We report a case of membranous nephropathy associated with type 1 autoimmune pancreatitis. A 58-year-old man presented with anorexia. Work-up revealed a mass in the pancreatic head, which was subsequently resected. Pathological examination showed diffuse infiltration of immunoglobulin (Ig) G4-positive plasma cells, which was compatible with the diagnosis of type 1 autoimmune pancreatitis. Serum IgG4 was elevated. He developed nephrotic syndrome around the time of the surgery. Kidney biopsy confirmed the diagnosis of membranous nephropathy. Immunofluorescent staining showed predominant glomerular IgG4 deposit among IgG subclasses. Tubulointerstitial nephritis, which is usually a dominant feature of renal involvement in IgG4-related disease, was not observed. The patient was treated with prednisolone and several immunosuppressants. During the course, the degree of proteinuria was associated with the serum IgG4 level. Serum antibody against phospholipase A2 receptor was negative. These findings together with IgG4-dominant glomerular deposit suggest that IgG4 may play a unique role in the pathogenesis of secondary membranous nephropathy caused by IgG4-related diseases. Immunoglobulin (Ig) G4-related disease (IgG4-RD) is characterized by increased serum IgG4 levels, caused by the infiltration of IgG4-positive cells into various organs –3. RenalWe describe a unique case of MN associated with IgG4-RD. The patient presented with IgG4-dominant deposit in the glomerular capillary wall and no associated TIN. The degree of proteinuria and serum IgG4 levels were associated and serum anti-PLA2R antibody was negative.The patient is a 58-year-old Japanese man with no previous medical history. In September 2010, he presented with anorexia. He was not on any medications. Serum transaminase and alkaline phosphatase levels were elevated. Urinalysis, renal function, serum total protein, and albumin levels were as follows: urinalysis: 3+ protein and 3+ occult blood, blood urea nitrogen 17 mg/dL (6.0 mmol/L), serum creatinine 0.67 mg/dL (51.1 μmol/L), total protein 6.2 g/dL (62 g/L), albumin 3.1 g/dL (31 g/L). Enhanced computed tomography of the abdomen showed localized tumor on the pancreatic head with no kidney abnormalities. Magnetic resonance cholangiopancreatography showed tumor on the pancreatic head without pancreatic duct dilation and stenosis. He underwent subtotal stomach-preserving pancreatoduodenectomy for localized tumor on the pancreatic head. Histological examination of the pancreas showed diffuse infiltration of plasma cells and lymphocytes without evidence of malignancy  and 24-h urinary protein excretion was 15.7 g. He also developed portal vein thrombosis.In December, he was transferred to our hospital for the evaluation of nephrotic syndrome. On admission, he was taking candesartan, furosemide, spironolactone, warfarin, aspirin, famotidine, and voglibose. On examination, he was normotensive and had bilateral lower extremity edema. Urinalysis showed 4+ proteinuria and 2+ occult blood. Other laboratory data were as follows (reference range in parentheses): blood urea nitrogen: 13 mg/dL (4.6 mmol/L), serum creatinine: 0.7 mg/dL (53.4 μmol/L), albumin: 1.8 g/dL (18 g/L), total cholesterol: 140 mg/dL, C-reactive protein: 0.01 mg/dL (9.52 nmol/L), hepatitis B surface antigen: negative, hepatitis C antibody: negative, antinuclear antibody: negative, C3: 85.3 (65–135) mg/dL, C4: 22.0 (13–35) mg/dL, IgG: 934 (870–1700) mg/dL (9.34 g/L), IgA: 148 (110–410) mg/dL (1480 mg/L), and IgM: 70 (35–220) mg/dL (700 mg/L). His serum IgG4 level was elevated to 377 (8–105) mg/dL.In January 2011, a renal biopsy was performed. Light microscopy showed normocellular glomeruli with mild thickening of the glomerular capillary walls and normal mesangial matrix  per well as an antigen. The patient’s plasma diluted 1:1000 was incubated with the antigen. After washout, horseradish peroxidase-conjugated anti-human IgG1–G4 diluted 1:1500 was added . Anti-PLA2R antibodies were not detected in the patient’s serum. Absorbance at 450 nm was 0.093 for IgG4 . Absorbance at 450 nm for IgG1–3 was not significantly elevated compared with the negative control.The patient was treated with prednisolone  and cyclosporine. There was improvement in the urine protein to creatinine ratio, but serum albumin remained low Fig. . Three mWe reported a case of MN associated with IgG4-RD. This case has several distinct features compared with previous reports: (1) the severity of proteinuria was associated with the serum IgG4 level; (2) anti-PLA2R antibody was negative; (3) immunofluorescence staining showed IgG4-dominant deposition among IgG subclasses in the glomerular capillary wall; (4) no associated TIN was observed.A literature review revealed 10 cases of biopsy-proven MN associated with IgG4-RD –24. A clA pathological feature of renal involvement of IgG4-RD is usually TIN, and MN was observed in only 8 % of cases . As far MN could be idiopathic or secondary to various diseases. The diagnosis of IMN is usually by the exclusion of secondary causes, but several features could help the differentiation of idiopathic from secondary MN. Anti-PLA2R antibody was positive in 68.5–81.7 % of IMN, whereas it was positive in only 0–12.2 % of secondary MN –28. HoweIn summary, we reported a case of MN without TIN associated with IgG4-RD. The association of serum IgG4 and proteinuria and negative anti-PLA2R antibodies suggested that MN was secondary to IgG4-RD. There was dominant IgG4 deposit in the glomeruli. Our case suggests the possible role of IgG4 in the pathogenesis of MN more clearly than previous cases. The antigen against IgG4 in our case needs to be identified in a future study.


Piper attenuatum is used as a traditional medicinal plant in India. One of the substances in P. attenuatum has been suggested to have anti-inflammatory effects. However, there is insufficient research about the anti-inflammatory mechanisms of action of P. attenuatum. The effects of P. attenuatum methanol extract (Pa-ME) on the production of inflammatory mediators nitric oxide (NO) and prostaglandin E2 (PGE2), the expression of proinflammatory genes, the translocation level of transcription factors, and intracellular signaling activities were investigated using macrophages. Pa-ME suppressed the production of NO and PGE2 in lipopolysaccharide- (LPS-), pam3CSK4-, and poly(I:C)-stimulated RAW264.7 cells without displaying cytotoxicity. The mRNA expression levels of inducible NO synthase (iNOS) and cyclooxygenase 2 (COX-2) were decreased by Pa-ME. P-ME reduced the translocation of p50/NF-κB and AP-1 (c-Jun and c-Fos), as well as the activity of their upstream enzymes Src, Syk, and TAK1. Immunoprecipitation analysis showed failure of binding between their substrates, phospho- (p-) p85 and p-MKK3/6. p-p85 and p-MKK3/6, which were induced by overexpression of Src, Syk, and TAK1, were also reduced by Pa-ME. Therefore, these results suggest that Pa-ME exerts its anti-inflammatory effects by targeting Src and Syk in the NF-κB signaling pathway and TAK1 in the AP-1 signaling pathway.  Inflammatory cells, such as macrophages and neutrophils, let leukocytes and plasma components come to sites where infection or injury has occurred during inflammation to eliminate dangers , 2. Althfor AP-1 –8. ThesePiper attenuatum is a plant in the Piperaceae family that inhabits the eastern tropical Himalayas, Assam, Khasi Hills, and the Nilgiris in India. The whole plant is used to cure headache and muscular pain [ Piper attenuatum has antibacterial [ P. attenuatum fruit are candidate free radical scavengers [ P. attenuatum [ P. attenuatum. In this study, we aimed to explore the molecular mechanism of P. attenuatum with its methanol extract (Pa-ME) with respect to the NF-κB and AP-1 signaling pathways.lar pain . Piper aacterial  and antiacterial , and neoavengers . In additenuatum . Cepharatenuatum . However P. attenuatum (Pa-ME) was purchased from the Plant Extract Bank in the Plant Diversity Research Center . RAW264.7 cells, a transformed macrophage cell line derived from the BALB/c mouse (ATCC number TIB-71), were purchased from ATCC . Dimethyl sulfoxide (DMSO), L-NG-nitroarginine methyl ester (L-NAME), indomethacin, lipopolysaccharide , pam3CSK, and -2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma Chemical Co. . Poly I:C was obtained from Calbiochem . The enzyme immune assay (EIA) kits used to quantitate the levels of PGE2 were purchased from Amersham . Specific PCR primers for iNOS, TNF-α, COX-2, and GAPDH were synthesized from Bioneer Inc. . Antibodies that specify phosphorylated and total forms of p65, p50, c-Jun, c-Fos, Lamin A/C, IκBα, IKKα/β, AKT, Src, Syk, ERK, p38, JNK, IRAK1, IRAK4, TAK1, MKK3/6, and β-actin were obtained from Cell Signaling .Methanol extraction ofMale C57BL/6 mice  were obtained from DAEHAN BIOLINK  and were housed in groups of 6–8 mice under a 12-hour light/dark cycle (lights on at 6 a.m.). Water and pellet diets  were supplied ad libitum. Animals were cared for in accordance with the guidelines issued by the National Institute of Health for the Care and Use of Laboratory Animals . Studies were performed in accordance with guidelines established by the Institutional Animal Care and Use Committee at Sungkyunkwan University.6 cells/ml) were washed using RPMI1640 medium with 10% FBS and were plated in 100 mm tissue culture dishes for 4 h at 37°C in 5% CO2 in a humidified incubator.To obtain peritoneal macrophages, we used C57BL/6 male mice lavaged 4 days after intraperitoneal injection of 1 ml of sterile 4% thioglycollate broth . Then, peritoneal macrophages (1 × 10μg/ml streptomycin). RAW264.7 cells (2 × 106 cells/ml) were incubated at 37°C in a 5% CO2, humidified incubator . For experiments, RAW264.7 cells were detached from the plate with a cell scraper.RAW264.7 cells, HEK293 cells, and peritoneal macrophages were cultured or maintained in RPMI1640 medium with 10% heat-inactivated FBS, 2 mM of L-glutamine, and antibiotics  were added to RAW264.7 cells or peritoneal macrophages and incubated for 24 h after a 30 min Pa-ME pretreatment. 100 eviously . The abseviously .μl MTT solution (10 mg/ml in PBS pH 7.4) was added and incubated for 3 h as reported previously [Pa-ME was added to testing cells  and incubated for 24 h. Then, 10 eviously . The rea3PO4 in H2O) and solvent B (acetonitrile). The gradient step of the solvent was solvent A to solvent B/min, and a Phenomenex Gemini C18 ODS (5 μm) column was used.High-performance liquid chromatography (HPLC) analysis was utilized for confirmation of the phytochemical characteristics of Pa-ME with the standard compounds quercetin, luteolin, and kaempferol . The anaμg/ml) after 30 min of Pa-ME pretreatment. Total RNA was obtained using TRIzol reagent (Gibco BRL) according to the manufacturer's instructions. Total mRNA was frozen at −70°C for future use. First, 1 μg of RNA was incubated at 70°C with oligo-dT for 5 minutes. After that, it was incubated for 5 more minutes after mixing with 5x first-strand buffer, 10 mM dNTPs, and 0.1 M dithiothreitol (DTT). Then, we added MuLV reverse-transcriptase (2 U) and incubated the mixture at 37°C for 60 min and 70°C for 10 minutes. The remaining RNAs were removed by RNase H. The polymerase chain reaction (PCR) was conducted with the incubation mixture ) in a 10 μl premix using the RT-thermal cycler . The primer  sequence is listed in μg/ml ethidium bromide (EtBr) and run at 100 V for 1 h. The relative quantities were visualized with the DNR Bio-imaging system .To measure the expression level of mRNA related to inflammatory signaling, RT-PCR was conducted. RAW264.7 cells were incubated for 6 h with LPS  containing protease inhibitors  for 30 min with rotation at 4°C. Lysates were refined by centrifugation at 16,000 ×g for 10 minutes at 4°C and stored at −20°C until use. Nuclear fractions were prepared in a three-step procedure. First, after treatment with Pa-ME, we collected cells with a rubber policeman and lysed them with 500 ml lysis buffer  on ice for 4 min. Lysates were centrifuged at 16000 ×g for 1 min. Second, the pellet was washed with the washing buffer (lysis buffer without Nonidet P-40). Finally, we incubated the nuclei with extraction buffer (lysis buffer with 500 mM KCl and 10% glycerol), froze the nuclei/extraction buffer mixture at −80°C, and centrifuged the mixture at 16000 ×g for 5 minutes. The supernatant was collected as a nuclear extract. Soluble cell lysates (30 μg/lane) were immunoblotted.RAW264.7 cells were washed with cold PBS containing 1 mM sodium orthovanadate and lysed using a sonicator  in ice-cold modified RIPA buffer (50 mM Tris-HCl (pH 7.4), 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM NaκBα, IKKα/β, AKT, Src, Syk, ERK, p85, p38, JNK, IRAK1, IRAK4, TAK1, MKK3, MKK3/6, and β-actin were visualized by Western blotting. Nuclear and whole cell extracts containing protein were subjected to 7–15% sodium sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Then, the gel was transferred onto a polyvinylidene difluoride (PVDF) membrane and blocked using BSA. The membrane was rotated overnight with the first antibody in BSA. It was washed with Tris-buffered saline with Tween 20 (TBST) and then probed with a second antibody conjugated with horseradish peroxidase in BSA for 1 h. Using an enhanced chemiluminescence kit , immunoreactive bands were detected.The phosphorylated or total levels of p65, p50, c-Jun, c-Fos, Lamin A/C, Iμg) from RAW264.7 cells (1 × 107 cells/ml) treated with or without LPS (1 μg/ml) for 3 min were precleared with 10 μl protein A-coupled Sepharose beads (50% v/v)  for 1 h at 4°C, as reported previously [μl antibodies to Syk, Src, or TAK1 overnight at 4°C. The antigen-antibody complexes were mixed with 10 μl protein A-coupled Sepharose beads (50% v/v) and rotated for 3 h at 4°C. After that, the immunoprecipitates were analyzed by immunoblotting analysis.Cell lysates containing equal amounts of protein  were transfected with 1 μg of HA-TAK1 alone or plasmids containing β-galactosidase  (as a control) and AP-1-Luc in the presence or absence of an inducing molecule, TAK1. Transfections were performed using the PEI method in 6-well plates, as previously outlined [HEK293T cells .Data presented herein are the mean ± SD of in vitro experiments performed with two samples Figures . For staμg/ml). As μg/ml) downregulated the release of PGE2 as shown in the case of COX inhibitor, indomethacin (Since NO is a representative mediator of inflammation, we examined whether Pa-ME was able to suppress inflammatory responses by determining NO levels in culture supernatant prepared from activated RAW264.7 cells in the presence or absence of Pa-ME (0 to 200 methacin . Viabilimethacin . MeanwhiκB and AP-1 were detected by Western blotting with nuclear fractions. As κB was reduced by Pa-ME at 15 and 60 min of LPS treatment. In addition, c-Jun and c-Fos, major AP-1 subunits, were also suppressed by Pa-ME at 15, 30, and 60 min (The effect of Pa-ME on inflammatory gene expression was examined by analysis of iNOS and COX-2 mRNA levels by reverse-transcription polymerase chain reaction. As d 60 min .κB signaling pathway immunoblotting analysis. Interestingly, the phosphorylated forms of IκBα, IKKα/β, and AKT were reduced by Pa-ME at 30 and 60 min after LPS treatment, while their total forms remained constant (To verify the immunopharmacological targets of Pa-ME, we elucidated the upstream signaling molecules that were involved in the NF-constant . Moreoveconstant . To ensuconstant .We evaluated whether Pa-ME could modulate the AP-1 activation pathway by measuring MAPK protein phosphorylation. As 2 production in a dose-dependent manner (Figures μg/ml concentration range (NO, which stands for nitric oxide, is secreted as a result of inflammation and is involved in the innate response and cytotoxicity . LPS, paon range . When weon range , we idenon range . This enμg/ml. Therefore, the ideal concentration of Pa-ME for inhibiting inflammation is 200 μg/ml, and we used this concentration in experiments afterwards. It is generally known that NF-κB regulates iNOS and AP-1 regulates COX-2 [κB consists of p65 and p50, while AP-1 consists of c-Fos and c-Jun [κB signaling pathway and tried to identify molecules linking to Pa-ME pharmacology. In the NF-κB signaling pathway, IκBα, IKKα, IKKβ, AKT, Src, and Syk are involved. By Western blotting, we found that the phospho-forms of IκBα, IKKα/β, and AKT were decreased at 30 and 60 min (κB signaling pathway.Under inflammatory conditions, NO is generated by iNOS in macrophages . Variouses COX-2 . NF-κB cnd c-Jun , 31. Thed 60 min . In addid 60 min . Becaused 60 min . OverallWe also explored whether Pa-ME could modulate the AP-1 signaling pathway, as the COX-2 mRNA level was decreased . There aκB and AP-1 pathways, as summarized in In conclusion, we revealed that Pa-ME can relieve inflammation by inhibiting Src, Syk, and TAK1 to suppress both the NF-


The principle of implanting a calcar-guided short stem consists of an individual alignment alongside the medial calcar providing the ability of reconstructing varus and valgus anatomy in a great variety. However, still, there are broad concerns about the safety of extensive varus and valgus positioning in regard to stability, bony alterations, and periprosthetic fractures.216 total hip arthroplasties using a calcar-guided short stem  in 162 patients were included. Depending on postoperative CCD angle, hips were divided into five groups (A–E). Varus- and valgus tilt and axial subsidence were assessed by “Einzel-Bild-Roentgen-Analyse” over a 2-year follow-up. The incidence of stress-shielding and cortical hypertrophy as well as clinical outcome [Harris Hip Score (HHS)] were reported.p = 0.502). Axial subsidence after 2 years was 1.20, 1.02, 1.44, 1.50, and 2.62 mm, respectively (p = 0.043). No periprosthetic fractures occurred and none of the stems had to be revised. Rates of stress-shielding and cortical hypertrophy as well as HHS showed no significant difference between the groups.Postoperative CCD angles ranged from 117.9° to 145.6° and mean postoperative CCD angles in group A–E were 123.3°, 128.0°, 132.4°, 137.5°, and 142.5°, respectively. After 2 years, the mean varus/valgus tilt was −0.16°, 0.37°, 0.48°, 0.01°, and 0.86°, respectively (Valgus alignment results in increased subsidence but does not affect the clinical outcome. There is no difference in stress shielding and cortical hypertrophy between the groups. The authors recommend long term monitoring of valgus aligned stems. In modern primary total hip arthroplasty (THA), short stems are increasingly regarded as implants of first choice , especiaIn the last decade, many different short-stem designs have been introduced , 9, resuSo-called calcar-guided short stems have been developed to optimally adapt to the anatomy of the proximal femur and to allow a restoration of hip biomechanics , 15. TheTo date, little is known about the outcome of extensive varus- or valgus positioning in calcar-guided short-stem THA. There still are broad concerns particularly regarding implant stability. It might have an impact on the rate of axial subsidence as well as on stem tilting. In addition, the influence of different stem alignment on bony alterations like stress-shielding and cortical hypertrophy, as a result of abnormal stress distribution in the loaded proximal femur, to date, is not fully understood. Consequently, it remains unclear that how different stem alignments affect stability and load transfer.The objective of the present analysis was to address the following research questions:Does extensive varus- and valgus alignment in calcar-guided short-stem THA impact on postoperative varus- and valgus tilt and subsidence?Does different stem alignment affect the incidence of stress-shielding and cortical hypertrophy?Is there a difference in postoperative functional outcome?n = 198) primary osteoarthrosis, 5.1% (n = 11) femoral head necrosis, 2.3% (n = 5) congenital dysplasia, and 0.9% (n = 2) secondary osteoarthrosis. Mean patient age was 63.0 years (standard deviation (SD) 10.0).In the present retrospective investigation, 216 consecutive hips in 162 patients were included after institutional review board approval  from an ongoing prospective observational study. Preliminary results of the same study group have already been published involving the same collective of patients in a different context , 19. PriIn all patients, the meta-diaphyseal anchoring calcar-guided short stem optimys  was implanted as described in Kutzner et al. , 19.n = 177 Fitmore, Zimmer; n = 39 RM Pressfit vitamys, Mathys Ltd Bettlach) with a ceramic-polyethylene bearing couple. All surgeries were performed in supine position using a modified, minimally invasive anterolateral approach [The investigated stem was combined with cementless press-fit cups  was used . A totalFor the EBRA-FCA measurements, a series of at least three radiographs was needed. Thus, radiographs during hospital stay as well as after 6, 12, and 24 months were analyzed. For radiological follow-up, including bone resorption and cortical alterations, only radiographs after surgery and after 24 months were considered.In the postoperative radiograph, CCD angles  were measured retrospectively, using the digital templating software MediCAD  Fig. . PatientUsing a modification of the zones described by Gruen , bone reIn addition, HHS was assessed in all patients after 2 years as well as the rate of periprosthetic fractures.Mean follow-up time was 2.2 years (range 2.0–3.0 years). Ten hips were either lost to follow up or the EBRA-FCA software failed to accept the radiograph and three patients (4 hips) had deceased unrelated to the operation with prosthesis in situ. In one patient, only a clinical follow-up could be performed. In total, 201 hips in 66 female and 84 male patients fulfilled the inclusion criteria . Correlations between subsidence and CCD postoperative were evaluated using Pearson correlations supplemented by Spearman rank correlations, the latter to evaluate the effect of extreme observations. In addition, regression analyses were carried out relating subsidence to CCD postop together with age and weight as potential covariates. To this end, robust regression was applied to account for extreme observations. Associations between CCD angle categorizations and discrete variables (such as stress-shielding occurrence) were examined by Chi-square tests.p value of less than 0.05 was considered. The SAS software 9.4 was used for all analyses .For statistical significance, a Postoperative range of CCD angles was 117.9°–145.6° with a mean of 132.5° (SD 4.9). Patients were divided into five groups (A–E) regarding postoperative stem alignment measured by CCD angles. Mean postoperative CCD angles for each group were (A) 123.3° (SD 1.8); (B) 128.0° (SD 1.3); (C) 132.4° (SD 1.4); (D) 137.5° (SD 1.4); and (E) 142.5° (SD 2.1), respectively (Table p = 0.502). Mean axial subsidence in the investigated groups after 2 years summed up to (A) 1.20 mm (SD 1.81); (B) 1.02 mm (SD 1.21); (C) 1.44 mm (SD 1.34); (D) 1.50 mm (SD 1.30); and (E) 2.62 mm (SD 2.17), respectively. Pearson as well as Spearman Correlation showed that the higher the CCD angles are the more pronounced the subsidence was. Regression analysis relating axial subsidence to CCD category together with age and weight as potential covariates accounted for 6.4% of the variation. Age and weight as covariates turned out to be significant. Axial subsidence increased by about 0.26 mm for a 10 year age increase and by about 0.13 mm for a 10 kg weight increase.Mean varus/valgus stem tilt after 2 years was (A) −0.16° (SD 3.26); (B) 0.37° (SD 2.72); (C) 0.48° (SD 2.28); (D) 0.01° (SD 2.94); and (E) 0.86° (SD 2.70), respectively .The rate of radiological alterations, such as bone resorption and cortical hypertrophy, is summarized in Table p = 0.458)  96.8 (SD 9.2); (B) 98.1 (SD 4.6); (C) 98.6 SD 3.5); (D) 97.8 (SD 4.8); and (E) 97.9 (SD 2.5), respectively  9No periprosthetic fractures occurred and none of the stems had to be revised in the observation period.This study analyzed the outcome of extensive varus- and valgus stem alignment of a calcar-guided short stem regarding implant stability and radiological alterations in a 2-year follow-up. While pronounced femoral varus alignment does not cause increased instability in terms of varus/valgus tilt and axial subsidence, extensive valgus positioning is followed by significantly increased initial subsidence.The philosophy of calcar-guided short stems emphasizes on the alignment of the stem alongside the medial calcar in different varus- and valgus position, according to the patient’s anatomy, using individual levels of neck resection , 15. In Furthermore, it remains unclear how different resection heights and stem alignments affect load transfer and stress-shielding patterns, which are, besides micromovement of the implant, also generally thought to have a considerable effect on aseptic loosening . Lerch eAligning the stem in an extensive varus position results in an increase in offset and subsequent enhanced lever-arm compared to the extensive valgus alignment, in turn, leading to an increased strain in the medial part of the proximal femur and the region around the distal tip of the stem . Given iThis study has several limitations. First to be mentioned is the short follow-up of 2 years. Although only long-term results should be considered valid, the initial evaluation of bony alterations is necessary to identify undesirable results . Early mThe results confirm a wide range of CCD angles to be adequately reconstructed using the investigated calcar-guided short stem. After 2 years, extensive varus positioning does not cause increased instability in terms of varus/valgus tilt and axial subsidence suggesting no restrictions regarding indications. Extensive valgus positioning is followed by significantly increased subsidence without any clinical correlation. Undersizing the stem providing insufficient cortical contact could be identified as the main cause leading to axial subsidence, especially in extensive valgus alignments. The overall rate of stress-shielding and cortical hypertrophy is low without any clinical consequences. Short-term clinical results in all groups are encouraging. Further monitoring, especially of the valgus hips, is mandatory.


Shigella flexneri and Shigella sonnei bacteria cause the majority of all shigellosis cases worldwide. However, their distributions differ, with S. sonnei predominating in middle- and high-income countries and S. flexneri predominating in low-income countries. One proposed explanation for the continued range expansion of S. sonnei is that it can survive in amoebae, which could provide a protective environment for the bacteria. In this study, we demonstrate that while both S. sonnei and S. flexneri can survive coculture with the free-living amoebae Acanthamoebae castellanii, bacterial growth is predominantly extracellular. All isolates of Shigella were degraded following phagocytosis by A. castellanii, unlike those of Legionella pneumophila, which can replicate intracellularly. Our data suggest that S. sonnei is not able to use amoebae as a protective host to enhance environmental survival. Therefore, alternative explanations for S. sonnei emergence need to be considered.IMPORTANCE The distribution of Shigella species closely mirrors a country's socioeconomic conditions. With the transition of many populous nations from low- to middle-income countries, S. sonnei infections have emerged as a major public health issue. Understanding why S. sonnei infections are resistant to improvements in living conditions is key to developing methods to reduce exposure to this pathogen. We show that free-living amoebae are not likely to be environmental hosts of S. sonnei, as all Shigella strains tested were phagocytosed and degraded by amoebae. Therefore, alternative scenarios are required to explain the emergence and persistence of S. sonnei infections. Shigella is a genus of Gram-negative enteric pathogens comprised of four species. All species can cause severe diarrhea, and Shigella is estimated to cause 165 million infections and 120,000 deaths annually, accounting for 10% of deaths due to diarrheal disease worldwide  and two S. sonnei isolates . All strains remained culturable at 106 to 107 CFU/ml over the 18 days  at 22°C were allowed to phagocytose bacteria for 1 h, which was followed by 1 h gentamicin treatment to kill extracellular bacteria. The numbers of total bacteria (prior to gentamicin treatment) were similar for all strains and indicated that, on average, 5 × 106 CFU/ml, or approximately 50 bacteria/amoeba, were present  to increase the rate of phagocytosis by amoebae. As anticipated, significantly higher numbers of phagocytosed bacteria could be enumerated than in low-nutrient medium . On averflexneri . ExtraceShigella by CFU determination at 1 h, 4 h, and 20 h. Cell counts for all strains decreased over this time frame similarly to those of the negative control of nonpathogenic E. coli, indicating they were efficiently digested by A. castellanii. The same trend of reduced intracellular numbers over time was observed in both high-  for slocated . ShigellShigella with amoebae.If the T3SS facilitated intracellular survival within amoebae, we would expect to see increased intracellular bacterial counts at 4 h and 20 h during incubation at 37°C. Instead, we saw a decrease in viable intracellular bacteria numbers, similar to those at 22°C and 30°C, suggesting that an active T3SS could not facilitate intracellular survival in amoebae . To furtS. flexneri used its T3SS to kill amoebae (S. flexneri (E. coli MG1655 and Shigella T3SS mutants) and any of the wild-type Shigella strains. Therefore, in our assays neither S. flexneri or S. sonnei were able to induce cell death in amoebae.It was previously reported that  amoebae , 26. Hav amoebae  and B, oflexneri , we deciflexneri . At all V. cholerae was recently shown to resist intracellular killing by A. castellanii, and at low frequency it can be released intact by the amoebae   . The undive host . We showirements . HoweverS. sonnei, it remains to experimentally test additional hypotheses. One popular suggestion is that exposure to unsanitized water in developing countries can result in Plesiomonas shigelloides infection and hence in natural immunity against S. sonnei. P. shigelloides serotype O17 has a lipopolysaccharide O-antigen identical to that of S. sonnei , which provides a niche-specific competitive advantage for S. sonnei over S. flexneri  of 0.5 was reached. Bacteria were washed in phosphate-buffered saline (PBS), resuspended in the appropriate medium, and added to cells.Isolates of Shigella  were plaShigella . ColonieLegionella cells were plated on buffered charcoal-yeast extract (CYE) agar plates for 3 days at 37°C. Bacterial colonies were diluted to OD600 = 0.1 in ACES [N-(2-acetamido)-2-aminoethanesulfonic acid] yeast extract (AYE) broth and incubated for 21 h at 37°C and 200 rpm.Acanthamoeba castellanii cells  were cultured in ATCC 712 peptone-yeast-glucose medium [PYG] medium 2% protease peptone, 0.1% yeast extract, 0.1 M glucose, 4 mM MgSO4·47H2O, 0.4 mM CaCl2, 0.05 mM Fe(NH4)2(SO4)2·6H2O, 2.5 mM KH2PO4, 2.5 mM Na2HPO4·7H2O, and 0.01% Na citrate·2H2O) in tissue culture flasks at 22°C. During infections, amoebae were seeded in PAS medium  or PYG.2.HeLa cells were maintained and seeded in Dulbecco's modified Eagle medium (DMEM), 1,000 mg/ml glucose, supplemented with 10% fetal bovine serum (FBS) and 5% Glutamax and incubated at 37°C and 5% COmxiD using lambda red recombination (mxiD (with primer pair 5′-tagggataacagggtaatcagtggtgctctagtagagc-3′ and 5′-gaagcagctccagcctacacaggtaacaatacaatcaagag-3′) and 500 bp downstream of mxiD (with primer pair 5′-ctaaggaggatattcatatgggaagacgaaaaatcattgg-3′ and 5′-tagggataacagggtaatcgacttgttcagcagtaagatc-3′), followed by overlapping PCR (I-SceI sites are underlined). This fragment was cloned into pGEM T-Easy and transformed into S. sonnei 53G alongside pACBSR. mxiD was then replaced with the kanamycin resistance gene with the help of lambda red recombinase and I-SceI induced from pACBSR.The T3SS mutant was created by deletion of bination , 40. A DA. castellanii was seeded at 106 cells/flask in T75 tissue culture flasks in PAS medium. Amoebae were infected with Shigella at a multiplicity of infection (MOI) of 10. Every other day 107 heat-killed (100°C for 20 min) E. coli DH5α was added to flasks to prevent amoebae starvation. At time points indicated, 1 ml of culture was removed. Ten microliters was used to count amoebae on a hemocytometer. Twenty microliters was used to perform serial dilutions to calculate coculture CFU. Remaining sample was washed with PAS and treated with 150 μg/ml gentamicin for 1 h. Amoebae were washed again with PAS and lysed with 0.25% sodium deoxycholate. Serial dilutions were performed to calculate intracellular CFU.A. castellanii was seeded at 1 × 105/ml in 24-well plates and infected with an MOI of 100. To synchronize infection, plates were centrifuged at 600 × g for 10 min and then incubated for 50 min at 22°C or 37°C. Cells were washed with PAS, and 150 μg/ml gentamicin in PAS/PYG was then added to cells for 1 h. If required, medium was replaced with 20 μg/ml gentamicin in PAS/PYG for a further 3 h or 19 h. At the time points indicated, cells were washed with PAS and lysed with 0.25% sodium deoxycholate for 10 min. Serial dilutions were carried out and plated to calculate intracellular CFU. Experiments using L. pneumophila were performed at 30°C in PYG, and amoebae were infected with L. pneumophila at an MOI of 1. To enumerate intracellular CFU in these experiments, amoebae were washed in PAS, detached from the well, and lysed by vortexing for 10 s. Serial dilutions were performed and plated on appropriate agar. All other experimental steps were as above.S. sonnei 53G and GFP-expressing S. flexneri M90T. Amoebae were then washed with low-fluorescence (LF) medium (S. sonnei (Remel agglutinating sera) or S. flexneri 5a  in 2% BSA in LF medium for 30 min. Amoebae were washed with ice-cold LF medium, followed by anti-rabbit Cy3 (Jackson ImmunoResearch) in 2% BSA in PAS for 30 min. Amoebae were washed with LF medium before being overlaid with agarose and immediately imaged on a Zeiss Axio Observer inverted microscope.Amoebae seeded on 4-well μ-slides (Ibidi) were infected as above with GFP (green fluorescent protein)-expressing ) medium  and placg). Amoebae were overlaid with agarose and immediately imaged on a Zeiss Axio Observer inverted microscope.For neutral red staining, amoebae were incubated in 125 μM neutral red in LF medium for 20 min at room temperature (RT). Amoebae were washed in LF medium before addition of bacteria harboring GFP and centrifuged briefly .5 cells/ml 24 h prior to infection. A. castellanii was seeded at 107 cells/flask in T75 flasks in PAS medium, infected with Shigella at an MOI of 100, and incubated at 22°C. After 24 h, amoebae were detached, centrifuged at 500 × g for 5 min, and resuspended in 150 μg/ml gentamicin in PAS for 1 h. Amoebae were then washed with PAS and lysed with 0.25% sodium deoxycholate. Released bacteria were centrifuged, washed, and resuspended in DMEM. Broth-cultured Shigella cells were prepared as described above and resuspended in DMEM. Prior to infection, medium was replaced with serum-free DMEM, and HeLa cells then infected at an MOI of 100 with Shigella cells released from amoebae or grown in broth at 37°C or 22°C. Cells were centrifuged at 600 × g for 10 min and incubated for 30 min at 37°C and 5% CO2. Medium was replaced with 150 μg/ml gentamicin in supplemented DMEM and incubated for a further 1 h or 3 h. At these time points, cells were washed with PBS and lysed with 0.5% Triton X-100. Serial dilutions were performed and plated to calculate intracellular CFU.HeLa cells were seeded in 96-well plates at 1 × 10


Streptomyces M37 growth and its acarbose biosynthesis ability. We observed that low pH was beneficial for cell growth, whereas high pH favored acarbose synthesis. Moreover, addition of glucose and maltose to the fermentation medium after 72 h of cultivation promoted acarbose production. Based on these results, a two-stage fermentation strategy was developed to improve acarbose production. Accordingly, pH was kept at 7.0 during the first 72 h and switched to 8.0 after that. At the same time, glucose and maltose were fed to increase acarbose accumulation. With this strategy, we achieved an acarbose titer of 6210 mg/L, representing an 85.7% increase over traditional batch fermentation without pH control. Finally, we determined that the increased acarbose production was related to the high activity of glutamate dehydrogenase and glucose 6-phosphate dehydrogenase.In this work, we investigated the effect of pH on  Actinoplanes and Streptomyces [Acarbose  is a competitive α-glucosidase inhibitor ,3. Acarbptomyces ,6. It waptomyces .Structurally, acarbose consists of moieties of acarviose and maltose. Work on acarbose biosynthesis has demonstrated that glucose is converted to glucose-6-phosphate, which is then metabolized to sedoheptulose 7-phosphate via the hexose monophosphate (HMP) pathway, subsequently forming valienamine . GlutamaStreptomyces M37.Medium pH is one of the most important environmental parameters affecting cell growth and product formation. Controlling medium pH is a common way to optimize metabolite and enzyme production. Although previous studies have investigated various physiological factors that influence acarbose production –14, the Streptomyces M37 (GenBank KJ888155), a high acarbose-producing strain, was used in this study. The strain was isolated and mutated in our previous study [Streptomyces M37 were stored at -80°C in 25% (v/v) sterile glycerol. Prior to the study, Streptomyces M37 was activated on agar plates at 28°C for about 5 days until visible white colonies emerged.2HPO4·3H2O, 1.0; FeSO4·7H2O, 0.02; MgSO4, 1.0; NaNO3, 2.0; peptone, 1.0; and agar, 20. Unless otherwise stated, the initial pH of the medium was adjusted to 7.0 with 2 M NaOH and then sterilized by steam autoclaving at 121°C for 20 min.Agar plate medium was composed of (g/L): sucrose, 20; K4Cl, 3.0; peptone, 4.0; yeast extract, 2.0; K2HPO4·3H2O, 1.5; FeSO4·7H2O, 0.02; MgSO4, 1.0; and CaCO3, 3.0.The seed culture for fermentation inoculation was prepared in a medium consisting of (g/L): soluble starch, 15; sucrose, 40; NH2HPO4·3H2O, 1.5; NH4Cl, 6.0; MgSO4, 1.0; and FeSO4·7H2O, 0.02. Glucose and maltose were sterilized separately to prevent Maillard reactions and added to the medium before inoculation. Unless otherwise stated, the feeding medium was composed of (g/L): glucose, 134; and maltose, 266.The fermentation medium contained (g/L): sucrose, 40; glucose, 15; maltose, 10; peptone, 2.0; yeast extract, 1.0; KStreptomyces M37 spores was inoculated onto an agar plate and incubated at 28°C in an electro-thermal incubator. After 5 days of incubation, seed cultures were prepared by inoculating 1-cm2 mycelia into 250-mL shake-flasks containing 100 mL fresh medium. Cultivation was performed for 3 days at 28°C with stirring at 200 rpm.A loop of 2SO4, while precursors for acarbose biosynthesis  were added to the fermentation broth. All fermentations were carried out at 28°C for 7 days.The effect of initial pH on acarbose fermentation was assessed in 250-mL Erlenmeyer flasks containing 90 mL medium shaken at 200 rpm. The effect of pH (constant pH control or two-stage pH control strategy) on acarbose fermentation was studied in a 5-L fermentor  with a working volume of 3.0 L. A 10% (v/v) seed culture was inoculated into the fermentor. Aeration rate was kept at 1.0 L/L/min and agitation speed was set to 200 rpm. Procedures for the two-stage fermentation strategy were carried out as follows. In the first stage (72 h), pH was kept at 7.0; in the second stage, pH was maintained at 8.0 using 2 M NaOH and 2 M HMycelia were harvested by centrifugation  and then dried at 85°C in an oven to obtain a constant dry cell weight (DCW). The resulting supernatants were transferred to sterile microcentrifuge tubes and stored at -20°C until analysis. Acarbose titer was measured by high performance liquid chromatography according to Li et al. . The pH ®. A p value < 0.05 was considered to be statistically significant throughout the study.Data are expressed as means of triplicate determinations, unless otherwise stated. Statistical significance was assessed with one-way analysis of variance (ANOVA) using SPSS 20.0 software  for WindowsStreptomyces M37 growth and acarbose production, we performed cultivations in 250-mL Erlenmeyer flasks containing 100 mL fermentation medium for a period of 168 h. The initial pH ranged from 6.5 to 8.5. As shown in max(mg/L DCW)) increased along with initial pH (in the 6.5 to 8.0 range). It should be noted that in all experiments the final pH after 168 h of cultivation was lower than 6.0  were estimated from experimental or fitted cell growth data (The effect of pH on acarbose batch fermentation was further investigated by controlling the pH of the medium (from 6.5 to 8.5) in a 5-L fermentor using 2 M NaOH and 2 M Hwth data  and acarwth data , respectwth data . Specifiqp was obtained at pH 8.0. It should be noted that acarbose production was not accompanied by increased DCW. A similar result was observed by Lou et al. during β-carotene fermentation [As can be seen in entation . HoweverIt has been reported that medium pH affects enzyme synthesis, enzymatic processes, and transport across cell membranes ,20,21. OBiosynthetic studies have demonstrated that glutamate is the primary source of N-glycosidic bonds in acarbose . Some reStreptomyces M37 acarbose production, they were added at different concentrations (5–30 g/L) to the fermentation broth at 72 h, 96 h, 120 h, and 144 h. Addition of 20 g/L of reducing sugars led to an acarbose yield of 5160 mg/L, corresponding to a 17.0% increase with respect to the control  and 6.4% (p < 0.05) compared to batch fermentations, respectively. In the case of a 1:2 glucose-to-maltose ratio, an acarbose yield of 5472 mg/L was obtained, representing a 24.1% increase with respect to no glucose or maltose addition. Therefore, we concluded that a feed of 20 g/L with a 1:2 mass ratio of glucose-to-maltose favored acarbose production by Streptomyces M37.To further define the optimal mass ratio of reducing sugars during acarbose production, we fed six different mixtures of glucose and maltose to the cultivation medium. During the fed-batch fermentation process in a 5-L fermentor at pH 8.0, 20 g/L of reducing sugars were fed at 72 h, 96 h, 120 h, and 144 h . According to the results in Our results indicated that pH played an important role in cell growth and acarbose biosynthesis during the fermentation process. We investigated whether a two-stage cultivation strategy could maximize the yield of the target metabolite. Accordingly, optimal cell growth and acarbose production would be obtained by separately optimized conditions.The following two-stage strategy was developed based on the foregoing results in a 5-L fermentor: pH was maintained at 7.0 during the first 72 h of fermentation to enhance cell growth; pH was then switched to 8.0 to facilitate acarbose biosynthesis. Meanwhile, 20 g/L of reducing sugars  was added to the fermentation broth at 72 h, 96 h, 120 h, and 144 h . The effThe obtained maximum acarbose titer yield was 27.3% higher than that reported by Wang et al. during fed-batch fermentation (4878 mg/L) . SimilarStreptomyces M37  is suitable for cell growth, whereas a high pH (8.0) favors acarbose synthesis. A two-stage fermentation strategy whereby pH was kept at 7.0 for the first 72 h and then shifted to 8.0 enhanced acarbose production. At the same time, glucose and maltose were fed in to promote acarbose accumulation. The specific activities of GDH and G6PDH were higher than in traditional batch fermentation . The information obtained in this study may be valuable for large-scale production of acarbose.In conclusion, we investigated the effect of pH and precursors  on acarbose biosynthesis by


Silsesquioxanes are promising precursors in the production of silica nanoparticles by thermolysis, due to their structural similarities with silica materials. This manuscript describes the production of a new Eu Luminescent nanoparticles hold great promise as biomarker tools in the emerging field of nanomedicine. Whilst organic dyes and quantum dot based materials possess useful properties, their downsides, such as the ease of photobleaching of organic dyes or the toxicity of heavy metal containing quantum dots, can be severely limiting to their practical application. Rare earth (RE) metal doped oxide nanomaterials on the other hand, may offer advantages over these materials, due to their long luminescence lifetimes, resistance to photobleaching and sharp emission profiles. The incorporation of RE ions into silica and silicates is of interest for a wide variety of applications, including optics, catalysis and biomedicine122 particles which are typically hundreds of nanometres to micrometres in diameter7Silica-based RE-doped particles have great potential as luminescent probes for biological systems, due to their small sizes,  strong emission properties and silanol surfaces, which allow further modification with ease. The most common route to such materials is the grafting or incorporation of a lanthanide complex, whose emission properties are enhanced through an “antenna” effect45e.g. siloxanes or siloxides) in the presence of a silica or alumina support111213141517Thermolysis is a well-known solid state chemistry technique for the preparation of (bulk) silica, alumina or titania based materials, which is often carried out by thermal decomposition of a complex or material 7Si7(OH)3 was initially prepared through the kinetically controlled hydrolysis of trichlorocyclohexylsilane (29Si NMR of (c-C6H11)7Si7(OH)3 showed peaks at −60.36, −68.25 and −69.79 ppm in a ratio of 3:1:3, representing the 3 environments of Si in the incompletely condensed trisilanol (c-C6H11)7Si7(OH)3 (−1), Si-O-Si stretching and bending vibrations (1050–1195 cm−1) and Si-C stretches (between 1260 and 1450 cm−1)403Li(μ-Cl)Eu[N(SiMe3)2]3] 3 , SI addiMe3)2]3]  was addicompound , SI show1) was prepared from these precursors following a silylamide route, reacting the incompletely condensed trisilanol (c-C6H11)7Si7(OH)3 with the europium precursor [(THF)3Li(μ-Cl)Eu[N(SiMe3)2]3] (29Si NMR of compound 1 displays a series of peaks (3)2]. The insets of 1. These peaks, at −69.50, −69.20, −67.90, −67.21, −67.15, −67.00, −66.70, −60.00, −58.37, −57.34, −55.61 ppm, are in an approximate ratio of 1:2:1:1:1:1:1:1:2:1:1. The two peaks which are near to one another and appear similar to a doublet signal (at −67.21 and −67.15 ppm) are close in proximity possibly due to a very fast transfer of a proton between two of the OH− groups in the structure. The presence of THF molecules coordinated to the Eu-metal in the complex can cause a distortion of the shape and symmetry of the molecule. The 11 peaks present in the 29Si NMR are all associated with the suggested structure in A europium-based metallasilsesquioxane compound (compound Me3)2]3] . The 29Sof peaks . A peak 1 (23536−1 are due to CH stretches of the cyclohexyl substituent−1) are due to C-C bonds of cyclohexyl groups. Peaks between 1015–1190 cm−1 are representative of Si-O-Si stretching and bending vibrations40−1 representative of asymmetric silicon-phenyl stretches2− groups when compared with the spectrum for the trisilanol (c-C6H11)7Si7(OH)3 3 , SI corr1 described above, thermolysis experiments were carried out at 300 °C under a nitrogen atmosphere (see Methods) and the resulting white powder was collected for analysis. Transmission electron microscopy (TEM) of 1 after thermolytic treatment demonstrated the presence of spherical nanoparticles with diameters of 13.5 ± 4.0 nm (1) due to the rearrangement of silicon atoms during thermal treatment and the incorporation of multiple Eu centres in the particles.Thermolysis of this rare earth metallasilsesquioxane was performed as a new route to produce luminescent rare earth doped silica nanoparticles. Thermogravimetric analysis (TGA) was initially carried out in order to assess the thermal stability of the prepared compound . Mass lo1312± 4.0 nm . Energy ± 4.0 nm , SI. Thi−1 representing Si-O-Si stretching and bending vibrations40−1, though these appear reduced compared to the compound prior to thermolytic treatment21 before and after thermal treatment are shown in via calcination), where peaks around 800 cm−1 are indicative of Si-O-Si stretches and those at ~600 and 485 cm−1 represent tri and tetracyclosiloxane rings of siloxanes−1 can be assigned to O-bending vibrations in the SiO4 tetrahedral silica matrixFTIR spectroscopy of the thermolytically-produced nanoparticles , SI show3+ ions present in the compound . This correlates well with other lanthanide-POSS species described in the literature, where characteristic Eu3+ emission is preserved in complex siloxane species and materials32Solid state photoluminescence spectra taken before thermolysis demonstrate characteristic emission peaks as expected from Eucompound ; with pe3+ peaks, peaks at 612 and 700 nm still remain visible after thermal treatment, indicative of the remaining presence of the Eu3+ species. Broadening of Eu3+ emission peaks can also result from clustering and phonon-assisted energy transfer effects between adjacent Eu3+ ions in the Eu3+ clusters, present herein due to rearrangements of the silsesquioxane structure during thermal treatmentBroad emission in the visible range of the spectrum is well documented in bulk silica gels and nanostructures prepared using thermal and high energy treatments454647453+-doped silica nanoparticles with small sizes and spherical structures. The siloxane structure of the parent species provides an appropriate inorganic cluster to allow the formation of small sized silicate nanoparticles. The nanoparticles retain the characteristic Eu3+ lanthanide luminescent properties of their parent compounds and a broad luminescent baseline due to the presence of hydrocarbon defects, which becomes enhanced by thermal treatment4546Thus, we have demonstrated that new rare earth based metallasilsesquioxane compounds can be used as single source precursors in the thermolytic preparation of rare earth-doped silica nanoparticles. The thermolytic approach presented herein is a convenient and promising technique for preparing silica nanoparticles from a single source precursor, yielding EuAll starting materials have been supplied by Sigma-Aldrich unless stated otherwise. Anhydrous tetrahydrofuran (THF) was purified by heating under reflux for 2–3 hours over a sodium-potassium alloy and benzophenone, followed by distillation under argon and then condensation into a reaction flask. Benzophenone forms a blue ketyl once the solvent is dry.6D6 under a N2 atmosphere and analyzed immediately after preparation. A JEOL JEM-2100, 200 kV LaB6 transmission electron microscope operated at 120 kV with a beam current of ~65 mA was used to image nanoparticle samples. Aqueous suspensions were drop-cast onto a formvar coated copper grid for imaging. Size analysis was carried out using ImageJ software by measuring a minimum of 100 particles. An energy dispersive X-ray spectroscopic (EDS) system attached to a JEOL JEM-2100 transmission electron microscope was used to collect elemental data on the prepared nanoparticles; EDS was carried out on several areas of the TEM sample, sampling a minimum of 100 particles. FTIR spectra were recorded by diffuse reflectance in dry Nujol (dried over molecular sieves) between NaCl discs using a Perkin Elmer Spectrum One FT-IR spectrophotometer. Raman spectra and solid state photoluminescence spectra were measured with a Renishaw 1000 micro-Raman system with a Leica microscope. The excitation wavelength was 457 nm from an Ar+ ion laser (Laser Physics Reliant 150 Select Multi-Line) with a typical laser power of ~10 W cm2. Samples were placed as a solid powder on a silicon substrate and pressed flat using manual pressure. The laser was focussed on the sample, with a spot size of ~10 μm and emission was collected. As such, the data presented should not be taken as quantitative and directly comparable between samples in terms of emission intensity. Thermogravimetric analysis (TGA) and thermolysis was carried out using a Perkin Elmer Pyris 1 TGA machine operated with a heating rate of 10 °C/min under an atmosphere of N2. X-ray diffraction was performed using an Empyrean diffractometer equipped with a Co Kα lamp fitted with a beam knife, 6 h scans were carried out measuring 5–50 degrees 2θ. Bright field and fluorescence microscope images were taken using an Olympus CKX41 optical system fitted with an Olympus XC30 camera and CoolLED pE300-white light source. Images were taken with a x10 objective lens under bright field  or excited with 365 nm wavelength (operated at 80 W). Camera settings were set at 371.6 ms exposure time with 4.9 dB gain and 2080 × 1544 resolution.NMR spectra were collected from Bruker 400-Avance-3 (9.4 T) spectrometer with samples prepared in Cc-C6H11)7Si7(OH)3 was carried out according to published procedureThe preparation of : δ = −69.79, −68.23, −60.36 ppm.1H : δ = 7.28, 2.27, 2.25, 1.93, 1.77, 1.76, 1.74, 1.72, 1.47, 1.45, 1.43, 1.41, 1.39, 1.37, 1.19, 1.17, 1.15, 0.99, 0.58, 0.41 ppm.13C NMR : δ = 27.54, 27.51, 27.38, 27.13, 27.05, 27.02, 26.93, 26.89, 26.84, 24.24, 23.91, 23.42 ppm.IR : 3149 (br), 1445 (s), 1286 (s), 1193 (s), 1080 (s), 890 (s), 846 (w), 824 (w), 750 (w), 677 (w).3)2)  was gradually added to the stirring solution at room temperature. The reaction was stirred at room temperature for several days. The white precipitate was allowed to settle before filtration of the orange liquid containing [(THF)3Li(μ-Cl)Eu[N(SiMe3)2]3]  was dissolved in anhydrous THF (30 mL). Li(N(SiMeMe3)2]3] . The ora29Si : δ = 54.78 ppm.IR : 2880 (w), 1405 (w), 1240 (s), 1180 (s), 1040 (m), 979 (m), 827 (m) 766 (m), 750 (m), 671 (w).c-C6H11)7Si7(OH)3 (3Li(μ-Cl)Eu[N(SiMe3)2]3]  was slowly added to the solution with stirring at room temperature. The solution was refluxed under argon at 70–80 °C for two days. The pale yellow liquid containing compound 1 : δ = 7.27, 2.20, 1.86, 1.69, 1.37, 1.33, 0.97, 0.38 ppm.13C NMR : δ = 27.52, 27.11, 26.87, 24.96, 24.21, 23.76, 23.37, 1.49, 0.96 ppm.29Si : δ = −69.50, −69.20, −67.90, −67.21, −67.15, −67.00, −66.70, −60.00, −58.37, −57.34, −55.61, −21.67, 11.39 ppm.IR : 2917 (w), 2848 (w), 1445 (w), 1260 (s), 1190 (s), 1080 (s), 1015 (s), 890 (s). 796 (s).1 was placed in a ceramic boat and heated under an atmosphere of N2 at a heating rate of 10 °C/min using a Perkin Elmer Pyris 1 thermogravimetric analysis machine and held at 300 °C for 30 mins. Samples were retained after burning for characterization as described in the main text.Compound http://wrap.warwick.ac.uk/85408/.Datasets are available open access at How to cite this article: Davies, G.-L. et al. Rare Earth Doped Silica Nanoparticles via Thermolysis of a Single Source Metallasilsesquioxane Precursor. Sci. Rep.7, 45862; doi: 10.1038/srep45862 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Plasmodium falciparum are responsible for the spread of the parasite in malaria endemic areas. The cysteine-rich Pfs48/45 protein, exposed on the surface of sexual stages, is one of the most advanced antigens for inclusion into a vaccine that will block transmission. However, clinical Pfs48/45 sub-unit vaccine development has been hampered by the inability to produce high yields of recombinant protein as the native structure is required for the induction of functional transmission-blocking (TB) antibodies. We have investigated a downstream purification process of a sub-unit (R0.6C) fragment representing the C-terminal 6-Cys domain of Pfs48/45 (6C) genetically fused to the R0 region (R0) of asexual stage Glutamate Rich Protein expressed in Lactococcus lactis.The sexual stages of Pfs48/45. The recombinant protein induced high levels of functional TB antibodies in rats.A series of R0.6C fusion proteins containing features, which aim to increase expression levels or to facilitate protein purification, were evaluated at small scale. None of these modifications affected the overall yield of recombinant protein. Consequently, R0.6C with a C-terminal his tag was used for upstream and downstream process development. A simple work-flow was developed consisting of batch fermentation followed by two purification steps. As such, the recombinant protein was purified to homogeneity. The composition of the final product was verified by HPLC, mass spectrometry, SDS-PAGE and Western blotting with conformation dependent antibodies against The established production and purification process of the R0.6C fusion protein provide a strong basis for further clinical development of this candidate transmission blocking malaria vaccine.The online version of this article (doi:10.1186/s12934-017-0710-0) contains supplementary material, which is available to authorized users. Plasmodium falciparum malaria from one person to another requires the production of male and female gametocytes in the human host that can be taken up by blood-feeding mosquitoes. Recent studies have indicated that a large proportion of individuals in malaria endemic areas carry gametocytes and may contribute to malaria transmission [P. falciparum Pfs48/45 is one of the leading transmission blocking vaccine (TBV) candidates that has entered the pipeline of clinical development (for a review see [Pfs48/45 (6C) containing three disulfide bonds as major target of transmission blocking (TB) antibodies [Pfs48/45 region is targeted by a monoclonal antibody (mAb) mAb45.1 which promotes strong TB activity in the standard membrane feeding assay (SMFA), the gold standard for assessing transmission blockade ex vivo [The transmission of smission . Controlsmission . P. falcview see ). This pPfs48/45 in bacterial and eukaryotic expression systems has been problematic due to insufficient protein folding capabilities of these systems. Proper folding of cysteine-rich Pfs48/45 depends on correct formation of disulfide bridges. Since eukaryotic cells possess a sophisticated machinery for disulfide bond formation, recombinant Pfs48/45 has been produced in a range of Baculovirus (Spodoptera frugiperda Sf9) cells [Vaccinia virus [Saccharomyces cerevisiae [Pichia pastoris [Chlamydomonas reinhardtii [Nicotiana benthamiana [Lactococcus lactis expression system represents a significant advancement in the production of recombinant Pfs48/45 [L. lactis cell factories generally recognized as safe (GRAS status) are well suited for the production of heterologous proteins and used for a wealth of food applications. In the recent years, L. lactis has also been used in modern biotechnology within the fields of mucosal delivery [L. lactis do not produce endotoxins or extracellular proteases. Moreover, gene expression can be controlled by a set of tightly regulated promoters in a simple and scalable fermentation process from a few ml up to thousands of liters. Recombinant proteins can be secreted into the culture medium in the absence of spore formation which clearly facilitates downstream processing. Accordingly, L. lactis has been used for the manufacturing of the GMZ2 malaria vaccine candidate [The production of ia virus , Saccharrevisiae , and Picpastoris , Chlamydnhardtii , and Nicthamiana . HoweverPfs48/45 , 15. L. delivery  generatidelivery  and recodelivery . L. lactandidate –22.Pfs48/45, we established a manufacturing process for R0.6C in L. lactis. The product was characterized by mass spectrometry (MS)-based and by HPLC-based methods and tested for functional immunogenicity in rats. The specificity of induced antibodies was assessed by ELISA, and functional capacity for transmission blockade in the standard membrane-feeding assay (SMFA).To advance development of a protein-vaccine based on Pfs48/45 6-Cys domain to address whether expression levels of secreted R0.6C was affected by: (1) codon optimization, (2) different signal peptides, and (3) the presence and/or position of a range of affinity-tags. All constructs were transformed into L. lactis MG1363 and grown in 5 ml of LAB medium at 30 °C without shaking. Firstly, the codon optimized construct generated the same amount of recombinant R0.6C fusion protein as did the non-optimized construct  pH 7.4 supplemented with 15 mM imidazole. R0.6C was captured on a HisTrap HP column and bound protein was eluted with a linear imidazole gradient Fig. b. Fracti27–500–Pfs48/45291–428 fusion protein.The mass of non-reduced and reduced full-length R0.6C was 71325.4 and 71331.3 Da as determined by LC–MS respectively , representing the active monomer state  in Pfs48/45. Serum pools from each dosing group were tested for functional activity in the SMFA showing >99% TB activity at a 1/9 dilution.To ascertain that R0.6C elicit adequate levels of gametocyte-specific antibodies with the capacity to inhibit parasite fertilization, Wistar rats (N = 8) were immunized 3 times at 3-week intervals with increasing doses  of R0.6C inducing similar levels of vaccine-specific  but also process economics are key to keeping the price of the vaccine as low as possible, especially for vaccines targeting low-income countries.Pfs48/45 is production of correctly folded protein with sufficient yield . The purity of the R0.6C batch is 87% as determined by RP-HPLC. Impurities in the final R0.6C batch are likely, at least in part, product-related consisting of misfolded protein species or degradation products.From a manufacturing perspective, the major challenge with recombinant iewed in ). One wa0 region , 15. The enzymes . Thus, ded R0.6C  suggestsAlthough the final yield of purified recombinant R0.6C is almost 25 mg per L culture broth, we suggest that further improvements are possible. Such improvements may include procedures to increase the biomass through systems where lactate is removed from the culture broth during fermentation using for example the REED™  technology . AlternaPfs48/45-based vaccine candidates using immune purified protein [Pfs48/45 fusion protein purified by conventional chromatographic procedures together with Montanide ISA 720 VG elicits TB antibodies at the lowest dose of 2.5 µg. Previously, doses below 4.7 µg failed to elicit TB antibodies when adjuvanted in Alhydrogel [Pfs25, which was covalently coupled to VLPs [L. lactis expression system is able to accommodate a range of affinity tags added to either end of the recombinant protein.Previously we have investigated the immunogenicity of  protein , 15, 27.hydrogel . Improvehydrogel , 29 or m to VLPs , 24. In L. lactis microbial factories are highly suited for the production of Pfs48/45. A manufacturing process for soluble and correctly folded R0.6C was developed and the resulting vaccine antigen induces high levels of functional antibodies. The high yield of correctly folded protein and the straightforward production process offers the possibility to investigate the vaccine potential of Pfs48/45 in human clinical trials.In conclusion, we have demonstrated that L. lactis pSS1 plasmid vector [BamHI-BglII was synthesized by  and inserted into pSS1. For construction of chimeric N-terminal (mSA-R0.6C.6H) or C-terminal (R0.6C-mSA.6H) R0.6C, a synthetic DNA fragment encoding the monomeric Streptavidin (mSA) (GeneBank: 4JNJ_A) with a small Gly–Gly-Ser linker was synthesized by (GeneArt® Life Technologies) and cloned into R0.6C.6H. The USP45-R0.6C.6H construct was generated by replacing the mutD310 signal peptide in pSS1 with the USP45 signal peptide [® Life Technologies) and cloned into R0.6C. All the constructs were verified by sequencing and subsequently transformed into L. lactis MG1363 by electroporation as described [All the constructs are based on the d vector . The R0.d vector . Non-tagd vector  containiescribed .L. lactis MG1363, containing R0.6C constructs was grown overnight at 30 °C in 5 ml LAB medium [g for 20 min and analysis of all the constructs was performed by Coomassie stained SDS-PAGE gel and Western blotting with mAb45.1 against conformational epitope I. Fermentation of L. lactis MG1363/R0.6C.6H was performed in a 1 l lab scale bioreactor at 30 °C with gentle stirring (150 rpm). For the time-course experiment 10 ml samples were withdrawn and used for analysis. Optical density at 600 nm (OD600) was used to assess cell density. Cell-free culture-filtrates were concentrated tenfold and buffer exchanged into phosphate buffered saline (PBS) pH 7.4 supplemented with 15 mM imidazole) using a QuixStand Benchtop system  followed by filtration through a Durapore filter  and applied to a 5 ml HisTrap HP column . Bound protein was step gradient eluted with 500 mM imidazole in tris buffer pH 8.0  at a flow rate of 4 ml/min and fractions containing the desired protein were applied to a 5 ml HiTrap Q HP column . Bound protein was eluted through step gradient elution in tris buffer pH 8.0  and fractions containing monomers with the highest amount of mAb45.1-reactive protein were concentrated by a VIVA spin column 30 kDa cutoff , and kept in 55 mM tris, 300 mM NaCl, 0.025% Tween 80 and 1 mM EDTA, pH 8.0 at −80 °C until use. Immune purification of R0.6C was performed as described earlier [B medium  suppleme earlier , 15. Anal-arginine, 0.05% NaN3, pH 6.7) at room temperature. For RP-HPLC, 210 pmol of protein was loaded on the RP-column and eluted with a linear gradient of 3–95% of 0.1% trifluoroacetic acid (TFA), 20% isopropanol and 70% acetonitrile over 30 min. The absorbance was measured at 280 or 215 nm for SEC–HPLC or RP-HPLC, respectively, and chromatographic peaks were integrated by HPLC ChemStation .Native size exclusion high-performance liquid chromatography (SEC–HPLC) or reversed-phase HPLC (RP-HPLC) of intact R0.6C were performed using an Agilent 1100 Series HPLC System  equipped with a TSKgel G3000SWXL SEC column, 5 µm, 7.8 × 300 mm  or equipped with a Vydac 214TP C4 reversed-phase column, 5 µm, 4.6 × 250 mm , respectively. 210 pmol of protein was loaded on the SEC column and eluted with a 0.15 ml/min flow of elution buffer  and eluted onto a Q-TOF mass spectrometer  with a chromatographic gradient. LC–MS data where recorded and analyzed by MassLynx software . Mass spectra were deconvoluted using the MaxEnt 1 algorithm in the MassLynx software. Peptide mapping of R0.6C was performed by LC–MS/MS of tryptic digests. Guanidine hydrochloride, dithiothreitol and iodoacetamide were added to 100 pmol of protein. This was followed by addition of iodoacetamide and the protease trypsin. Tryptic peptides were desalted and chromatographically separated over 40 min on a UHPLC C18 pre column and column, respectively  using a nanoACQUITY UPLC system . The eluate ionized by electrospray ionization and data were acquired on the same mass spectrometer software as described above. LC–MS/MS data were analyzed using PLGS  and peptide maps were created using DynamX . Cysteine connectivity characterization was done with identical UPLC conditions as for peptide mapping. Tryptic digestion was additionally performed under similar conditions, with and without addition of guanidine hydrochloride, dithiothreitol or iodoacetamide to produce peptides without and without intact cysteine bonds, respectively. Collision induced dissociation (CID) fragmentation was performed on the two identified cross-linked peptides to produce MS/MS spectra used for manual analyzing the MS/MS fragments by MassLynx software for characterization of cysteine connectivity.Female Wistar Hannover rats  and kept in the Laboratory Animal Facility Centre at Panum, University of Copenhagen, Denmark for 7 days before the first immunization. All procedures regarding animal immunizations complied with European and National regulations. Groups of eight rats were immunized by the s.c. route 3 times at 3-week intervals with increasing doses  of R0.6C. Protein preparations were emulsified in Montanide ISA720 VG (Seppic) immediately before use. Responses were measured using sera taken three weeks after the third immunization (Day 63).Pfs48/45 (6C) and R0.6C was 0.5 µg/ml and bound antibodies were detected with HRP-conjugated rabbit-anti rat IgG-HRP  as described in detail [® ELISA plates, The Netherlands) were coated at 4 °C overnight with 100 µl per well of 5 µg/ml of mAb45.1 in PBS pH 7.4. Wells were blocked with 150 µl of 5% non-fat skimmed milk powder in PBS for 1 h followed by three washings with PBS containing 0.05% Tween 20 (PBST). Sera at threefold dilutions (50 µl) were mixed with 50 µl R0.6C (1 µg/ml) in PBST, added to the wells and incubated for 4 h at room temperature. Wells were washed with PBST and bound antigen was revealed with anti-His-HRP (1/6000 in PBST) for 2 h at room temperature. Midpoint (EC50) values were calculated using GraphPad Prism 7, .The mAb45.1 sandwich ELISA was performed as previously described , 15. Then detail . The mAbThe biological activity of anti-R0.6C antibodies was assessed in the SMFA as previously described in detail . EstimatThe Mann–Whitney rank sum test was used for analyzing differences between groups. P < 0.05 was considered to be statistically significant.Additional file 1. Production and purification of R0.6C.Additional file 2. Intact mass analysis of R0.6C. Mass spectra of full-length antigens by LC–MS and peptide maps from LC–MS/MS of trypsin digested antigens. (a) Deconvoluted mass spectra of non-reduced R0.6C. (b) Deconvoluted mass spectra of reduced R0.6C. (c)Tryptic peptide maps of R0.6C. In brackets is the calculated accuracy of the measurement to the predicted mass in ppm. All measurements were done in duplicates.Additional file 3. Stability of R0.6C. R0.6C was incubated at 4 °C for 0, 15 and 30 days. Three and 1 µg of R0.6C with (+) and without (−) DTT was analyzed by (a) SDS-PAGE coomassie staining, (b) Immune-blotting using mAb 45.1 and (c) Sandwich ELISA. Antigens were captured with mAb45.1 and detected with anti-His-HRP.


Mycobacterium tuberculosis which is persisting inside mature adipocytes. It remains unknown whether this holds true for Mycobacterium canettii, a rare representative of the M. tuberculosis complex responsible for lymphatic and pulmonary tuberculosis. Here, we infected primary murine white and brown pre-adipocytes and murine 3T3-L1 pre-adipocytes and mature adipocytes with M. canettii and M. tuberculosis as a positive control. Both mycobacteria were able to infect 18–22% of challenged primary murine pre-adipocytes; and to replicate within these cells during a 7-day experiment with the intracellular inoculums being significantly higher in brown than in white pre-adipocytes for M. canettii (p = 0.02) and M. tuberculosis (p = 0.03). Further in-vitro infection of 3T3-L1 mature adipocytes yielded 9% of infected cells by M. canettii and 17% of infected cells by M. tuberculosis (p = 0.001). Interestingly, M. canettii replicated and accumulated intra-cytosolic lipid inclusions within mature adipocytes over a 12-day experiment; while M. tuberculosis stopped replicating at day 3 post-infection. These results indicate that brown pre-adipocytes could be one of the potential targets for M. tuberculosis complex mycobacteria; and illustrate differential outcome of M. tuberculosis complex mycobacteria into adipose tissues. While white adipose tissue is an unlikely sanctuary for M. canettii, it is still an open question whether M. canettii and M. tuberculosis could persist in brown adipose tissues.Adipose tissues were shown to host  In mammals, brown adipose tissue (BAT) and white adipose tissue (WAT) are mainly composed of mature adipocytes, pre-adipocytes and immune cells , resistance to anti-mycobacterial drugs , Mycobacterium canettii is a peculiar member specifically diagnosed in dozens of tuberculosis patients with reported contacts to the Horn of Africa  supplemented with 10% oleic acid-albumin-dextrose-catalase (OADC) (Becton Dickinson). Fluorescent M. canettii CIPT 140010059 mCherry (CSURP3621) was constructed by transforming the pMV261 mCherry vector  into the M. canettii strain , 0.5 mM isobuthyl-methylxanthine (Sigma), and 1 μM dexamethasone (sigma) into the culture medium. After 48 h of induction, the medium was replaced by DMEM-10% FBS containing 1 μg/mL of insulin and maintained each 2 days in order to achieve a full differentiation as previously described  were cultured in DMEM  supplemented with 10% heat-inactivated FBS at 37°C under a 5% CO5 cells per well were infected with 1 × 105 mycobacteria. After 4-h incubation, cells were washed 3 times with serum-free DMEM to remove all extracellular bacteria. Only mature adipocytes were then incubated with 200 μg/mL amikacin for 2 h and then washed thrice. The last wash was plated to validate the correct elimination of extracellular mycobacteria. The inoculated cultures were incubated with fresh complete medium up to 7 days for primary cultures and 12 days for 3T3-L1 cell lines at 37°C under a 5% CO2 atmosphere. At various time points, infected cells were lysed in PBS containing 0.1% Triton X-100 and the lysate was plated at 10-fold dilutions from 10−1 to 10−4 onto Middlebrook 7H10 agar plates incubated at 37°C under a 5% CO2 atmosphere. The number of colonies enumerated after 20-day of incubation was used to estimate the number of colony-forming units (CFUs) per 105 cells. All experiments were conducted in triplicate: three independent plates the same day for primary cell infection experiments and three replicates at different days with different bacterial cultures for 3T3-L1 cells infection experiments.Pre-adipocytes and mature adipocytes were infected with a multiplicity of infection (MOI) of 1 per cell at day 6 after plating and at day 10 post-induction respectively. Accordingly, 1 × 10Ziehl-Neelsen staining  was performed on infected cells (MOI 1:1 and 5:1) at day 3 p.i. Observations were acquired using the slide scanner Axio Scan.Z1 (Zeiss) (Magnification 20X) with a color Hitachi tri-CCD  camera. The number of intracellular mycobacteria per one cell was recorded on image acquisition by ImageJ software.M. canettii mCherry was used to localize intracellular bacteria using confocal microscopy. Infected adipocytes (MOI 10) were fixed using 4% formaldehyde at day 3 p.i. Lipid droplets were stained with BODIPY 493/503 (Sigma). The images were acquired using a confocal microscope  at magnification 63X/1.4 oil objective.2, and MgCl2 5 mM, washed with complete cacodylate buffer and postfixed for 1 h at room temperature with 1% osmium tetroxide in the same buffer without sucrose  containing 0.1 M sucrose, 5 mM CaClM. canettii at MOI 5:1 on 12-well plate. At day 7 p.i. all wells were lysed in 500 μL water containing 0.1% Triton X-100. The pooled lysate was sonicated and centrifuged at 9,000 g for 25 min. The M. canettii cells were washed thrice with 0.1% Triton X-100 and the pellet was resuspended in 500 μL of PBS containing 0.05% Tween 80. Nile red staining was performed as previously described  were collected at day 3, 7, and 12 post-inoculation. The release of LDH from negative controls and infected cells was measured spectrophotometrically at 340 nm on a Cobas 8000 .LDH, a soluble cytoplasmic enzyme released into extracellular space when the cell membrane is damaged, was used as an estimator of cell lysis  but the number of bacilli in brown pre-adipocytes was significantly higher than in white pre-adipocytes (p = 0.02 for M. canettii and p = 0.03 for M. tuberculosis).The ratio of infected cells relative to the total number of observed cells as well as the average number of intracellular bacilli per one cell were measured on a random selection of 10 microscope fields on stained infected cells at day 3 p.i.  and 10% (9 × 103 ± 4 × 103 CFUs) of the initial 1 × 105 inoculum of M. canettii and M. tuberculosis, respectively. As for brown pre-adipocytes, 45% (4.5 × 104 ± 2.3 × 103 CFUs) of the M. canettii inoculum and 25% (2.47 × 104 ± 4 × 103 CFUs) of the M. tuberculosis inoculum was phagocytized. By scoring CFUs, intracellular M. canettii and M. tuberculosis organisms were more abundant in primary brown pre-adipocytes than in white pre-adipocytes (p = 0.01 for M. canettii and p = 0.008 for M. tuberculosis) which is consistent with our microscopic observations.In order to confirm these observations and to study the viability of bacilli into the infected cells, the number of intracellular mycobacteria was quantified by scoring CFUs/10s Figure  . Under the same conditions, only 9% and 17% of adipocytes were infected with M. canettii and M. tuberculosis, respectively. The ratio of infected cells was significantly higher for pre-adipocytes compared to mature adipocytes .To explore M. canettii within 3T3-L1 mature adipocytes, cells were inoculated with M. canettii CIPT 140010059 mCherry (CSURP3621) at MOI = 10. Three days p.i. infected cells were treated with BODIPY 493/503 stain and fixed. Confocal microscopy observations showed the presence of 15 ± 6 intracellular M. canettii inside adipocytes, as compared to negative controls which remained free of detectable mCherry-fluorescence  in 100% of the observed adipocytes  and 50% of the initial M. tuberculosis inoculum (5 × 104 ± 8 × 103 CFUs). In mature adipocyte infection experiments, 12% (1.2 × 104 ± 4 × 103 CFUs) of the infecting M. canettii inoculum and 37% of the infecting M. tuberculosis inoculum (3.7 × 104 ± 4 × 103 CFUs) was internalized. Initial infection of mature adipocytes was significantly lower with M. canettii (p = 0.001) than with M. tuberculosis, as observed with infected pre-adipocytes (p = 0.006), but numbers of intracellular mycobacteria from day 0 were significantly higher in pre-adipocytes than in mature adipocytes (p = 0.01 for M. canettii and p = 0.03 for M. tuberculosis).As for internalization, at day 0, pre-adipocytes contained 25% of the initial M. tuberculosis and M. canettii showed two different kinetics: intracellular M. tuberculosis inoculum stabilized between day 3 and 12 p.i. (4 ± 3.66 CFUs gain), whereas M. canettii replicated with a 2 × 101 ± 2.5 × 101 CFUs gain between day 3 and 12 p.i. which is significantly higher than M. tuberculosis (p = 0.01)  in the supernatant of infected and non-infected control adipocytes (see Supplementary Data M. canetti-infected adipocytes than in negative control adipocytes at day 3 p.i. (p = 0.002) and day 12 p.i. (p <0.001) .Microscopic examination indicated that ) Figure . In M. tt Figure . PositivM. canettii is able to infect, survive and replicate in primary murine white and brown pre-adipocytes which are constantly detected in adipose tissues  funded by the “Investissements d'Avenir” French Government program, managed by the French National Research Agency (ANR).This study was financially supported by URMITE, IHU Méditerranée Infection, Marseille, France; CNRS and by the AThe 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.


Background: Chronic inflammation is one of the important mediators of colitis-related colon cancer (CRC). Abundant mast cells (MCs) were observed in the tumor microenvironment and mediators released upon MC activation play an important role in the process of chronic inflammation. Previously, we found that activation of intestine mucosal MCs recruited and modulated the inflammatory CD11b+Gr1+ cells to promote the CRC development. In the current study we investigated the effects of Vam3, a resveratrol dimer with potent anti-inflammatory effects, on CRC development.Methods: RBL-2H3 cells, a basophilic leukemia cell line, were pretreated with 2.5 or 5 µM Vam3 and then stimulated with dinitrophenol-conjugated bovine serum albumin (DNP-BSA) plus lipopolysaccharide (LPS). The MC degranulation was determined by measuring β-hexosaminidase release. Generation of TNF-α and IL-6 in RBL-2H3 cells or in peritoneal macrophages was determined by ELISA and real-time qPCR. NF-κB p65 and phospho-NF-κB p65 expression was determined by Western blotting. NF-κB activity in RAW264.7 cells was determined by luciferase reporter assay. CRC was induced in C57BL/6 mice by intraperitoneal injection of azoxymethane (AOM), followed by oral exposure to dextran sodium sulfate (DSS). Vam3 at 50 mg/kg, or disodium cromoglycate  at 100 mg/kg, or vehicle were administrated to the mice 4 weeks after DSS withdrawal. Levels of TNF-α, IL-6, and mouse MC protease-1 were determined by ELISA. Infiltration of CD11b+Gr1+ cells was determined by flow cytometry analysis. One-way ANOVA was used to compare difference between groups.Results: Pretreatment with Vam3 significantly inhibited RBL-2H3 cell degranulation and inflammatory cytokine production from RBL-2H3 cells and from peritoneal macrophages. After Vam3 treatment, NF-κB activity in RAW264.7 cells, and expressions of phospho-NF-κB p65 in RBL-2H3 cells and in peritoneal macrophages were significantly down-regulated. In the AOM plus DSS-induced CRC murine model, the Vam3 and DSCG-treated mice had less tumor numbers than those treated with vehicle. Expression of phospho-NF-κB p65, production of inflammatory cytokines, and infiltration of MCs and CD11b+Gr1+ cells were attenuated in the Vam3-treated mice.Conclusion: Vam3 treatment could attenuate the CRC development. This effect may be due to its inhibition on NF-κB signaling pathway in MCs and macrophages of the inflamed intestines. Intrinsic genetic lesion is critical in tumor formation, nevertheless, the importance of chronic inflammation in cancer development has been highlighted recently . EpidemiAs the important sentinel cells to the inflammatory stimuli, the role of mast cells (MCs) in promoting the CRC development has been identified in recent years . Based oLipopolysaccharide (LPS)/Toll-like receptor-4 (TLR4)/NF-κB pathway plays a crucial role in the development of CRC . TLR4 isVitis amurensis Rupr., which grows in northeastern and central China. Previously we found that Vam3 possessed potent anti-inflammatory and anti-oxidant effects  and murine macrophage cell lines (RAW264.7) were obtained from the American Type Culture Collection . LPS (serotype O127:B8), AOM, disodium cromoglycate (DSCG), dinitrophenol (DNP)-specific IgE, and DNP-conjugated bovine serum albumin (DNP-BSA) were purchased from Sigma–Aldrich . DSS  was purchased from MP Biomedicals, LLC . FITC-conjugated anti-mouse CD45 antibodies, PE.Cy7-conjugated anti-mouse CD11b antibodies, and APC-conjugated anti-mouse Gr1 antibodies were purchased from eBioscience . Recombinant mMCP-1 was purchased from Sino Biological Inc. . Anti-CD117 antibody was purchased from Dako . FuGENE HD Transfection Reagent was purchased from Promega . Anti-phospho-NF-κB p65 (Ser536) antibodies, anti-NF-κB p65 antibodies, and anti-β-actin antibodies were purchased from Cell Signaling Technology .Compound Vam3, synthesized from resveratrol, was provided by the Institute of Materia Medica, Chinese Academy of Medical Sciences . The cells were then cultured at 37°C in an air atmosphere containing 5% CO2 for 2 h. The non-adherent cells were removed by washing with warmed PBS.Peritoneal macrophages were isolated according to the reference . Briefly. Peritoneal macrophages isolated as described above were grown in RPMI-1640 medium containing 10% FBS. Cell viability was assessed using the MTT assay. RBL-2H3, peritoneal macrophages, and RAW264.7 were plated into 96-well plates at a density of 1 × 104 cells per well. Following overnight culture, the cells were then treated with Vam3  for 12, 24, 36, or 48 h. The same volume of DMSO was added to control wells. The cells were subsequently incubated in fresh medium containing MTT solution (0.5 mg/mL) for 4 h at 37°C. The culture medium was removed, and the formazan dye was dissolved in DMSO. The optical densities (OD) at 570 nm were measured with a microplate reader. Relative cell viability was expressed as percentage of controls that were cultured in the absence of Vam3.RBL-2H3 and RAW264.7 cells were grown in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% FBS4 cells per well and sensitized with 0.1 μg/mL DNP-specific IgE for 24 h. The cells were then treated with Vam3  for 2 h followed by adding 1 μg/mL LPS for 48 h. The same volume of DMSO was added to control wells. To test degranulation, the cells were stimulated with 500 ng/mL DNP-BSA for 30 min. Fifty microliters of cell culture supernatant or cell lysates  were mixed with an equal volume of substrate solution  and incubated for 1 h at 37°C. The enzyme reaction was terminated by adding 200 μL of stop solution , and the absorbance was measured at 405 nm with a microplate reader. Release of β-HEX was calculated as a percentage of β-HEX release in the culture medium to the whole β-HEX release in the culture medium and in the cell lysate.RBL-2H3 cells, a rat basophilic leukemia cell line that was widely used in MC-associated studies , were pl4/well) and peritoneal macrophages (5 × 105 cells/well) were pretreated with Vam3  for 2 h followed by stimulating with 1 μg/mL LPS for 6 h  or for 24 h . The same volume of DMSO was added to control wells. Total RNA was extracted using TRIzol. First-strand cDNA was synthesized using Prime Script RT Reagent kit . Real-time qPCR was performed using SYBR Premix Ex Taq . Primers used for real-time qPCR (Table 1) was purchased from SinoGenoMax . The relative mRNA levels of TNF-α and IL-6 were determined with β-actin or GAPDH as the control and represented as 2-ΔΔCt. The cell culture supernatant TNF-α and IL-6 levels were determined using ELISA kits .RBL-2H3 cells  for 2 h and then stimulated with 1 μg/mL LPS for 24 h. The same volume of DMSO was added to control wells. The cells were lysed with 1x passive lysis buffer. Luciferase activity was measured using the luciferase assay kit .Murine macrophage RAW264.7 cells were plated in 48-well plates at 1 × 10RBL-2H3 cells and peritoneal macrophages were pretreated with Vam3  for 2 h followed by stimulating with 1 μg/mL LPS for 12 h. The same volume of DMSO was added to control wells. Total protein was extracted using protein extraction kit . NF-κB p65 and phospho-NF-κB p65 expression was examined by Western blotting. The density of each band was quantified by QuantiScan Version 11 . Densities of NF-κB p65 and phospho-NF-κB p65 bands were normalized against β-actin.n = 9) received DSCG (n = 9) received Vam3 daily at 50 mg/kg of body weight, and one group (n = 9) were treated with vehicle control (0.1% Tween 80 in distilled water). All the reagents were administrated by gavage for 2 weeks. Mice were sacrificed by CO2 asphyxiation 8 weeks after completing the treatment and the colon tissues were collected. Each colon was opened longitudinally and any fecal contents were cleared out with ice-cold saline. The colon weight was measured, and the numbers of polyps were macroscopically assessed individually. The colons were then fixed in 10% formaldehyde and analyzed after staining with hematoxylin and eosin. MCs were stained as reported using toluidine blue  and Lamina propria lymphocytes (LPL) were prepared as previously described . Isolaten = 3): the naive control group was pretreated with the vehicle control (0.1% Tween 80 in distilled water) and intraperitoneally injected with 0.5 mL DMEM medium; the mMCP-1 group was pretreated with the vehicle control and intraperitoneally injected with 10 ng recombinant mMCP-1 diluted into 0.5 mL DMEM medium; the Vam3 group was pretreated with 50 mg/kg Vam3 and intraperitoneally injected with 10 ng recombinant mMCP-1 diluted into 0.5 mL DMEM medium. Cells in peritoneal cavity were collected and counted 22 h after the mMCP-1 injection. Isolated cells were stained with FITC-conjugated anti-mouse CD45, PE.Cy7-conjugated anti-mouse CD11b, and APC-conjugated anti-mouse Gr1 antibodies for FACS. All procedures involving mice  were approved by the Institutional Animal Care and Use Committee at the Cancer hospital, Chinese Academy of Medical Sciences.Male C57BL/6 mice, 8–10 weeks, were randomly divided into three groups . Therefore, we chose at least 2.5 μM Vam3 for the following work. The cell viability of RBL-2H3, fresh isolated peritoneal macrophages, and RAW264.7 cells was not affected when all these cells were treated with Vam3  for 24 h . No significant cytotoxicity was observed after incubation with 5 μM Vam3 for up to 48 h . However, 10 μM Vam3 reduced the cell viability of RBL-2H3 cells to 81.71 ± 3.28% of control . Therefore, 2.5 and 5 μM were selected for the present study.The chemical structure and in vitro model of mucosal MCs. Since mucosal MCs are mainly reside within the intestinal mucosa where they are readily exposed to the microbial products, we used DNP-BSA plus LPS-induced degranulation in RBL-2H3 cells as a preliminary screening method to investigate the effects of Vam3 on MC degranulation. Compared with the cells stimulated with DNP-BSA plus LPS, pretreatment with 2.5 and 5 μM Vam3 reduced β-HEX release from an average of 35.21 ± 3.05 to 30.44 ± 3.37% (p = 0.023) and 26.61 ± 1.90% (p < 0.01), respectively . However, the levels of β-HEX release were still higher in the Vam3-treated groups than baseline (5.46 ± 2.54%).RBL-2H3 cells have been used as a model of intestinal mucosal MCs . The celp = 0.05) and 76.13 ± 11.55 pg/mL (p < 0.01), respectively , but not to baseline level (43.63 ± 8.90 pg/mL). These data suggested that pretreatment with Vam3 could inhibit MC degranulation.We then determined the effects of Vam3 on TNF-α release, the main preformed inflammatory cytokine in MCs. The TNF-α production in RBL-2H3 cells stimulated with DNP-BSA plus LPS was 110.37 ± 9.85 pg/mL. When the cells were pretreated with 2.5 and 5 μM Vam3 for 2 h, TNF-α production was reduced to 85.77 ± 11.19 pg/mL (p = 0.044) and 2.06 ± 0.08 (p = 0.025), respectively , and reduced the protein levels of TNF-α from an average of 130.63 ± 6.49 pg/mL to 106.17 ± 5.79 pg/mL (p = 0.022) and 92.20 ± 3.05 pg/mL (p < 0.01), respectively . However, the mRNA and protein levels of TNF-α in the Vam3-treated groups were still higher than baseline. These data suggested that pretreatment with Vam3 could inhibit LPS-stimulated TNF-α production in MCs unrelated to degranulation.Mast cells could produce pro-inflammatory cytokines, such as TNF-α, in response to LPS without degranulation occurring. So we further investigated the effects of Vam3 on TNF-α production in LPS-stimulated RBL-2H3 cells. Compared with the cells stimulated with LPS alone, pretreatment with 2.5 and 5 μM Vam3 reduced the relative mRNA levels of TNF-α from an average of 2.32 ± 0.04 to 2.09 ± 0.12 . Pretreatment with 2.5 and 5 μM Vam3 reduced the protein levels of TNF-α from an average of 1268.02 ± 25.82 pg/mL to 964.01 ± 119.39 pg/mL (p = 0.032) and 718.88 ± 129.13 pg/mL (p < 0.01), respectively . Meanwhile, when the cells were pretreated with 2.5 and 5 μM Vam3 for 2 h, the relative mRNA levels of IL-6 decreased from an average of 2.42 ± 0.01 to 2.24 ± 0.05 (p = 0.038) and 2.06 ± 0.02 (p < 0.01), respectively , and the protein levels of IL-6 decreased from an average of 1393.46 ± 30.55 pg/mL to 1242.89 ± 64.33 pg/mL (p = 0.038) and 1056.01 ± 71.28 (p < 0.01), respectively . However, the mRNA and protein levels of TNF-α and IL-6 in the Vam3-treated groups were still higher than baseline. These data suggested that pretreatment with Vam3 could inhibit LPS-stimulated cytokine production in macrophages.Primary peritoneal macrophages are a common source of macrophages for various and IL-6 . At the and IL-6 . Moreoveand IL-6 . Lamina and IL-6 . So we fFigure 5A). All of the mice developed colon tumors 14 weeks after DSS withdrawal. However, Vam3 and DSCG treatment reduced total tumor numbers from an average of 6.50 ± 2.07/mice in the vehicle-treated mice to 3.40 ± 1.58/mice (p < 0.01) and 3.20 ± 2.49/mice (p = 0.018), respectively . Treatment with Vam3 and DSCG reduced colon weight from an average of 0.48 ± 0.03 g to 0.38 ± 0.02 g (p = 0.050) and 0.35 ± 0.02 g (p = 0.010), respectively . The effect of Vam3 was not different from that of DSCG in Figures 5C,D (p > 0.05). Histologically, the vehicle-treated mice showed more destruction of the colonic epithelium, a thicker muscularis layer and adenocarcinoma development. However, the structure of colonic epithelium was improved in the Vam3 and DSCG-treated mice . These results collectively indicated that Vam3 treatment could inhibit CRC development in the murine model.The CRC murine model was induced by intraperitoneal injection with procarcinogen AOM followed by one cycle of DSS exposure. Four weeks after DSS withdrawal, the mice were treated with Vam3, DSCG, and vehicle for 2 weeks  and DSCG (p = 0.018)-treated relative to the vehicle-treated mice. We then stained the colon tissues with antibody against CD117. CD117 is an early marker for MC precursors and expressed throughout their lifetime (p = 0.050) and DSCG (p = 0.017)-treated relative to the vehicle-treated mice . The effect of Vam3 was not different from that of DSCG in reducing MC infiltration (p > 0.05).As Vam3 could inhibit RBL-2H3 cell degranulation and decrease cytokine production in RBL-2H3 cells and macrophages, we hypothesized that the inhibitory effect of Vam3 on CRC development may be due to its inhibition on MC degranulation and cytokine production. As shown in lifetime . The numin vivo  and 2098.89 ± 1559.16 pg/mL (p = 0.016), respectively . The effect of Vam3 was not different from that of DSCG in Figure 6E (p > 0.05). The mMCP-1 levels in serum did not decrease significantly after Vam3 treatment . However, as mentioned above, it is important to monitor biomolecules in the compartment where they are biologically active, such as interstitial fluid. These data indicated that Vam3 treatment inhibited MC infiltration and activation in vivo.The mMCP-1 is predominantly expressed by intestinal mucosal MCs. After activation and degranulation of the mucosal MCs, mMCP-1 is released from mucosal MCs. Treatment with Vam3 and DSCG reduced interstitial liquid mMCP-1 levels from an average of 4266.94 ± 329.48 pg/mL in the vehicle-treated mice to 2433.57 ± 896.16 pg/mL . Meanwhile, the mice treated with Vam3 revealed a lower IL-6 level, averaging 18.37 ± 13.33 pg/mL, than the mice treated with vehicle (52.10 ± 40.60 pg/mL) (p = 0.034) . These results collectively indicated that Vam3 treatment could inhibit the MC infiltration and degranulation, and attenuated cytokine production in vivo.TNF-α and IL-6, expressed in the tumor microenvironment, are important for the development of inflammation-associated intestinal tumorigenesis. Treatment with Vam3 reduced TNF-α levels from an average of 36.65 ± 8.80 pg/mL in the vehicle-treated mice to 19.61 ± 3.71 pg/mL . The effect of Vam3 was not different from that of DSCG (p > 0.05).CD11bactivity . mMCP-1 mulation . We then+Gr1+ cell infiltration, mice were treated with Vam3 and recombinant mMCP-1 was injected into mouse peritoneal cavity, where no mucosal MCs reside. Consistent with previous study . However, there were no significant differences in CD11b+Gr1+ cell numbers between the vehicle and Vam3-treated mice that received mMCP-1 . These results indicated that Vam3 treatment did not directly inhibit CD11b+Gr1+ cell infiltration. It is most likely that Vam3 inhibited CD11b+Gr1+ cell infiltration through suppression of MC degranulation.To test whether Vam3 may directly inhibit CD11bus study , the CD1p < 0.01; Figures 8A,B). In a similar pattern, LPS significantly increased phospho-NF-κB p65 expression in peritoneal macrophages (p < 0.01) whereas Vam3 pretreatment at 2.5 μM (p = 0.044) and 5 μM (p < 0.01) reduced this change . Consistent with the in vitro studies above, treatment with Vam3 reduced relative phospho-NF-κB p65 expression in colon tissues from an average of 0.77 ± 0.20 to 0.58 ± 0.08 . However, NF-κB p65 levels were not changed in LPS-stimulated RBL-2H3 cells and peritoneal macrophages and in colon tissues.NF-κB plays a central role in DNP-BSA plus LPS-induced MC degranulation and in LPS-induced cytokine production in MCs and macrophages. In the NF-κB signaling cascade, phosphorylation of p65 at the serine 536 position is required for nuclear translocation . Therefop = 0.050) and 263.77 ± 19.06-fold (p = 0.022) of the control, respectively . All these findings indicated that Vam3 treatment inhibited NF-κB activation.In addition, we used an NF-κB-dependent luciferase assay to further determine the effect of Vam3 on NF-κB activity in LPS-stimulated RAW264.7 cells. Compared with the cells stimulated with LPS alone, pretreatment with 2.5 and 5 μM Vam3 reduced the relative luciferase activity from 700.41 ± 260.86-fold of the control to 331.59 ± 32.65-fold . Previously we found that oral administration of Vam3 at about 50 mg/kg inhibited inflammatory cell recruitment, cytokine production and NF-κB activation in ovalbumin (OVA)-induced asthma murine model (unpublished data), and showed anti-oxidative effect on cigarette smoke-induced damage . Therefo+Gr1+ in mice. MDSCs are responsible for T cell suppression and the formation of the immunosuppressive tumor microenvironment that favors tumor growth. Recent studies highlight the importance of the bidirectional axis between MDSCs and MCs in creating tumor-inflammatory and tumor-immunosuppressive microenvironments . Vam3 might be a potential prophylactic agent for CRC therapy.In summary, our results showed that administration of Vam3 could attenuate the CRC development in an AOM plus DSS-induced CRC murine model. This effect may be due to its inhibition on NF-κB signaling pathway in MCs and macrophages of the inflamed intestines (in vitro experiments. ZW and HY assisted in animal experiments. CY carried out chemical synthesis. QH designed the study and revised the paper. CQ designed the study, drafted and revised the manuscript. All authors read and approved the final manuscript.LX designed and performed the experiments, analyzed the data, and drafted the manuscript. RJ assisted in 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.