Lens culinaris in chromium polluted soils by using two different chromium salts i.e., K2Cr2O7 and K2CrO4 in varying concentrations . The results revealed that Bacillus species are efficient in significantly reducing the deleterious effects of Cr. These effective bacterial strains were able to stimulate the growth of metal effected plants of Lens culinaris which were grown in chromium contaminated environment. Therefore, these plant growth promoting rhizobacteria PGPRs, having both auxin production potential and chromium-resistance ability, are considered as efficient micro-factories against chromium pollution.Bacteria are tiny organisms which are ubiquitously found in the environment. These microscopic living bodies are responsible for the flow of nutrients in biogeochemical cycles and fertility imparted to the soil. Release of excessive chromium in agricultural soils due to rapid growth of industries may result in minimizing the fertility of soil in future, which will lead to reduction in crop production. Plant growth promoting bacteria (PGPB) are beneficial to the environment, some of which can tolerate chromium and protect plants against heavy metal stress. The current study aims to identify such chromium-tolerant auxin-producing rhizobacteria and to investigate their inoculation effects on the growth characteristics of  Microorganisms living in the rhizospheric regions are better able to naturallly degrade hazardous soil compounds into less toxic forms. Beneficial interactions of these microscopic creatures with soil nutrients are essential for a balanced nutrient flow in the soil. Therefore, being primary producers, bacteria play an important role for sustaining life on earth .Rapid industrialization over the past many years has resulted in many environmental problems worldwide . As a re2Cr2O7 is more toxic than K2CrO4 so, wherever possible in the industries, it should be replaced with other Cr-salts like K2CrO4 which are less toxic.Many reports suggest that microbial activities may counteract negative effects of chromium (Cr) and other heavy metals (HMs) in plants growing in contaminated areas through certain mechanisms . These m2Cr2O7 and K2CrO4. Bacteria which were resistant to chromium, were then screened for their auxin production potential. Three chromium-tolerant auxin-producing bacterial strains  were selected and characterized for their morphological as well as physiological characteristics. Molecular identification of these bacterial isolates was carried out using 16S rDNA sequencing and obtained DNA sequences were then searched for their homology with other sequences through BLAST and submitted to GenBank to obtain their accession numbers.In the present study, different soil and water samples were collected from contaminated areas of Kasur, Pakistan. MIC (minimum inhibitory concentration) of chromium salt stress for the growth of selected bacterial isolates was determined using KLens culinaris (var. masoor-2009) seeds with and without chromium stress. The effect of selected bacterial cultures with and without chromium stress (K2Cr2O7 and K2CrO4 (500 µgml−1)) on the growth of plants inoculated with bacterial isolates was observed by comparing their growth parameters with the non-treated plants. Certified seeds of Lens culinaris (var. masoor-2009) were procured from a Government seed organization  and these seeds were then surface sterilized to remove dust and other bacteria by treating these seeds with standard solution of 0.1% HgCl2 for 3 to 4 min and then washed several times with autoclaved distilled water. These sterilized lentil seeds were treated with chromium-tolerant bacterial liquid culture (inoculum) after adjusting the optical density of the culture to 106–107 CFU ml−1 at 600 nm. For control treatment, seeds were treated with autoclaved distilled water for the same period of time. The inoculated seeds of Lens culinaris (var. masoor-2009) were spread in a uniform manner in autoclaved petri-plates on sterilized filter paper with the help of forceps. Ten ml of both chromium salt solutions i.e., K2Cr2O7 and K2CrO4 with varying concentrations  was supplied to each respectively labeled autoclaved petri-plate containing sterilized seeds separately. Petri-plates were kept in dark at 25 ± 2 °C for three days. After germination, germinated seedlings were transferred to the labeled pots each containing about 140 gm autoclaved sieved soil i.e., seven seeds per pot and chromium stress solution with varying concentration  of both chromium salts (K2Cr2O7 and K2CrO4 ml−1) were given to the respective labeled pots separately. The pots were placed in light  at 25 ±  2 °C. After 21 days of growth, plants were harvested and different growth parameters recorded which include shoot length, root length, fresh weight and number of leaves of plants, auxin content, pigment content and protein content. The experiment was repeated thrice. Auxin content, pigment content and protein content were estimated by following Three chromium-tolerant auxin-producing isolates  were used to treat Bacillus anthracis with accession number KT321455 while the other two isolates (EIV and 3a) have shown 99 and 96% homology with Bacillus cereus. These bacterial isolates were given accession numbers i.e.,  KT025249 and KM409709, respectively , on the basis of their ability to produce auxin and chromium-resistance potential, were selected for the present study. 16S rDNA sequencing of the bacterial species named as EII, EIV and 3a was carried out and the sequences obtained were then submitted to GenBank. The bacterial isolate EII has shown 98% similarity to ectively . All thealine pH .B. cereus (EIV) and B. cereus (3a), respectively, over control treatment. Increase in concentration of chromium from 0–500 µgml −1 of K2Cr2O7 and K2CrO4 also resulted in significant reduction of plant shoot length. Severe damage was observed in case of chromium stress of K2Cr2O7 but bacterial inoculation with Bacillus anthracis (EII) caused significant increase in shoot length of the treated plants from 50 –500 µgml−1 i.e., 24, 17, 12, 35 and 6% in case of K2Cr2O7 as compared to treatments with K2CrO4 affected plants when compared with respective controls. The effect of chromium toxicity was considerably higher in plant roots than in plant shoots for both bacterial inoculated and non-inoculated treatments exposed to chromium in higher amount  and Bacillus cereus (3a) respectively, when results were compared with non-inoculated control plants. Similarly under chromium stress, treatment with B. cereus (EIV) caused 177, 233, 231, 216 and 248% increment in root length with stress of K2Cr2O7  respectively. Similarly, B. anthracis (EII) caused 221, 211, 250, 427 and 352% proliferation in root length of treated plants under K2CrO4 stress from 50, 100, 200, 400 and 500 µgm l−1 respectively, over non- inoculated control. Similar enhancements were noticed in the number of leaves i.e., 85, 6 and 81%, respectively, when lentil plants were grown in the absence of chromium (Bacillus anthracis (EII) was used for inoculation under chromium stress, the number of leaves of inoculated lentils were significantly increased by 11, 21, 9, 28 and 29% (with K2Cr2O7) and 81, 52, 44, 51 and 55% (with K2CrO4) ranging from 50, 100, 200, 400 and 500 µgm l−1 respectively, when compared with plants without inoculation treatment under the identical experimental conditions. This indicates chronic damage of infrastructure of lentil plants due to K2Cr2O7 contamination as compared to K2CrO4 as number of leaves were increased in case of plants with Potassium chromate stress. Similar findings were observed in fresh weight of Lens culinaris in inoculated and non-inoculated treatments in the absence and presence of K2Cr2O7 and K2CrO4 stress (B. anthracis (EII) as compared to non-inoculated control without stress. While, when lentil plants were inoculated with B. cereus (EIV) and B. anthracis (EII) in the presence of chromium, the fresh weights of the inoculated lentil plants significantly increased by 31, 16, 0, 7 and 47% (K2Cr2O7) and 450, 289, 213, 257 and 285% (K2CrO4), respectively, under uniform experimental conditions  was applied with chromium, the amount of chlorophyll ‘a’ in treated plants significantly increased by 275, 179, 212, 158 and 233% (with K2Cr2O7) and 91, 82, 73, 246 and 399% (with K2CrO4) in stress concentrations of 50, 100, 200, 400 and 500 µgml−1 respectively, as compared with plants without inoculation treatment under the same conditions (Bacillus cereus (3a) compared to control in the absence of chromium. Chlorophyll b’ content of the treated plants also showed significant increase in the presence of both chromium salts , as a result of bacterial treatment with B. cereus (EIV) under chromium stress of K2Cr2O7 in concentrations ranging from 50,100, 200, 400 and 500 µgml−1 i.e., 135, 125, 108, 109 and 61% respectively, as compared to control. On the other hand, inoculation treatment with B. cereus (3a) in the presence of chromium resulted in significant increase in the chlorophyll ‘b’ content of the inoculated lentil plants by 70, 78, 78, 102 and 87% (K2Cr2O7) and 158, 140, 131, 149 and 90% (K2CrO4) respectively, when compared with non-inoculated plants (B. cereus (EIV) when compared with non-inoculated plants. However, under chromium stress, treatments with Bacillus cereus (3a) and Bacillus cereus (EIV) caused almost similar enhancements i.e., 62, 28, 1, 24 and 23% increase (under chromium stress of K2Cr2O7) and 24, 15, 82, 55 and 206% increase (under chromium stress of K2CrO4) in carotenoid content with chromium stress ranging from 50.100, 200, 400 and 500 µgml−1 respectively. In the absence of chromium stress, treatment with bacterial isolate Bacillus cereus (EIV) caused significant improvement of protein content (126%) with respect to control. Plants grown in the presence of hexavalent chromium caused significant stress on lentil seedlings (B. cereus (EIV) caused increase in protein content upto 60, 26, 10, 9 and 15% with addition of K2Cr2O7 and 72, 129, 174, 498 and 383% with addition of K2CrO4 in concentration ranging from 50, 100, 200, 400 and 500 µgml−1 respectively, when compared with control. Microbial inoculation caused significant augmentation in the total amount of auxin in bacterial-treated plants when they were compared with their respective control treatments. Increase in chromium concentration of both K2Cr2O7 and K2CrO4 from 0 to 500 µgml−1 caused reduction in auxin content as compared to respective control treatment. Seedling length of the treated plants was again increased by bacterial inoculation. The amount of auxin produced by inoculated plants significantly increased upto 241% as a result of bacterial treatment with B. cereus (EIV) over respective control under similar conditions. However, under chromium stress, treatment with B. cereus (EIV) caused 63, 69, 52, 36 and 14% increment in auxin content with chromium stress of K2Cr2O7 (50–500 µgml−1) respectively. Moreover, under chromium stress of K2CrO4, inoculation with Bacillus cereus (EIV) caused 117, 82, 42, 114 and 67% increase and B. cereus (3a) exhibited 156, 88, 42, 100 and 83% enhancement in auxin content of treated plants under stress condition ranging from 50, 100, 200, 400 and 500 µgml−1, respectively, over non-inoculated control  in both inoculated and non-inoculated treatments. In the absence of chromium, all the inoculated plants have shown 70 and 80% enhancement in length of plant shoot with selected isolates i.e., r amount . Prominechromium . On the 4 stress . Fresh wnditions . Significhromium . On the nditions . Chloropd plants . The amod plants . The careedlings . Bacteri control .B. anthracis (EII), B. cereus (3a) and B. cereus (EIV) through 16S rDNA sequencing (2Cr2O7 and K2CrO4 (500 µgml−1). Temperature and pH both play an important role in the regulation of variation in environmental conditions that may cause physiological changes in plants. All the three liquid cultures of selected strains have shown their highest growth in growth medium at 37 °C with pH 9 after 24 hours of incubation in the presence of both chromium salts i.e., K2Cr2O7 and K2CrO4(500 µgml−1). Maximum bacterial growth at pH 9 showed the alkaline nature of these bacterial species. Similar growth rates in alkaline environments have also been reported by other workers (2Cr2O7 and K2CrO4) in the presence and absence of stress of chromium. Similarly, microbial treatment also caused enhancement in root length of the treated plants which may be due to ability of roots exudates to produce auxin. This auxin production potential is a key factor in root enhancement which resulted in increase in roots lengths of the treated plants (B. cereus (EIV) i.e., 150% in the absence of chromium  in the absence and presence of chromium stress (P. vermicola inoculation in the presence of copper stress (B. anthracis (EII) showed highest increment in the chlorophyll a, b and carotenoid contents, in different chromium stresses i.e., 50–500 µgml−1 of K2Cr2O7 and K2CrO4, compared to non-inoculated control which may be due to ACC-deaminase and nitrogen fixing ability  resulted in significant production of auxin (67 and 241%) in chromium-free as well as chromium supplemented growth media. Bacterial isolate B. cereus (EIV) also showed a higher auxin production (114% at 400 µgml−1) in chromium supplemented media rather than media without chromium  exhibited best growth production potential under toxic chromium stress upto 500 µgml−1 in both chromium treatments of K2Cr2O7 and K2CrO4. It is very important to increase the productive efficiency and working potential of a specific PGPR to reduce toxic effects of different heavy metals by establishing resistance mechanism with the proper acclimatization and optimization stability. This study illustrates greater toxicity with enhanced deleterious impact of K2Cr2O7 as compared to K2CrO4 on plants, therefore, use of chromium in the form of K2Cr2O7 in the industrial processes should be replaced with K2CrO4 which is less toxic and less hazardous as compared to K2Cr2O7.Microbial assisted treatment of agricultural crops with PGPR is a promising approach to utilize chromium contaminated soils and to increase the area of available land for agricultural practices. PGPRs protect the plants from deleterious effects of heavy metals like chromium by producing certain phytohormones and several antioxidant enzymes thereby converting Cr (VI) into Cr (III) which is less toxic, less mobile and insoluble. Bacterial isolate 10.7717/peerj.6076/supp-1Supplemental Information 1Click here for additional data file.


IKs current, while KCNE2 functions as a dominant negative modulator to suppress the current amplitude of KCNQ1. Any anomaly in these channels will lead to serious myocardial diseases, such as the long QT syndrome (LQTS). Trafficking defects of KCNE1 have been reported to account for the pathogenesis of LQT5. However, the molecular mechanisms underlying KCNE forward trafficking remain elusive. Here, we describe an arginine/lysine-based motif ([R/K](S)[R/K][R/K]) in the proximal C-terminus regulating the endoplasmic reticulum (ER) export of KCNE1 and KCNE2 in HEK293 cells. Notably, this motif is highly conserved in the KCNE family. Our results indicate that the forward trafficking of KCNE2 controlled by the motif (KSKR) is essential for suppressing the cell surface expression and current amplitude of KCNQ1. Unlike KCNE2, the motif (RSKK) in KCNE1 plays important roles in modulating the gating of KCNQ1 in addition to mediating the ER export of KCNE1. Furthermore, truncations of the C-terminus did not reduce the apparent affinity of KCNE2 for KCNQ1, demonstrating that the rigid C-terminus of KCNE2 may not physically interact with KCNQ1. In contrast, the KCNE1 C-terminus is critical for its interaction with KCNQ1. These results contribute to the understanding of the mechanisms of KCNE1 and KCNE2 membrane targeting and how they coassemble with KCNQ1 to regulate the channels activity.KCNE β-subunits play critical roles in modulating cardiac voltage-gated potassium channels. Among them, KCNE1 associates with KCNQ1 channel to confer a slow-activated  The KCNE family (KCNE1–KCNE5) functions as auxiliary β-subunit to modulate the subcellular localization, ion selectivity, gating and pharmacology of Kv channels in muscles and epithelia (S)[R/K][R/K], in the proximal C-terminus of KCNE1 and KCNE2 that is essential for efficient ER export and regulation of KCNQ1 functions. This motif is highly conserved in the KCNE family. Besides, co-immunoprecipitation assays indicated that the KCNE2 C-terminus may not physically interact with KCNQ1, while the KCNE1 C-terminus is important for its interaction with KCNQ1. Since many mutations in the C-terminus of KCNE1 and KCNE2 have been reported to result in LQTS , comprehFor the constructs KCNE2(E2)-EGFP and KCNE1(E1)-EGFP, the EGFP cDNA was amplified by PCR using Pfu polymerase  and cloned into pcDNA3.1 (+), then KCNE2 or KCNE1 cDNA lacking stop codon were amplified and fused in frame to the N-terminus of EGFP. Using the same method, the truncations of KCNE2 or KCNE1 were made by deleting appropriate amino acids, fused to the N-terminus of EGFP and cloned into pcDNA3.1 (+). The construct Myc-KCNQ1 (Q1) was prepared as previously described . Q1-Myc HEK293 cells were cultured and transfected as previously described . For immAfter transfection for 22–24 h, HEK293 cells were washed and fixed with 2% paraformaldehyde (PFA) in PBS for 12 min, followed by 5 min × 3 washes with PBS. Then cells were blocked with 2% BSA in PBS for 1 h and incubated with mouse monoclonal anti-HA antibody  or mouse monoclonal anti-Myc antibody  for 3 h. Cells were washed with PBS 5 times and 1% BSA once. After incubation with Alexa Fluor 488-conjugated goat anti-mouse IgG (H + L)  for 1–1.5 h, the cells were washed with PBS for 6 times. All the procedures were performed at room temperature (22–25°C).Fluorescence images were visualized by using a spinning-disk confocal imaging system  equipped with an Olympus IX-71 inverted microscope . 16-bit digital images were obtained with oil-immersion objective  and an EM CCD camera .2, 1 MgCl2, 10 HEPES (pH 7.0)) at room temperature (22–25°C). Patch pipettes were pulled from borosilicate glass capillaries with resistance of 2–3 megohms and filled with the pipette solution ). The recordings were carried out using a Multiclamp 700B amplifier with a Digidata 1440A A/D converter and pClamp software (Axon Instruments). The currents were typically digitized at 20 kHz and filtered at 5 kHz. All the chemicals were purchased from Sigma-Aldrich. When recording IKs, an IKs selective blocker  was applied to test the non-IKs current. Briefly, whole-cell recording was performed on Q1/E1-expressing HEK293 cells first, then HMR 1556 was added to the bath solution at a final concentration of 1 μM and incubated for 5 min before the non-IKs current was tested.The day after transfection, whole-cell patch clamp recordings were carried out in HEK293 cells in the bath solution /κ))−1, where V50 is the voltage at which the conductance (G) is half the maximum conductance (Gmax) and κ is a factor affecting the steepness of the activation. For IKs analysis, the non-IKs current was subtracted from the recorded IKs current using an offline subtraction strategy. Unless stated otherwise, the data were presented as mean ± SEM. Student’s t-test was used to evaluate the statistical significance. P < 0.05 was considered significant .The quantification of the fluorescence intensities of Myc-Q1, HA-E2 or HA-E1 was performed with ImageJ 1.45 . Electrophysiology data were analyzed with Clampfit 10.3  and SigmaPlot 14.0  software. The conductance-voltage (G-V) relationship were fitted by the Boltzmann equation: G/GAfter transfection for 36–48 h, HEK293 cells were lysed in 700 μL lysis buffer ) for 15 min and then scraped into 1.5 mL tube, incubated for 30 min with gentle agitation at 4°C. Insoluble material was removed by centrifugation  at 4°C for 20 minutes. Supernatants were collected and pre-cleared with dynabeads protein G (Novex by Life Technologies), 40 μL sample was used for input and the rest for Co-IP. Extracted proteins were incubated with 2 μg of mouse monoclonal anti-HA tag (Millipore) at 4°C overnight with gentle agitation on a platform shaker, followed by incubation with dynabeads protein G for 3 h. The beads were washed with lysis buffer 3 times to remove nonspecific binding. Immunoprecipitates were prepared in 40 μL of 2 x SDS-PAGE loading buffer (2% SDS (w/v), 20% glycerol (v/v), 2% β-mercaptoethanol (v/v), 0.2% bromphenol blue (w/v), and 100 mM Tris-HCl (pH 6.8)) for 30 min at room temperature and resolved on a 10% or 15% SDS-PAGE gel. Immunoprecipitations with mouse normal IgG (IgG-IP) were used as negative controls.For immunoblotting, immunoprecipitates were transferred to nitrocellulose membrane. Membranes were incubated with 5% milk in 1× TBS-0.05% Tween-20 for 2 h at room temperature, and then incubated with HRP-conjugated Mouse anti-HA antibody  or rabbit polyclonal anti-Myc antibody  in 1× TBS-0.05% Tween-20. The membranes were subsequently washed four times for 10 minutes each with 1× TBS-0.05% Tween-20. Immunoreactivity was visualized by using the ECL detection system directly or after incubation with the peroxidase-conjugated goat anti-rabbit IgG antibody  for 2 h.Experimental Procedures) and E2-EGFP or E2ΔC-EGFP. The results showed that E2ΔC-EGFP clearly colocalized with the ER marker, as shown in the merged image and the pixel intensity profile of crossed section indicated by the white line (All the five KCNE β-subunits harbor one transmembrane (TM) segment, an extracellular N-terminus and an intracellular C-terminus  37,38].,38.37,38ite line . Thus, wProtein transport is often governed by molecular recognition of trafficking signals at different stages of the secretory pathway. To explore whether a forward trafficking signal resides in the C-terminus of KCNE2, we made a series of C-terminal truncations of KCNE2  that werConsidering that the use of fusion fluorescent protein EGFP might influence the trafficking of KCNE2 subunits, we inserted a small tag, HA tag, at the extracellular N-terminus of KCNE2 and its mutants  to visuaThe phosphorylation state of membrane proteins has been reported to determine their export from ER ,40. SincPositively charged amino acids have also been reported to facilitate ER export ,43. ThusKCNE2 can interact with KCNQ1 and decrease KCNQ1 current amplitude . Our preRoura-Ferrer et al. found that the membrane targeting of KCNE2 was also impaired when co-expressed with KCNQ1 . SimilarBased on these results, we conclude that the KSKR sequence determining the ER export of KCNE2 is essential for prevention of KCNQ1 membrane targeting to suppress KCNQ1 activity.KCNE1, another important member of the KCNE family, is also expressed clearly in the cell membrane in HEK293 cells, but the C-terminal (amino acid residues 67–129) deletion mutant E1ΔC showed intracellular retention and clearly colocalized with the ER marker . SimilarIn addition, mutation of the residue S68 did not significantly alter the membrane targeting of E1(RSKK) . HoweverIKs modulation (S)[R/K][R/K], in the juxtamembranous C-terminal region that positively regulates the ER export of KCNE1 and KCNE2 in HEK293 cells. The results indicated that ER export of KCNE1 and KCNE2 is vital for regulating KCNQ1 function. Additionally, the KCNE1 C-terminus is crucial for its interaction with KCNQ1, whereas the KCNE2 C-terminus is not. Our study provides significant insight into the trafficking determinants of KCNE β-subunits and how they bind and modulate KCNQ1 channels.


Background The signaling protein p38 mitogen-activated protein kinase (MAPK) regulates the tumor cell microenvironment, modulating cell survival, migration, and invasion. This phase 1 study evaluated the safety of p38 MAPK inhibitor LY3007113 in patients with advanced cancer to establish a recommended phase 2 dose. Methods In part A , LY3007113 was administered orally every 12 h (Q12H) at doses ranging from 20 mg to 200 mg daily on a 28-day cycle until the maximum tolerated dose (MTD) was reached. In part B (dose confirmation), patients received MTD. Safety, pharmacokinetics, pharmacodynamics, and tumor response data were evaluated. Results MTD was 30 mg Q12H. The most frequent treatment-related adverse events (>10%) were tremor, rash, stomatitis, increased blood creatine phosphokinase, and fatigue. Grade ≥ 3 treatment-related adverse events included upper gastrointestinal haemorrhage and increased hepatic enzyme, both occurring at 40 mg Q12H and considered dose-limiting toxicities. LY3007113 exhibited an approximately dose-proportional increase in exposure and time-independent pharmacokinetics after repeated dosing. Maximal inhibition (80%) of primary biomarker MAPK-activated protein kinase 2 in peripheral blood mononuclear cells was not reached, and sustained minimal inhibition (60%) was not maintained for 6 h after dosing to achieve a biologically effective dose (BED). The best overall response in part B was stable disease in 3 of 27 patients. Conclusions The recommended phase 2 dosage of LY3007113 was 30 mg Q12H. Three patients continued treatment after the first radiographic assessment, and the BED was not achieved. Further clinical development of this compound is not planned as toxicity precluded achieving a biologically effective dose. The signaling protein p38 mitogen-activated protein kinase (MAPK) is activated by cancer cells downstream of oncogenic receptor tyrosine kinases and in response to both radiation and chemotherapy . It phosRecent studies have indicated that p38 MAPK down-regulates fibulin 3 expression and leads to migration and invasion in some cancers . FurtherIn p38 MAPK knockout mice, phosphorylated MAPK-activated protein kinase 2 (p-MAPKAP-K2) levels are significantly reduced, and circulating tumor necrosis factor α and IL-6 levels are greatly attenuated . This phThe primary objective of this study was to evaluate the safety and tolerability of LY3007113 when administered orally to patients with advanced cancer to establish a recommended dose for phase 2 studies. The secondary objectives were to assess the pharmacokinetics (PK), pharmacodynamics (PD), and tumor response rate in patients who received LY3007113.This multicenter, nonrandomized, open-label, dose-escalation phase 1 study had 2 parts: dose escalation (part A) and dose confirmation (part B).The study enrolled adults aged 18 years or older with histologic or cytologic evidence of advanced or metastatic cancer. Other inclusion criteria included an Eastern Cooperative Oncology Group performance status score of ≤2 and adequate organ function. Exclusion criteria included major surgical resection involving the stomach or small bowel, symptomatic central nervous system malignancy or metastasis, diagnosis of acute leukemia, history of any other cancer (except nonmelanoma skin cancer or carcinoma in situ of the cervix) unless in complete remission and not treated for at least 3 years, or an autologous or allogeneic stem-cell transplant within 75 days of starting study drug. The study was conducted in accordance with the Declaration of Helsinki and good clinical practice guidelines, and the protocol was approved by each participating institution’s ethics review board. All patients provided written informed consent.In part A, LY3007113 was to be administered orally at dosages ranging from 20 mg to 200 mg every 12 h (Q12H) daily on a 28-day cycle, with modifications during cycle 1  to enable PK sampling for 72 h after a single dose. Dose escalation was guided by safety assessments during cycle 1 using the Common Terminology Criteria for Adverse Events, Version 4.0, and continued until the maximum tolerated dose (MTD)  that was possibly related to the study drug and met any of the following criteria: grade ≥ 3 nonhematological toxicity event ; grade ≥ 3 nausea, vomiting, diarrhea, or electrolyte disturbance lasting more than 2 days despite maximal supportive intervention; grade 4 hematological toxicity event lasting more than 5 days; grade ≥ 3 thrombocytopenia with bleeding (except epistaxis); or grade ≥ 3 febrile neutropenia. If a DLT occurred in more than 1 patient at any dose level, dose escalation ceased at that dose level, and the previous dose was declared to be the MTD.After the last patient in part A had completed cycle 1 and the MTD was determined, the dose-confirmation phase (part B) began. In part B, approximately 15 evaluable patients were treated with the MTD on days 1 through 28 of a 28-day cycle, with modifications during cycle 1 to enable PK sampling after a single dose and repeated doses.Patients received 2 cycles of LY3007113 unless they met one or more of the criteria for discontinuation. Patients who experienced clinical benefit, in the investigator’s opinion, were permitted to receive additional cycles.LY3007113 was supplied by Eli Lilly and Company  as 10-mg and 40-mg capsules for oral administration.Plasma concentrations were determined for LY3007113 and its metabolites, LSN3025641 and LSN3047151, using a validated liquid chromatography with tandem mass spectrometry method. The analytes were extracted from human plasma by protein precipitation and chromatography was performed using an Onyx Monolithic C18 column (Phenomenex). Analysis was performed with positive ion electrospray for the analytes. For LY3007113, the lower and upper limits of quantification were 1 ng/mL and 1000 ng/mL. Interassay accuracy (percent relative error) during validation ranged from −0.9% to 7.0% for LY3007113, from 0.2% to 6.0% for LSN3025641, and from −1.4% to 4.0% for LSN3047151. Interassay precision (percent relative SD) during validation ranged from 2.8% to 5.6% for LY3007113, from 3.2% to 7.5% for LSN3025641, and from 3.9% to 8.7% for LSN3047151. Plasma sample analysis was conducted at Covance Bioanalytical Services, LLC .max), area under the concentration-versus-time curve (AUC), terminal half-life (t1/2), apparent volume of distribution, apparent clearance, and other relevant parameters calculated after administration of the first dose on day −3 as single-dose PK and then on days 1, 14, and 28 of cycle 1.The PK analyses included but were not limited to maximum plasma concentration  from patients, 20 μg/ml of anisomycin for 20 min. The biologically effective dose (BED) was defined as the lowest dose that would achieve at least 80% maximal inhibition of p-MAPKAP-K2 and at least 60% inhibition for up to 6 h after dose in PBMCs.Tumors were radiographically assessed according to Response Evaluation Criteria in Solid Tumors, Version 1.1, at baseline (day −28 to day −4) and in every other cycle beginning with cycle 2 before the start of the subsequent cycle for patients in part B. The overall response rate  was determined for patients in Part B only.Twenty-seven patients (12 patients in part A and 15 patients in part B) were enrolled and received at least 1 dose of study drug. Table n = 3), 30 mg (n = 4), or 40 mg (n = 5) Q12H. The mean (SD) numbers of received cycles and completed cycles  were 1.3 (0.87) and 0.5 (0.67) respectively ,. The majority of patients did not complete the first cycle.In part A, patients received LY3007113 dosages of 20 mg . The mean (SD) numbers of received cycles and completed cycles were 1.4 (6.3) and 0.4 (0.63), respectively .Overall, 5 patients (18.5%) received more than 2 cycles of treatment: 1 patient  received 3 cycles, 3 patients  received 4 cycles, and 1 patient (gastric adenocarcinoma) received 5 cycles.Overall, 25 of 27 patients (92.5%) discontinued study drug. In part A, all 12 patients discontinued study drug: 10 patients (83.3%) had progressive disease, 1 patient (8.3%) died because of progressive disease, and 1 patient (8.3%) was discontinued because of physician decision. In part B, 13 patients (86.6%) discontinued study drug: 11 patients (84.6%) had progressive disease, 1 patient (6.7%) chose to discontinue, and 1 patient (6.7%) was discontinued due to physician decision. Two patients (13.3%) in part B did not give a reason for discontinuation of study drug.The most frequent treatment-emergent adverse events (TEAEs) reported in ≥10% of patients were tremor (11 patients [40.7%]); rash, including the terms rash, rash maculopapular, rash papular, and dermatitis acneiform (10 patients [37.0%]); fatigue (8 patients [29.6%]); dyspnoea, including the terms dyspnoea and dyspnoea exertional (7 patients [25.9%]); constipation and diarrhea (5 patients each [18.5%]); anaemia, nausea, peripheral edema, and stomatitis (4 patients each [33.3%]); and abdominal pain, increased blood creatine phosphokinase, decreased appetite, cough, dehydration, epistaxis, upper respiratory tract infection, hypotension, vertigo, and vomiting (3 patients each [11.1%]).n = 16) were reported in a total of 9 patients. These TEAEs included gastrointestinal haemorrhage (n = 3), intestinal obstruction (n = 2), pneumonia (n = 2), anaemia, peptic ulcer, pericardial effusion, unilateral blindness, constipation, abdominal pain, thrombocytopenia, hypercalcaemia, and increased hepatic enzyme. Grade 4 TEAEs (n = 3) were reported in 2 patients. These included sepsis and hypovolaemia with hypotension.Most TEAEs were reported as mild (grade 1) or moderate (grade 2). Grade 3 TEAEs (Study-drug–related TEAEs were reported in 18 patients (66.7%), including 10 patients (83.3%) in part A and 8 patients (53.3%) in part B Table . OverallTwo treatment-related TEAEs, upper gastrointestinal hemorrhage and increased hepatic enzyme, were grade 3 and both were considered DLTs at the 40 mg Q12H dose level. No grade 4 events were considered possibly related to study treatment; thus, none were considered DLTs. Because of the DLTs observed at the 40 mg Q12H dosage, the MTD for use in part B was defined as 30 mg Q12H. Serious adverse events (SAEs) were reported in 9 patients (33.3%): 4 patients (33.3%) in part A and 5 patients (33.3%) in part B. In part A, the reported SAEs included large intestinal obstruction, lower gastrointestinal hemorrhage, small intestinal obstruction, pneumonia, and hypercalcemia ; peptic ulcer, hypovolemia, and hypotension ; sepsis; and upper gastrointestinal hemorrhage. In part B, the reported SAEs included staphylococcal pneumonia and dyspnea (both in the same patient), pericardial infusion, abdominal pain, rectal hemorrhage, and convulsion. The only SAE considered possibly related to study drug was the upper gastrointestinal hemorrhage reported in part A in a patient treated with 40 mg Q12H.Six patients died during the study. One patient  died during the treatment period of progressive disease, and five patients died of progressive disease during the 30-day follow-up period. It was the investigator’s opinion that none of these deaths were caused by TEAE.max for LY3007113 was approximately 2 h  after both single and repeated dosing. On cycle 1 day 28, the estimated geometric mean t1/2 was approximately 10 h . Therefore, as expected with this value of t1/2, the LY3007113 accumulation ratio was approximately 1.8. The geometric means for apparent clearance and apparent volume of distribution were 14 L/h  and 179 L . The t1/2, apparent clearance, volume of distribution, and the metabolite-to-parent exposure ratios were similar after a single dose (day −3) and after one cycle of Q12H dosing (day 28).Mean concentration-versus-time profiles for LY3007113 and its metabolites LSN3025641 and LSN3047151 are presented in Fig. n = 25) after a single dose and 1.38  after repeated dosing (Table (0–12) and AUC(0-inf). Because the 90% CI for the ratio of dose-normalized means for all dose-proportionality assessments always included 1, these findings do not suggest a large departure from linearity with an approximately dose-proportional increase in exposure across this range of doses.In dose-proportionality analyses for the 20-mg to 40-mg dose range in this study, LY3007113 exhibited dose-normalized means ratios of 0.75  had a best response of stable disease for tumor types, including renal cell carcinoma, gastric adenocarcinoma, and epithelial ovarian carcinoma. In these patients, radiographically assessed stable disease was maintained for periods ranging from 58 days to 115 days. Ten patients (76.9%) had a best response of progressive disease for tumors including carcinoma , adenocarcinoma (rectum and prostate), squamous cell carcinoma (head and neck), cholangiocarcinoma, and sarcoma. Two patients discontinued early and were not assessed for response.This phase 1 study evaluated safety, PK, PD, and tumor response in patients with advanced cancer after oral administration of LY3007113. Nine patients (33.3%) experienced an SAE, and 18 patients (66.7%) experienced at least one TEAE. The MTD was 30 mg Q12H, although the expected dose ranged from 20 mg to 200 mg. Because of toxicities observed early in the study, such as tremors (33.3%) and rash (22.2%), dose escalation was stopped at 40 mg Q12H; thus, the predetermined targets of either maximal inhibition (80%) or sustained minimal inhibition (60%) for up to 6 h after dosing were not achieved. The relationship between exposure and safety is not clear because the DLTs observed at 40 mg Q12H  occurred in patients whose relative exposures were no higher than those of other patients receiving the same dose  nor sustained minimal inhibition (60%) for up to 6 h after dosing. In addition, no explanation was found for the pattern of higher interpatient variability and lower inhibition seen after repeated versus single dosing. Given the high incidence of tremors, fatigue, and rash, it was not clinically feasible to continue with dose escalation as planned. Thus, these factors may have contributed to a lack of clinical responses.The recommended dosage of LY3007113 was established at 30 mg Q12H, although a biologically effective dose was not achieved due to tolerability concerns. With only 3 of 27 patients (11.1%) continuing treatment after the first radiographic assessment, further clinical study of this compound is not planned.


Osteoporosis is a debilitating skeletal disorder that is characterized by loss of bone density over time. It affects one in two women and one in four men, age 50 and older. New treatments that specifically drive bone formation are desperately needed. We developed a peptide, CK2.3, that acts downstream of the bone morphogenetic protein receptor type Ia and it induces osteogenesis in-vitro and in-vivo. However, its mechanism of action, especially its mode of uptake by cells remains unknown. To demonstrate CK2.3 internalization within a cell, we conjugated CK2.3 to Quantum Dot®s (Qdot®s), semiconductor nanoparticles. We purified CK2.3-Qdot®s by size exclusion chromatography and verified the conjugation and stability using UV/VIS and Fourier transform infrared spectroscopy. Our results show that CK2.3 was conjugated to the Qdot®s and the conjugate was stable for at least 4 days at 37 °C. Moreover, CK2.3-Qdot®s exerted biological response similar to CK2.3. Addition of CK2.3-Qdot®s to cells followed by confocal imaging revealed that CK2.3-Qdot®s were internalized at 6 h post stimulation. Furthermore, using pharmacological inhibitors against endocytic pathways, we demonstrated that CK2.3-Qdot®s were internalized by caveolae. These results show for the first time that the novel peptide CK2.3 is taken up by the cell through caveolae mediated endocytosis. Osteoporosis is a silent disease due to its gradual progression of loss in bone mass and deterioration of the microarchitecture of the bone tissue. It predisposes patients to an increased risk of fractures at either the hip, spine, or wrist ,2. ApproMajority of the existing treatments for osteoporosis such as recombinant parathyroid hormone (PTH) , bisphosOur lab developed a peptide, CK2.3, by using the protein kinase 2 (CK2) phosphorylation consensus sequence found on bone morphogenetic protein receptor type Ia (BMPRIa), the phosphorylation motif is conserved and distinct from other consensus sequences of protein kinases that have been characterized so far . CK2 is In order to determine these parameters, a fluorescent probe was designed by using Quantum Dot®s (Qdot®s) . Qdot®s N,N′-Dicyclohexylcarbodiimide (DCC) was used for the coupling [The Qdot®s used in this study were coated with carboxyl groups, which were used to conjugate it to the amino groups of the peptide CK2.3. There are five lysine amino acids in the CK2.3 sequence, and it also has the N-terminal amino group. coupling .Conjugated CK2.3-Qdot®s were separated from non-conjugated reactants by size exclusion chromatography (SEC). The separation of the sample was determined by UV/VIS spectroscopy and the conjugation was confirmed by Fourier transform infrared (FTIR) spectroscopy. FTIR was also used to determine the stability of CK2.3-Qdot®s. CK2.3-Qdot®s were stable for at least 4 days at 37 °C. To characterize the activity of CK2.3-Qdot®s in-vitro, C2C12 cells were used as the model cell line, as it is one of the most commonly used mesenchymal stem cell- derived cell line to study osteoporosis in-vitro ,27,28. FSignificance of the study: Currently prescribed drugs against osteoporosis  have failed to find a lasting treatment for this disease, and in many cases, have led to adverse side effects . We haveCK2.3 (1.915 μM) was designed using the CK2 phosphorylation sequence found on BMPRIa, which was identified using the prosite search and it was ordered from GenScript . 16 amino-acid long (2.246 kDa) Antennapedia homeodomain signal sequence  is incor™ carboxyl quantum dots, catalog # Q21341MP, 8 μM) were purchased from Life technologies, , the Qdot®s used here are made up of cadmium selenide core with zinc sulfide shell, coated with non-toxic carboxyl groups and are excitable at 488 nm. The Qdot®s have an ellipsoid core/shell with diameters of 6 nm (minor axis) by 12 nm (major axis). The hydrodynamic diameters were measured by high-performance size exclusion chromatography with a major axis to be 18 nm for carboxylic acid coated Qdot®s [Qdot®s  (36 mg dissolved in 1 mL of DMSO) from Sigma-Aldrich  was added to initiate the conjugation. As control, eight different reactions consisting of all possible combinations of reactants were used. The reactions were carried out at room temperature for 30 min. After 30 min, 200 μL of 1 × PBS pH 7.4 was added to quench the reaction and the samples were immediately put on ice. We added equal moles of Qdot®s and CK2.3, to ensure a 1:1 conjugation ratio.To conjugate CK2.3 to Qdot®s, a solution mixture consisting of 2 μL of Qdot®s (8 μM), 10 μL of CK2.3 (1.915 μM), and 2 μL of 10 × PBS were mixed in 84 μL of dimethyl sulfoxide (DMSO) purchased from Fisher Scientific . 2 μL of g rpm for 5 min to tightly pack the beads and remove excess water from the columns. 300 μL of the conjugation solution, as well as the eight other control reactions were gradually added to their respective filtration columns. Later, 100 μL of distilled water was gently added one at a time to each filtration columns, to collect 30 fractions of samples from each column. The samples were then analyzed using UV/VIS spectroscopy to identify the fraction containing the conjugated CK2.3-Qdot®s. On an average, we obtained about 160 nM of CK2.3-Qdot®s solution per conjugation reaction, as determined using the standard curve. Absorbance of CK2.3-Qdot®s is background corrected by subtracting the absorbance value of the control PBS sample.Conjugated CK2.3-Qdot®s were separated from unspecific reactants based on SEC. Sephadex G-50 fine beads were purchased from Sigma-Aldrich . 2.5 g of Sephadex G-50 beads were suspended in 50 mL of distilled water and allowed to swell overnight, the slurry was then packed into 3 mL syringe columns and centrifuged at 2000× UV/VIS spectra were collected by drop-casting 2 μL (×3 times) of sample onto the pedestal of NanoDrop® (ND-1000 Spectrophotometer). UV/VIS spectra were gathered by plotting absorbance of sample against its respective range of wavelength (220–300 nm). The concentration of the conjugation was calculated using the standard curve. For this curve, the absorbance of Qdot®s at 223 nm was plotted against known concentrations of Qdot®s. After conjugation, the optical density of the conjugate was determined at 223 nm and eventually the concentration of CK2.3-Qdot®s was calculated using the standard curve.−1 resolution. OPUS v6.0 was used for spectral acquisition. Essential FTIR software was used to view and baseline correct each spectrum.Mid-infrared spectra were collected in specular reflectance mode by drop-casting 5 μL (×3 times) of sample onto gold-coated round coverslips from Substrata  and the sample was analyzed using Bruker Optics vertex FTIR spectrometer  equipped with Hyperion 2000 microscope and liquid nitrogen cooled Mercury-Cadmium-Telluride (MCT) detector. Each spectrum consists of 100 scans averaged at a 4 cmv/v) heat in-activated fetal bovine serum (FBS) , 1% (v/v) penicillin/streptomycin . Phenol-free DMEM media  supplemented only with 1% (v/v) penicillin/streptomycin , but no FBS. 1 × Trypsin  was used to detach cells from flasks during sub-culturing.C2C12 cells (murine myoblast cells) were purchased from American Type Culture Collection . Cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM)  supplemented with 20%  paraformaldehyde from Sigma-Aldrich  for 20 min and washed with cold 1 × PBS pH 7.4. 5% (w/v) silver nitrate from Chem-Impex international  dissolved in diH2O was applied to each well and the plate was placed under the UV light for 1 h. Cells were then washed with diH2O and were allowed to dry overnight. Twelve random images of the cells were taken per well using Zeiss Axiovert 10 microscope at 5×/12 Achrostigmat objective and images were analyzed using ImageJ software . Images were first converted to 8 bit and threshold was set to the positive control. The same threshold was used for all treatments. Mineralized areas were quantified using the analyzing particles plugin function of ImageJ. Data was analyzed using one-way Anova and Tukey-Kramer post hoc statistical test.C2C12 cells were grown in 24-well plate at a cell density of 1 × 104 cells/well for two days in DMEM media containing 20% FBS and 1% penicillin/streptomycin and at the end of the 2nd day, cells were serum starved with phenol-free DMEM media supplemented with 1% penicillin/streptomycin, but without FBS for 18 h.C2C12 cells were grown in 35 mm dishes containing 18 mm × 18 mm square coverslips at a cell density of 1 × 10w/v) paraformaldehyde purchased from Sigma-Aldrich  for 20 min and permeabilized with 0.05% (w/v) saponin from Sigma-Aldrich  diluted in diH2O and incubated for 10 min on ice, and later the nucleus was stained using 100 μL (0.5 ng/mL) of Hoechst  from Sigma-Aldrich . The coverslips were then mounted on the slides using an anti-fading mixture called as Airvol. It is made up of 20 g polyvinyl alcohol from Chemistry Connection LLC  diluted in 80 mL of diH2O, the mixture is stirred at 60–70 °C for 2 h. Followed by the addition of 39.6 mL of Glycerol, 2 g of n-propyl gallate from Sigma-Aldrich , and 0.4 mL of 0.2 M Tris (pH 8.5) from Fisher Scientific . The mixture is centrifuged at 18,000× g for 30 min and stored in −20 °C for long term storage. Slides were imaged using Zeiss LSM 710 at 20×/0.8 and 63×/1.4 oil Plan-Apochromat objectives.(a) Visualization of CK2.3-Qdot®s uptake within cells. C2C12 cells were either left unstimulated or stimulated with 100 nM of CK2.3-Qdot®s for 2 h, 6 h, 12 h, and 18 h. After each time point, plasma membrane was stained using 100 μL (10 ng/μL) of FM™ 4-64FX membrane dye  from Fisher Scientific  diluted in 1× PBS pH 7.4 and incubated for 1 min on ice, followed by fixation of the cells using 4.4% (w/v) paraformaldehyde purchased from Sigma-Aldrich  for 20 min and permeabilized with 0.05% (w/v) saponin from Sigma-Aldrich  diluted in diH2O and incubated for 10 min on ice. Cells were blocked with 3% (w/v) protease-free Bovine Serum Albumin (BSA) from Fisher Scientific  diluted in 1× PBS pH 7.4, at room temperature for 1 h. Following blocking, cells were labelled for Antennapedia homeodomain using 100 μL of mouse monoclonal anti-Antp 4C3 from Developmental Studies Hybridoma Bank (University of Iowa) diluted in the ratio of 1:100 in 3% protease-free BSA, at room temperature for 1 h. The primary antibody was then stained against using 100 μL (1 μg/μL) of fluorescently tagged-secondary antibody, donkey anti-mouse IgG H&L  from Abcam  diluted in the ratio of 1:500 in 3% protease-free BSA, at room temperature for 1 h. Later the nucleus was stained using 100 μL (0.5 ng/mL) of Hoechst  from Sigma-Aldrich . The coverslips were then mounted on the slides using Airvol [(b) Confirmation of conjugation between CK2.3 and Qdot®s. C2C12 cells were either left unstimulated or stimulated with 100 nM of CK2.3-Qdot®s for 12 h. After 12 h, cells were fixed using 4.4% (g Airvol . Specifiw/v) paraformaldehyde purchased from Sigma-Aldrich  for 20 min and permeabilized with 0.05% (w/v) saponin from Sigma-Aldrich  diluted in diH2O, for 10 min on ice. Cells were blocked with 3% (w/v) protease-free Bovine Serum Albumin (BSA) from Fisher Scientific  diluted in 1× PBS pH 7.4, at room temperature for 1 h. Following blocking, cells were either labelled for Caveolin-1 or Adaptin α proteins using 100 μL (2.5 ng/μL) of purified mouse anti-Caveolin-1  and purified mouse anti-Adaptin α ; respectively from BD Transduction Laboratories™ , diluted in the ratio of 1:100 in 3% protease-free BSA, at room temperature for 1 h. The primary antibody was stained against using 100 μL (20 ng/μL) of fluorescently tagged-secondary antibody, goat anti-mouse IgG (H + L)  from Fisher Scientific  diluted in the ratio of 1:500 in 3% protease-free BSA, at room temperature for 1 h. Later the nucleus was stained using 100 μL (0.5 ng/mL) of Hoechst  from Sigma-Aldrich . The coverslips were then mounted on the slides using Airvol [(c) Co-localization of CK2.3-Qdot®s with either endogenous Adaptin α or Caveolin-1 proteins. C2C12 cells were either left unstimulated or stimulated with 100 nM of CK2.3-Qdot®s for 6 h. After 6 h, cells were fixed using 4.4%  to inhibit CCPs-mediated endocytosis. Chlorpromazine is a cationic amphiphilic drug and it functions by inhibiting the formation of CCPs by reversible translocation of clathrin and its adapter proteins from plasma membrane to intracellular vesicles [w/v) paraformaldehyde purchased from Sigma-Aldrich  for 20 min and permeabilized with 0.05% (w/v) saponin from Sigma-Aldrich  diluted in diH2O, for 10 min on ice. Later the nucleus was stained using 100 μL (0.5 ng/mL) of Hoechst  from Sigma-Aldrich . The coverslips were then mounted on the slides using Airvol [C2C12 cells were grown in 35 mm dishes containing 18 mm × 18 mm square coverslips at a cell density of 1 × 10vesicles ,40. C2C1vesicles . Lovastavesicles . C2C12 cvesicles ,42. Methvesicles . After 2g Airvol . Slides The carboxyl group on the Qdot®s was conjugated to the amino group of CK2.3 in the presence of DCC  as descr−1 and 1546 cm−1 which represent amide I bond and amide II bond respectively [−1 represented the asymmetric bending of proteins [−1 and 1355 cm−1, which signify the carboxylate  peaks [−1 represented the OH bending of absorbed water [−1, 1414 cm−1, and 1344 cm−1. Absorption peaks of amide I and amide II moved to a lower frequency [−1 and 1466 cm−1; respectively. Presence of a shoulder suggests that some of the CK2.3 are no longer conjugated to Qdot®s and thus, CK2.3-Qdot®s were breaking into their separate components. However, even at day 7 about 44.44% of CK2.3 and Qdot®s are still conjugated.Next, FTIR was used to verify the conjugation between CK2.3 and Qdot®s. In the spectra of CK2.3 A, peaks proteins . In the t) peaks ,49. The requency ,51. Conjrequency  due to rrequency . Thus, cIn order to expose C2C12 cells to equimolar amounts of either CK2.3 or CK2.3-Qdot®s, we determined the concentration of the CK2.3-Qdot®s fraction. We determined the absorbance of unconjugated Qdot®s at 223 nm wavelength to its respective known concentrations (0–800 nm) A and subCK2.3 induces significant mineralization at 100 nM in C2C12 cells ,17, therCK2.3 induces osteogenesis and bone formation in-vitro ,17 and iTo further verify the conjugation between CK2.3 and Qdot®s, we stained for the Antennapedia homeodomain. The Antennapedia homeodomain signal sequence was incorporated in the peptide sequence of CK2.3. The third α-helix of the Antennapedia homeodomain, a 16-amino acid long peptide, is well known for its ability to translocate across the plasma membrane of a cell in a receptor-independent manner ,57,58,59Thus far, we have confirmed that CK2.3-Qdot®s were taken up by C2C12 cells as early as 6 h post stimulation. Next, we investigated the mechanism of its internalization, either via CCPs- or caveolae-mediated endocytosis. C2C12 cells were either left unstimulated or stimulated with 100 nM of CK2.3-Qdot®s for 6 h. Following stimulation, one set of cells were stained for the Adaptin α of CCPs- and another set of cells were stained for Caveolin-1, a marker for caveolae-mediated endocytosis. In case of CCPs , there wNext, we employed pharmacological inhibitors against specific endocytic pathways. We used chlorpromazine and lovastatin to inhibit CCP- and caveolae mediated endocytosis; respectively . To inhiIn this paper we report for the first time the development of a biologically active fluorescent CK2.3 analog. We chose Qdot®s as our fluorescent label due to their photostability and brightness compared to traditional organic dyes, fluorescent proteins, and lanthanide chelates ,22,23. MNext, we investigated the mechanism of uptake of CK2.3-Qdot®s into the cells. Clathrin dependent endocytosis is the most well characterized method of endocytosis, it is receptor-dependent and can internalize molecules of up to 200 nm in size . The proIt has been reported that the Antennapedia homeodomain signal sequence incorporated in CK2.3 sequence, mediates cellular uptake in a receptor-independent mechanism ,70. HoweOur long-term goal is to visualize the distribution and localization of CK2.3-Qdot®s within living organisms using non-invasive, real-time in-vivo fluorescence imaging. Some of the limitations associated with using conventional organic dyes are short lifetimes, photodegradation, limited fluorescence brightness, along with intrinsic tissue autofluorescence ,23,24. HIn conclusion, we have developed a biologically active, fluorescently tagged CK2.3 imaging probe. We have shown that CK2.3 is conjugated to the Qdot®s, and the bioconjugate is stable for at least 4 days at 37 °C and remains conjugated within the cell for at least a day. We have also confirmed that the biological activity of CK2.3 is retained in the CK2.3-Qdot®s conjugate. Furthermore, using confocal microscopy, we have determined that CK2.3 is taken up by C2C12 cells as early 6 h post stimulation and it is internalized via caveolae mediated endocytosis. It is significant that CK2.3 co-localizes with Caveolin-1 since it is reported that BMPRIa receptors aggregate in the caveolar microdomains ,62 and i


SJ16 (1.0 mg/ml)” should have read as “SJ01 (0.1 mg/ml).”In the original article, there was an error. “Materials and Methods, Virulence Factor Analysis,Paragraph 1:A correction has been made to P. aeruginosa strains by quantifying pyocyanin and rhamnolipid, and analyzing elastase and protease activities. Briefly, P. aeruginosa PAO1 and P. aeruginosa PAH were grown overnight in 5 ml of PB medium  supplemented with extract of strain SJ01 (0.1 mg/ml) and without extract (control) at 37°C (180 rpm). The culture was centrifuged at 10,000 × g for 10 min, and pyocyanin was extracted first from the supernatant in 3 ml of chloroform, followed by 1 ml of 0.2 N HCl. The absorbance was measured spectrophotometrically at 520 nm  was studied on the production of virulence factors of reference “After incubation, the mixture was centrifuged at 30,000×g for 10 min” should have read as follows: “After incubation, the mixture was centrifuged at 3,000× g for 10 min.”In the original article, there was an error. Materials and Methods, Virulence Factor Analysis,Paragraph 2:A correction has been made to P. aeruginosa) were grown in nutrient broth supplemented with bacterial extract  or without extract (control). The culture was centrifuged at 10,000 × g for 10 min, supernatants were collected, acidified with HCl (to pH 2) and absorbance was measured at 570 nm  at 37°C, 180 rpm for 16 h. After incubation, the mixture was centrifuged at 3,000 × g for 10 min, and absorbance was measured at 490 nm for elastase activity (Zhu et al., For rhamnolipid, reference strains (The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated.


Valproic and arundic acids are astrocytes-modulating agents with potential effects in the treatment of Alzheimer’s disease (AD). S100B is an astrocytic cytokine with a possible role in the pathogenesis of AD. In this study, we aimed to assess the glioprotective effects of valproic and arundic acids against amyloid-β-peptide (Aβ)-induced glial death and contribution of S100B to the glioprotective effects of these agents in an astrocytic culture. We used Aβ25–35 at a concentration of 200 μM in 1321N1 astrocyte cells. We treated the cells with valproic acid (0.5 and 1 mM) and/or arundic acid 50 µM for 24 hr. Methylthiazolyldiphenyl-tetrazolium bromide (MTT) test was used to measure cell viability. The intracellular and extracellular S100B levels were measured using an ELISA kit. The data were analyzed using one-way analysis of variance followed by the Tukey’s test. P<0.001). Valproic acid (0.5 and 1 mM) and arundic acid (50 µM) ameliorated the gliotoxic effects of Aβ (P<0.05). The Aβ-treated group had higher S100B levels (both intracellular and extracellular) compared to the negative control groups (P<0.001). Arundic and valproic acids (0.5 and 1 mM) decreased both the intracellular and extracellular S100B levels compared to the Aβ-treated group (P<0.001).Aβ (200 µM) decreased the cell viability compared to the control group (By considering homeostatic and neuroprotective functions of astrocyte, the astroprotective effects and the attenuation of S100B level may be responsible, at least in part, for the beneficial effects of valproic and arundic acids in AD. Alzheimer’s disease (AD) is a progressive neurodegenerative disorder which affects several brain regions responsible for learning and memory . The maiIn vitro and in vivo studies have shown the close association of Aβ with astrocytes . Cell culture materials including DMEM/F12, FBS , and penicillin-streptomycin were obtained from Gibco® life technologies™ . We obtained Aβ, valproic acid, methylthiazolyldiphenyl-tetrazolium bromide (MTT), phosphate buffered solutions (PBS), and dimethyl sulfoxide (DMSO) from the Sigma-Aldrich Corporation . We purchased arundic acid (ONO-2506) from Tocris Bioscience (USA). Astrocyte cell culture°C in 95% humidified atmosphere/5% CO2. Upon reaching confluency, we detached the cells from the flask by 0.25% Trypsin/EDTA. We then centrifuged the cell suspension for 10 min at 1200 rpm and 22 °C. The cells then were seeded on 6 or 96 well plates. We kept 1321Na cells in a t75 flask and maintained it at 37 Aβ25–35 preparation °C) until use. Aβ25–35 was aggregated by incubation for 4 days at 37 °C before administration in the cell culture. Aβ25–35 at a concentration of 2 μg/μl was dissolved in sterile distilled water and kept in the freezer . We calculated the effective concentration of Aβ25-35, valproic acid, and arundic acid by dose-response experiments and MTT assay. On the day of the experiment, we treated the cells with the chosen concentrations of each agent. We incubated the cells with Aβ (200 μM), valproic acid (0.5 and 1 mM), and arundic acid (50 µM), or both for 24 hr. Cell viability assay We used MTT reagent to measure cell viability, and added 5 mg/ml MTT reagent to the cell culture media 24 hr after the treatments. Four hours later, we removed the cell culture media and dissolved the precipitation of each well in 100 μl of DMSO. Then, we measured the absorbance of each well at 570 nm by a microplate reader .Sample preparation for measurement of the S100B protein °C. The cell culture supernatants were centrifuged at 1000×g and 4 °C and used for measurement of the extracellular level of S100B. After the dose selection, astrocytes were cultured in the 6-well plates in a condition similar to the cell viability assay. The intracellular S100B level was measured using the astrocytic cell lysate. We used pre-cooled PBS and trypsin to detach cells from the plates. The suspended cells were centrifuged for 5 min at 1000×g and then lysed with three cycles of freeze-thawing. Then the cell mixture was centrifuged at 1500×g, 4 Measurement of S100B level°C. Then, 100 μl of the biotinylated detection Ab was added to the separated supernatant and incubated for 1 hr at 37 °C. After washing, 100 μl of the HRP conjugate was added and incubated for 30 min at 37 °C. After washing, we poured the substrate reagent into wells and incubated for 15 min at 37 °C. Finally, we poured the stop solution (50 μL) into each well and measured the absorbance at 450 nm using a microplate reader . The concentration of the S100B protein was determined by a standard curve. Twenty-four hours after the treatment, intracellular and extracellular S100B levels were measured using a commercially available ELISA kit . The experimental procedure was compatible with the manufacturer’s instructions. In brief, 100 μL of the standard or samples were added to each well and incubated for 90 min at 37 Statistical analysisP<0.05 was considered as significance cutoff. All analyses were performed using the SPSS software, version 23. The data were analyzed using the one-way analysis of variance (ANOVA) followed by the Tukey’s test. Effects of different treatments on the glial cell viabilityP<0.001) (P<0.001) (P=0.021), valproic acid , or arundic acid  to Aβ ameliorated the gliotoxic effects of Aβ (P<0.001) (P=0.998) in the absence of Aβ exerted no effect on the survival of glial cells.This study showed that cell viability was different in various treatment groups  = 10.139, P<0.001) . The admP<0.001) . Additiots of Aβ . AdditioP<0.001) . HoweverEffects of different treatments on the intracellular S100B levelsP<0.001) (P<0.001). The treatment with arundic acid suppressed the S100B level compared to the Aβ and control groups (P<0.001). Addition of arundic acid (P<0.001) + valproic acid  or valproic acid ) attenuated the intracellular S100B level compared to the Aβ-treated group. We summarized the results of intracellular S100B protein levels in In this study, various treatments altered the intracellular S100B levels  =988.95, P<0.001) . The Aβ-Effects of different treatments on the extracellular S100B levels P<0.001). The administration of Aβ elevated the extracellular S100B level in the astrocyte culture compared to the control group (P<0.001) (P<0.001) and control (P<0.001) groups. Addition of arundic acid or valproic acid (0.5 and 1 mM) suppressed the extracellular S100B rising induced by Aβ (P<0.001). The astrocytes that received arundic acid + valproic acid + Aβ had a lower extracellular S100B level compared to Aβ and Aβ + arundic acid treated groups (P<0.001). We summarized the results of extracellular S100B protein levels in The present study showed that different treatments changed the extracellular S100B levels  =1824.88 (7), P<0.001) . In contThe present study showed that Aβ induced astrocytes apoptosis. In agreement, treatment of rat primary astrocyte cultures with Aβ decreased astrocyte viability . HoweverAstrocytes have critical functions in the CNS . ConsequOur study showed that Aβ increased the apoptosis of astrocytes and raised the intracellular and extracellular S100B levels. S100B is mainly synthesized and released by astrocytes. Astrocytic activation induces S100B production and further enhances microglial activation and inflammatory responses . The sevin vitro and in vivo studies have shown neuro- and astroprotective properties of valproic acid against cytotoxic insults (Recent evidence has shown the valproic acid ability to restore learning and memory deficit in the animal models of neurodegeneration , 35. Mor insults . Thus, v insults . However insults . Arundic insults . Our stu insults . HoweverOur study showed that Aβ increased S100B while arundic and valproic acids decreased the extracellular and intracellular S100B level. Our results may imply that the cytoprotective effects of valproic and arundic acids against Aβ may be related to the suppression of S100B production and secretion. According to the present study, Aβ increased both intracellular and extracellular S100B levels. In contrast, arundic and valproic acids decreased both intracellular and extracellular S100B levels. These results may imply that Aβ enhances synthesis and secretion of S100B while arundic and valproic acids reduce the synthesis and secretion of S100B from astrocytes. Previous reports have shown that arundic acid suppressed S100B synthesis in astrocytes . HoweverLimitationsWe evaluated the effects of arundic and valproic acids on astroprotection and S100B in an astrocytic cell line. For the extrapolation of these results, it seems necessary to confirm the results of this study in animal models. Aβ induced astrocytic apoptosis and enhanced the S100B level. Arundic and valproic acids attenuated the S100B level and protected astrocytes against Aβ-induced toxicity. By considering homeostatic and neuroprotective functions of astrocytes, the astroprotective effects and the attenuation of S100B level may be responsible, at least in part, for the beneficial effects of these drugs in AD.


Subsequent sintering of this mixture for 3 h at 1000–1350°C resulted in the formation of graphitic carbon and SiC. Meanwhile, sintering without a cobalt catalyst resulted in the formation of graphitic carbon, regardless of the temperature. The use of a cobalt catalyst allowed the formation of a pure SiC phase at 1350°C. The formed SiC had an irregular worm-like morphology, with a particle size of ~5 µm. The Brunauer-Emmett-Teller surface areas of graphitic carbon and SiC were 28–150 and ~7.0 m2/g, respectively. We concluded that graphite and SiC were produced at this low sintering temperature because of the cobalt catalyst, which facilitated nanomixing of carbon and SiO2 by sandwiching the carbon films between the silicate layers.Silicon carbide (SiC) was prepared by carbothermal reduction of a crystalline-layered sodium silicate (δ-Na  Therefore, SiC has been widely used in various industrial applications, such as grinding materials, polishing paste, wear-resistant materials, catalyst supports, filters for molten metals or hot gases, high-temperature structural materials, and reinforcement in composites –7.2 on the production of SiC powder 2Co, special grade) was purchased from Sigma-Aldrich, USA. PFO  had an average molecular weight of ~8000 was composed of long-chain hydrocarbons and various polyaromatics (above 60%) with high viscosity (3800–4200 cps).Amorphous sodium silicate (molar ratio of SiO2.2.2.2.1.2Si2O5) was prepared using amorphous sodium silicate as a starting material through a procedure previously described by J. K. Suh et al. [2Si2O5. The final product was crushed and used to prepare the nanocomposite with PFO.Crystalline-layered sodium silicate  contained 0% and 0.1% cobalt, respectively. The nanocomposites were then loaded onto an alumina boat with a cover and sintered in a programmable quartz tube furnace, where they were kept under 1 L/min He flow for 3 h at the diverse holding temperature range of 1000–1350°C (heating rate: 5°C/min), allowing for the conversion to SiC to take place. The resulting mixture was dispersed for 12 h in an HF (24%) solution to remove the unreacted silicate framework, and then filtered, washed with deionized water, and air-dried to obtain SiC.δ-Na2.3Powder X-ray diffraction (XRD) measurements were performed using a Rigaku Rotaflex 200B diffractometer equipped with Cu Kα X-ray radiation and a curved crystal graphite monochromator. The scanning electron micrographs (SEMs) were recorded with a JEOL JSM-840A scanning electron microscope. The transmission electron micrographs (TEM) were obtained with a JEOL JEM-200 CX transmission electron microscope operated at 200 kV, using a thin-section technique. The powder samples were embedded in epoxy resin and then sectioned with an ultramicrotome. Nitrogen adsorption/desorption isotherms were determined at 77 K using Micromeritics ASAP 2020. All samples were outgassed at 300°C under vacuum for 4 h. The specific surface areas were determined by the Brunauer-Emmett-Teller (BET) equation.3.2Si2O5. The X-ray powder diffraction pattern of δ-Na2Si2O5 exhibited several reflections  produced a δ-Na2Si2O5/carbon nanocomposite with carbon films sandwiched between the silicate layers. Here, the removal of silicate framework from LCN by HF can leave a pure-layered carbon. A successive sintering converted this nanocomposite into SiC via graphite.In the formation of the intercalation compound, δ-NaPreviously, we have reported on the preparation of porous-layered carbon by using Na-kenyaite and PFO . Here, a2Si2O5/PFO nanocomposite (LCN) result in layered silicate/graphite nanocomposite, which is sandwiched with graphite films between the silicate layers. The removal of silicate framework leaves only sandwiched graphite films with galleries about 1 nm wide. These galleries result in a peak at a diffraction angle (2θ) of 11.5°. The peak is broad and its intensity is low because graphite exists with graphitic carbon comprising amorphous carbon.Previously, we reported that amorphous carbon in the interlayer space of magadiite and kenyaite could convert to graphite after sintering for 3 h at 900°C in the presence of a cobalt or nickel catalyst . The appA pure SiC phase with strong XRD peaks was obtained through the reaction for 3 h at 1350°C, elucidating the effect of the sintering temperature on the SiC crystallization. In particular, when compared with the XRD pattern of pure cubic β-SiC , the her2Si2O5, indicating that δ-Na2Si2O5 acted as a template during the formation of graphitic carbon.2Si2O5 was broken to bits by the sintering for 3 h at 1000–1100°C, causing the formation of graphitic-layered carbon particles. However, sintering at 1200–1350°C changed the particle morphology to irregular worm-like with a size of ~3 µm due to the formation of SiC. In particular, the size of the as-synthesized SiC particles was smaller than that of the commercial reagent. As mentioned earlier, commercial SiC powder is prepared via the Acheson process, which involves a carbothermal reduction of sand by petrol coke at about 2400°C [t 2400°C . This me2/g, depending on the presence of cobalt catalyst and the sintering temperature. The large surface area of graphitic carbon was attributed to the galleries, which were formed from the removal of the silicate templates. Furthermore, increasing the sintering temperature could bring about a large decrease in the surface area, due to the formation of the SiC phase.As shown in 2Si2O5/PFO nanocomposite (LCN) at 600°C produces a δ-Na2Si2O5/carbon nanocomposite. Here, removal of silicate framework left only carbon films sandwiched between the silicate layers . This palleries ). The in2Si2O5 was composed of two-dimensional spaces at the nano-scale, which can act as nanoreactors for chemicals introduced into the gallery. This confirms that, in this nanoreactor, PFO could easily be transformed into graphite by sintering at 1000–1100°C, affording a δ-Na2Si2O5/carbon nanocomposite with a carbon film sandwiched between the silicate layers. The removal of the δ-Na2Si2O5 template resulted in graphitic carbon particle with layered structure. Moreover, in the presence of a cobalt catalyst, LCN could also be converted to a well-crystallized SiC phase by sintering above 1350°C. This is attributed to the nanomixing by sandwiching of the δ-Na2Si2O5 framework and carbon source, in which the δ-Na2Si2O5 frame composed of silicate layers acts as a silica source.4.2/g, respectively.Graphitic carbon and SiC were synthesized through a cobalt-catalyzed carbothermal conversion of LCN in a short sintering time at a temperature range of 1000–1350°C. In the absence of cobalt, the SiC phase did not form even at 1300°C. However, in the presence of cobalt, SiC formed at 1200°C thereby elucidating the effect of cobalt on the formation of the SiC phase. Here, a pure SiC phase was obtained through the reaction for 3 h at 1350°C. The particle morphology of SiC with cobalt was irregular worm-like with a size of ~5 µm. The surface areas of the graphitic-layered carbon and SiC were 28–150 and ~7.0 m


The rotation of the coordinating plane of the square-antiprismatic environment induces a magnetic relaxation path through higher excited states, offering a new way to modulate the geometries of lanthanides to facilitate magnetic relaxation climbing up to higher energy levels. 2(o-vanilin)2]·X·solvent , NO3– (2), CF3SO3– (3)), were realized by changing the nature of the counter-anion. The DyIII ions in all complexes are eight-coordinate and in approximate D4d symmetry environments. The magnetic relaxation and anisotropy of these complexes were systematically investigated, both experimentally and from ab initio calculations. All complexes exhibit excellent single-molecule magnetic behavior. Remarkably, magneto-structural studies show that the rotation of the coordinating plane of the square-antiprismatic environment in complex 2 induces a magnetic relaxation path through higher excited states, yielding a high anisotropy barrier of 615 K (696 K for a diluted sample). Additionally, obvious opening of the hysteresis loop is observed up to 7 K, which is the highest blocking temperature ever reported for dysprosium single-molecule magnets.Geometry and magnetic relaxation modulations in a series of mononuclear dysprosium complexes, [DyLz For example, in the polyoxometalateD4d coordination environment,D4d coordination polyhedron in the β-diketonate series gives rise to strong Ising ground states, leading to significant relaxation blockages for dysprosium derivatives.cis–trans isomerism, could influence the relaxation behavior.Aside from low-coordinate systems, high symmetry cases, such as 2(o-vanilin)2]·X·solvent , NO3– (2), CF3SO3– (3)), with the metal ions in a distorted D4d coordination environment : 3462 (m), 3393 (m), 3273 (b), 3130 (b), 1611 (m), 1536 (m), 1394 (s), 1253 (m), 993 (m), 830 (m), 792 (s), 687 (w).All chemicals and solvents were commercially obtained and used as received without any further purification. IR spectra were recorded with a Perkin-Elmer Fourier transform infrared spectrophotometer. Elemental analysis for C, H, N and S was carried out on a Perkin-Elmer 2400 analyzer. All doped samples were analyzed using Inductively Coupled Plasma Optical Emission Spectrometry (ICP OES). The ligand Lz  was prepared according to a previously published method1 is described here in detail. The reaction of DyBr3·6H2O (0.15 mmol) with Lz (0.1 mmol) and o-vanilin (0.1 mmol) in the presence of triethylamine (0.1 mmol), with 5 : 5 ml methanol/acetonitrile as the media, produced yellow crystals of 1 after 7 days. Anal. calcd for 1 : C, 42.01%; H, 3.50%; N, 18.74%. Found: C, 42.12%; H, 3.61%; N, 18.69%. For the synthesis of 2 and 3, Dy(NO3)3·5H2O and Dy(CF3SO3)3·6H2O were used to replace DyBr3·6H2O with methanol (15 ml) as the solvent. Anal. calcd for 2 : C, 42.52%; H, 3.32%; N, 20.15%. Found: C, 41.93%; H, 3.51%; N, 20.26%. Anal. calcd for 3 : C, 38.73%; H, 2.93%; N, 16.43%; S, 3.13%. Found: C, 38.55%; H, 3.06%; N, 15.91%; S, 3.20%. Doping of complex 2 was performed by adding Dy(NO3)3·5H2O and Y(NO3)3·5H2O together  in the reaction. ICP OES analysis found the components of [Dy0.671Y0.329Lz2(o-vanilin)2]·NO3, [Dy0.076Y0.924Lz2(o-vanilin)2]·NO3 and [Dy0.024Y0.976Lz2(o-vanilin)2]·NO3 corresponding to the 2 : 1, 1 : 12 and 1 : 39 samples.All complexes were prepared by similar procedures; therefore only the synthesis of complex λ = 0.71073 Å) at 293(2) K. The structures were solved by direct methods and refined by full-matrix least-squares methods on F2 using SHELXTL-97.Single-crystal X-ray data of the title complexes were collected on a Bruker Apex II CCD diffractometer equipped with graphite monochromated Mo-Kα radiation  using the pseudo-spin S = 1/2 approximation as defined in the SINGLE_ANISO routine.III ion spanning the seven 4f orbitals, i.e. CASSCF. The state-averaged CASSCF calculations were performed for all the 21 sextet roots, all the 224 quadruplet roots and 300 out of the 490 doublet roots, due to software limitations. In the RASSI-SO calculation, the 21 sextet roots were allowed to mix through spin–orbit coupling with the first 128 quadruplet roots and the first 107 doublet roots. All atoms were described by ANO-type basis sets from the ANO-RCC library of MOLCAS. The following contractions were used: [8s7p4d3f2g1h] for the DyIII ion, [4s3p2d] for the four N and the four O atoms of the first coordination sphere, [3s2p1d] for the remaining N and O atoms and all C atoms, and [2s] for the H atoms.Wavefunction-based calculations were carried out on the X-ray structures of o-vanilin (0.1 mmol) in the presence of triethylamine (0.1 mmol), with 5 : 5 ml methanol/acetonitrile as the reaction media leads to the formation of mononuclear dysprosium complexes, [DyLz2(o-vanilin)2]·X·solvent , NO3– (2), CF3SO3– (3)). The structures of 1, 2 and 3 are depicted in Fig. S1–S3.III ion is in a N4O4 square-antiprismatic environment . In all three complexes, the positive charge is balanced by one counter-anion, namely Br– in 1, NO3– in 2 and CF3SO3– in 3. Although 1, 2 and 3 have similar ligand sets, a closer look at the structural features reveals significant ligand rotation and considerable distortion from the D4d symmetry, which is most likely induced by the change of counter-anions. In order to evaluate these differences, the α angle between the pseudo S8 axis and the Dy–L directions, the Φ space angle between the two Lz ligands, as well as the θ angle between the upper and lower mean planes, were investigated (α angle is found in 2 (56.5°) with respect to 1 and 3 (57.0° in both systems), indicating an axial extension of the coordinating environment in 2. This can be explained by the insertion of the NO3– counter-anion between the coordination planes in 2, while in 1 and 3 the counter-anions reside outside these planes and relatively far from the molecular unit. For the Φ angle, small values are found in 1 and 3  and are characteristic of the two Lz ligands being in the cis position relative to each other, while the much larger value found in 2 (140.9°) is characteristic of the two Lz ligands being in the trans position  and π-stacking distance between the two triazinyl centers of the ligand Lz (3.56 Å) found in 3  with Lz (0.1 mmol) and ironment , where tstigated  and S4† position . Finally1–3 under a 1 kOe applied field from 2 to 300 K  vs. T curves for 1–3  close to the expected value of 20.00 for a pure MJ = ±15/2 eigenstate. Wave-function analysis also confirms greater than 80% percentages of the MJ = ±15/2 eigenstate in 1–3.Direct-current (dc) magnetic susceptibilities were investigated for to 300 K . The roo1–3, alternating-current (ac) magnetic susceptibility measurements were also performed under zero dc fields (χ′) and out-of-phase (χ′′) magnetic susceptibility signals at 1488 Hz for 1–3 exhibit peaks at 27 K, 42 K and 20 K, respectively. Upon cooling, a new tail peak appears below 2.5 K in the χ′ and χ′′ signals of 1 and 2, while a rapid increase is observed below 10 K for 3. This rapid increase in the low temperature region could be attributed to quantum tunneling effects at zero dc field, which is very common in 4f SMMs.3, as the temperature is lowered, the maximum peak in the out-of-phase χ′′ signal is shifted toward a lower frequency until 8 K, beyond which the same frequency was maintained, confirming the presence of the classical quantum regime  with a τ0 = 5.7(5) × 10–11 s. The Cole–Cole plots of χ′′ versus χ′ display semi-circular profiles and are fitted to a generalized Debye model , indicating a relatively wide distribution of the relaxation times, and thus multiple pathways for spin reversal.τ0 and Ueff are listed in Table S4.To investigate the SMM behavior of c fields . Tempera Fig. S10. Similares for 2  show gre1–3 . In contrast to 1 and 3, complex 2 displays distinct butterfly shape hysteresis. Herein, the hysteresis loops remain open until 7 K with a much larger opening near zero-field. More interestingly, non-coincidence was observed in the M vs. H curves at H = 0  and triazine analogues (Lz) is expected to significantly influence the energy spectrum and magnetic anisotropy of the low-lying states of DyIII, and thus lead to efficient Dy-based SMMs, which is indeed the case here. However, the different environment geometries and subsequent various magnetic behaviors induced by the change of counter-anions in our system with the same set of ligands are remarkable. Notably, the energy barrier observed in complex 2 is extraordinary larger than those in 1 and 3, which might be ascribed to the rotation of the coordinating plane of the square-antiprismatic environment.All complexes presented herein show large energy barriers, which is rare among the dysprosium-SMM family, and is most likely due to the unique  Table S5. Additio6H15/2 ground multiplet of DyIII are computed from ab initio for all complexes . This value is much higher than that obtained from ac measurements (221 K), but it has to be kept in mind that this kind of calculation does not account for all possible relaxation mechanisms , and relies on several approximations.3, the situation is almost the same, except that large transversal components already appear at the 2nd excited state (Table S6–1 (430 K). Here again, the computed value is much higher than that from ac measurements (120 K), but still smaller than that of 1, which is in good agreement with the experimental tendency. Finally, for 2, calculations evidence a relaxation pathway going through the 3rd excited state, leading to an energy barrier of approx. 600 cm–1 (860 K). This pathway is supported by the high axiality of the magnetic anisotropy of the three lower states and the small angular deviation between the associated magnetic easy-axis directions, while for the 3rd excited state, the transversal components become very large, with a large deviation to the direction of the ground state easy-axis  in all complexes, suggesting that the main factor responsible for the difference in energy barriers in 1–3 is the total energy splitting of the 6H15/2 ground multiplet, which itself depends on the structural features of each complex, and most likely on the cis or trans configuration of the Lz ligands. Indeed, in the cis configuration, contrary to the trans configuration, π-stacking interactions are operative and contribute towards reducing the energy splitting of the 6H15/2 ground multiplet. This explains why the splitting is much larger in 2 (trans configuration) than in 1 and 3 (cis configuration). In more detail, this also explains why the energy splitting of 1 is larger than that of 3, since π-stacking interactions are much more effective in 3 than in 1, thus having a larger stabilizing effect. Additionally, the axial extension in 2 might also, to some extent, be responsible for the associated larger splitting, as well as the exact C2 symmetry held by 3  but discriminative counter-anions. The distortions in the D4d symmetry environment led to large energy gaps between the ground and first excited states, and strong axial anisotropies for complexes 1–3. Upon changing the counter-anion, significant differences in the structure and thus magnetic behavior were observed: for instance, when going from 3 to 1, the π–π stacking interactions between the Lz ligands lead to axial constriction in the environment geometry, from which fast quantum tunneling arises because the symmetry is lowered; proper rotation of one coordinating plane, as well as relative axial extension in complex 2 with respect to complex 3, allows magnetic relaxation to pass through higher excited states, due to an increase in molecular symmetry. Ab initio calculations substantiate the diversity of the magnetic behaviors in complexes 1–3 and suggest that the ground states are almost isolated, magnetically speaking. Notably, the efficient magnetic relaxation pathways of complex 2 probably go through the fourth and fifth Kramer doublet states. This conforms a high anisotropy barrier of 696 K (484 cm–1) and magnetic blocking up to 7 K. This work offers a new way to modulate the geometries of lanthanides in order to facilitate magnetic relaxation climbing up to higher energy levels.In conclusion, we have structurally isolated three dysprosium SMMs with similar square-antiprismatic environments (Supplementary informationClick here for additional data file.Crystal structure dataClick here for additional data file.