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Research ArticleCancer
Targeting p53-dependent stem cell loss for intestinal chemoprotection
Brian J. Leibowitz1,2,
Liheng Yang2,3,
Liang Wei1,2,
Monica E. Buchanan2,3,
Madani Rachid2,
Robert A. Parise2,
Jan H. Beumer2,3,4,5,
Julie L. Eiseman2,3,5,
Robert E. Schoen2,4,
Lin Zhang2,3 and
Jian Yu1,2,*
1Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA.
2UPMC Hillman Cancer Center, Pittsburgh, PA 15213, USA.
3Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA.
4Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA.
5Department of Pharmaceutical Sciences, University of Pittsburgh School of Pharmacy, Pittsburgh, PA 15261, USA.
↵*Corresponding author. Email: yuj2{at}upmc.edu
Science Translational Medicine 07 Feb 2018:
Vol. 10, Issue 427, eaam7610
DOI: 10.1126/scitranslmed.aam7610
Brian J. Leibowitz
Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA.UPMC Hillman Cancer Center, Pittsburgh, PA 15213, USA.
ORCID record for Brian J. Leibowitz
Liheng Yang
UPMC Hillman Cancer Center, Pittsburgh, PA 15213, USA.Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA.
ORCID record for Liheng Yang
Liang Wei
ORCID record for Liang Wei
Monica E. Buchanan
ORCID record for Monica E. Buchanan
Madani Rachid
UPMC Hillman Cancer Center, Pittsburgh, PA 15213, USA.
Robert A. Parise
ORCID record for Robert A. Parise
Jan H. Beumer
UPMC Hillman Cancer Center, Pittsburgh, PA 15213, USA.Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA.Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA.Department of Pharmaceutical Sciences, University of Pittsburgh School of Pharmacy, Pittsburgh, PA 15261, USA.
ORCID record for Jan H. Beumer
Julie L. Eiseman
UPMC Hillman Cancer Center, Pittsburgh, PA 15213, USA.Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA.Department of Pharmaceutical Sciences, University of Pittsburgh School of Pharmacy, Pittsburgh, PA 15261, USA.
Robert E. Schoen
UPMC Hillman Cancer Center, Pittsburgh, PA 15213, USA.Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA.
ORCID record for Robert E. Schoen
Lin Zhang
Jian Yu
ORCID record for Jian Yu
For correspondence: yuj2@upmc.edu
Safer without PUMA
Gastrointestinal toxicity is a major cause of side effects from chemotherapy and radiation, which can greatly decrease the quality of life and limit the dosing of cancer treatments. Tumor suppressor p53 plays a major role in chemotherapy-induced intestinal cell death, but inhibiting p53 is not safe because a loss of p53 would worsen cancer growth. Instead, Leibowitz et al. showed that one can block only the detrimental function of p53 by inhibiting its downstream effector, PUMA. A small-molecule inhibitor of PUMA protected intestinal cells in mice and in human colon organoids but did not protect cancer cells, suggesting that this could be a viable strategy for intestinal protection in cancer patients.
The gastrointestinal (GI) epithelium is the fastest renewing adult tissue and is maintained by tissue-specific stem cells. Treatment-induced GI side effects are a major dose-limiting factor for chemotherapy and abdominal radiotherapy and can decrease the quality of life in cancer patients and survivors. p53 is a key regulator of the DNA damage response, and its activation results in stimulus- and cell type–specific outcomes via distinct effectors. We demonstrate that p53-dependent PUMA induction mediates chemotherapy-induced intestinal injury in mice. Genetic ablation of Puma, but not of p53, protects against chemotherapy-induced lethal GI injury. Blocking chemotherapy-induced loss of LGR5+ stem cells by Puma KO or a small-molecule PUMA inhibitor (PUMAi) prevents perturbation of the stem cell niche, rapid activation of WNT and NOTCH signaling, and stem cell exhaustion during repeated exposures. PUMAi also protects human and mouse colonic organoids against chemotherapy-induced apoptosis and damage but does not protect cancer cells in vitro or in vivo. Therefore, targeting PUMA is a promising strategy for normal intestinal chemoprotection because it selectively blocks p53-dependent stem cell loss but leaves p53-dependent protective effects intact.
Gastrointestinal (GI) side effects are a major dose-limiting factor in chemotherapy and abdominal radiotherapy and can cause long-term complications in cancer survivors (1, 2). Colorectal cancer patients are commonly treated with irinotecan hydrochloride (CPT-11) in combination with 5-fluorouracil (5-FU). Radiation and most chemotherapeutic agents damage multiple tissues and organ systems, whereas CPT-11 causes selective GI injury, with severe diarrhea and nausea in more than 50% of patients (3). This acute toxicity is caused by the accumulation of high concentrations of SN-38, the active metabolite of CPT-11, in the intestine (4), as well as the conversion of nontoxic SN-38 glucuronide back to SN-38 by gut bacteria, followed by intestinal reabsorption (5). SN-38 is an inhibitor of topoisomerase I, a key enzyme in both DNA replication and RNA transcription, and causes replication stress and DNA damage in proliferating cells (6). Antidiarrhea medication can help alleviate CPT-11–induced diarrhea but has a limited effect on reducing long-term GI dysfunction (1). There is currently no U.S. Food and Drug Administration–approved agent that prevents or treats CPT-11–induced GI injury or complications.
Chemotherapy or radiation-induced acute enteropathy is characterized by loss of proliferating crypt cells, epithelial barrier breakdown, and inflammation during or shortly after the treatment. In many patients, delayed enteropathy can occur months or later after therapy and is characterized by intestinal dysfunction associated with pathological changes in the epithelial and stromal compartments, such as vascular sclerosis and progressive intestinal wall fibrosis. Emerging evidence suggests that chronic intestinal damage is likely a consequence of early toxicity (1, 7). Enterotoxicity caused by chemotherapeutics, such as CPT-11 and 5-FU, is associated with rapid crypt apoptosis in mice and humans (8, 9). The small intestinal epithelium is the fastest renewing adult tissue, and intestinal stem cells (ISCs) located in the bottom of crypts drive the renewal and regeneration after injury. ISCs include the columnar cells at the crypt bottom (CBCs) and some cells at position 4 relative to the crypt bottom (+4 cells) (10, 11). Genetic ablation of leucine-rich repeat-containing G protein (heterotrimeric guanine nucleotide–binding protein)–coupled receptor 5–expressing (LGR5+) CBCs is well tolerated in healthy mice but strongly exacerbates radiation-induced intestinal injury, although underlying mechanisms remain unknown (12).
Activation of p53 after DNA damage results in tissue- and target-specific outcomes such as tumor suppression or acute toxicity (13–16). p53 activation is a double-edged sword in controlling intestinal regeneration after high-dose radiation. On one hand, p53-dependent PUMA induction accounts for most radiation-induced apoptosis and acute loss of intestinal and hematopoietic stem and progenitor cells, and PUMA deficiency or down-regulation protects against radiation-induced lethality (17–20) and lymphomagenesis (21, 22). However, p53-dependent induction of p21, and perhaps other targets, is essential for productive intestinal regeneration by preventing DNA damage accumulation, replication stress, and delayed nonapoptotic cell death, and p53/p21 deficiency exacerbates intestinal injury (20, 23). The role of p53 in chemotherapy-induced GI injury is not well understood. The p53 inhibitor pifithrin-α reduced CPT-11–induced intestinal apoptosis within the first several hours in rats but did not affect delayed cell death or the onset of mucositis (8).
Here, we sought to determine the role of p53-dependent apoptosis in chemotherapy-induced intestinal injury. We demonstrated that genetic or pharmacological inhibition of Puma by a small-molecule PUMA inhibitor (PUMAi), but not p53 deficiency, provides potent protection against GI injury induced by single and repeated exposures to CPT-11 but does not compromise its antitumor activity in vivo. This protection is associated with selective preservation of LGR5+ stem cells, stem cell niche, and genome integrity. Our findings provide a mechanism-based approach and a compound for intestinal chemoprotection through selective inhibition of p53-dependent apoptosis and enterotoxicity.
PUMA mediates chemotherapy-induced crypt apoptosis and lethality
To investigate a potential role of p53-dependent apoptosis in chemotherapy-induced acute intestinal injury, we treated wild-type (WT), p53 knockout (KO), and Puma KO mice with a single dose of irinotecan (200 mg/kg; CPT-11). CPT-11 induced robust crypt apoptosis in WT mice, as measured by terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick end labeling (TUNEL) and active caspase-3 staining, which peaked at 6 hours and decreased gradually by 48 hours. Apoptosis was observed in both the stem cells at the crypt base (CBCs at positions 1 to 3 relative to the crypt bottom) and +4 to 9 cells (relative to the crypt bottom), but it was abrogated in p53 KO and Puma KO mice (Fig. 1, A and B, and fig. S1, A to D). CPT-11 treatment induced p53 and its targets PUMA and p21 within 6 hours in the intestinal mucosa of WT but not p53 KO mice (Fig. 1C and fig. S1E). Puma KO did not significantly affect the expression of p53 or p21 (Fig. 1C). BH3-only proteins BIM and NOXA were not induced by CPT-11 or affected by Puma KO (fig. S1F). RNA in situ hybridization (ISH) confirmed that Puma mRNA was undetectable in the crypts of untreated WT mice and strongly induced in the CBCs and some +4 cells by CPT-11 treatment (Fig. 1D).
Fig. 1 PUMA mediates CPT-11–induced intestinal injury.
Mice with indicated genotypes were treated with CPT-11 (200 mg/kg) once or as specified and analyzed at indicated times. (A) Representative images of terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick end labeling (TUNEL) staining in intestinal crypts at 6 hours. DAPI (4′,6-diamidino-2-phenylindole) was used to stain nuclei. Scale bar, 50 μm. (B) Quantitation of TUNEL+ columnar cells at the crypt bottom (CBCs) and +4 to 9 cells in (A). (C) Expression of indicated proteins was analyzed by Western blotting at 6 hours. Lysates were pooled from the intestinal mucosa of three mice. Representative results are shown, and similar results were obtained in at least three independent experiments. (D) Puma RNA in situ hybridization (ISH) in the crypts at 6 hours. Scale bar, 20 μm. In (A) and (D), arrows indicate CBCs at positions 1 to 3 below Paneth cells, and asterisks indicate +4 to 9 cells at positions 4 to 9 above the CBCs. In (A) to (D), n = 3 mice per group. In (B), values are means + SEM. ***P < 0.001 (two-tailed Student's t test), knockout (KO) versus wild type (WT). (E) Survival of mice treated with three consecutive daily doses of CPT-11 (215 mg/kg per day) on days 0, 1, and 2. WT versus Puma KO, P = 0.0004; WT versus p53 KO, P = 0.382 (log-rank test). (F) Hematoxylin and eosin (H&E) staining of the small intestine from mice on day 5 treated as in (E). Scale bar, 200 μm.
CPT-11 causes dose-dependent lethal GI injury in mice (24). To determine the sensitivity of C57BL/6J mice, we monitored survival after three consecutive daily doses of CPT-11. The 180, 215, and 250 mg/kg per day doses resulted in 30, 90, and 100% lethality, respectively, and all deaths occurred between days 5 and 8, characteristic of lethal GI injury (fig. S2). At the 90% lethal dose (LD90) of WT mice (215 mg/kg per day for 3 days), all Puma KO mice survived for 30 days, whereas all p53 KO mice died between days 6 and 9 (Fig. 1E). Histological analysis confirmed lethal GI injury in WT and p53 KO mice on day 5, showing severe loss of crypts and shortening of villi, which was abrogated in Puma KO mice (Fig. 1F). Consistent with a dual role of p53 in intestinal recovery from radiation (20), our data demonstrate that selective inhibition of p53-dependent induction of PUMA and apoptosis, but not p21 or other functions, results in intestinal chemoprotection.
A small-molecule PUMAi protects against chemotherapy-induced crypt apoptosis and lethality
We previously identified a number of potential PUMAis using a pharmacophore model of the key interactions of the PUMA BH3 domain with BCL-2 family proteins, in silico compound screening, and apoptosis assays (25, 26). One of these compounds (PUMAi) was confirmed to inhibit interaction of PUMA/BCL-xL but not of BIM/MCL-1 (fig. S3, A to C). PUMAi did not reduce camptothecin (CPT)–induced apoptosis in colon cancer cell lines with different p53 or PUMA genotypes, including p53 WT (HCT 116), null (p53 KO), mutant (HT 29, SW480, and DLD1), or PUMA null (PUMA KO) (fig. S3D). PUMA was only induced by CPT treatment in p53 WT HCT 116 cells, not in any p53-deficient line (fig. S3E) (27–29). The basal amount of PUMA was much higher in HCT 116 cells, compared to very low or undetectable expression in mouse intestinal mucosa (Fig. 1, C and D) (17) and in normal human small intestine and colon (30).
To examine the efficacy of PUMAi in vivo, we treated WT mice with vehicle or PUMAi (10 mg/kg, an empirical dose) 2 hours before a single dose of CPT-11 (200 mg/kg) and analyzed crypt apoptosis 6 and 24 hours after CPT-11 administration. PUMAi reduced TUNEL and active caspase-3 staining preferentially in the CBCs, compared to transit-amplifying (TA), +4 to 9, cells (Fig. 2, A and B, and fig. S4, A to D). PUMAi treatment did not further reduce crypt apoptosis in Puma KO mice (fig. S4E). We then developed a high-performance liquid chromatography (HPLC) assay and assessed PUMAi distribution in the intestinal mucosa and plasma at three time points, −1.5, +1, and +6 hours of CPT-11 administration (0 hour) (fig. S4F). PUMAi concentration remained at 67 and 30% in the intestinal mucosa at +1 and +6 hours, respectively, compared to −1.5 hours, but dropped more quickly in the plasma (Fig. 2C). CPT-11 is rapidly cleared from the plasma, with a half-life around 36 min (31). We found that PUMAi given 1 hour after CPT-11 treatment still reduced apoptosis effectively, suggesting that PUMAi does not affect CPT-11 conversion or intestinal absorption (Fig. 2D).
Fig. 2 PUMAi protects against CPT-11–induced intestinal injury.
Mice were treated with CPT-11 with or without PUMA inhibitor (PUMAi). PUMAi (10 mg/kg) or vehicle was given intraperitoneally for 2 hours before CPT-11 or as specified. The small intestine or indicated tissue was analyzed at the indicated times. (A) Representative images of TUNEL staining in intestinal crypts of mice treated with CPT-11 (200 mg/kg). DAPI was used to stain nuclei. Scale bar, 50 μm. Arrows indicate CBCs, and asterisks indicate +4 to 9 cells. (B) Quantitation of TUNEL+ CBCs and transit-amplifying cells from (A). Inh, inhibitor. (C) Tissue distribution of PUMAi at different times after a single injection. (D) Quantitation of TUNEL+ crypt cells in mice 6 hours after CPT-11 (200 mg/kg) treatment. PUMAi (10 mg/kg) was given 2 hours before or 1 hour after CPT-11 treatment. n = 3 mice per group (B to D). In (B) and (D), values are means + SEM. *P < 0.05 (two-tailed Student's t test). Veh, vehicle. (E) Survival of WT mice after three doses of CPT-11 (215 mg/kg per day) (done with the control in Fig. 1E). PUMAi (10 mg/kg) was given 2 hours before each dose of CPT-11. P = 0.0047 (log-rank test). (F) H&E staining of the small intestine from mice treated as in (E) on days 0, 5 and 30. Scale bar, 200 μm.
Further, PUMAi decreased CPT-11–induced lethality at three different doses (Fig. 2E and fig. S5, A and B). At the LD90, PUMAi improved survival from 10 to 70%. Histological analysis confirmed reduced GI injury on day 5 in the PUMAi and Puma KO groups, compared to WT mice (Figs. 1F and 2F). PUMAi also increased the survival of mice after 9.5- and 15-gray total body irradiation (fig. S5, C and D), further supporting the idea that targeting p53-dependent apoptosis, but not other functions, selectively reduces treatment-induced normal tissue toxicity.
PUMA inhibition does not reduce antitumor activity of CPT-11
To determine whether PUMA inhibition provides selective intestinal chemoprotection, we established subcutaneous Lewis lung carcinoma (LLC) tumors in immunocompetent WT and Puma KO mice. After tumors reached an average of 100 mm3 (day 11), mice received six doses of CPT-11 (200 mg/kg, intraperitoneally) over 2 weeks (fig. S6A). Tumor engraftment and CPT-11 response were indistinguishable in WT and Puma KO hosts (Fig. 3A and fig. S6B). In contrast, CPT-11–induced weight loss was reduced in Puma KO mice (Fig. 3B), correlated with preserved intestinal structure and barrier evident by higher villus height, crypt numbers, and lower neutrophil infiltration (Fig. 3, C to G).
Fig. 3 PUMA deficiency protects tumor-bearing mice from chemotherapy-induced GI injury.
WT and Puma KO mice bearing Lewis lung carcinoma (LLC) tumors were treated with CPT-11 (200 mg/kg) six times over 15 days, on days 11, 14, 17, 19, 23, and 25. Mice or tumors were analyzed at the indicated times. (A) Tumor volumes were measured every other day from days 11 to 25. CT, vehicle control. (B) Body mass was expressed as a percentage of that on day 11 for each mouse from days 11 to 25. (C) H&E staining of the small intestine tissue on day 25. Scale bar, 100 μm. (D) Villus height from (C). Measurements were from a minimum of 40 villi per mouse. (E) Quantitation of intestinal crypts per field from (C). (F) Intestinal neutrophils detected by immunofluorescence on day 25. DAPI was used to stain nuclei. Scale bar, 100 μm. (G) Quantitation of neutrophils per 400× field from the same slides as (F). In (A), (B), (D), (E), and (G), values are means + SEM; n = indicated (A and B) or 3 to 4 mice per group (D, E, and G). **P < 0.01 and ***P < 0.001 (two-tailed Student's t test). Puma KO, CPT versus control (A); CPT-11–treated, Puma KO versus WT (B).
The effects of PUMAi were then determined in tumor-bearing mice. PUMAi (10 mg/kg) or vehicle was given 2 hours before and 20 hours after each dose of CPT-11. The PUMAi group showed comparable tumor response compared with the vehicle group (Fig. 4A and fig. S6B) but was highly resistant to CPT-11–induced weight loss, intestinal damage (Fig. 4, B to G), and lethality (table S1). Furthermore, PUMAi or PUMA KO groups exhibited improved grooming and physical activity throughout the study based on daily inspections. LLC tumors in all three CPT-11–treated groups showed similar effects on proliferation or apoptosis (fig. S6, C and D) but lacked induction of p53, PUMA, or p21 (fig. S6E), consistent with mutant p53 (32). These studies demonstrate that PUMA inhibition attenuates CPT-11–induced enterotoxicity but not the establishment or therapeutic responses of p53 mutant tumors in immunocompetent hosts.
Fig. 4 PUMAi protects tumor-bearing mice from chemotherapy-induced GI injury.
WT mice bearing LLC tumors were treated with CPT-11 (200 mg/kg) six times over 15 days on days 11, 14, 17, 19, 23, and 25. Vehicle or PUMAi was given 2 hours before and 20 hours after each dose of CPT-11. Mice or tumors were analyzed at the indicated times. (A) Tumor volumes were measured starting on day 11. (B) Body mass was expressed as a percentage of that on day 11 for each mouse from days 11 to 25. (C) H&E staining of the small intestine tissue on day 25. Scale bar, 100 μm. (D) Villus height from (C). Measurements were from a minimum of 40 villi per mouse. (E) Quantitation of intestinal crypts per field from (C). (F) Intestinal neutrophils detected by immunofluorescence on day 25. DAPI was used to stain the nuclei. Scale bar, 100 μm. (G) Quantitation of neutrophils per 400× field from the same slides as (F). In (A), (B), (D), (E), and (G), values are means + SEM; n = indicated (A and B) or 3 to 4 mice per group (D, E, and G). *P < 0.05, **P < 0.01, and ***P < 0.001 (two-tailed Student's t test). PUMAi, CPT versus control (A); CPT-11–treated mice, PUMAi versus CT (B).
Targeting PUMA potently protects the LGR5+ stem cells and niche against chemotherapy
Little is known about the role of the ISC niche in injury-induced regeneration. We hypothesized that the loss of CBCs, not of more chemosensitive TA cells, sends "injury" signals via niche "emptying" to activate remaining stem cells. PUMAi provides a pharmacological way to test this because it selectively blocks CPT-11–induced CBC apoptosis (Fig. 2B). Using LGR5–EGFP (enhanced green fluorescent protein) reporter mice, we found that Puma KO or PUMAi decreases CPT-11–induced apoptosis of LGR5+ cells by more than 90 or 70%, respectively (Fig. 5, A and B). Puma KO also blocked LGR5+ cell apoptosis induced by another commonly used chemotherapy drug, 5-FU (fig. S7).
Fig. 5 Targeting PUMA protects the LGR5+ stem cells against CPT-11.
WT and Puma KO mice were treated with CPT-11 (200 mg/kg) and analyzed at the indicated times. PUMAi (10 mg/kg) was given once 2 hours before CPT-11 treatment. (A) Green fluorescent protein (GFP) (LGR5) and TUNEL immunofluorescence staining in intestinal crypts. Scale bar, 50 μm. Arrow indicates double positive cell. (B) Quantitation of GFP/TUNEL–double-positive cells per GFP-positive crypt from (A). (C) Immunofluorescence staining for CD166 in the intestinal crypts. Scale bar, 50 μm. (D) Quantitation of CD166 cells in the +4 to 9 region in crypts. (E) Indicated mRNAs were analyzed by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Complementary DNAs (cDNAs) were synthesized from RNA pooled from three mice. Values were normalized to Gapdh expression and expressed relative to each gene's own 0-hour control. (F) 53BP1 immunofluorescence staining in intestinal crypts. Scale bar, 50 μm. (G) Quantitation of 53BP1+ crypt cells at 6 and 48 hours after single CPT-11 treatment or #120 hours with three daily doses of CPT-11 (215 mg/kg per day) as in Fig. 2E. In (C) and (F), arrows indicate CBCs, and asterisks indicate +4 to 9 cells. In (A), (C), and (F), DAPI was used to stain the nuclei. In (B), (E), and (G), values are means + SEM; n = 3 mice per group. *P < 0.05, **P < 0.01, and ***P < 0.001 [one-way analysis of variance (ANOVA) with Tukey post hoc test performed separately for each time point]. In (D), values are means + SEM; n = 3 mice per group. **P < 0.01 (two-tailed Student's t test).
CD166 is a WNT target that marks the ISC niche, including CBCs and Paneth cells at the crypt bottom, in homeostasis (33). CPT-11 treatment induced a marked expansion of CD166+ cells from 48 to 96 hours in WT mice, which was fully suppressed by PUMAi treatment by 96 hours (Fig. 5, C and D). This was preceded by transient but strong activation of WNT (Lgr5, CD44, and Wnt3A)– and NOTCH (Olfm4, Math1, Hes1, Hes5, and Dll1)–responsive genes as early as 6 hours, which was largely suppressed in Puma KO and PUMAi-treated mice (fig. S8). The only exception is the TA maker CD44, which was strongly suppressed by Puma KO but not by PUMAi (Fig. 5E). We then monitored crypt DNA damage by 53BP1 foci, which was comparable in Puma KO, PUMAi, and control groups at 6 hours. However, DNA damage in Puma KO and PUMAi groups was lower by 48 hours after a single dose of CPT-11 and, more so, by 120 hours after three doses of CPT-11 (Fig. 5, F and G). These data strongly suggest that the loss of LGR5+ cells disrupts the niche and triggers WNT and Notch signaling and intestinal regeneration after chemotherapy and that blocking LGR5+ cell loss likely delays compensatory proliferation and improves DNA repair.
LGR5+ cells are the critical target in intestinal chemoprotection by PUMAi
We further hypothesized that WNT and NOTCH activation triggered by LGR5+ cell loss will likely sensitize remaining stem cells to repeated exposures and stem cell exhaustion. After six doses of CPT-11 treatment, the LGR5+ crypt cells and the fraction of LGR5+ crypts (34) were reduced by more than 90% in WT mice, compared to only 40 to 60% in the Puma KO and PUMAi groups (Fig. 6, A and B). Olfm4 (10) RNA ISH indicated a 93% drop in Olfm4+ crypts in WT mice, compared to 8 and 49% in the Puma KO and PUMAi groups, respectively (Fig. 6C). Paneth cells are the niche for LGR5+ cells and marked by MMP7 expression. Repeated CPT-11 treatment, but not single treatment, diminished MMP7+ cells within the crypts, with prominent mislocalization scattered through the epithelium and nearly complete loss of the close contact of MMP7+ and LGR5+ cells, which was suppressed in the Puma KO and PUMAi groups (Fig. 6, D to F). Overall, these data demonstrate LGR5+ cell exhaustion as the key mechanism underlying chemotherapy-induced enterotoxicity, which can be effectively targeted by PUMAi.
Fig. 6 Targeting PUMA prevents LGR5+ stem cell exhaustion after repeated CPT-11 exposure.
WT and Puma KO mice carrying the Lgr5-EGFP-IRES-creERT2 marking allele were treated with CPT-11 and PUMAi over 2 weeks as in Fig. 5 and analyzed for intestinal phenotypes. (A) GFP (LGR5) immunofluorescence in intestinal crypts. Arrows indicate LGR5+ (GFP) crypts. Scale bar, 100 μm. (B) Quantitation of intestinal crypts containing at least one GFP+ (LGR5+) cell. (C) Top: Olfm4 RNA ISH in intestinal crypts. Scale bar, 50 μm. Asterisks indicate Olfm4+ crypt cells. Bottom: Percentage of crypts containing at least one Olfm4+ cell. (D) MMP7 immunofluorescence in intestinal crypts. Scale bar, 100 μm. (E) Quantitation of MMP7+ crypt cells at the crypt base (CBC region). (F) Top: GFP (LGR5) and MMP7 immunofluorescence in intestinal crypts. Scale bar, 20 μm. Asterisks indicate LGR5+/MMP7+ cell pairs. Bottom: Quantitation of LGR5+/MMP7+ cell pairs per crypt. In (A), (D), and (F), DAPI was used to stain nuclei. In (B), (C), (E), and (F), values are means + SEM; n = 3 mice. *P < 0.05, **P < 0.01, and ***P < 0.001 (one-way ANOVA with Tukey post hoc test).
PUMAi protects mouse and human colonic organoids against CPT-induced injury
To determine whether PUMAi acts directly on crypt cells, we used a three-dimensional epithelial organoid (35) or enteroid (36) system previously used to demonstrate radiation-induced, PUMA-dependent LGR5+ cell apoptosis (37). CPT induced growth suppression and activation of caspase-3 in mouse colonic organoids, which was strongly blocked by PUMAi (Fig. 7, A to C). PUMAi also suppressed CPT-induced activation of WNT and NOTCH targets (Lgr5, CD44, and Olfm4) (Fig. 7D). The suppression of TA marker CD44 was less pronounced compared to Lgr5 or Olfm4, similar to what we observed in vivo.
Fig. 7 PUMAi protects mouse and human colonic culture against CPT-induced damage.
(A) Representative images of mouse colonic organoids 6 days after CPT treatment. Organoids were treated with vehicle control (CT) or 500 nM CPT (day 1) for 24 hours with or without 50 μM PUMAi and monitored for growth until day 7. Scale bar, 500 μm. (B) Quantitation of organoids 100 μm or greater in diameter per field from (A). (C) Western blots for active (cleaved) caspase-3 (Casp3) from organoids 24 hours after CPT treatment. (D) Indicated mouse mRNAs were analyzed by qRT-PCR 24 hours after CPT treatment. NS, not significant. (E) Representative images of human colonic organoids treated as in (A). Scale bar, 500 μm. (F) Quantitation of organoids 100 μm or greater in diameter per field from (E). (G) Western blots for active (cleaved) caspase-3 from human organoids 24 hours after CPT treatment. (H) Indicated human mRNAs analyzed 24 hours after CPT treatment. In (C) and (G), lysates were prepared from three wells. Actin was used as a control for protein loading. In (D) and (H), cDNAs were synthesized from RNA pooled from three cultured wells. Values were normalized to Gapdh and expressed relative to vehicle controls. In (B), (D), (F), and (H), values are means + SEM. *P < 0.05, **P < 0.01, and ***P < 0.001 (one-way ANOVA with Tukey post hoc test).
To demonstrate the translational potential of PUMAi, we used primary human colonic organoids. PUMAi enhanced organoid growth (Fig. 7, E and F) and inhibited CPT-induced caspase-3 activation (Fig. 7G). PUMAi suppressed CPT-induced expression of WNT and NOTCH targets (Fig. 7H), establishing that the p53- and PUMA-dependent loss of LGR5+ cells triggers a substantial activation of ISC-related pathways within the epithelial compartment.
GI complications are a major dose-limiting factor in patients receiving chemotherapy and abdominal radiation and can reduce the quality of life in cancer survivors. Identification of druggable targets is likely to have a major impact on cancer care by alleviating debilitating side effects, as well as improving outcomes by potentially increasing maximum tolerated doses. Our current study establishes p53- and PUMA-dependent LGR5+ stem cell loss as a major mediator of chemotherapy-induced enterotoxicity. Intestinal injury induced by repeated cytotoxic treatment is likely a consequence of the acute toxicity (1, 7), attributable to p53-dependent ISC loss, defective intestinal recovery, differentiation, and barrier, and persistent DNA damage and inflammation that further perpetuate ISC dysfunction and exhaustion (11). We speculate that compromised ISC functionality underlies "leaky gut syndrome" seen in some patients after pelvic or abdominal radiotherapy or chemotherapy, which is characterized by increased mucosal permeability, chronic fatigue, digestive problems, and systemic toxicities associated with persistent DNA damage (1).
p53 loss (38), but not Puma loss (26, 39, 40), strongly predisposes to spontaneous cancer development in mice. Our current work complements and extends several studies using single high-dose radiation models to uncouple the destructive and protective arms of p53 responses (17, 20, 23, 41). These findings strongly support the notion that targeting pathologic effects and apoptotic mediators of p53, not p53 itself, is a safer and more selective way to protect against cancer treatment–induced enterotoxicity (15, 16). This is consistent with stimulus and tissue-specific roles of BH3-only proteins in apoptosis and compromised apoptosis in cancer cells even with WT p53 due to oncogenic drivers such as WNT, phosphoinositide 3-kinase, and RAS/RAF (26, 42, 43). We discovered a promising lead compound, PUMAi, for intestinal chemoprotection, and its intestinal accumulation and normal tissue selectivity warrant further investigation.
Maintenance of intestinal epithelial integrity and function is vital, and it is driven by ISCs (10, 11, 44). Our data support position-sensitive loss of LGR5+ cells as the key pathology in chemotherapy- and radiation-induced enterotoxicity and the trigger for WNT and NOTCH activation and intestinal regeneration. This helps explain intestinal radiosensitization by genetic ablation of LGR5+ cells (12), and radio- or chemoprotection via LGR5+ cell preservation afforded by structurally and functionally diverse agents, including growth factors, Toll-like receptor 4 (TLR4) and TLR9 agonists, WNT agonists [reviewed in (11)], an hypoxia-inducible factor (HIF) activator (45), or inhibitors of glycogen synthase kinase β (37) and cyclin-dependent kinases 4 and 6 (46). Most, if not all, of these agents inhibit crypt apoptosis, and some also cause LGR5+ expansion (37, 47). These findings caution against the use of pro-proliferating agents, such as growth factors and WNT agonists, during repeated exposures to cytotoxic agents, which might result in ISC exhaustion via exacerbated DNA damage and prolonged proliferation. The sensitivity of WNT and NOTCH targets to CPT or PUMAi differed between human and mouse colonic organoids, as well as between organoids and mice, suggesting potentially species-specific differences in target regulation (37) and involvement of nonepithelial factors. Considering that PUMA mediates both p53-dependent and p53-independent apoptosis (26), it would be interesting to determine the effects of PUMAi in other injury models including DNA damage and inflammation (48–51).
Rapid renewal predisposes the GI epithelium to common side effects seen in cancer patients receiving radio- or chemotherapy, bone marrow transplant (1, 11), and even immunotherapy, with limited options for prevention or mitigation, representing a major unmet medical need. Here, we demonstrate PUMA as a promising target for selective intestinal chemoprotection and identify a small-molecule PUMAi active in vivo. Future studies will be needed to further define the mechanisms and pharmacokinetic and pharmacodynamic properties of PUMAi in normal and cancer tissues and to develop improved analogs.
The goals of this study were to determine whether PUMA- and p53-dependent apoptosis mediates chemotherapy-induced intestinal stem cell loss and enteropathy and whether its genetic and pharmacological inhibition provides intestinal chemoprotection. Non–tumor-bearing and tumor-bearing mice, p53 and Puma KO mice, a small-molecule PUMAi, and in vitro mouse and human colonic organoids were used to measure the effects of PUMA inhibition on intestinal injury and regeneration, as well as tumor responses after chemotherapy. Sample size was determined using published work and power calculations. Experimenters were not blinded to treatment groups during the acquisition of data.
Mice and treatment
The procedures for all animal experiments were approved by the Institutional Animal Care and Use Committee of the University of Pittsburgh. Puma+/+ (WT) and Puma−/− (Puma KO) mice (52) were generated in-house by heterozygote breeding. WT and Puma KO alleles were genotyped from genomic DNA isolated from tail snips, as described (52). p53−/− (p53 KO) mice were purchased from the Jackson Laboratory. The LGR5 marking mice Lgr5-EGFP (Lgr5-EGFP-IRES-creERT2) have been described (53). All strains are in or have been backcrossed with the C57BL/6 background for more than 10 generations (F10). Mice were housed in microisolator cages in a room illuminated from 07:00 to 19:00 hours (12:12-hour light/dark cycle), with access to water and chow ad libitum. CPT-11 (irinotecan hydrochloride; Camptosar, Pfizer) treatments were administered at 200 mg/kg in saline, unless otherwise indicated, by intraperitoneal injection (200 μl for a 20-g mouse). PUMAi was custom-synthesized by ChemBridge with a minimum purity of 95% by HPLC/mass spectrometry (MS), prepared as a stock (50 mg/ml) in dimethyl sulfoxide, then freshly diluted in phosphate-buffered saline (PBS) (2 mg/ml), and given at 10 mg/kg intraperitoneally (200 μl for a 20-g mouse).
For tumor experiments, 4 million LLC cells [American Type Culture Collection (ATCC)] were injected into the flanks of WT and Puma KO mice and allowed to grow for 11 days. Mice were then treated with CPT-11 (200 mg/kg) three times per week for 2 weeks. PUMAi (10 mg/kg) or vehicle control was administered by intraperitoneal injection 2 hours before and 20 hours after each dose of CPT-11. Mice were sacrificed 4 hours after the final CPT-11 treatment. Details on the dosing of CPT-11 and PUMAi, tumor measurements, and 5-FU and radiation models are found in Supplementary Materials and Methods.
Tissue processing and histological analysis
Immediately after sacrifice, about 10-cm portion of the jejunum was removed and carefully rinsed with ice-cold saline. The tissue was opened longitudinally and tacked to a foam board for fixation overnight in 10% formalin. Tissues were then rolled up into "swiss rolls" and embedded in paraffin. For tumor analysis, flank tumors were removed, measured, and cut into pieces for either formalin fixation or flash freezing in a dry ice/ethanol bath. Tissue sections (5 μm) were deparaffinized and rehydrated through graded ethanols. Histological analysis was performed by hematoxylin and eosin (H&E) staining.
Average villus height was determined by measuring 40 to 50 villi from different locations of the small intestine, from at least three different animals per group, and reported as means ± SEM. Measurements were made from the top of the crypt to the tip of the villus using 100× H&E images and SPOT 5.1 Advanced software (Diagnostic Instruments Inc.).
Immunohistochemistry and immunofluorescence
Rehydrated sections were treated with 3% hydrogen peroxide (immunochemistry only), followed by antigen retrieval for 10 min in boiling 0.1 M citrate buffer (pH 6.0) with 1 mM EDTA. Apoptosis was analyzed by TUNEL staining with the ApopTag Peroxidase In Situ Apoptosis Detection kit (Chemicon International) according to the manufacturer's instructions. Details on other staining are found in Supplementary Materials and Methods.
ISH for Olfm4 was carried out with the RNAscope 2.0 BROWN kit (Advanced Cell Diagnostics) according to the manufacturer's instructions. Briefly, tissue sections were baked for 60 min at 60°C, then deparaffinized in xylene, and rehydrated through graded ethanols. Sections then underwent three pretreatment steps, probe hybridization, six amplification steps, development, and counterstaining. ISH for Puma was carried out as previously described (17).
Fresh mucosal scrapings or minced tumor tissues were washed in 1 ml of ice-cold PBS and pelleted at 400g. Pellets were resuspended in 700 μl of homogenization buffer (0.25 M sucrose, 10 mM Hepes, and 1 mM EGTA) supplemented with protease inhibitors (cOmplete EDTA-free mini, Roche) and homogenized in a Dounce homogenizer with 50 strokes of the pestle. After clearing by centrifugation at 16,000g, protein concentrations in the supernatant were determined by a spectrophotometer (NanoDrop 2000, Thermo Fisher Scientific).
Proteins (30 μg) were separated by SDS–polyacrylamide gel electrophoresis using the NuPAGE system (Invitrogen) and transferred to polyvinylidene difluoride membranes (Immobilon-P, Millipore). Representative results are shown, and similar results were obtained in at least three independent experiments. Antibodies used include PUMA (ab9643, Abcam), p53, p21, hemagglutinin (HA) (sc-6243, sc-397, and sc-805; Santa Cruz Biotechnology), V5 (R960-25, Invitrogen), tubulin (CP06, Oncogene Science), and actin (A5541, Sigma-Aldrich).
Quantitative real-time polymerase chain reaction
Fresh mucosal scrapings from 10 cm of jejunum were washed in cold PBS, resuspended in 700 μl of RNA lysis buffer, and homogenized in a Dounce homogenizer. RNA was isolated using the Quick-RNA MiniPrep kit (Zymo Research) according to the manufacturer's instructions. Organoids were freed from Matrigel using Cell Recovery Solution (Corning), as described in (37), before RNA isolation. Complementary DNA was generated from 2 μg of total RNA from mice or ~100 ng of total RNA from colonic organoids and was pooled from three mice or three wells of organoid culture per treatment group using SuperScript III reverse transcriptase (Invitrogen) and random primers. Gene expression was normalized to Gapdh. Representative results are shown, and similar results were obtained in at least three independent experiments. Details on mouse and human primers are found in tables S2 and S3.
Cells and treatment
HCT 116, DLD1, SW480, and HT29 human colon cancer cells (ATCC), as well as HCT 116 p53 KO (54) and PUMA KO (27), were maintained at 37°C and 5% CO2 in McCoy's 5A medium (Invitrogen) supplemented with 10% defined fetal bovine serum (HyClone), penicillin (100 U/ml), and streptomycin (100 μg/ml) (Invitrogen). Human embryonic kidney 293 cells (ATCC) were maintained in Dulbecco's modified Eagle's medium (Invitrogen) with the same supplements. For treatments, cells were plated in 12-well plates at ~30% density for 24 hours and then treated with CPT (Sigma-Aldrich). PUMAi was added to cells at 25 μM at the same time as CPT.
Measurement of apoptosis
After treatment, floating and adhering cells were collected and stained in a solution containing 3.7% formaldehyde, 0.5% NP-40, and Hoechst 33258 (10 μg/ml) (Molecular Probes) in PBS. Apoptosis was assessed through microscopic visualization of condensed and fragmented nuclei, as previously described (25, 27). A minimum of 300 cells per treatment were analyzed in triplicate. Representative results are shown, and similar results were obtained in at least three independent experiments.
HEK293 cells were transfected with previously described plasmids expressing HA-tagged PUMA (HA-PUMA), HA-tagged inactive PUMA (ΔBH3), or V5-tagged Bcl-xL (V5-Bcl-xL) (55) using Lipofectamine 2000 according to the manufacturer's instructions. To test the PUMAi, 293 lysates containing HA-PUMA or HA-ΔBH3 were incubated for 15 min with 25 μM PUMAi, then mixed with V5-Bcl-xL lysates for 1 hour, and subjected to immunoprecipitation with an anti-HA antibody (sc-805, Santa Cruz Biotechnology) (25, 30). BIM/Mcl-1 interaction was tested in a similar fashion using lysates containing HA-BIM and V5-Mcl-1. Representative results are shown, and similar results were obtained in at least three independent experiments.
Measurement of PUMAi in tissues
PUMAi {1-[4-(2-hydroxyethyl)-1-piperazinyl]-3-(2-naphthyloxy)-2-propanol dihydrochloride} was synthesized by ChemBridge, with a purity of 95% by HPLC-MS, and an internal isotopic standard PUMAih7 (D5, five internal hydrogens replaced by deuterium) was synthesized by Alsachim, with purity approaching 100% by HPLC-MS. Nuclear magnetic resonance confirmed the isotope enrichment by 99.4% or more. Pooled plasma from untreated mice was used as control plasma. Detailed methods on LC-MS/MS (liquid chromatography–tandem MS) quantitation of PUMAi are found in Supplementary Materials and Methods.
Mouse and human colonic organoids
Mouse crypt isolation, organoid development, and passage were carried out as previously described (37, 56). Human normal colon crypt isolation and organoid development were performed as previously described (57). Modifications included the use of conditioned medium from L-WRN cells (ATCC CRL-3276) to supplement growth factors. Detailed protocols, reagents, and drug treatment are found in Supplementary Materials and Methods.
Statistical analyses were performed with GraphPad Prism IV software. Multiple comparisons of the responses were analyzed by one-way analysis of variance (ANOVA) and Tukey's post hoc test, whereas those between two groups were made by two-tailed, unpaired t test. Survival data were analyzed by log-rank test. Differences were considered significant if the probability of the difference occurring by chance was less than 5 in 100 (P < 0.05). Sample size was determined using a combination of published work and power calculations. For ANOVA, we have computed the power for a test of interaction in a two-way factorial design applied by constructing mixed linear growth models to calculate the needed sample size. We estimated that usually 5 to 10 per group will provide 80% power to detect a standardized interaction of 1.5 SDs.
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Fig. S1. PUMA KO inhibits CPT-11–induced crypt apoptosis.
Fig. S2. CPT-11 causes dose-dependent lethality in mice.
Fig. S3. A small-molecule PUMAi does not protect colon cancer cells against CPT-11–induced apoptosis.
Fig. S4. PUMAi inhibits CPT-11–induced CBC apoptosis.
Fig. S5. PUMAi protects against chemotherapy- and radiation-induced lethality.
Fig. S6. Targeting PUMA does not compromise tumor response to CPT-11.
Fig. S7. 5-FU–induced LGR5+ cell apoptosis is PUMA-dependent.
Fig. S8. Puma KO and PUMAi suppress CPT-11–induced expression of WNT and NOTCH targets.
Table S1. Treatment-associated lethality in LLC tumor experiments.
Table S2. Mouse-specific primers used for real-time reverse transcription polymerase chain reaction.
Table S3. Human-specific primers used for real-time reverse transcription polymerase chain reaction.
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Acknowledgments: We would like to thank P. Buckhaults (University of South Carolina) for sharing the protocols on colonic cultures. Funding: This work is supported, in part, by American Cancer Society grant RGS-10-124-01-CCE, NIH grant U19-A1068021, Pennsylvania Tobacco funds and institutional funds (to J.Y.), CA172136 and CA203028 (to L.Z.), and U01 CA152753 (to R.E.S.). This project used the UPMC Hillman Cancer Center shared cancer pharmacokinetics and pharmacodynamics facility, glassware, animal, and cell and tissue imaging facilities that were supported, in part, by National Cancer Institute award P30CA047904. Author contributions: B.J.L., L.Y., L.W., J.H.B., J.L.E., L.Z., and J.Y. designed the experiments. B.J.L., L.Y., L.W., M.E.B., M.R., and R.A.P. performed the experiments. L.Z. and R.E.S. provided the key reagents including access to human tissues. B.J.L., L.Y., L.W., J.H.B., and J.Y. analyzed the data. B.J.L. and J.Y. wrote the manuscript. J.Y. conceived and supervised all experiments. Competing interests: The authors declare that they have no competing interests.
Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works
Vol 10, Issue 427
You are going to email the following Targeting p53-dependent stem cell loss for intestinal chemoprotection
By Brian J. Leibowitz, Liheng Yang, Liang Wei, Monica E. Buchanan, Madani Rachid, Robert A. Parise, Jan H. Beumer, Julie L. Eiseman, Robert E. Schoen, Lin Zhang, Jian Yu
Science Translational Medicine 07 Feb 2018
Targeting of p53- and PUMA-dependent apoptosis protects intestinal stem cells, but not cancer cells, against chemotherapy.


Explore Dubrovnik In Croatia, The Pearl Of The Adriatic!
Dubrovnik, the heart of Croatia's tourism and a UNESCO World Heritage, is worth every penny, that you spend to visit this mesmerizing city. Amongst the hustle-bustle of the tourists, you will find gigantic city walls overlooking at the endless crystal blue water, orange tilled roofs, big cruise ships, fortress amalgamated with the intoxicating aroma of mouth-watering Mediterranean cuisine, that you can relish while watching the most soothing sunsets. These things are well blended and quite integral to each other, well this play a pivotal role in making this city one of most popular tourist destinations across the World.
Lately, this city has been the talk of the town for Indian travelers, as it has been captured in many Hollywood movies/ TV series, the list is unending, but what grabbed my attention, were the ecstatic locations of one of my favorite TV series, Game of Thrones and the Hollywood movie Star wars. Apart from this, one of the Bollywood's most loved actors, Shah Rukh Khan's movie FAN, was shot in this beautiful City and how can we forget, the chasing scene in the walled city.
Travel to Dubrovnik with us!
Well, if you are travelling to Dubrovnik from India, then you can reach this mystic city without must hassle, as there is direct flight from India to Dubrovnik. If, one is not getting a good deal or want to explore other parts of Croatia then one can take flight to Split or Zagreb. There are multiple direct flights from Zagreb to Dubrovnik as well as a couple of direct flights from Split too. One can also take a bus or take a long road trip along the coastal road by the side of pristine blue waters of the Mediterranean, which can certain be an experience to remember.
Travel tips: You can also travel inside Croatia using their excellent bus service. You can book your tickets online using websites getbybus.com and www.buscroatia.com.
The period from May-July and September-October, are the best time to visit. During your visit within this period, you can expect pleasant sunny days making it perfect for swimming and gazing at the blue waters. During this time, the sea water is perfectly warm and the weather is ideal for you to enjoy this city at its best version.
You are spoilt for choices, when it comes to stay in this city. You can choose your accommodation depending on whether you want to stay within the walls, in Old Town, in a beach resort, hotel or in an apartment. Being an ardent GOT fan, I stayed inside the Old Town and I must tell you the experience of staying in the setting of King`s Landing was something I would cherish for life.
Travel tips: Staying in the Old town has many advantages. Since you are at the center of the main activity in Dubrovnik, popular restaurants are at walking distance, the ropeway entry is close by and lastly all the tours have pick-up points from the main gate of the Old town.
Moreover, there are several grocery stores (e.g. Konzum) inside and in the vicinity of the Old Town, so you can buy all your necessary items including food and drinks from there.
The entire Old Town is quite walkable and you can easily cover the entire place in a few hours. It is great fun walking around the old town. Accommodation inside the old town is a bit expensive, but the experience of staying within the historic walls of the old town is unparalleled.
Game of Thrones Walking Tour, is one of the biggest highlights of Dubrovnik. Here you will explore the locations, where various scenes of King`s Landing in GOT were shot. The guide will take you around the Old town and the humongous walls that encircle it, adding to this, they will show you the photographs of various scenes from GOT, where they were shot. The tour also includes visit to Fort of St. Lawrence. If you don`t want to take the tour, then you can explore the Old town and the city walls on your own as well. There is an entry ticket to the city wall, that includes the St. Lawrence Fortress too.
Dubrovnik Cable Car ride to the summit of Mount Srd will give you a breathtaking view of the Old Town and surrounding islands. The starting point of the cable car is right next to the old town and one can buy one-way or both-way tickets from there. In addition, to the great view of the city and crystal clear Adriatic sea from the hill top, you can visit the souvenir shop or enjoy drinks and snacks at the Panorama Restaurant. Don't miss the sunset, as it is one of the most mesmerizing things that you will witness during your visit.
If you are seeking some adventure, then I would highly recommend you take Kayaking and snorkeling tour. You can take this tour even if, you are kayaking for the very first time. The instructor will guide you and after some minor difficulty, you will get a hang of it and enjoy the entire experience of kayaking in the turquoise Adriatic Sea and nearby islands.
Since the day I started planning for Dubrovnik, Buza Bar was at the top of my list. I used to visit this place everyday during my stay in Old town. A small passageway through the Old Town walls will take you to the cliff side Buza (hole in wall), one of the most gorgeous as well as jaw-dropping view. Buza bar is situated on the sea side of the city walls over the rocks.
This place is a secret heaven, as it is difficult to find the way to this place, during the walking tour of the walled city. In Buza, you can not only enjoy the beautiful view, drinks, and the mouth-watering savories, but you can also take a dip in the Adriatic Sea, as this bar is hanging by the cliff, it is a treat for cliff jumping (tip: don`t forget to pack your swimwear during your visit). Despite the great view and experience, this place will not burn a hole in your pocket.
You would love to explore the cobblestone streets of Old town in the evening. You can enjoy a nice meal with drinks in some of the great restaurants at this place and you can also take a stroll through numerous souvenir shops.
Dubrovnik, is an abode for many good food joints, cafes and restaurants. We have tried to pick out the ones based on our needs, taste, view, and most importantly the price. Most of the good restaurants had similar pricing. When a food is fresh & prepared nicely, it`s always a treat. The menu was in general very good and had many items to choose from, even for people like us who do not eat meat, octopus etc.
What more one can ask for? When they can treat their taste buds and eyes at the same time, from the top of a Café & restaurant called Dubravka estd. in 1836. Located in pile, Dubravka restaurant café overlooks Lovnjenac fortress, sea & the beautiful old city walls.
They offer delicious food, with a mind-boggling view of Adriatic Sea. The view overlooking King`s Landing from the Game of Thrones, is a treat for people like us, who are diehard fans of the show. This place is certainly the best one to kickstart your day with some amazing breakfast and intoxicating aroma of the Adriatic Sea. You can also see people Kayaking in the sea or if you are lucky enough, you can get a magnificent view of the Karaka ship sailing to the Old Town.
Here you can reach either by trekking, ATV bike or cable car. (Note: Cable car point is situated very close to the Dubrovnik old town). As I wanted to be decked-up for my evening meal, I opted for the cable car option. Mind you, this place only opens in the evening. Located at the top of the hill, Panorama restaurant gives you a fabulous view of the Adriatic Sea, nearby islands, and Dubrovnik Old Town. It is a perfect place to spend the evening over salad & wine, while enjoying the mesmerizing view.
Travel tip: Don`t forget to take a light jacket as it can be very windy at the top.
Restaurant Lokanda Peskarija is located by the city walls of Old town, in the picturesque backdrop of the harbor. This place offers a simple menu, that includes delicious sea food, salad and wine. After a long day of walking in the scorching sun, exploring the city walls, this place caught our attention for a romantic dinner with wine, cool breeze and great view of the sea. We ordered Risotto & red wine, and the food as well as the ambiance was worth every penny.
My sweet tooth is there, where sweets are, and marshmallows are my favorite. Well, that was the main reason behind my visit to this shop. It is just the perfect spot for the candy lovers, like me, who crave sweets anytime and every time. Uniqueness of this amazing place inside the town walls is that, their marshmallows are crafted in different shapes & sizes, ranging from pirates to Macaw, you will find every interesting shaped marshmallow here.
All of these restaurants are at a walking distance from the hotels inside the Old Town.
There are lots of souvenir shops selling GOT themed items and one of them also has the GOT iron throne, where you can get clicked with the infamous sword.
Even tough Croatia is a part of European Union, the accepted currency is Kuna.
There are ATMs available everywhere, so you won`t have problem in transacting in local currency.
Renting a car is not required in Dubrovnik, as everything is at a considerable walking distance.


text:
Bucks boycott Game 5; NBA postpones all Wednesday games
Milwaukee players did not take the floor for the 3:00 pm tipoff in Orlando, Florida
Milwaukee Bucks' Giannis Antetokounmpo (34) dunks the ball over Orlando Magic's Nikola Vucevic (9) during the second half of an NBA basketball first round playoff game Monday, Aug. 24, 2020, in Lake Buena Vista, Fla. (AP Photo/Ashley Landis, Pool)(Ashley Landis | AP)
Published: Aug. 26, 2020 at 3:23 PM CDT|Updated: Aug. 26, 2020 at 5:57 PM CDT
ORLANDO, Fla. (WBAY) - The NBA has postponed all of Wednesday's games in the wake of the Milwaukee Bucks boycotting their game against the Orlando Magic on Thursday afternoon.
The Bucks boycotted Game 5 of their first round NBA Playoffs series to protest the police shooting of Jacob Blake in Kenosha, Wisconsin, earlier in the week.
Sterling Brown and George Hill read aloud a team statement.
.@Bucks team statement moments ago pic.twitter.com/8YWZbIXb19
— Dave Schroeder (@SchroederWBAY) August 26, 2020
The NBA says Game 5 will be rescheduled, meaning Milwaukee won't forfeit the game.
The NBA and the NBPA today announced that in light of the Milwaukee Bucks' decision to not take the floor today for Game 5 against the Orlando Magic, today's three games – MIL-ORL, HOU-OKC and LAL-POR have been postponed. Game 5 of each series will be rescheduled.
— NBA (@NBA) August 26, 2020
The Bucks team owners issued a joint statement on the boycott:
"We fully support our players and the decision they made. Although we did not know beforehand, we would have wholeheartedly agreed with them. The only way to bring about change is to shine a light on the racial injustices that are happening in front of us. Our players have done that and we will continue to stand alongside them and demand accountability and change."
- Milwaukee Bucks owners Marc Lasry, Wes Edens, Jamie Dinan
The team was not present on the court as the scoreboard countdown struck 0.0. It remains to be seen how the NBA will react to the boycott. The Bucks had held a 3-1 series lead and could have closed out the series with a win on Wednesday.
Guard George Hill reportedly told reporters in Florida: "We are tired of the killings and injustice."
Before the boycott, many Bucks players and coaches had spoken out after the Jacob Blake shooting.
"Watching stuff that happened in Wisconsin the other day really breaks my heart," Hill said. "We are down here playing in the bubble to do things for social justice and to see it still going on, and us still playing the games like it is nothing is a really messed up situation."
"We had a great talk today before our film with Coach Bud leading, giving us a few minutes to say what is on our minds," said Brook Lopez. "A lot of guys: Wes, Vin Baker, and others brought up good points. The most important thing and the consensus was not being complacent in these times."
"There is nothing more important than getting social justice and getting the wrongs that are going on in our country right and creating real lasting change," said coach Mike Budenholzer. "There is literally nothing more important."
The Bucks players are not expected to make any comment today according to General Manage Jon Horst, who says there will be a team statement.
Bucks Senior Vie President Alex Lasry tweeeded: "Some things are bigger than basketball. The stand taken today by the players and org shows that we're fed up. Enough is enough. Change needs to happen. I'm incredibly proud of our guys and we stand 100% behind our players ready to assist and bring about real change."
According to Shams Charnia of The Athletic, the Rockets and Thunder plan to boycott their playoff game today as well.
The Brewers Josh Hader said boycotting MLB games is something the team will discuss.

redpajama_set_name: RedPajamaCommonCrawl


text:
SPEC Summary
Proposed problems
1. Mantle structure, composition, and temperature
2. H2O in the mantle
3. Exoplanetary materials
Main theoretical research reference:
Theoretical and computational methods in mineral physics: geophysical applications.
Vol. 71. Mineralogical Society of America, 2010.
Wentzcovitch, Renata and Stixrude, Lars
http://static.msi.umn.edu/rreports/2010/25.pdf (current unavailable)
The principal investigator's (PI) contribution to this field has been seminal. In particular, she developed computational methods that opened to way to first principles calculations in mineral physics. She introduced techniques for calculations of thermodynamics and thermal-elastic properties of materials in general. These properties are of first order importance in geophysics and planetary modeling. Thermal-elastic properties are fundamental for calculations of acoustic velocities in crystalline and poly-crystalline materials (rocks). These are being successfully used to interpret seismic tomography data.
Thermodynamics properties - more specifically thermal expansion coefficient, specific heat, compressibility, and thermodynamics phase boundaries - are being used to improve the realism of geodynamics simulations. These detailed calculations of mineral properties are enabling modeling of Earth and other planets from the atomic to the global scale. It is allowing for direct integration of these three branches of geophysics, i.e., seismology, geodynamics, and mineral physics. Novel methods to compute electrical conductivity, dielectric, magnetic, and optical properties in bulk materials also promise to make an impact in geodynamo modeling and paleo-magnetism. This integration, described in details in the proposal, provides the foundation and long term vision for an original research program in Brasil and hopefully in the world, a "Virtual Earth Institute". The proposed research aims to initiate this project and will focus on computational mineral physics.
Here we describe three classes of Earth and planetary science problems to be addressed by this research program. High throughput first principles calculations are essential to advance them. Extensive results will be generated and made available online using new technology proposed in the next section.
Advances in seismic imaging of the Earth's deep interior are providing structural information about convective and thermal patterns in the lower mantle. Several fascinating structures holding keys to the nature of the deep mantle are currently being seismologically mapped in detail. These require self-consistent interpretations compatible with mineralogically and geodynamically feasible contexts. Examples are: i) two nearly antipodal large low-shear-velocity provinces (LLSVPs) with abrupt lateral boundaries in the deep mantle, which likely represent chemically distinct and denser material whose nature is still unclear; ii) isolated pockets (zones) of ultra-low seismic velocities (ULVZs) in the deepest tens of kilometers of the mantle, may indicate the presence of deep magma chambers or accumulations of materials highly enriched in iron; iii) the long-standing problem of mantle composition, inseparable from large scale convection patterns in the Earth. This problem needs to be re-investigated in light of novel results on the Fe spin-state crossover in lower mantle minerals [43-45,56]. It has recently become clear that their manifestation might be interpreted as chemical heterogeneity [56].
Three broad themes in which H2O ¬ plays a critical role in geophysics are: i) cycling of water through the Earth's deep interior [261-264] (see Fig. 13), ii) the influence of water on melting and other phase transitions in the deep mantle [265,266], and iii) seismic detection of water in the mantle [277], which would be very helpful to advance understanding of i) and is intimately related with ii). In recent years there has been a tremendous effort by the deep Earth community to determine the influence of H2O on mantle properties, but key questions remain unanswered. We still do not have strong constraints on the inventory of H2O in different layers of the Earth, nor do we know the fluxes of H2O between these layers or the locus of melting induced by H2O in the deep mantle. We have made significant progress toward understanding the microscopic role played by trace substitutions of H in nominally anhydrous minerals (NAMs) [268], particularly on the effect of hydration on phase transitions in MORB at mantle pressures [269]. Such microscopic effects must be pervasive in mantle minerals.
This research concerns a novel challenging area of computational materials theory: properties of matter at extreme conditions of pressure (P) and temperature (T), i.e., T ~ 3 eV, P ~ 10 TPa, and beyond. This research is fundamental for advancing understanding of the internal structure of giant planets [111] and exoplanets [99,100], particularly of the terrestrial type [101]. This is clearly an area that presents remarkable opportunities for interdisciplinary discoveries of high scientific impact and methodological developments broadly applicable to other areas of materials discovery. Controlled experiments at these conditions are virtually impossible. However, it is possible to connect this research with experimental data by investigating low pressure analogs.
Geophysics stands on a synergistic tripod consisting of seismology, geodynamics, and mineral physics and advances by close cooperation between these fields that can be computationally very intensive.
Mineral physics' role is to provide information on mineral properties that are needed i) to interpret seismic tomography and ii) to bolster advanced and more refined geodynamics simulations. Computational mineral physics has contributed greatly to the integration of these fields by complementing experiments by expanding the pressure (P) and temperature (T) range in which properties can be obtained. With the discovery of extra-solar planets, particularly of the terrestrial type, its role has expanded in range of physical conditions, type of materials, and materials properties to be investigated. Today mineral physics interfaces also with planetary sciences. For many problems, first principles computations based on density functional theory (DFT) [1,2] and its successful implementations and extensions [3-8] is the most or only practical method of investigation. This capability has been changing the scientific landscape in Earth and planetary sciences. Successes and contributions of computational mineral physics are already too numerous to cite. They cover thermodynamics properties and phase boundaries, discovery of new phases relevant for Earth and exoplanets, and thermal elastic and transport properties of minerals and melts. A snapshot of this evolving field was captured in a special volume published in 2010 [9]. Since then progress has accelerated as will be demonstrated in this proposal. These contributions have been possible by a combination of methodology and algorithmic innovations, availability of reliable commercial [10-13] or free open source [14,15] first principles codes, and steady increase in computational power. We have now entered another transformative stage in computational mineral physics. Cyberinfrastructure (CI) [16] is enabling a leap in computational capability. It is helping to produce huge amounts of results tens of times faster than were produced five years ago and to communicate them thoroughly and in detail online.
The main goals of this proposal are:
1) to strengthen the synergy between computational mineral physics, seismology, geodynamics, and planetary sciences. PI, co-PIs, and collaborators bring to this project a rich history of breakthroughs, accomplishments, and synergies. Some of the PI's Previous accomplishments reviewed in the next section illustrate technological possibilities and recent scientific advances. We wish to continue using and developing them in the context of projects outlined in the Proposed problems section tapping local expertise in materials theory and simulations and Earth and planetary sciences. The Departamento de Física dos Materiais e Mecânica from Instituto de Física, USP, has a long tradition of research in first principles calculations. Prof. Luiz Guimareães Ferreira, a pioneer in this field, Profs. Lucy C. Assali and Helena Petrilli, with their extensive research experience in semiconductor physics, solid solutions, polymers, and defects in solids and in using/developing first principles calculations are co-investigators in this project. Prof. João Francisco Justo, from the Polytechnic School, is another co-investigator. He has collaborated with the PI in mineral physics and is the scientist in São Paulo that most closely relates to the PI's research. They will be instrumental in building a bridge to the local seismology, geodynamics, geochemistry, and planetary sciences communities, here represented by Prof. Ricardo Trindade, a geochemist, Dr. Victor Sacek, a geodynamicist, and Dr. Marcelo Bianchi, a seismologist from Instituto Astronômico e Geofísico (IAG), USP. They provide the motivation for our research are are the "consumers" of our results, since they will make direct use of our "data" in their modeling;
2) to enable cyber-infrastructure applications in this field. Planetary interior modeling requires detailed information on mineral properties in a continuum of pressure-temperature-composition-phase fields. This is essentially impossible to report in written form or to calculate using standard technology for materials simulations. These are high throughput calculations and we have developed a special cyber-infrastructure consisting of a portal, web services, and databases to obtain and report these properties – the VLab CI [16-22]. It is essential for Earth and planetary sciences applications requiring mineral physics data and we want to have it locally supported. Prof. Sérgio Ferraz Novaes, from the Institute of Theoretical Physics Institute (IFT), UNESP, will host the VLab cyber-infrastructure in his data center. He has….;
3) training of exchange students and post-docs and developing the future generation of scientists working in these related field. Organization of workshops, conferences, and tutorial courses in São Paulo will also connect scientists in São Paulo to new communities abroad and will hopefully activate other collaborations as well. PI and co-PIs have extensive experience with organization of such events.
-- winckler - 2015-09-11
Topic revision: r1 - 2015-09-11 - winckler


Ramiro has launched an Android app for all its customers. It allows users to contact ramiro car services on any device including mobile phones. This commercial app, created and registered by AppBuilderOnline.com, is loaded with multiple features, including photo albums, social media share, direct call feature, push notification, and more!
We all are aware of apps, and we every day come across in our day to day life. Yes, they are right in our pockets, on your phones. We use apps in a numerous way like Google Maps helps us get around, whereas Facebook enables us to be in touch with our friends and Candy Crush becomes our guilty pleasure. But are you aware that there are two different types of apps? Yes, Native and Web apps. Though they might seem to be very similar, these two types of apps have relatively different experience for users.
Native apps are something we come across very frequently on a regular basis. It is designed for an explicit device that need to be downloaded directly to the mobile device. Once tapped into a device, its native features allows for more interactive experience for your clients. For instance, native apps enable user to access their device's camera, accept push notes or share their current location through the app. It is one of the great as they are available right there on your device's home screen that can be readily available for the app users.
The Web apps that are usually accessed through the browser available on a mobile device are known as Web apps. It makes an excellent option if your client base holds a big deal of Blackberries and Windows phones as well as iPhones and Androids. The web apps enable you to reach a broader audience with their limited features. It, however, lacks in some abilities when compared with the native app.
AppBuilder mobile websites provide a similar experience to a web app. We would suggest you a native app for your clients. Its rich experience will surely make your app to be one of go-tos on the user's home screen.
With the rising transition of customers to mobile apps from websites, it has become necessary for online marketers to discover new strategies to put on their existing trading tactics to the mobile.
One out of five people are shifting towards the mobile app at a time in the world. Marketing Charts in U.S. has revealed that total time spent on the mobile apps has exceeded the desktop web access, with an increasing 21% of app viewership.
The funnel is the best tool to analyze marketing regarding tracking user engagement. It is just like moving a consumer from identifying an app- downloading it- encouraging engagement through its contents, and then carrying out the desired action.
It mainly concentrates on metrics that represents the awareness of user's for an app.
Site Traffic – Downloads: Just like traffic of a site would assist in estimating a website's SEO, similarly, downloads aid to determine the apps from App Store Optimization along with the fixed figure of people who have installed the app.
Visitors – Users: They are ones who actively used an app. However, they tend to show extra commitment to the website than a newly visited user would show. This supports you to trace user sections and discover broader user's engagement.
Pageviews – Sessions: It only calculates the time from when a consumer accesses an app and interacts when it is closed.
With the help of metrics, you can evaluate the approach of your users engaging with your app.
Monthly Unique Site Visitors – Monthly Active Users: It enables you to analyze the number of users previously used your app in the last 30 days and also the trends of those users.
Target Audience – User Segments: It sets of user's group to share relevant features. You can use these groups for customized marketing promotions, and they mostly target within-app strategies like push notifications.
Time on Page – Time on Screen: It measures the duration of each user spending on each page of your app. It enables you to keep the check on where the users are navigating, the time of spending, and from where they are navigating away swiftly.
At the bottom, it appears with a window into all the important alteration or the user's action taken place within the app. This leads to the eventual conversion and the desired actions of the users.
Conversions – Events: These actions are taken by users within an app that depend on offered features and platforms.
Bounce Rate – Funnel Drop-Off Rate: It estimates the total amount of users entering in one step of adaptation development and escaping without concluding it. You will need to examine the rate at every level of the funnel to recognize the state of your app where it is least and most effective.
Revenue per Visitor – User Lifetime Value (LTV): It gives an evaluation of how much a user is worth during the entire engagement of the app. It can be primarily used as a gauge of revenue that goes further than profits especially through incorporating loyalty of a user and downloads to an app at a time.
There are swift and reasonable methods to improve apps and get them into the market for test-firing analytics.
AppBuilder allows you to create professionals apps for several platforms. It helps to deliver an exceptional onboard CRM system that enables you to permit current user metrics and download data figures through platforms and markets.
With the dawn of smartphones and its apps, many of us have found new ways of using the handheld gadget in their daily life. This also includes businesses who have started identifying various benefits offered by smartphone apps.
There are varied advantages due to the development of smartphone apps. Smartphones have become more convenient and effective in today's world than the normal personal computers. The amazing features of a touch screen, mobility, and the user screen friendly size makes it best for use as well as preferable choice for many. With many people joining the kingdom of smartphone users, the advertising strategies used are more effective in reaching a large number of users. This transition has made most of the business transactions and communications easier and effective.
Some business benefits offered by smartphone app development?
There are numerous benefits of using a smartphone app for your business.
Such apps make it easier to build brand awareness. You can publicize your brand in a much better way with the quicker and better app development. The best performing app will stand out best in its audience with always being mentioned in the online market and attracting a high number of new users daily. However, this indirectly means a better advertisement for your business.
App development has another great benefit by offering customer interaction. The different and innovative developments in apps will only make all the business communication pleasant and appealing to all your clients. You can actually win a good edge over your competitors through this. As the organization creates a business app and makes further development to it, you are given the option of impacting the client's mind impressively, leading to increasing the face value of your business.
Customer loyalty is also a key benefit that is established through an app development as clients may find it easier to interact with your company. Providing people an easy and better access to your company and its related information with having some fun apps will aid in increasing the clientele. Moreover, it can be an amazing way of creating newer revenues and avenues for your business. Along with some promotional benefit, a subscription will also provide a good source of profits. Hence, all kinds of development of Smartphone related applications help in the business increase. Therefore, there is a two-way benefit that includes promoting the business and generate a fair income. You can find out specialized experts who can exclusively deal with apps development.
House Near You has launched an Android app for all its customers. It allows users to browse, share and buy real estate properties on any device including mobile phones. This Real Estate Sales & Property Management app, created and registered by AppBuilderOnline.com, is loaded with multiple features, including photo albums, social media share, direct call feature, push notification, and more!
Before you think off creating a mobile app for your business, it is essential to be clear about your aims from the start and plan according to it.
The best feature of a mobile app is that it enables companies to engage with their customers in real-time, by location and complete with profile information.
If it is done in the right way, you can actually turn those engaged customers into revenue generating ones. For an instance, Kraft's iFood Assistant offers over 2000 recipes then a lot of people will use Kraft products. Giving out shopping lists, interactive deals, and coupon features to incentivize shoppers can make an enormous impact on customer engagement. This app also includes social sharing capabilities that allow consumers to share recipes with friends on social networks.
A mobile app makes it simpler and more efficient for your customers to get in touch with your company. Offering them tools to ease their life while using your products or services can be the excellent reason to develop an app.
For instance, Marlborough-based Digital Federal Credit Union had launched an app called Mobile PC Deposit that enables members to deposit payment using the iPhone and Android-based smartphones. This made lives of customers much easier and it's value-added to their existing service.
One of the most popular means to make the profit is by offering coupons. The mobile gives an interesting layer of push notifications based on locations to the coupon economy.
For instance, imagine you being the owner of an apparel store in New York where thousands of people directly walk-in within a one-mile radius of your outlet. If they would get notified on their cell phones about the coupon offers from your store, then you would increase the likelihood of having them visit your store as well by providing a proper reason.
If you are trading your services or products online, a mobile app can be the best place that you can consider. It gives your customers the mobility to do the same things that they would usually have performed sitting at their desks, but on their phones.
There are several examples in which online retailers have extended their offerings to customers who want to shop on-the-go using their mobile phones. If you are in a service company, you too can capture a larger share of the market, just like, Dropbox did.
With these ways, you can think of some creative ways to achieve your potential or existing customer's attention, enhance your offerings, attract people to buy, make life easier for your customer and you reap the benefits too.
Nowadays every individual uses smartphones especially people from the field of doctors, lawyers, engineers, IT professionals, miners, consultants, bankers, etc. A smartphone is one such device that is a universally is becoming powerful with each passing day. Apps have the potential to perform a broad range of tasks that was before only limited to desktops. Brands can use apps to promote their products/services. By getting a mobile app, you can actually take your business one step ahead towards a successful future.
In today's technology-obsessed world, building a strong brand image can be critical. The brand image makes your clients aware of your services with an increase in customer loyalty. A brand without a mobile app seems to be an outdated and isolated from the modern day trends.
A mobile app enables you to send information to your potential customers based on their past activities and choices. However, e-commerce apps help you broadcast custom advertisements to increase the sales.
A mobile app provides a platform where you can showcase the services and products of your business at one place. Displaying discounts and offers can encourage more people to visit an enterprise and spread the word.
83% of smart phone users do first thing in the morning is access various apps on their phone. Mobile apps have the higher level of engagement than that of ordinary websites. An app, however, facilitates real-time engagement with customers and making it possible for companies to build deeper relations with customers.
An app enables the users with enhanced experience, excellent customer support and service vehicle. It allows users to post their queries and feedback anywhere and anytime making it easy to get in touch with a company. An app can even have query-solving tools and information on board for the customer's instant solutions to everyday problems.
A mobile app gives you instant access to all the mobile features. Therefore, users can make a phone call on one button access to a business.
You might be thinking that is it essential to have a mobile app for the growth of the business? The extent of your business doesn't matter nowadays because going mobile is the only way. It is the era when people, especially the younger crowd, are continuously using their mobile devices to browse and if you aren't there, then you may end up missing out the opportunities. You can think more beyond having a mobile website. A mobile app enables you to achieve results you simply cannot relay on mobile-friendly website.
Boosts interest: Creating an app for your business can build an easy platform for you to showcase your products or services to your potential customers. Your customers can get all the necessary information on one stop whenever and wherever. Updating the content of your app can give them a glimpse of the latest offer. This will actually excite your customers and show some interest to check out your regular updates.
Enhances engagement: Try to engage your customers with great deals or discount, offers and incentives in real time. Using their profile information and location can help to customize your offers and make them more attractive too.
Aids promotion: In case you have apparel and accessories boutique at a prime location, and you know that many customers walk by your store every day. Try to design an app that attracts them to your store with an irresistible offer and notify about it through the app.
Offers support: Often in a rush to receive and then respond to a customer support inquiry, the same becomes simple when you have an appropriate app to do it. A mobile app enables you to offer basic instructions for your customers. Creating a mobile app can ease to connect you with your customers and provide necessary information on using your product or service with ease.
Ensures ease: Users wish for a fuss-free experience, whether they access your site, your app, or they do it on their PC. This will only be possible with better planning and clear vision. Design your app in such a way that it comprises what the users would prefer.


Multimodal container traffic as a logistics system nowadays constitutes the backbone of world economy. And the important loading method FCL (Full Container Load) is the core competence of the Leschaco Group.
We organize the complete transport including all downstream and upstream services. This includes in the country of departure the way from the loading station to the port of loading or from the port of discharge to the unloading station respectively. We provide for all industries the standard and special container that are necessary for the respective assignment or type of cargo, e.g. reefer container or flexitanks. On the basis of our certified expertise in hazardous goods we also treat very sensitive goods in an extremely professional way.
The Leschaco Group offers FCL transports as complete door-to-door services, i.e. including the organization of intermodal pre and on carriage via train, barge, or truck. Our long-term experience in container traffic on all continents guarantees highest product quality. Our optimized processes ensure a balanced price-performance ratio in the interest of our customer. The personal consulting and support allows short decision-making processes and contributes to the high flexibility of our FCL services.