected. It had a parent ion [M-H]− at m/z 513.1759. The fragment ion at m/z 367.1115 indicated the loss of rhamnosyl. The anomeric signals of 5.44/102.2 ppm in NMR spectra also confirmed the presence of rhamnosyl. A cyclization between C-3″ of prenyl and 6-OH was observed by the NMR signals 2.84/16.8 (H-1″/C-1″), 1.88/32.1 (H-2″/C-2″), 77.5 (C-3″), 1.36/26.4 (H-4″/C-4″ and H-5″/C-5″). Therefore, chemical 5 was identified as anhydroicaritin 3-O-rhamnoside.A minor amount of chemical 2 and 5 contained rhamnosyl, but no glucosyl, while 3 and 4 contained glucosyl but no rhamnosyl. Rhamnosyl was easier to be cleaved than glucosyl by acid. In acidic conditions, the prenyl was unstable and readily to be hydrated or cyclized. No products without both rhamnosyl and glucosyl were detected. It indicated that TFA hydrolysis could not directly generate icaritin. Therefore, combination of cellulase and TFA hydrolysis was carried out in the following work.The four chemicals generated by TFA hydrolysis of icariin were compared. 6 was the dominant product which had a parent ion [M-H]− at m/z 367.1142. No rhamnosyl and glucosyl signals were observed in the NMR signals , icariside I (3), maohuoside A (4), icaritin (6), anhydroicaritin (7), wushanicaritin (8) and wushanicaritin 3-O-rhamnoside (9). Anhydroicaritin 3-O-rhamnoside (5) was not detected as a product. The yields of icaritin, anhydroicaritin and wushanicaritin were calcalated to be 28.8%, 16.9% and 25.3%, respectively.When icariin was hydrolysed by TFA firstly and then cellulase, seven chemicals were produced as the main reaction products Fig. . These c2) was not detected in the products. It was cyclized into anhydroicaritin 3-O-rhamnoside (5). The yields of icaritin, anhydroicaritin and wushanicaritin were calcalated to be 61.1%, 12.2% and 3.7%, respectively. Therefore, hydrolysis by cellulase firstly and then TFA would be a good choice for icaritin preparation.When icariin was hydrolysed by cellulase firstly and then TFA, baohuoside I . In the estrogen receptor α/wushanicaritin complex, H12 was predicted to seal the ligand-binding cavity and to generate a competent AF-2 that could interact with coactivator. This could be helpful to explain how wushanicaritin acted as an agonist.Figure Figure 30. It was easy to carry out and to produce at a large scale when comparing synthesis from kaempferol31. Icaritin, wushanicaritin and anhydroicaritin are good drug candidates due to their good performance against cancer and osteoporosis. Developing a cost effective technqiue is of great interest for medicine industry. As the prenylation pattern produced a significant effect on the activity performance, it is required to conduct more bioactivity evaluations, including the estrogen receptor modulatory mechanisms in vitro and in vivo, to understand the potents of these chemicals in medicines. Some bioactivites, like anti-breast cancer, anti-osteoporosis or memory improving capabilities, should be evaluated, as estrogen receptor modulatory behaviours are involved in the mechanism of action.A combination of cellulase and TFA hydrolysis was confirmed to be an efficient technique to synthesize icaritin, wushanicaritin and anhydroicaritin. This technique extended the application of cellulase in icariside preparation and made production of three aglycones in one pot possiblesupplementary information without marked changes


Despite recent progress in the preparation of feeder cells for human induced pluripotent stem cells (hiPSCs), there remain issues which limit the acquisition of feeder cells in large scale. Approaches for obtaining feeder cells quickly on a large scale are in immediate need. To reach this goal, we established suspension-adhesion method (SAM) and three-dimensional (3D) suspension method (3DSM). In SAM, mouse embryonic fibroblast (MEF) growth were fully inhibited by 10 μg/ml mitomycin-C (MMC) in 0.5 hours, and the feeder cells generated display higher adherent and recovery rates as well as longer survival time compared to conventional method (CM). 3DSM, an optimized method of SAM in which MEFs were cultured and MMC treated in suspension, was developed to lower the costs and workload using CELLSPIN System. The yield of feeder cells is several times the yield of SAM while the adherent and recovery rates and the capacity of supporting hiPSCs growth were not sacrificed. Hence, 3DSM is an economical and easy way to generate large-scale feeder cells for hiPSCs cultures. We previously reported to obtain iPSCs from human hair follicles-mesenchymal stem cells (hHF-MSC-derived iPSCs) using four Yamanaka factors 5. These iPSCs are capable of self-renewal and differentiate into various cell types, feeder cells are required to support their growth while maintaining pluripotency.Induced pluripotent stem cells (iPSCs) can be obtained from somatic cells by forced expression of a defined set of reprogramming factors, including either the combinations of Oct4, Klf4, Sox2, and c-Myc, or of Oct4, Sox2, Nanog, and Lin287. In that system, mouse feeder cells are replaced with human cells such as human fetal and adult fibroblasts8, human fetal muscle fibroblasts9, foreskin fibroblasts10, amniotic mesenchymal cells11, adipose-derived mesenchymal stem cells12, bone marrow mesenchymal stem cells15, placenta-derived mesenchymal stem cells16, multipotent mesenchymal stem cells of desquamated endometrium17, and decidua-derived mesenchymal cells18.Feeder cells are known to produce growth factors, adhesion molecules, and extracellular matrix. The most widely used feedder cells include mouse embryonic fibroblasts (MEFs). Recently, a xeno-free cell culture method was established to avoid contamination by pathogens and animal proteins19. To achieve large-scale production of hiPSCs, a large-scale culture system for hiPSCs expansion using the E8 chemically defined and xeno-free medium has recently been developed20. However, the efficiency of human feeder layers in the maintenance of undifferentiated human embryonic stem cells (hESCs) growth is not as high as that of mouse feeder cells due to the lower level of secretion of activin A21. Although there are numerous chemically defined and xeno-free media such as mTeSR and StemPro conducive to the production of hiPSCs, the inclusion of human serum albumin and human sourced matrix proteins makes those conditions prohibitively expensive, impractical for routine use, and not truly completely defined, which limits their use in large-scale amplification of hiPSCs23. Thus, the feeder-based system remains an important method of hiPSCs propagation.In spite of recent progress in hiPSCs culture conditions, large-scale production of hiPSCs by robust and economical methods has been one of the major challenges for the translational realization of hiPSCs technology30 or MMC34. Gamma irradiation can treat more cells than MMC at one time, but the γ-ray radiation source of Cobalt-60 is rare and costly. The affordability, flexibility, and convenience of MMC make it a good routine protocol to prepare feeder cells. For the feeder-based culture system, MEFs of CF-1 strain mice characteristically exhibit active proliferation, high-density dependence, and being aging-prone at low-density, and are still the most common feeder source for hiPSCs cultures.Currently, feeder cells are mitotically inactivated either by gamma irradiation35, CF-1 MEFs of 80–90% confluence were inactivated and used as feeder cells to maintain hiPSCs or for the production of conditioned medium. However, low yield with high costs need to be optimized as individual dishes or flasks accommodate limited numbers of cells in CM. Failure to fully inactivate MEFs in stratified growth by MMC is another problem. At low density, however, MEFs are aging-prone and their supportive capacities for iPSCs are compromised. Hence, MMC processing time is inflexible. Therefore, it is necessary to find new approaches that not only can be used for the production of feeder cells on a large scale in a short time, but also can ensure that MEF proliferation is sufficiently inhibited. To this end, we recently established a suspension-adhesion method (SAM) and a three-dimensional (3D) suspension method (3DSM) by optimization of CM. These new methods for feeder preparation will promote the advances and applications of induced pluripotent stem cell technology.In the conventional method (CM) for feeder cells preparationAll methods were carried out in accordance with relevant guidelines and regulations of the Ethics Committee of the Norman Bethune College of Medicine, Jilin University. All experimental protocols were approved by the Ethics Committee of the Norman Bethune College of Medicine, Jilin University. Informed consent was obtained from all subjects. Animal experiments were performed in accordance with a protocol approved by Jilin University School of Medicine Animal Care and Use Committee [Animals use license: SYXK (Jilin) 2013-0005]. All mice were housed in a sterile environment and could access food and water commodiously as outlined in the institutional guidelines.5. hHF-MSC-derived iPSCs were maintained on mitotically inactivated CF-1 MEFs in hESCs culture medium  . The hHF-MSC-derived iPSCs were split with 1 mg/ml collagenase type IV  for 30 min at 37 °C, at a ratio of 1:5 every 6–7 days. Conditioned medium was collected as described36. All manipulations and cultivations were performed in a good manufacturing practice (GMP)-compliant facility.CF-1 mouse embryonic fibroblasts (MEFs) were derived from day-12.5 embryo pools of CF-1 strain mice. The cells were cultured in MEF medium  and maintained at 100% confluence. hHF-MSC-derived iPSCs were cultured as described previouslyg for 5 min, and re-suspended in MEF medium. Cells were counted and frozen for later use.CF-1 MEFs of passage 3 (P3) at 80–90% confluence were inactivated with 10 μg/ml of MMC  for 0, 0.5, 1.0, 1.5, and 2.0 h at 37 °C. After the incubations, the cells were washed with PBS 6 times, trypsinized, centrifuged at 180 × 4–1.1 × 105 cells/cm2 in 10 cm-dishes. MMC (10 μg/ml) was added after 2.0–3.0 h at 37 °C. Medium containing MMC was discarded 0.5–3.5 h post-treatment. The cells were then washed with PBS 6 times, trypsinized, centrifuged at 180 × g for 5 min, and resuspended in MEF medium. Cells were counted and frozen for later use.We prepared feeder cells by SAM according to Fig. After connecting the CELLSPIN System  to the incubator, spinner flasks were sterilized by autoclaving.6 cells/ml. MMC were added at 10 µg/ml. After incubation for 0.5, 1.0, 1.5, and 2.0 h at 37 °C, the cells were centrifuged at 180 × g for 5 min, washed with PBS 3 times, resuspended in MEF medium, counted, and cryopreserved for later use.We prepared feeder cells by 3DSM according to Fig. 5 cells per well in 24-well plates and counted using trypan blue staining solution on days 1, 3, 5, and 7.The feeder cells of CM, SAM and 3DSM were plated at 1 × 104 cells per well in 24-well plates. The cells were exposed to 50 μM EdU for 8–24 h at 37 °C. The cells were fixed with 4% formaldehyde for 15 min at room temperature and treated with 0.5% Triton X-100 for 20 min at room temperature for permeabilization. After 3× washes with PBS, the cells were treated with 100 μM 1 × ApolloR reaction cocktail for 30 min. Subsequently, cells were stained with 200 μl/well of Hoechst 33342 (5 μg/mL) for 30 min and visualized under a fluorescent microscope . The number of EdU-positive cells was counted using open-source digitizing software . The percentage of EdU-labeled cells was calculated as follows: EdU-positive cell number/Hochest-positive cell number.The inhibitory effect of MMC on the proliferation of MEFs was measured using an EdU assay kit  according to the manufacturer’s instructions. Briefly, inactivated MEFs were cultured in triplicate at 2 × 106 cells/T25-flask. Non-adherent cells were collected and counted on the second day. Direct adhesion rates were then evaluated.Inactivated MEFs freshly prepared with CM, SAM and 3DSM were plated at 1 × 10The frozen MMC-inactivated MEFs of CM, SAM and 3DSM in T25-flasks were resuscitated on days 1, 7, 21, 28 respectively after cryopreservation and the non-adherent cells were collected and counted the following day to analysis the recovery rates of MMC-inactivated MEFs.Frozen MMC-inactivated MEFs of CM, SAM and 3DSM were resuscitated and cultured at the same density. Replace the media every three days and observe cell survival time and status.assay was determined by flow cytometry . Briefly, 1 × 106 inactivated and cryopreserved MEFs of CM, SAM, and 3DSM were plated in T25 flasks. The cells were harvested and fixed in 70% ice-cold ethanol for 24 h, then stained with propidium iodide (PI). The different cell cycle phases were analyzed using a FACS Calibur instrument.Cell cycle 37 with modifications. Briefly, a set of MMC calibration solutions with different concentrations were made from a standard stock solution of 1 mg/ml MMC by dilution with methanol: water  solution. Aliquots (400–500 μl) were analyzed by HPLC-MS/MS to construct a standard curve. All chomatographic experiments were carried out at room temperature using HPLC  equipment. MS/MS analysis was performed using LCQ Deca XP plus equipment  with an ESI source in the positive ion mode. The daughter ions with m/z 274 for MMC were monitored via an ion trap mass analyzer.High performance liquid chromatography-Mass Spectrometry/Mass Spectrometry (HPLC-MS/MS) was performed as describedThe hHF-MSC-derived iPSCs pluripotency markers were assayed using a Fluorescent Human ES/iPS Cell Characterization Kit from EMD Millipore  according to the manufacturer’s instructions.hHF-MSC-derived iPSCs were harvested by collagenase type IV. After settling, the supernatant was aspirated and the MEF medium was replaced to remove the MEF. hHF-MSC-derived iPSCs were transferred to petri dishes in the MEF medium. After an 8 days floating culture, embryoid bodies were transferred to gelatin-coated plates and were then incubated for another 16 days. After the incubation, the cells were fixed with 4% paraformaldehyde in PBS and then incubated in PBS containing 5% normal goat serum , 1% bovine serum albumin , and 0.2% Triton X-100. The primary antibodies were as follows: anti-alpha smooth muscle actin , anti-alpha fetoprotein polyclonal antibody , and anti-Nestin . vimentin, desmin , βIII-tubulin . The secondary antibodies were Alexa 555-labeled anti-mouse IgG . Nuclei were stained with 1 mg/ml Hoechst 33342 .Karyotype of the hHF-MSC-derived iPSCs analysis was evaluated and performed at the Reproductive Center of the First Hospital of Jilin University using standard protocols for high-resolution G-banding.6 cells per site). After 12 weeks, teratomas were retrieved from the injection site, dissected, and fixed with 10% formaldehyde in PBS. Paraffin embedded tissue sections were then prepared and analyzed with Hematoxylin and Eosin (H&E) staining.The hHF-MSC-derived iPSCs were injected intramuscularly into non-obese diabetic/severe combined immune deficient (NOD/SCID) mice in DMEM containing 10% FBS  and were used as feeders. MEFs were treated with 10 μg/ml MMC for 0, 0.5, 1.0, 1.5, or 2.0 h respectively. Then, the culture media were replaced with MEF media and cell numbers were counted on days 1, 3, 5, and 7. As shown in Fig. P < 0.01) in the culture on days 1, 3, 5, and 7 after being treated with 10 µg/ml MMC for 0.5 h by CM, indicating that MMC treatment for 0.5 h failed to inhibit MEF proliferation in CM. However, the number of cells significantly decreased in CM when using MMC for 1.0, 1.5, and 2.0 h, and statistically significant differences were found between days 1, 3, 5, and 7 (P < 0.01). It is worth mentioning that some of the cells were unhealthy in the MMC 1.0–2.0 h groups and died in three days.We prepared feeder cells by conventional method (CM) as previous reports45. To identify the optimal time of inhibition in suspension-adhesion method (SAM), we performed a time course experiment, exposing the MEFs to 10 µg/ml MMC for 0–6.0 h. Phase contrast microscopy images and cell counts . MEFs exposed to MMC for 0.5–4.0 h did not change in number over 7 days. The number of cells did not perceptibly increase or decrease on days 1, 3, 5, or 7 for cells treated with MMC for 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, or 4.0 h, and no statistically significant differences were found in cell numbers on days 1, 3, 5, or 7. However, the number of cells decreased on days 1, 3, 5, and 7 for MEFs treated with MMC for 4.5, 5.0, and 6.0 h (P < 0.05) probably due to cell death. Take together, our data suggest that treatment with 10 µg/ml MMC for 0.5–4.0 h is sufficient to suppress the proliferation of MEFs but maintains acceptable viability of feeder cells. Thus, we prepared feeder cells with SAM using 10 µg/ml MMC for 0.5–4.0 h.To optimize CM, we first prepared feeder cells according to Fig. nts Fig.  showed tP < 0.001). However, there was no significant difference between the gamma ray group and cells treated with MMC for 0.5–3.5 h (data of 2.0–3.5 h are not shown). Together, these results further confirmed that 10 μg/ml MMC for 0.5–3.5 h was sufficient to inhibit MEF proliferation.To further validate the suppressive effect of MMC on the proliferation of MEFs, we labeled the cells with EdU and determined the cell proliferation rate after MMC treatment Fig. . There wP < 0.01). The number of cells did not change on days 1, 3, 5, or 7 after treatment with MMC for 0.5–2.0 h.Both the SAM and CM require MEFs to attach to the flasks/dishes, which limits the processing efficiency of the feeder cells. To increase the processing efficiency and reduce costs Table , we usedTo determine the quality of feeder cells prepared by SAM and 3DSM, we compared the shortest processing time (SPT) of inhibition MEF proliferation, the direct adherent rates (DAR), recovery rates (RR) and survival time (ST) of MEFs harvested from SAM, 3DSM and CM.The aforementioned data showed that MMC for 0.5 h was sufficient to inhibit the proliferation of MEFs in SAM and 3DSM Figs  and 5A,BNext, we compared the DAR of inactivated MEFs treated with MMC for 0.5, 1.0, 1.5, and 2.0 h in CM with SAM and 3DSM Fig. . The DARSimilarly, the RR of inactivated MEFs treated with MMC for 0.5–2.0 h in SAM was up to 89% ± 5% and 3DSM was 86% ± 3%, whereas the rate in CM was 71% ± 3% Fig. , indicatNotably, the RR of 3DSM-feeder cells treated with MMC for 0.5, 1.0, or 1.5 h were higher (86% ± 5%) than that for 2.0 h (81% ± 3%) Fig. . These fWe then cultured the MEFs inactivated with MMC for 2.0 h by CM, SAM and 3DSM at a high density Fig. . The surThus, our data suggest that SAM and 3DSM were more efficient than CM in inhibiting cell proliferation. SAM/3DSM-derived feeder cells can better maintain long-spindle-cell growth, and exhibit an improved DAR, RR, and ST compared to those derived using the CM. Therefore, these data further support our conclusion that the qualities of feeder cells prepared in SAM and 3DSM are better than that in CM. Furthermore, SAM and 3DSM may be able to address the issues important for the application of feeder cells for hiPSCs culture, such as failure of full mitotic inactivation, poor quality of the feeder cells, and over-growth of the MEFs.46. However, there is no statistically significant difference in the distribution of cells prepared with CM , digestive tract epithelium (endoderm), bone tissue and osteoid tissue (mesoderm), cartilage (mesoderm), smooth muscle tissue (mesoderm), pavement epithelium (ectoderm), pigmented epithelium (ectoderm), neural tissues (ectoderm) Fig. .Our data suggest that the qualities of feeder cells prepared in SAM and 3DSM are better uniformity, more standardized than that in CM. SAM and 3DSM could not only increase the processing efficiency of feeder cells but also reduce costs, and lower workload Table . FlexibiIn SAM, MEFs are in single cell suspension and have more accessibility to MMC than CM, therefore are easier to be inhibited by MMC. Only those healthy MEFs that adhere to culture dish/flask bottom are collected. Besides, additional 4-day culture of P3 MEFs increased 300% cell yield. Additionally, no 80%-90% growth restriction for MMC treatment is necessary in SAM or 3DSM.3DSM has additional advantages over SAM: no subculture is needed, hence less workload; and less culture dishes/flasks are used. In the 3DSM, the MEFs are in suspension during the entire process and access MMC thoroughly, which could improve the efficiency of inhibiting cell proliferation. In addition, since enzymatic digestion is omitted during feeder cell preparation, no damaged cells caused by enzyme digestion exist in our culture system which may also contribute to the high yield of MEFs in 3DSM. According to the density of the treated cells, the yield of a single spinner flask in 3DSM was greater than that achieved using a 10-cm dish, in the same volume of culture medium. Therefore, 3DSM can be used to rapidly produce feeder cells on a large-scale which could be very helpful in mass production of hiPSCs.47. Feeder cells at high density  may cause rapid depletion of nutrients and oxygen and physically hinder the attachment and growth of hiPSCs. MEFs at low density may result in insufficient levels of extracellular matrix, secreted factors, and intercellular contacts. Newly prepared feeder cells are used, DAR of the cells would affect the feeder cell density. That is, the higher DAR, the higher feeder cell density. Variability in feeder cell density may affect the outcome of hiPSCs culture. Our data showed that the adhesion rates of freshly prepared and frozen feeder cells in SAM and 3DSM were far higher than in CM. Thus, less SAM and 3DSM feeder cells are needed to support hiPSCs.hiPSCs were feeder-density dependentIt usually takes the cells 4–20 days for hHF-MSC-derived iPSCs to revive from cryopreservation and the routine cultures were split every 5–7 days. Our data Fig.  showed fThe development of reprogramming technology promotes the advance of stem cell biology research. Adult cell reprogramming by ectopic coexpression of transcription factors and gene editing brings new hope to the treatment of many human diseases, especially personalized regenerative medicine treatments. In response to the requirement of rapid and large-scale hiPSCs production, it has become a very active research topic preparing a large number of feeder cells of high-quality. We developed SAM and 3DSM to prepare feeder cells on a large scale in this study. However, how feeder cells maintain the self-renewal capacity and pluripotency of hiPSCs remains unclear and further studies are required to elucidate these issues.In summary, we successfully established SAM and 3DSM for the preparation of feeder cells for hiPSCs culture. In SAM, MEF growth is fully inhibited by MMC which is essential for hiPSCs cultures using MEFs as feeder cells. Feeder cells prepared by SAM and 3DSM possess a high direct adherent and recovery rate, and show a high quality in supporting hiPSCs. To increase the processing efficiency and reduce costs, 3DSM was developed to prepare the feeder cells and high processing efficiency of healthy cells was harvested in a short time. Therefore, 3DSM is a more economical way to generate feeder cells for large-scale production of hiPSCs.Supplementary Information


Arnica montana L on the gene expression of the human monocytic leukemia cell line THP-1, reporting that homeopathic preparations of Arnica upregulated the expression of genes, such as CCL7, CXCL1, CXCL2, interleukin 8, and bone morphogenetic protein 2 (BMP2) , a percentage of 0.036% sesquiterpene lactones, should correspond to 0.72 μg, i.e., 72 pg in the dilution 2c  and to 8 and 12  for THP-1 monocytes treated with IL-4 (M2-type), where p < 0.01 where 13 (M2) vs. 4 (M0) (2) and addressed to drive any molecular machinery to a highly controlled response to a stimulus  amplified signals vs. noises, i.e., reduced the difference between noises and statistical variability.We evaluated statistic distribution of SEM variability to ascertain if any reported outliers due to bias error affected the parametric or non-parametric behavior of the investigated samples. The authors used Wilcoxon test, although no special requirements to call for a non-parametric test were requested . Effectsp = 0.676; 3c, p = 0.279: 5c, p = 0.254; 9c, p = 0.792. Data were confirmed in a Kolmogorov–Smirnov test and the Shapiro–Wilk test, and only the D’Agostino–Pearson test gave 15c, p = 0.07) (z = 3.5), showed, respectively 18 and 14 outliers in the SEM distribution of M0-type TPH-1 cell line and 4 and 0 for SEM distribution in M2-type THP-1 cell line, confirming a more constant dynamics of gene expression in M2-cells , while in the M2 group they occurred only in the 2 h cluster   . A possi = 0.07) . A RosneM2-cells . Outlier < 0.04) . Althougp = 0.000865; 5c, p = 0.0134, 15c, p = 0.00020) but also that regression data for the other comparisons contained some bias, regarding either normal fit, Durbin–Watson test or the constant variance test (CVT)  . The prest (CVT) , 22.Statistic significance appeared as highly frequent in a more stable experimental condition and in dilutions with active principles very far from a pharmacological bioactivity and without an apparent dose–response behavior. Probably, not all the distributions should be evaluated with a Wilcoxon due to the existence of outliers much more frequent only in certain experimental conditions.Arnica water/ethanol extracts should be, therefore, replicated.In conclusion, data arrays as presented by the authors may contain some discordant outlying observations, thus affecting results reliability. The effects of Arnica is really effective on inflammatory gene upregulation in innate immune cells.Our consideration is that by replicating the data, we could appreciate if homeopathic SC read the original paper, discussed it with GB, statistics were re-evaluated by SC with GB, GB gave insights from the literature, SC re-wrote the paper, GB revised it, and SC reviewed the revision and submitted the paper once GB has given his agreement.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


To investigate the safety and optical quality of small-incision lenticule extraction (SMILE) combined with monovision, and patient satisfaction with the procedure.The present study assessed a non-random case series involving 60 eyes of 30 patients (mean age 45.53 ± 3.20 years [range 41 to 52 years]) treated bilaterally using the VisuMax 500 system  between January and July 2016. The target refraction was plano for the distance eye, and between − 0.5 and − 1.75 diopters (D) for the near eye. Visual acuity, refraction errors, ocular aberrations, and satisfaction questionnaire scores were calculated 1 year after surgery.P = 0.036). Binocular uncorrected distance visual acuity was related to preoperative spherical diopter .All surgeries were uneventful, with a mean safety index of 1.03 and 1.04 in dominant and nondominant eyes, respectively. Binocular uncorrected distance visual acuity of all patients was ≥20/32, while binocular uncorrected near visual acuity was ≥20/40 1 year postoperatively. Higher-order aberration , spherical  and coma aberration  were identical between dominant and nondominant eyes after surgery. The overall satisfaction rate was 86.7% (26/30), with large contributions from age (OR = 1.76 95% CI: 1.03–2.53; Monovision appears to be a safe and effective option for myopia patients with presbyopia who are considering the SMILE procedure. Patients with younger age were more satisfied with the procedure. Presbyopia refers to an impairment of near vision that is common among adults > 40 years of age, resulting from declined amplitude of accommodation . CurrentWith advances in refractive surgery technology, small incision lenticule extraction (SMILE) is becoming more prevalent due to its excellent safety, efficiency, and good preservation of corneal biomechanics , 7. To tThe present study was a non-comparative case series, and was approved by the Ethics Committee of the Eye and ENT Hospital of Fudan University  and a written informed consent from each patient was obtained before surgery as a standard protocol preoperatively. All procedures were adhered to Declaration of Helsinki. Patients who underwent bilateral SMILE (performed by the same surgeon [ZX]) between January and July 2016, with available 1-year follow-up data, were reviewed. A total of 30 patients  were enrolled. The cohort had a mean preoperative spherical diopter (D) of − 6.12 ± 2.39 D (− 1.5 to − 10 D), cylinder of − 0.79 ± 0.62 D (− 3.0 to 0 D), binocular uncorrected near visual acuity ranging from 20/32 to 20/20, and add 0.85 ± 0.56 D (0 to 2.25 D).Inclusion criteria were as follows: ≥40 years of age; best corrected visual acuity ≥20/20 in either eye; spherical diopter ≤ − 10.0 D; add > 0 D; and cylinder ≤ − 3.0D. Exclusion criteria included severe eye comorbidities such as diabetic retinopathy, age-related macular degeneration, cataract causing visual impairment, or glaucoma with significant field loss, and a history of severe amblyopia or strabismus.Regular preoperative examinations, including cycloplegic refraction, corrected visual acuity, slit-lamp examination, corneal topography , ocular aberration , and fundus examination were performed. The dominant eye was determined using the “hole test” . PatientThe 1-year examinations typically included manifest refraction, assessments of monocular and binocular uncorrected distance visual acuity (UDVA) (at 4 m), uncorrected neat visual acuity (UNVA) (at 33 cm) and corrected distance visual acuity (CDVA) under the same illumination. In addition, we constructed a questionnaire considering patient satisfaction including spectacle dependence for daily activities, halo, glare, visual fatigue, dry eye, and overall satisfaction . Each quThe surgical procedure was similar to the standard SMILE treatments described by the authors in a previous study . The domP < 0.05 was considered to be statistically significant.Statistical analysis was performed using SPSS version 22.0 , and all data are presented as mean ± SD. Visual acuity data are in LogMAR units. The paired t test was performed to compare root mean square (RMS) differences in ocular aberration, and the Wilcoxon signed-rank test was performed to compare safety indexes, which were nonlinear values between the dominant and nondominant eye. For satisfaction was subjectively graded on 4 ordered levels, orderly regression analysis was used to detect factors affecting satisfaction. Factors included in this analysis are age, sex, and preoperative spherical equivalent, which are independent variables.Spearman’s test was used to determine relationships between visual acuity and other parameters; P > 0.05) in the dominant and nondominant eye, respectively. In the dominant eyes, the percentage of UDVA ≥20/32 were 96.7%; in the nondominant eyes, the percentage of UDVA ≥20/40 was 76.7%. Predictability and accuracy are presented in Fig. All surgeries were uneventful, with no intraoperative or postoperative complications. The mean safety index was 1.03 and 1.04  (60 eyes).Ocular aberrations in both eyes are summarized in Table Results of the satisfaction survey revealed that 63.3, 6.7, and 3.3% patients experienced mild, moderate, and severe halo, respectively; 86.7% patients complained of dry eye, of which 69.2% was mild dry eye. Three patients still required reading glasses occasionally, and 6 required glasses when driving. The completely “glasses-off” rate was 76.7%. The mean satisfaction score was 8.32 ± 1.27 (range 5 to 10).P = 0.02). On that premise, age  was related to the satisfaction, while sex (P = 0.67) was not significant.In orderly regression analysis, age, and sex constitutes a significant mode .Spearman’s test revealed that preoperative spherical D was related to postoperative binocular UDVA (Monovision excimer laser correction has a considerable history in photorefractive keratectomy and laser-assisted in situ keratomileusis (LASIK), although various degrees of satisfaction have been reported in previous studies , 10. WitIn our study, the mean safety index was 1.04 and 1.03 in the dominant and nondominant eye, respectively, with no statistically significant differences found between eyes. The percentage of remaining or gained BCVA was 83.3 and 86.7% in the dominant and nondominant eyes, respectively. This result is consistent with previous SMILE results . LevingeAll patients in this study achieved a binocular UDVA ≥20/32, and the percentage of binocular UDVA ≥20/25 was 76.7%, which was a significant improvement from preoperative values. In terms of binocular UNVA, 100% of patients achieved UNVA ≥20/40, and the percentage of patients with UNVA ≥20/25 was 83.3%. Accordingly, SMILE combined with monovision was effective in both far and near vision. Similar results were also found in the study by Goldberg et al. , in whicSpherical aberration and HOA were unchanged after surgery, while coma increased significantly. Ocular was usually associated with postoperative glare and halo. It has been reported that SMILE-induced aberration can be restored over a long period . TherefoThe satisfaction rate in this study was 86.7%, which is different from that of contact lens with monovision, which ranged from 60 to 80% in a previous study,  and alsoAlthough age has been considered to be unrelated to the success of monovision,  we foundOne limitation of the present study was the lack of a control group and, given the relatively small sample size, it was difficult to make comparisons between subgroups. Further comparison between groups stratified according to different target refraction or sex would be interesting areas of investigation. Furthermore, we only analyzed data 1-year postoperatively, and consecutive observation would be helpful to further understand the adaption period of these patients.In conclusion, SMILE combined with monovision appeared to be safe and effective in a population of presbyopic patients. Patients with younger agewere more satisfied with the procedure.


Previous study showed that pyroglutamate aminopeptidase 1 (PGP‐1) has a relationship with the immune response in cells. However, whether PGP‐1 is involved in inflammatory response in vivo and can serve as a new inflammatory cytokine are still unclear. To address these issues, a new near‐infrared fluorescent probe, which exhibits high selectivity and super sensitivity, is developed. With this probe, the up‐regulation of PGP‐1 (evidenced by western blot) in BALB/c mice legs and livers under the stimulation of two main immunopotentiators is revealed for the first time. The occurrence of inflammatory process (including tissue necrosis) in mice is determined by up‐regulation of tumor necrosis factor‐α and hematoxylin‐eosin staining. Interestingly, it is revealed for the first time that knocking down PGP‐1 leads to the weakness of inflammatory process in RAW264.7 cells. These new findings suggest that PGP‐1 is indeed involved in inflammatory response in vivo and can be a new inflammatory cytokine. The new probe  but also high selectivity and super sensitivity to PGP‐1 under the physiological conditions. Using the probe as well as animal imaging device, the up‐regulation of PGP‐1 in BALB/c mice legs and livers under the stimulation of immunopotentiators is revealed for the first time, which is evidenced by Western blot (WB). And the occurrence of inflammatory process (including tissue necrosis) in mice is determined by up‐regulation of tumor necrosis factor‐α (TNF‐α: a common inflammatory factor) and hematoxylin‐eosin staining (H&E staining). Very interestingly, it is revealed for the first time that inhibiting PGP‐1 with small interfering RNA (siRNA) leads to the weakness of inflammatory process in RAW264.7 cells (weak up‐regulation of TNF‐α). These important findings as well as the previous resultsInflammation, a protective response to damage factor that involves immune cells, blood vessels, and molecular mediators, plays a significant role in many diseases,be Figure1a exhiex/em = 670/700 nm) and good biocompatibility,We first prepared probe Figure a as an N−1  – 338.37 (R = 0.998), where ∆F is the difference of fluorescence intensity of probe after and before reaction with PGP‐1. The detection limit−1 of PGP‐1, which is the lowerst as far as we know. Notably, the probe also exhibits rather high selectivity to PGP‐1 . According to our hypothesis, the enzymatic cleavage reaction of the probe by PGP‐1 will cause the release of hemicyanine fluorophore . In addition, inhibitor experiments with iodoacetamide  also supported that the fluorescence off–on response of the probe arises from the enzymatic action of PGP‐1. On the basis of Michaelis–Menten equation,Km) of probe for PGP‐1 were determined to be 7 × 10−6m , which is much lower than that of commercial probe , indicating probe has stronger affinity for PGP‐1. Moreover, the probe shows good biocompatibility .Then, spectroscopic properties and analytical performance of probe were studied in detail. As shown in Figure Figurehttp://immunopotentiatorNext, we explore whether PGP‐1 has a relationship with inflammatory processes in living bodies by detecting the variation of PGP‐1 during the processes with our probe and WB analysis. As shown in FigureIn order to further verify the relativity between up‐regulation of PGP‐1 and inflammatory processes in living bodies, the liver inflammation model (one common model) experiments were performed. Mice were first intraperitoneal injected with lipopolysaccharides/D‐galactosamine[[qv: 2a]]  and then prepared to image. As shown in FigureFinally, to further investigate the relationship between PGP‐1 and inflammation, the small RNA interference (siRNA) experiment was used to deeply explore this issue. As shown from In conclusion, we have developed a new NIR fluorescent probe, which exhibits good selectivity and super sensitivity. Using the probe, the up‐regulation of PGP‐1 in BALB/c mice legs and livers under the stimulation of immunopotentiators is revealed for the first time, which is evidenced by WB analysis. And the occurrence of inflammatory process (including tissue necrosis) in mice is determined by up‐regulation of TNF‐α or H&E staining. Very interestingly, it is revealed that inhibiting PGP‐1 with siRNA leads to the weakness of inflammatory process in RAW264.7 cells (weak up‐regulation of TNF‐α). These important findings as well as the previous results suggest that PGP‐1 may be a new inflammatory cytokine, and provide us a new viewpoint for inflammation‐related medicinal development and disease treatment. And the relationships between some other important inflammation‐related bioactive substances or signaling molecules and PGP‐1 can be explored based on our new findings. Besides, it is believed that with our probe more in vivo biological functions of PGP‐1 can be revealed.Reagents: N‐(tert‐butoxycarbonyl)‐L‐pyroglutamic acid (BOC‐L‐pyroglutamic acid) was obtained from Tokyo Chemical Industry Co., Ltd. N,N‐Diisopropylethylamine (DIPEA) and 3‐nitrophenol were purchased from Acros Organics. Stannous chloride (SnCl2) was obtained from J. K. Chemical Co., Ltd., Beijing, China. 2‐(7‐Aza‐1H‐benzotriazole‐1‐yl)‐1,1,3,3‐tetramethyluronium hexafluorophosphate (HATU) and trifluoroacetic acid  were purchased from Alfa Aesar Chemicals. 11‐Chloro‐1,1′‐di‐n‐propyl‐3,3,3′,3′‐tetramethyl‐10,12‐trimethyleneindatricarbocyanine iodide (IR‐780 iodide), FCA, LPS, D‐Gal, dimethyl sulfoxide (DMSO), iodoacetamide, esterase, prolidase, trypsin, leucine aminopeptidase, dipeptidyl peptidase IV, and commercial PGP‐1 probe (L‐pyroglutamic acid 7‐amido‐4‐methylcoumarin) were purchased from Sigma‐Aldrich. Fibroblast activation protein was purchased from R&D Systems Inc. PGP‐1  was obtained from State Key Laboratory of Antibody Medicine and Targeted Therapy, Shanghai, China. Lipidosome 3000, Roswell Park Memorial Institute‐1640 medium (RPMI‐1640), new‐born calf serum (CS), and RAW264.7 cell lines were purchased from KeyGEN BioTECH Co., Ltd., Nanjing, China. Proteins were pure as judged by Coomassie‐stained sodium dodecyl sulfate (SDS)‐polyacrylamide gel electrophoresis. Radio immunoprecipitation assay lysis buffer (CW2333) was purchased from CWbiotech Co. Ltd., Beijing, China. Agarose, poly(vinylidene fluoride) membranes, gel electrophoresis kits, WB kits, enhanced chemiluminescence kits (ECL), PGP‐1 siRNA (sequence: GGUUACAAAGGACUGGAUATTUAUCCAGUCCUUUGUAACCTT), and Braford protein assay kits were purchased from KeyGEN BioTECH Co. Ltd., Nanjing, China. Anti‐TNF‐α and Anti‐PGP‐1 antibody were purchased from Proteintech, USA. All other chemicals used were local products of analytical grade. Ultrapure water (over 18 MΩ cm) from a Milli‐Q reference system (Millipore) was used throughout. The stock solution (500 × 10−6m) of probe was prepared by dissolving requisite amount of it in DMSO. Stock solutions of other substances were prepared by dissolving in PBS or water.Apparatus: Fluorescence measurements were made on a Fluoromax‐4 Spectrofluorometer (France). UV–vis absorption spectra were measured in 1 × 1 cm quartz cells with a TU‐1900 spectrophotometer (China). 1H NMR and 13C NMR spectra were measured with a Bruker Avance 400 spectrometer in CD3OD. Electrospray ionization mass spectra (ESI‐MS) were measured with an LC‐MS 2010A (Shimadzu) instrument. High‐resolution ESI‐MS (HR‐ESI‐MS) were recorded on a TripleTof 4600 instrument . Cell imaging experiments were operated on a TSC SPS‐II confocal fluorescence microscope . Mice imaging experiments were operated on PerkinElmer IVIS Lumina XRMS instrument . Flow cytometer results were obtained from a BD LSRFortessa flow cytometry (USA). The WB signal was detected using an ECL kit. Cytotoxicity assay was made on a microplate absorbance reader .Synthesis of Probe: Hemicyanine fluorophore was obtained according to the previous method.[[qv: 5b]] BOC‐L‐Pyroglutamic acid , HATU , and DIPEA  were added to 30 mL of dichloromethane (CH2Cl2) at 0 °C. After stirring for 40 min, hemicyanine fluorophore  was added slowly, and the reaction mixture was further stirred at room temperature overnight. Then, the mixture was diluted with CH2Cl2, and washed three times with water (100 mL × 3). The solvent was removed by evaporation under reduced pressure, and the crude product was purified by silica gel chromatography eluted with CH2Cl2/methanol , affording intermediate S1 as a bluish violet solid . The 1H NMR and 13C NMR spectra of intermediate S1 are shown below in Figures S1 and S2 (Supporting Information), respectively. 1H NMR : 8.76–8.79 , 8.14 , 7.33–7.69 , 6.57–6.61 , 4.36–4.39 , 2.42–2.80 , 2.08–2.11 , 1.93–1.99 , 1.83 , 1.45 , 1.07‐1.10 . 13C NMR : 178.46, 175.37, 171.30, 160.99, 153.44, 149.47, 145.98, 142.18, 141.72, 141.54, 132.38, 128.88, 128.07, 127.34, 122.50, 118.24, 116.47, 114.43, 112.83, 105.71, 104.28, 83.31, 60.70, 50.83, 30.81, 28.77, 26.75, 23.59, 21.55, 20.98, 20.19, 10.20. HR‐ESI‐MS, calcd for C38H44N3O5 [M]+: m/z 622.3281; found: m/z 622.3245.2Cl2 (2.5 mL) was added dropwise to a solution of the above intermediate S1 in 5 mL of CH2Cl2 at 0 °C, and the reaction mixture was stirred at room temperature for 3 h. The solvent was removed by evaporation under reduced pressure, and the crude product was purified by flash silica gel chromatography eluted with CH2Cl2/methanol , affording probe as a violet solid . The 1H NMR and 13C NMR spectra of intermediate S1 are shown below in Figures S3 and S4 (Supporting Information), respectively. 1H NMR : 8.77–8.81 , 8.12 , 7.35–7.69 , 6.57–6.60 , 4.35–4.40 , 2.73–2.79 , 2.38–2.53 , 2.21 , 1.93–1.97 , 1.84 , 1.28–1.33 . Note that the peaks around 1.0 are grease peaks from purification.13C NMR : 180.18, 178.62, 171.90, 168.53, 160.96, 153.37, 145.81, 142.02, 141.57, 132.43, 128.83, 127.81, 127.26, 122.23, 118.03, 116.74, 114.39, 112.54, 105.80, 104.30, 61.23, 57.42, 50.65, 41.47, 31.52, 29.32, 26.75, 25.38, 24.52, 23.58, 20.99, 20.19, 10.17. HR‐ESI‐MS, calcd for C33H36N3O3 [M]+: m/z 522.2757; found: m/z 522.2746.Then, the probe was prepared as follows. Trifluoroacetic acid (2.5 mL) in CHGeneral Procedure for PGP‐1 Detection: Unless otherwise stated, all the fluorescence measurements were made according to the following procedure. In a test tube, 20 µL of stock solution of probe and appropriate volume of PBS were mixed, followed by addition of an appropriate volume of PGP‐1 or other substance solutions. The mixed solution was adjusted to 2 mL with PBS. After incubation at 37 °C for 25 min, the reaction solution was transferred to a quartz cell of 1 cm optical length to measure fluorescence with λex/em = 670/700 nm (both excitation and emission slit widths were set to 2.5 nm). For absorbance measurements, the same method was used. At the same time, a blank solution without PGP‐1 was prepared and measured under the same conditions for comparison. Data are expressed as mean ± standard deviation (SD) of three separate measurements.Molecular Docking: The molecular docking was carried out according to the previously describe.Cell Culture and Cytotoxicity Assay: RAW264.7 cells were cultured in RPMI‐1640 and supplemented with 10% CS. The cells were incubated at 37 °C with 5% CO2. The cytotoxicity of probe was tested on cells using a standard 3‐‐2,5‐diphenyl‐2‐H‐tetrazolium bromide assay (MTT assay), as described previously.Transfection: The transfection experiments were accomplished RAW264.7 according to the previous procedure.Cell Imaging and Flow Cytometer Experiment: Before cell imaging, the culture media were removed, and RAW264.7 cells  were washed using RPMI‐1640 for three times. Then, the cells were incubated with the probe (5 × 10−6m) at 37 °C for 25 min in RPMI‐1640, washed with RPMI‐1640 to remove the free probe, and finally subjected to fluorescence imaging or were preserved in PBS to flow cytometer experiment.Mice Imaging: All mice were purchased from the Card Vince Laboratory Animal Co. Limited  and animal experiments were approved by the animal care and use committee of the Ningbo University (Permit No. SYXK Zhe 2013‐0191). For dose experiment, female mice  were hypodermic injected with FCA  in right legs, and then fed with 7 d. For time experiment, female mice  were hypodermic injected with FCA (100 µL) in right legs, and then fed with 0, 4, 7, and 11 d, respectively. After these, mice were then hypodermic injected with 50 µL of probe (50 × 10−6m) to image. In liver inflammation experiments, female mice  were intraperitoneal injected with PBS (100 µL) and LPS/D‐Gal . After 2 and 16 h, mice were intravenous injected with 100 µL probe (50 × 10−6m) in the tail vain to image. Unless otherwise noted, data are expressed as mean ± standard deviation (SD) of three separate measurements.Histopathology Assessment of Mice Liver and Kidney: The modeled mice were sacrificed, the livers and kidneys were preserved in 4% formalin solutions and stained with H&E for histological analysis to evaluate the inflammatory conditions.WB: The cell lysates were prepared according to the previous method.[[qv: 8a]] The modeled mice were sacrificed, paws, livers, and kidneys were collected, and then the proteins were extracted. After these, the cell lysates and extracted proteins  were placed to WB experiments and WB results were obtained according to the previous method.Statistical Tests: The t analysis was operated according to the previous method.[[qv: 8a]]The authors declare no conflict of interest.SupplementaryClick here for additional data file.