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Macrophages serve as a major reservoir for HIV-1 because a large number of macrophages in the brain and lung are infected with HIV-1 during late stage disease. Recent evidence suggests that those HIV-1-infected macrophages play a key role in contributing to tissue damage in AIDS pathogenesis. Macrophages undergo apoptosis upon HIV-1 infection; however, the mechanisms of this process are not well-defined. Previously, we demonstrated that HIV-1 infection inhibits Akt-1, a critical protein for cell survival of macrophages. In the present study, we investigated the involvement of transcription factor FOXO3a in the regulation of HIV-1-mediated apoptosis in macrophages. HIV-1 infection significantly decreased phosphorylation of FOXO3a and promoted FOXO3a translocation to the nucleus in human monocyte-derived macrophages (MDM). Overexpression of a constitutively active FOXO3a increased DNA fragmentation with decreased cell viability in MDM, whereas a dominant-negative mutant of FOXO3a or small interfering RNA for FOXO3a to knockdown the function of FOXO3a in HIV-1-infected MDM decreased DNA fragmentation and protected macrophages from death in HIV-1-infected MDM. Overexpression of constitutively active Akt-1 increased FOXO3a phosphorylation, suggesting that FOXO3a phosphorylation in human MDM is dependent on Akt-1. We therefore conclude that FOXO3a plays an important role in HIV-1-induced cell death of human macrophage. Understanding the PI3K/Akt-1/FOXO3a pathway and its associated death mechanism in macrophages during HIV-1 infection would lead to identification of potential therapeutic avenues for the treatment of HIV-1 infection.
Macrophages reside in a variety of tissues including the liver, spleen, lymph nodes, lung, and brain, and link the innate immune system and the adaptive immune system. Macrophages are among the first cells infected by HIV (HIV-1) and act as a viral reservoir. Macrophage-tropic (CCR5-tropic) strains of HIV-1 are the predominant strains for viral transmission in vivo, but later in disease T cell-tropic (X4-tropic) strains evolve (1, 2). Macrophages are also important sources of proinflammatory cytokines that affect cell function and viral replication. Macrophages have been demonstrated to be critical to HIV-1 disease progression (3, 4, 5).
Pathogen-infected or -activated macrophages are targeted by the immune system for elimination by apoptosis. Macrophage apoptosis has been observed in various diseases and contribute to pathogenesis in pathological conditions. For example, in advanced atherosclerotic lesions, type A scavenger receptor activation triggered macrophage apoptosis and led to plaque necrosis. Bacterial and viral infections also induce macrophage cell death (6, 7, 8, 9). It has been reported that macrophage cell death contributes to atherosclerotic plaque formation and stability in atherogenesis (10, 11). Ebola virus infection causing massive macrophage and lymphocyte death also contributes to Ebola pathogenesis (12). HIV-1-infected macrophages are found in tissues including the brain and lung (13, 14, 15). Various host factors promote cell death of infected cells during the viral infection process (16, 17). The removal and elimination of activated macrophages is considered to be important to the prevention of chronic inflammation and promoting tissue repair. Further elimination of infected host cells particularly may control viral dissemination. Understanding the mechanism of HIV-1-induced macrophage cell death is crucial to the treatment of AIDS- and HIV-1-associated diseases.
FOXO3a, a member of the forkhead family of transcription factors, is the predominant FOXO member expressed in peripheral lymphoid organs and plays a pivotal role in regulating cellular differentiation, proliferation, and apoptosis in the immune system (18). Gene knockout of FOXO3a revealed spontaneous lymphoproliferation associated with inflammation of several organs in mice, suggesting FOXO3a regulates homeostasis of immune cells (19, 20). However, the detailed regulation and function of FOXO3a in peripheral immune cells is still not clear. In mammals, FOXO3a contains a highly conserved phosphorylation motif that is posttranslationally modified by phosphorylation, acetylation, and the nuclear transport machinery (21, 22). The most common upstream influence of FOXO3a is the PI3K/Akt-1 pathway. In the absence of insulin or growth factors, FOXO3a is activated and localized to the nucleus as a consequence of Akt-1 deactivation. This activation and nuclear localization of FOXO3a regulates a series of key target genes, causing cell cycle arrest, stress resistance, or apoptosis (23).
Our recent work demonstrated HIV-1 infection significantly down-regulates the Akt-1 activity in human macrophages (17). In this report, we investigated FOXO3a, a potential downstream factor of Akt-1, in HIV-1-mediated macrophage death. Our data found HIV-1 infection decreased phosphorylation of FOXO3a and promoted nuclear translocation in infected monocyte-derived macrophages (MDM).5Azidothymidine (AZT), an inhibitor for HIV-1 reverse transcriptase, blocked these effects. Overexpression of constitutively active FOXO3a decreased cell viability and increased DNA fragmentation in MDM. Moreover, both a dominant-negative (DN) mutant of FOXO3a and FOXO3a small interfering RNA (siRNA) knockdown partially reduced cell death in HIV-infected MDM. These studies provide insight into the apoptotic signaling events of HIV-1-induced cell death and identify FOXO3a as an important apoptotic factor in HIV-1-infected macrophages.
Materials and Methods
Isolation and culturing of primary human monocytes
Human monocytes were recovered from PBMC of HIV-1, HIV-2, and hepatitis B virus-seronegative donors after leukophoresis and purified by countercurrent centrifugal elutriation (24). Monocytes were cultured in DMEM (Sigma-Aldrich) supplemented with 10% heat-inactivated human serum, gentamicin (50 μg/ml), ciprofloxacin (10 μg/ml), and M-CSF (1000 U/ml, a gift from Wyeth, Cambridge, MA). Monocytes were allowed to differentiate for 7 days at which time they were considered MDM. All human subject studies were performed in full compliance with the University of Nebraska Medical Center and National Institutes of Health ethical guidelines.
HIV-1 infection of MDM
Seven days after plating, MDM were infected with HIV-1 virus at a multiplicity of infection (MOI) of 0.1 virus/target cell (24). Briefly, viral stocks were diluted 1/15 in medium for overnight incubation with MDM. On the second day, medium was removed and substituted with MDM culture medium that was half-exchanged every 2 days (24). Stock virus was screened for mycoplasma and endotoxin using hybridization and Limulus amebocyte lysate assays, respectively.
Measurements of reverse transcriptase (RT) activity
RT activity was determined in triplicate samples of cell culture fluids. For this assay, 10 μl of supernatant was incubated in a reaction mixture of 0.05% Nonidet P-40, 10 μg of poly(A)/ml, 0.25 μg of oligo(dT)/ml, 5 mM DTT, 150 mM KCl, 15 mM MgCl2, and [3H]TTP in Tris-HCl buffer (pH 7.9) for 24 h at 37°C. Radiolabeled nucleotides were precipitated with cold 10% trichloroacetic acid on paper filters in an automatic cell harvester and washed with 95% ethanol. Radioactivity was determined by liquid scintillation spectroscopy (13).
Immunofluorescence and confocal microscopy
Cells on coverslips were cultured 3 days following HIV-1ADA infection, fixed for 15 min in 4% paraformaldehyde in PBS, dried in methanol for 5 min, permeabilized, and blocked 30–60 min with 0.1% Triton X-100 with 2% BSA in PBS. Cells were incubated overnight with mouse anti-P24 mAb (DakoCytomation) and rabbit anti-FOXO3a Ab (Cell Signaling Technology), then conjugated with anti-mouse Alexa Fluor 488 nm (green; Invitrogen Life Technologies) and anti-rabbit Alexa Fluor 647 nm (red; Invitrogen Life Technologies) secondary Abs. Hoechst 33342 (Sigma-Aldrich) was used for nuclear staining. Triple immunostaining was examined by a Bio-Rad MRC1024ES laser scanning confocal microscope, using a triple laser line and simultaneous triple display mode of the Bio-Rad LaserSharp imaging program.
Western blot analysis
Cell lysates from MDM were prepared with M-PER Mammalian Protein Extraction Buffer (Pierce). Nuclear and cytoplasmic fractions were prepared from MDM with the Nucleus Protein Extraction kit according to the manufacturer’s instruction (Pierce). Protein concentration was determined by a BCA Protein Assay kit (Pierce). Protein (30 μg) was electrophoresed on 10% SDS-PAGE (Bio-Rad) and transferred to an Immune-Blot polyvinylidene difluoride membrane (Bio-Rad). Total FOXO3a, phospho-FOXO3a, Puma, Akt-1, β-actin, and TATA box binding protein (TBP) proteins were detected using anti-FOXO3a (Cell Signaling Technology), anti-phospho-FOXO3a at Ser318/321 (Cell Signaling Technology), anti-Puma (Cell Signaling Technology), anti-Akt-1 (Cell Signaling Technology), anti-β-actin (Sigma-Aldrich), and anti-TBP (Abcam) Abs. Membranes were treated overnight with primary Ab at 4°C followed by HRP-ligand anti-rabbit (Cell Signaling Technology) or anti-mouse (Cell Signaling Technology) secondary Ab for 1 h at room temperature. Ag-Ab complexes were visualized by ECL (Amersham Biosciences) and captured with CL-XPosure Film (Pierce). For data quantification, the films were scanned with a CanoScan 9950F scanner; the acquired images were then analyzed on a Macintosh computer using the public domain NIH image program (developed at the U.S. National Institutes of Health and available on the internet at http://rsb.info.nih.gov/nih-image/).
Replication-defective adenoviral vectors expressing dominant negative FOXO3a (Ad-DN-O3a) and constitutively active FOXO3a (Ad-AAA-O3a) were purchased from Vector Biolabs. The constitutively active FOXO3a was prepared by substituting alanine for amino acids threonine 32, serine 253, and serine 315. Both Ad-DN-O3a and Ad-AAA-O3a have a hemagglutinin tag at the N terminus and express GFP. Constitutively active Akt-1 (Ad-myrAkt-1) was generated as described (25) and provided by K. Walsh (Boston University School of Medicine, Boston, MA). The constitutively active Akt-1 construct has the c-src myristoylation sequence fused in-frame to the N terminus of the HA-Akt-1 (wild-type) coding sequence. Adenoviral constructs were amplified in HEK 293 cells and purified by ultracentrifugation through a CsCl gradient. MDM were infected with recombinant adenovirus at ∼90% confluency in serum-free DMEM for 2 h and then incubated for 48 h in a growth medium. Infection efficiency was close to 70% as determined by the GFP expression. For adenovirus and HIV-1 double infection, MDM were infected with HIV-1 for 24 h followed by infection with adenovirus. Apoptosis assays were performed 3 days after adenovirus infection.
Predesigned siRNA duplexes targeting FOXO3a (ID: 144672) and a control siRNA (catalog no. D-001206-01-05) were synthesized by Ambion and Dharmacon, respectively. Control siRNA is the number one siCONTROL nontargeting siRNA from Dharmacon that has been tested to have minimal off-target effects. At 24 h after HIV-1 infection, cells were transfected with 100 nM siRNA duplexes for 4 h in DMEM culture medium without serum, in the presence of siIMPORTER reagent (Upstate Cell Signaling Solutions) according to the manufacturer’s instructions. To evaluate transfection efficiency, cells were transfected with siGLO (red fluorescence tagged nonspecific siRNA; Qiagen). At 48 h posttransfection, cells were incubated with Hoechst 33342 (Sigma-Aldrich) for nuclear staining. Transfection efficiency was measured by counting siGLO-positive cells and total cells in fluorescence microscopy.
Assessment of cell viability and apoptosis
To assess apoptosis, MDM were plated in 24-well plates with 1.1 × 106 cells/well. After 48 h of adenoviral infection with or without HIV-1 infection, cells were lysed and assayed by Cell Death Detection ELISA (Roche), as previously described (26). After 72 h of adenoviral infection, a colorimetric MTT assay was performed to measure the cell viability as previously described (26).
After 3 days of adenoviral infection, MDM were dissociated with Accutase (Chemicon International) for 15 min. Cells were then washed with 3% FBS/PBS and incubated with 1 μg/ml propidium iodide (PI; catalog no. 537059; Calbiochem) for 15 min at 4°C. Cells were washed twice with 3% FBS/PBS and resuspended in PBS containing 4% paraformaldehyde. Cell death was determined by flow cytometry and analyzed with CellQuest software. At least 10,000 cells were analyzed per sample.
Data were analyzed as means ± SD of the mean (SD) unless specified. The data were evaluated statistically by the ANOVA, followed by the Tukey test for paired observations. Significance was considered to be <0.05 unless specified. To account for any donor-specific differences, all experiments were performed with at least three donors. All assays were performed at least three times with triplicate or quadruplicate determinations for each time.
HIV-1 infection significantly decreased phosphorylation of FOXO3a in human macrophages
Phosphorylation regulates the function of FOXO3a. When in an unphosphorylated form, FOXO3a resides inside the nucleus. Phosphorylation of Foxo3a in the forkhead domain leads to disruption of DNA binding, cytosol exportation, abrogating its transcriptional activity (27, 28, 29). To assess the regulation of FOXO3a in macrophages during HIV-1 infection, we used three macrophage-tropic HIV-1 strains (HIV-1ADA, HIV-1JRFL, and HIV-1BAL) to infect cultured human MDM. The phosphorylated FOXO3a, total FOXO3a, and β-actin were determined through Western blotting (Fig. 1, A and B). After densimetric quantification of the Western blotting, we found HIV-1ADA, HIV-1JRFL, and HIV-1BAL infection decreased phosphorylation of FOXO3a to 28.7, 34, and 45.7% of control MDM, respectively (Fig. 1,C), when normalized with total FOXO3a. HIV-1 infection did not change total FOXO3a and β-actin levels. AZT, a reverse transcriptase analog, was used to inhibit HIV-1 replication. AZT treatment blocked the observed decrease of FOXO3a phosphorylation (Fig. 1 D). Infection levels of MDM by HIV-1 were monitored by RT activity assay to confirm infection of MDM by all the viral strains tested (data not shown). To assess whether the inhibition of FOXO3a phosphorylation is in transcription level, we examined the mRNA expression of FOXO3a after HIV-1 infection. There were no significant changes in the mRNA levels of FOXO3a following HIV-1 infection (data not shown), suggesting the decrease of FOXO3a phosphorylation occurred posttranscriptionally. Among all HIV-1 strains tested, HIV-1ADA represented typical HIV-1 infection and was used thereafter.
Next, we examined phosphorylation of FOXO3a over time during HIV-1 infection. Phosphorylated FOXO3a decreased in a time-dependent manner following HIV-1 infection (Fig. 2,A). Densimetric quantification determined that phosphorylation of FOXO3a was reduced to 60% of control MDM 3 days after infection and to 23% 5 days after infection (Fig. 2,B). Furthermore, we infected MDM at different MOIs of HIV-1, and quantitated FOXO3a phosphorylation by Western blotting (Fig. 2,C). The infection levels by different MOIs of HIV-1 were monitored by RT activity assay, which showed a dose-dependent increase of RT activities as the MOI increased (Fig. 2,E). Densimetric quantification showed a dose-dependent decrease of FOXO3a phosphorylation as HIV-1 infection levels increased (Fig. 2,D). We observed a significant negative correlation between RT value and the ratio of p-FOXO3a to FOXO3a in HIV-1-infected MDM (Fig. 2 F, r2 = 0.7171), suggesting the inhibition of FOXO3a phosphorylation is dependent on the levels of HIV-1 infection. Together, these results indicate HIV infection decreases the phosphorylation of FOXO3a, an effect that is dependent on HIV-1 replication.
HIV-1 infection causes nuclear translocation of FOXO3a
Phosphorylation of amino acids tyrosine 32, serine 253, and serine 315 of FOXO3a retains FOXO3a in the cytosol; loss of phosphorylation at those sites causes nuclear translocation of FOXO3a (28). To confirm decreased FOXO3a phosphorylation promotes nuclear translocation of FOXO3a, we immunostained MDM with or without HIV-1 infection and determined the subcellular localization of FOXO3a by confocal microscopy. p24 Ab against HIV-1 viral Ag was used to differentiate infected cells from noninfected cells. In control and AZT-treated cells, HIV-1 was not present or unable to replicate thus p24 staining was absent (Fig. 3, A, I, and M). In these cells, the majority of FOXO3a staining was localized in the cytoplasm (Fig. 3, B, J, and N). In HIV-1-infected MDM, there was staining of FOXO3a in the nucleus with minimal FOXO3a detected in the cytoplasm (Fig. 3, E–H). These results indicate HIV infection increases FOXO3a nuclear translocation. Furthermore, when treated with AZT, the nuclear translocation was blocked, confirming the translocation of FOXO3a was reliant upon HIV-1 replication (Fig. 3, M–P).
We also used subcellular fractionation as another approach to study the nuclear translocation of FOXO3a. Whole cells, cytoplasm, and nuclear protein lysates were prepared 72 h postinfection. Levels of phospho-FOXO3a and total FOXO3a, along with their corresponding loading control, were determined by Western blotting. The phosphorylation of FOXO3a decreased in a manner consistent with Fig. 1 in HIV-1-infected MDM in the whole cell lysates. In the subcellular fractions, the phosphorylation of FOXO3a decreased in the cytoplasm, whereas nuclear FOXO3a increased in HIV-infected MDM (Fig. 4,A). After densimetric quantification of the Western blotting, we found HIV-1 infection significantly decreased phosphorylation of FOXO3a in the cytosolic fractions, while it increased the amount of total FOXO3a in nuclear fractions (Fig. 4, C and D). Decreased phosphorylation of FOXO3a in the cytosol and increased total FOXO3a in the nucleus was blocked by AZT treatment, suggesting this effect is reliant on HIV-1 replication. (Fig. 4 B). Together, these data further confirmed HIV-infection inhibited phosphorylation of FOXO3a, leading to FOXO3a translocation to the nucleus.
Constitutively active FOXO3a induces cell death in MDM
To investigate the function of FOXO3a in MDM, we used an adenoviral gene-delivery system to manipulate the activity of FOXO3a in MDM. Constitutively active FOXO3a (AAA-FOXO3a) delivered by adenovirus (Ad-AAA-FOXO3a) was used to increase activity of FOXO3a. The constitutively active FOXO3a was modified from native FOXO3a by mutating all three phosphorylation sites to alanine. Ad-DN-Foxo3a (DN-FOXO3a) was constructed by deletion of the transactivation domain from the C terminus of FOXO3a. Thus, DN-FOXO3a can competitively inhibit the regulatory function of the endogenous FOXO3a. GFP adenovirus was used as a vector control in all adenoviral infections. After 48h of adenoviral infection, the infection efficiency was monitored by fluorescence microscopy (Fig. 5,A). At 2000 MOI, the infection efficiency was ∼70% for each adenovirus. FOXO3a overexpression in Ad-AAA-O3a and Ad-DN-O3a was confirmed by Western blotting (Fig. 5,B), which also showed the endogenous FOXO3a of Ad-GFP group, 98 kDa of AAA-O3a, and 40 kDa of DN-O3a. We also examined Puma, one of possible downstream factors of FOXO3a. After infection with constitutively active FOXO3a for 72 h, the expression of Puma is increased as compared with the GFP vector control, whereas there are no significant changes in MDM infected with Ad-DN-Foxo3a (Fig. 5 B). This result indicated an induction of downstream factor Puma by FOXO3a overexpression in human MDM.
Next, cell viability and apoptosis were assessed by MTT, DNA fragmentation ELISA, nuclear condensation, and flow cytometry followed by adenoviral infection. Cell viability in AAA-FOXO3a cells was significantly decreased in a dose-dependent manner as compared with Ad-GFP and Ad-DN-FOXO3a (Fig. 5,C). DNA fragmentation in Ad-AAA-FOXO3a dramatically increased after 48 h (Fig. 5,D) and, consequently, nuclear condensation and nuclear fragmentation also increased (Fig. 5, E and F). Flow cytometric analysis showed PI-positive cells also dramatically increased following Ad-AAA-FOXO3a infection (Fig. 5 G). These results demonstrated constitutively active FOXO3a induced significant cell death in MDM.
DN-FOXO3a and FOXO3a knockdown protected MDM from HIV-induced cell death
To explore the role of FOXO3a in HIV-1-induced cell death, DN-FOXO3a was delivered to MDM by adenovirus after 1 day of HIV-1 infection. We tested DNA fragmentation and cell viability 3 days after adenoviral infection. As compared with non-HIV-infected cells, HIV infection elevated DNA fragmentation and decreased cell viability (Fig. 6, A and B). More importantly, in HIV-infected MDM, we observed DN-FOXO3a partially prevented the increase of DNA fragmentation and the decrease of cell viability induced by HIV-1 infection (Fig. 6, A and B).
We also used siRNA to knockdown the expression of FOXO3a. First, we assessed FOXO3a expression 72 h after siRNA transfection, and found the FOXO3a protein was reduced ∼70% by FOXO3a siRNA. Compared with nonspecific siRNA (Fig. 6, C and D), knockdown of FOXO3a by siRNA decreased DNA fragmentation by 50% in HIV-infected MDM (Fig. 6,E). FOXO3a knockdown partially prevented the decreased cell viability in HIV-1-infected MDM (Fig. 6 F). These results demonstrated that DN FOXO3a and FOXO3a siRNA knockdown prevented HIV-induced macrophage death, suggesting HIV-1-induced macrophage death is at least in part via FOXO3a transcription factor.
Reduced phosphorylation of FOXO3a in HIV-1-infected macrophages is mediated by inhibition of Akt-1
FOXO3a has been reported to be an important transcription factor downstream of PI3K/Akt-1 in various cell types (27). To test whether FOXO3a is a downstream factor of PI3K/Akt-1 pathway in MDM, PI3K inhibitor LY294002 was used at 0.5 and 5 μΜ to treat MDM (Fig. 7 A). Phosphorylation of FOXO3a dramatically decreased after 2 h of both 0.5 or 5 μM LY294002 treatment. These results demonstrated that the PI3K pathway is necessary for FOXO3a phosphorylation in human macrophages.
To further confirm FOXO3a regulation by the PI3K pathway, we delivered constitutively active Akt-1 with an adenoviral vector (Ad-myr-Akt) to control and HIV-1-infected MDM. First, we measured Akt-1 expression through Western blotting after 72 h of infection with Ad-myr-Akt and confirmed Akt-1 was overexpressed (Fig. 7,B). Then, we tested the phosphorylation of FOXO3a with or without Ad-myr-Akt infection. Densimetrical quantification of FOXO3a phosphorylation were shown in Fig. 7,C. Compared with Ad-GFP vehicle control, HIV-1 infection reduced the phosphorylation of FOXO3a by 30%. Ad-myr-Akt infection increases the phosphorylation of FOXO3a in both the control and HIV-1-infected MDM (p < 0.05) (Fig. 7 C). More importantly, there was no significant decrease of phosphorylated FOXO3a in HIV-1-infeced MDM after Ad-myr-Akt infection. These results further demonstrated that HIV-1 reduced phosphorylation of FOXO3a via down-regulating the PI3K/Akt-1 pathway and FOXO3a is a downstream factor of Akt-1 in human MDM.
Eliminating HIV reservoirs and combating long-term viral persistence is critical to the treatment of AIDS (30, 31, 32). In the present study, the regulation and function of FOXO3a in HIV-induced macrophage cell death was investigated. First, we demonstrated that phosphorylation of FOXO3a was decreased in HIV-1-infected macrophages facilitating FOXO3a translocation to the nucleus (Figs. 1–4). Second, we applied a gene delivery system implementing adenoviral delivery of constitutively active FOXO3a. This rFOXO3a contains three mutated phosphorylation sites maintaining a transcriptionally active FOXO3 and was found to induce apoptosis in macrophages (Fig. 5). Third, both DN FOXO3a and FOXO3a knockdown with siRNA prevented macrophages from HIV-induced cell death (Fig. 6). Fourth, the PI3K inhibitor LY294002 inhibited FOXO3a phosphorylation, whereas constitutively active Akt-1 increased FOXO3a phosphorylation (Fig. 7). Together, these results suggest HIV infection increases the activity of transcription factor FOXO3a, and elevated FOXO3a activity promotes HIV-1-infected macrophage cell death. Although FOXO3a has been shown to influence cell death signaling, to our knowledge, we are the first to report that FOXO3a is a pivotal transcription factor involved in HIV-1-induced human macrophage death.
The physiological relevance of FOXO3a-mediated macrophage death is significant because a large number of macrophages in the brain and lung are infected with HIV-1 during late stage disease (13, 14, 15). Although macrophage are usually resistant to HIV-1-mediated cell death, targeted cell death of infected macrophage in tissue is likely a host immune response to prevent viral dissemination (17). Nonetheless, excessive cell death may also promote chronic inflammation and thus contribute to AIDS pathogenesis. Clearance of activated or HIV-1-infected macrophages from inflamed tissue is important to the host-mediated immune response. Understanding the signaling pathways regulating these processes, such as Akt-1 inhibition and FOXO3a activation, provides important information about macrophage survival and is relevant to HIV-1 pathogenesis, and could potentially identify therapeutic avenues for the treatment of HIV-1 infection.
Forkhead transcription factors play key roles in immunoregulation. Members of the FOXO subfamily have been implicated in the regulation of the cell cycle and/or apoptosis. However, the function of FOXO3a in peripheral immune cells, particularly in macrophages, is not clear. Gene knockout of FOXO3a led to spontaneous lymphoproliferation associated with inflammation of several organs in mice, suggesting FOXO3a regulates homeostasis of immune cells (19, 20). Indeed, FOXO3a appears to be a critical factor regulating apoptosis in lymphocytes (33, 34, 35). Our results support a similar role for FOXO3a in human macrophages. Exogenously expressed FOXO3a induced apoptosis in human macrophages and was found to significantly reduce viability of macrophages in vitro (Fig. 5).
In mammals, FOXO3a contains a highly conserved motif that is posttranslationally modified by phosphorylation. PI3K/Akt activation is the major kinase for phosphorylation of FOXO3a on T32, S253, and S315 or on homologous sites in other family members (29, 36, 37). Akt-1-mediated phosphorylation of FOXO proteins leads to cytoplasmic retention and impairment of nuclear transcription (38, 39, 40). Our data demonstrated an increase of nuclear translocation of FOXO3a by HIV-1 infection (Figs. 3 and 4). We demonstrated that inhibition of the PI3K/Akt pathway caused a decrease of FOXO3a phosphorylation in human macrophages and overexpression of constitutive Akt-1 blocked the HIV-1-mediated decrease of FOXO3a phosphorylation (Fig. 7). Notably, phosphorylated Akt-1 is also critical for macrophage survival (41). We recently demonstrated that Akt-1 is inhibited by HIV-1 infection and this inhibition contributes significantly to TRAIL-induced apoptosis (17). Together, these observations suggest the decrease of FOXO3a phosphorylation is due to inhibition of Akt-1 after HIV-1 infection. Although downstream factors of Akt-1 inhibition in our model are not clear, our study suggests the Akt-1-related signaling pathway plays a critical role in macrophage survival and is relevant to HIV-1 pathogenesis.
Akt-1-associated pathways and signaling cross-talk between Akt-1 and different apoptotic molecules remain a complex story. Recent studies have identified interactions between molecules downstream of Akt-1 and apoptosis pathways (see review in Ref. 42). For example, phosphorylation of BCL2-antagonist of cell death at Ser136 by phospho-Akt-1 prevents mitochondrial translocation of BCL2-antagonist of cell death (43, 44); phospho-Akt-1 phosphorylates procaspase-9 at Ser196 (45); phosphorylation of the Forkhead family of transcription factors by phospho-Akt-1 leads to decreased transcriptional activity for proapoptotic genes including Fas, TRAIL, Bim, or Puma (46, 47, 48). Furthermore, Akt-1 phosphorylation activates NF-κB, a transcription factor promoting the transcription of many essential survival genes (49). In our study, we demonstrate the inhibition of FOXO3a phosphorylation during HIV-1 infection of human macrophage is at least in part responsible for HIV-1-mediated cell death. Thus, FOXO3a appears to be one downstream factor of Akt-1 mediating apoptosis in HIV-1-infected macrophages.
HIV-gp120 has been reported to up-regulate the PI3K pathway and, thus, enhanced viral replication (50). We observed phosphorylation of Akt-1 decreased following 3 days of HIV-1 infection in human macrophages (17). Activation of FOXO3a by productive HIV-1 infection further supports this earlier observation. Variation in Akt-1 phosphorylation status is likely due to the state of HIV-1 infection. In addition, although we found HIV-1-infection decreased Akt-1 phosphorylation, and concurrently inhibited FOXO3a phosphorylation, there are other kinases that regulate the phosphorylation and translocation of FOXO3a, such as serum/glucocorticoid regulated kinase and dual-specificity regulated kinase 1A. Among those kinases, only phosphorylation of the Akt site in the forkhead domain on FOXO3a protein is indispensable for FOXO3a translocation (38). The exact roles of other kinases involved in the FOXO3a regulation require further investigation.
How downstream factors of FOXO3a are involved in HIV-1-mediated macrophage death is also an interesting and important topic. FOXO3a has been reported to induce proapoptotic signaling by regulating the expression of TRAIL, FasL, Bim, and Puma (23, 48, 51), and regulates cell cycle arrest by activation of p27, p130, and p21, repression of cyclin D expression (G1/S arrest) (29, 52), or activation of cyclin G2 (G0/G1 arrest) (53). Intriguingly, recent reports suggest FOXO3a also promotes cell type-specific antioxidative stress of manganese superoxide dismutase, catalase, and growth arrest and DNA damage inducible gene 45 in addition to promoting apoptosis (29, 54). Our study supports the role of FOXO3a in the induction of Puma in human MDM (Fig. 5 B), and this would likely shift the cellular equilibrium of survival-apoptosis, and promote an apoptotic phenotype in macrophage. The specific mechanism by which Puma participates in FOXO3a- or HIV-1-mediated macrophage apoptosis is certainly interesting and remains further investigation.
In summary, the evidence presented in this study demonstrates that HIV-1 infection inhibits the phosphorylation and increases the activity of transcription factor FOXO3a activation, resulting in cell death of human macrophages. Our study may help unveil the mechanism of macrophage cell death in HIV-1 pathology, which facilitates the elimination of HIV-1 reservoirs during disease, making this pathway a potential therapeutic target during HIV-1 infection.
We acknowledge Dr. You Zhou, Dr. Hui Peng, Angelique Walstrom, and Yu Zhen for providing technical support for this work. Dr. Howard E. Gendelman, Dr. Terry Hexum, Nathan Erdmann, Nick Whitney, and Matthew Beaver provided valuable comments and suggestions about the manuscript. Dr. Charles Kuszynski, Linda Wilkie, and Victoria Smith performed the FACS analyses. Julie Ditter, Robin Taylor, Johna Belling, Na Ly, Myhanh Che, and Emilie Scoggins provided administrative support.
The authors have no financial conflict of interest.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
This work was supported in part by National Institutes of Health Research Grants R01 NS 41858, P20 RR15635, and P01 NS043985 (to J.Z.).
Abbreviations used in this paper: MDM, monocyte-derived macrophage; AZT, azidothymidine; DN, dominant negative; MOI, multiplicity of infection; RT, reverse transcription; TBP, TATA box binding protein; PI, propidium iodide; siRNA, small interfering RNA.

domain: health


text:
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domain: hobby


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For obvious reasons, Wall Street has been very excited about the tax reform package that was signed into law in the final days of 2017.
While the White House and Congressional Republicans have touted it as a major force in helping companies to grow, the economy to expand and job creation to accelerate, one has to wonder if the impact might be much more limited.
While I am less convinced that, after final revisions, the tax plan will be a drag on growth, given its relatively modest impact on individual incomes, what occurred to me this morning is that there could be a real issue for corporations that analysts have not yet admitted to themselves.
The true benefit to corporations from the large reduction in both the stated and effective tax rates is to boost profitability, to be sure.
That does not, however, mean that corporate profits will enjoy additional organic growth. It simply means that profit margins will expand, and after-tax profits will be larger.
Some could complain that this is mere semantics. But it is not. For tax reform to deliver excess growth that drives pre-tax profits higher, GDP would have to accelerate meaningfully, driving revenue higher, as well.
Most Wall Street analysts look at a company's pre-tax profits, and/or cash flow, to determine a firm's organic rate of growth.
It is altogether possible, though somewhat unlikely, that corporate revenue and profits could be flat in 2018 relative to 2017, but lower effective tax rates would still boost the bottom line and expand corporate profit margins without the benefit of organic growth.
Of course, the global and domestic economies appear to be accelerating in 2018, raising the chances that corporations will benefit from both stronger growth and lower tax rates, factors now being priced into what would be otherwise richly valued stocks.
In addition, the year-long decline in the value of the U.S. dollar, which suffered its worst performance in a decade last year, will boost the profitability of U.S.-based multinational corporations by making their exports more attractive in overseas markets. That's another plus for stocks, broadly speaking.
Together, those factors would explain the increasingly rapid pace of stock price appreciation and record closing highs for the major stock market averages. The Dow Jones industrial average notched 71 closing highs in 2017, the most ever. It also had 12 consecutive months of gains — the longest unbroken streak of appreciation on record.
Analysts have dramatically raised their earnings estimates for 2018, thanks to all those developments.
However, there will be limited benefits to the reduction in tax rates when it comes to apples-to-apples earnings comparisons in 2019.
If corporate profitability surges in 2018, it might be much harder to post year-over-year gains in profits a year from now. Certainly that's not something to worry about immediately, but as the second half of 2018 approaches, the market will begin to look ahead to the following year's prospects.
This is where the trouble could start.
If the economy continues to heat up, profits peak and the Federal Reserve raises interest rates more rapidly than expected as growth accelerates, the calculus begins to change markedly for the market.
I was mistakenly far too cautious about stocks beginning in March of 2017. I was concerned about the multiplicity of economic, political and geopolitical risks.
And while those issues remain, the real problems ahead for the stock market may be far more prosaic.
It could be that there's a more traditional end to a near-record business cycle expansion as the economy overheats and is cooled by a Fed finally having to react to rapid growth and rising inflation.
Hence, this early year strength in stocks may be more of a "blow-off top" than a continuation of the trend.
We are, at the very least, due for a correction. There are budding signs of speculative excesses in the markets, at home and abroad.
But my growing concern is that tax reform offers only a one-time lift to stocks and it may all be discounted by the stock market before this month is over.
Of course, I could be wrong. But the concern about a one-off upward adjustment to corporate profits merits some serious consideration as investors adjust their portfolios to yet another "new normal."

domain: news


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We are now officially in the last month of Summer and though I am always excited for the start of Fall, I wanted to create a look that truly captured the essence of the current season. For starters, I knew that this outfit could not have an ounce of black or any dark colors despite my love of contrast. I really wanted to keep the overall ensemble light, fresh, and airy while highlighting some of my favorite styles and pieces. The “milk maid” or bardot trend has remained a flirty staple during the hotter months, and I was instantly drawn to this perforated version from the moment I saw it. I was a bit hesitant because this was an online purchase, however, the quality left me pleasantly surprised. Recently, I also added a pair of cut-out jeans to my collection which were unlike anything I currently own. Though I wasn’t sure how flattering these would look on me, again, I was elated from the moment I tried them on. While this may be a matter of preference, two things that have seemed to go hand-in-hand during this time of year are vacations and gold jewelry. Being a huge fan of both, I really wanted to convey those vibes with my accessories. Heeled sandals are probably my most worn style of shoe during the summertime, so I used this as an opportunity to break out a pair I’ve been dying to wear. In efforts to keep my metals consistent, I grabbed my favorite oversized aviator frames and a dainty pair of hoops. Lastly I incorporated a light charcoal bag featuring a croc design for an added bit of texture and was quite pleased with the look overall! I could definitely see myself wearing this outfit for a variety of occasions while feeling chic, stylish, and on trend. Comment below your favorite featured item and until next time!
Outfit Details & Links:
Bag- & Other Stories

domain: design


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Rising British star Hannah Klugman is excited to be in the main draw of the Budgen W100 Shrewsbury tournament after impressively coming through two qualifying rounds, writes Stuart Dunn.
The talented 14-year-old produced a fine performance to beat experienced Turkish player Pemra Ozgen, 37, in straight sets at The Shrewsbury Club yesterday.
Klugman progressed 6-4, 6-3 against a player with a career-high world ranking of 182, and will now face Poland’s Gina Feistel in the first round of the ITF World Tennis Tour event today.
“It was a great match,” said Klugman. “I really enjoyed it and I’ve loved playing here. The crowd’s really good. I’m excited to play again.”
Klugman reached the quarter-finals of the US Open Junior Championship girls’ singles last month, having teamed up with Isabelle Lacy earlier this year to progress to the girls’ doubles final at Junior Wimbledon.
Now she’s pleased to be playing in Shrewsbury: “It’s my first time here and it’s a lovely tournament,” she said. “The venue’s really nice, they treat us really well, so I’m happy to keep going.”
On being in the main draw, Klugman added: “I’m really excited – it’s the first time in a main draw of a 100K.”
Reflecting on the year and the progress she’s made, Klugman said: “It’s been a good year. I finished the year with making quarters at the US Open Juniors.
“I got really lucky to play in the Wimbledon qualifiers, the main one, and I made the final of the doubles in the juniors, so overall it’s been a really great year. I’ve really progressed, so, yes, I’m really happy.”
The two other Brits to reach the second qualifying round in Shrewsbury both lost yesterday.
Katherine Barnes was beaten 6-1, 2-6, 6-3 by French player Emma Lene, while Alice Gillan lost 6-3, 7-5 to Feistel, Klugman’s next opponent.
The draw for the first round of the main draw was held during a sponsors and players’ party at The Shrewsbury Club last night, with matches, both singles and doubles, to be played either today or tomorrow (10am start both days).
Harriet Dart, a regular member of Great Britain’s Billie Jean King Cup team and currently third in the national rankings, leads the British challenge in Shrewsbury and is one of ten Brits in the main draw
This week’s number four seed, Dart has been handed a first round meeting against French qualifier Nahia Berecoechea.
Lily Miyazaki and Katie Swan, currently fifth and eighth respectively in the British rankings, are also playing in Shrewsbury this week.
Miyazaki, who recently reached the second round of the US Open, faces Emily Appleton, another British player today, with Swan paired against fifth seed Dalma Galfi from Hungary in today’s opening match in the DMOS People Arena.
Top seed Viktorija Golubic, a silver medallist playing doubles alongside Belinda Bencic at the last Olympics, has been drawn in round one against British teenager Isabelle Lacy.
Golubic, a member of the Switzerland team crowned Billie Jean King Cup champions in Glasgow last November, reached the Wimbledon singles quarter-finals in 2021 and is currently ranked 101, having been as high as 35 last year.
French player Oceane Dodin, ranked 116 in the world and the champion of lower level Shrewsbury tournaments in both 2014 and 2015, is the second seed and faces Katarina Stresnakova from Slovakia in the first round today.
International tournaments such as the W100 Shrewsbury are part of the LTA’s Performance Competitions Calendar which provides significantly enhanced opportunities for British players at each age and stage of the performance player pathway.
Tickets to watch today’s first round matches from court-side stands, priced £15, which includes a 40-page tournament programme, can be purchased via the tournament website at https://worldtennistourshrewsbury.com/tickets-and-events/#tickets
A tournament pass to watch all matches throughout the week, including Sunday’s singles and doubles finals, is also available for £59.


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The force is strong with these two.
It’s unclear if the two were just hanging around or getting their outfits ready ahead of The Force Awakens’ Dec. 18 debut, but the photo shows Munn wearing a Star Wars T-shirt while Rodgers opted to channel a specific character from the galaxy far, far away.
“Who’s the bigger #StarWars fan…The one who bought this awesome shirt at @oldnavy or the one who already owned his own Obi-Wan Kenobi robe?” the X-Men: Apocalypse star captioned the snapshot.
Keep up with all the latest Star Wars news by subscribing to our newsletter. Head here for more details.

domain: entertainment


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domain: game


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Inspiration for Your Bullet Journal Mood Trackers
Do you understand when or why you experience different moods each day?
If you would like to do more of what makes you happy, (and a lot less of what doesn’t), then keeping track of your mood and emotions is a great way to identify the triggers in your life and avoid those negative moods!
If you’ve reached the point in your Bujo journey, where you would like to start your own mood tracker, then you are in the right place!
This post is packed with our favorite mood tracker ideas and tons of different ways to get your mood tracker underway.
Why Keep A Mood Tracker?
So what is the point of a mood tracker?
Tracking your moods and having a better understanding of your own emotions, is an incredibly simple way to improve your quality of life.
A proven mental health technique, mood tracking enables you to remove the doubt around why you might be feeling a certain way, giving you back the control, to make healthier decisions for your own piece of mind and mental wellbeing.
Mood trackers are a great tool that anyone can benefit from, also recommended as a self-help method for people suffering with mental health and mood disorders, such as:
Mood trackers can also track physical health, as physical and mental wellbeing both complement one another greatly.
What Are Bullet Journal Mood Trackers?
A mood tracker bullet journal page is quite possibly the purest form of tracking your mood.
While some people use different ways, like Apps, notes on their smartphone and post-it notes, creating and completing your own bullet journal mood trackers is the ultimate in mindfulness and personal satisfaction.
A mood tracker in your Bullet Journal is a creative and easy way to track your mental states and moods in a personal way that works best for you, whether it’s on a daily, weekly, or monthly basis.
How it Works:
- Choose a mood tracker spread to best suit your needs (and Bujo style)
- Create a color / image coded key for your moods
- Fill in your daily trackers at the end of the day
- Embrace the therapeutic benefits of Journaling and mood tracking
For your inspiration, we’ve put together some of the most beautiful mood tracker ideas from across the internet, so whether it’s your first time creating one, or you have been keeping a Bujo mood tracker for some time, there are some wonderful ideas here for everyone.
What Will You Need?
Keeping track of your emotions and mood patterns on a daily basis may sound a little intimidating, but there really is nothing to it.
Once you get in the swing of creating and completing your mood trackers, there really is no end to the creative writing styles and techniques you can introduce, using different colors and different shades, to make your mood tracker page as personalized as your heart desires!
Click this link for a full run-down of Amazing Bullet Journal Supplies.
To get us started, let’s take a look at a simple mood tracker…
15 Mood Tracker Ideas
1. Minimalist Mood Tracker
Minimalist mood trackers are great ideas for anyone who doesn’t have much time, or if you’re just getting started with your mood trackers.
With just a black fineliner pen and a blank page, this minimalist dandelion mood tracker is a good example of how you can turn a simple idea into a really effective design to track your moods effectively.
As you start to shade each heart, you’ll see that even tracking your gloomy days can add a splash of color to your simple mood tracker spreads.
2. Somewhere Over The Rainbow
This is a lovely monthly mood tracker, and another minimalist spread.
This spread fits perfectly on a single page and it’s simple design makes it a mood tracker idea that anyone can have fun creating.
A key of bright colors to match your rainbow will leave you with a stunning, pastel-colored page in your Journal.
3. Picture Perfect
This polaroid themed mood tracker is a really fun idea, capturing your overall mood, instead of images.
Once you have decided on the color code for your key, you can start shading in your polaroid images at the end of each day.
4. Light Up Your Life
This light-bulb themed mood tracker idea is definitely one of our favorite mood trackers.
These hanging light bulbs give a sense of hope and brightness to any bad mood you might have experienced that day.
Even the large key itself at the bottom of the page, is playing its part in adding a splash of brightness to this mood journal page.
5. When Life Gives You Lemons
A bright and refreshing, citrus-themed mood tracker is a really fun way to make your mood tracker entries.
Different shades of yellow in the key depict a range of moods from happy to sad, but instead of words, it uses simple and fun icons of facial expressions that are a cute way to mix up your key ideas.
6. Tree Top Tracker
We love these tree bullet journal mood tracker ideas.
Despite being simple to create, adding small doodles to your mood tracker page are one of the more creative ways to personalize your spreads.
Each tree will only need a black fineliner and a few seconds to create, but by the time you finish, your page will look alive with wintery warmth.
7. Beautiful Bujo Supplies
This fun idea just goes to show – you can create a mood tracker based on any of your hobbies or interests, just label them as days of the month, choose your color code and you’re off!
There is no better way for Bullet Journalists to personalize their mood tracker pages. The only limit here is your own imagination…
8. Avocado Creation
This January, Avocado mood tracker is all about starting the year the right way.
But let’s be honest, healthy food alongside a sleep log, is perfect for any time of year.
Looking at your daily moods alongside your quality of sleep is a good idea, as sleep plays such an important role in sleep quality, and vice versa.
Looking back at the end of the month at any direct links between the two, will help you to see if you can make any adjustments to improve your quality of life.
9. Anyone For Ice Cream?
Good moods, bad moods, happy, sad. All of these emotions are possible in a single day, and these habit tracker ideas are a good way of capturing the range of feelings that you can experience each day.
You can see here, the mood tracker spread takes its place alongside a habit tracker spread, (much like our sleep log earlier), helps you see your mood alongside your healthy routines.
Maybe you missed some study time or your daily walk and now it’s made you feel stressed. These pictures can paint a thousand words and help you towards a more healthy balance of time.
10. Out Of This World
You can have an absolute blast, creating this zero-gravity mood tracker.
A dotted bullet journal will help you space-out (sorry), your stars, planets and rockets evenly, making it even easier for you to create this really fun mood tracker idea.
Each component is simple to draw, but the overall effect is out of this world! (Again, sorry).
11. Beacon Of Light
Be your own beacon of light, with this stunning lighthouse-themed, Bullet Journal mood tracker.
You can of course customize the color scheme to suit your personal style, but these striking blues really add to the ocean scene of this tracker.
12. Bloom With Grace
This sunflower mood tracker idea is a beautiful addition to your Bullet Journal.
Each petal on the flower is numbered with different days of the month, helping you work your way around the outside in sequence, with different shades of yellows and oranges that will brighten up your month, whatever the weather.
Have a go at these sunflower mood tracker ideas free-hand, and see where it takes you.
13. Printable Mood Trackers
Short on time? Not a problem.
These printable planners are some of the best ways that you can still have stunning mood tracker pages in your Bujo, without necessarily spending hours creating them yourself.
On these designs, you can still create your own color key to personalize the designs even further, just head over to RADishPaperCo.com to find more printable habit tracker spread ideas.
14. Rise And Shine
If there is ever a chance that recording a bad day can leave you in a more positive mood before bed, then this tracker might just be the one to do it.
This mood tracker presents every single day of the month as a ray of light, leaving the entire page full of sunshine, a good idea for anyone who feels like a little boost could go a long way.
15. Year In Pixels
This is a perfect way to track your mood across an entire year.
A neat, easy-to-read mood chart, with months across the top and days down the left hand side, you can place this mood tracker in one of the blank pages of your Journal, and take a few seconds each day to capture your mood.
You can begin this tracker in any month of the year and create a separate, smaller grid for an overview of your mood throughout the past year.
Either create your own, or head over to Foxprintsstudio.com for printable versions.
Mood trackers are a great thing that will have a huge influence on your journey to a happier, healthier daily life.
And remember, we all have good days and bad days, so don’t be too hard on yourself when you’re feeling low.

domain: psychology


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The hospitals of Aruba, Curaçao, Sint Maarten and Dutch Caribbean have established a cooperative under the name Dutch Caribbean Hospital Alliance (DHCA u.a.). This memorable moment took place during the Four Countries Consultation that was organized in Aruba from 20th to 24th of June. All ministries of health, health insurers and hospitals of the CAS-BES islands participated in this summit. The theme here was cooperation in the field of public health within the Kingdom.
The experience with Covid-19 was the direct reason for the establishment of the partnership. DCHA aims to strengthen the quality of hospital care on these islands. DCHA wants to be able to guarantee and improve medical specialist care in the future and to coordinate and optimise the quality and availability of medical specialist care.
The goal of the Dutch Caribbean Hospital Alliance is to provide the best possible medical care locally and regionally through a solid cooperation between the countries within the Kingdom and strategic partners. The well-being of our patients and employees is paramount, in addition to maintaining a financially – economically healthy situation and safeguarding our cultural values.
The hospital administrators of the four countries of the Kingdom have expressed their confidence that the intensification of cooperation between the hospitals will improve and strengthen the quality and continuity of care for the inhabitants on the islands in the Caribbean part of the Kingdom.
Giovanni Frans, director of Fundashon Mariadal and the current chairman, says: ‘After almost two years of preparation and intensive consultation, among themselves and with the four countries, DCHA is finally a fact. We are proud of this step and hope that this will be one of many for optimal healthcare on our islands.’
Ingemar Merkies, medical director of CMC: A milestone made possible by the efforts of many. Thank you for that. I expect that we will work together with DCHA on education and training with the aim of bringing back our human capital abroad. This will further improve the quality of care on our islands.
Felix Holiday, director of SMMC: ‘This is a great step forward and we see that a more intensive cooperation with the other islands is already bearing fruit during covid care. This is because we know how to find each other earlier, help each other and keep each other well informed of the most recent developments”.
Jacco Vroegop, medical and general director of HOH: The establishment is a small step realized by a motivated group of people from the islands and the Netherlands. The important thing now is to inspire others and thus further support a team, so that we not only stay together, but also achieve successes. Essential in this is to keep an eye on the people around whom it all revolves; our societies and our employees.


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About 200 km north of Ho Chi Minh City are Mui Ne and Phan Thiet. Together, these traditional fishing villages are fast becoming a popular leisure destination. Resort-style developments have been rising along the coastline, though so far the lives of the fishing communities remain unchanged and Mui Ne’s long beach is quiet and serene, great for walks, jogging and spectacular sunsets. Phan Tiet, which is rather larger, is famous for its fish sauce, an ingredient in most Vietnamese dishes. It, too, has a beautiful beach. There is a wide range of accommodation, and restaurants offer a huge selection of seafood at very affordable prices. There are golf courses, beautiful pagodas, spectacular red and white sand dunes, and a variety of water sports.
Upon arrival at Tan Son Nhat International Airport then you transfer to Phan Thiet. Check-in hotel, take a rest and free to visit.
Having breakfast buffet at the hotel. After that, you are free to explore the city of Phan Thiet with attractive tourist attractions: Sand Dunes, Hong Stream, Mui Ne mineral bath, Bau Sen, ...
After breakfast, you turn back to Ho Chi Minh City and transfer to the airport for a flight departure.

domain: geography


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The snakes can grow to 2m in length
Keepers at a Birmingham Nature Centre are welcoming the arrival of 12 baby Jamaican Boa snakes. The babies are part of a captive breeding programme at Birmingham Nature Centre and it is the first time their mother has given birth.
The snakes, an endangered species, are currently just 1cm (0.3in) long, but can reach 2m (6ft 7in) as adults. Their native habitat is moist limestone forests, caves and trees. Accurate numbers in the wild are not known. The nature centre, near Birmingham city centre, is home to more than 130 different species.
More info on Jamaican Boas:

domain: biology


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Generate a number from 1 to 2999 via:
You can skip movies 10 times but never go back.
After the events at Lake Victoria, the prehistoric school of blood-thirsty piranhas make their way into swimming pools, plumbing, and a newly opened water park.
Is it a bird??
Is it a plane??
It's Moron Man, who against better judgement and a ton of bad reviews still decided to watch his crap!
A quick summary:
Shot of gnarling and yapping fish
Dying generic teenagers
Yapping fish swimming by (which actually look faker than most of the boobs)
The Hoff (who plays himself. Badly.)
People I don't give a fuck about that are dying
The scariest bit of this film: A hint towards a sequel....
I'd like to close with a quote from the film that sums up nicely what level it sinks to:
'Josh cut off his penis because something came out of my vagina.'
”Josh cut off his penis because something came out of my vagina.”
I’m surprised this film has taken such a critical beating when it achieves its (admittedly very low) aspirations. It is obviously a bad film but as a schlocky B-movie it was always intended to be bad but by those standards it just about delivers the required T&A, bloody set pieces and pointless celebrity cameos to make it relatively engaging. In fact I enjoyed it a hell of a lot more than the rather boring first film.
It is a film that certainly plays up to convention with lots of knowing winks along the way. If anything the films major fault is that it isn’t as funny or knowing…
Probably, no definitely, the best title for a sequel of all time. Unfortunately, 3DD’s finale is peanuts in comparison to 3D’s, and not as this film dares to proclaim the main meal after 2010’s appetiser. Do not get me wrong, there is still a lot of fun to be had in here, and the goriest gags will surely have you caught out loud, but it is nevertheless clear that, overall, it lacks the creativity that went into Alexandre Aja’s production. This is perhaps most notably in the incredibly short runtime of 88 minutes, of which the last ten are after titles; they simply did not have enough dough to work with. On the slight upside: the title certainly does not lie, in terms of boobs it trumps the franchise’s previous instalment! It even has some great moobs: the Hoff’s to be precise.
One look at my ratings will show that I do not have much low rated films.
This is not because of perhaps me giving films higher ratings than I should.
It is because I try my hardest to skip over films that are considered bad/stupid etc.
There are so many classics and great films I have not seen, I try to limit myself to watching only interesting films, films by directors I like and films that are considered at least good.
Why am I saying this? Becuase I also try to reserve half a star rating to films that are seriously the lowest films made.
Even if a film has at least some good scense I will try and give…
Welcome to rock bottom.
The first thing most people seem to ask is "Well did you even like the first one?" and the answer is yes, in fact I loved the 2010 remake a great deal. It was fun, had great gross-out gags, self-aware enough to be funny and walked that fine line that some horror/comedies have a hard time to find.
I didn't find this one much fun at all. It's almost as if the casting director went out of her way to find worst actors then the first film. Admittedly the first one surprisingly had the great Elisabeth Shue in it, but even the supporting cast of mostly unknown actors came off as competent for the…
Una peli de monstruos de serie b tirando a z mucho más deficiente (en todos los aspectos) que la original.
Gulager me comentó que no había presupuesto (casi) ni para comidas y que no deberíamos creernos toda la información que vemos en Internet.
En el próximo número de Horror Vision entrevista con el director de Feast.
Its exactly what you think. David Koechner may have the best death scene ever.
A bizarre ripoff under the guise of a sequel. It feebly tries to push the envelope in the cheesy fake violence it presents past that of the first film - however, it fails. While Piranha 3D is barely a good movie anyway, it at least gave us hilariously scenarios (Ving Rhames walking into the water to shoot at the swarming piranhas with a shotgun). Piranha 3DD, on the other hand, gives us typical horror scenarios and plays it off like comedy. There's another character that gets his dick bit off, but it feels like they were merely trying to top the first film in how ridiculously it happens. However, what made the scenario in the first film so funny is…
Welcome to rock bottom - the 26th entry of Boobtoper - 27.10.2016
As The Hoff says, this might as well be rock bottom - if I hadn't experienced Zombeavers already. Both have boobs, breasts, tits, bumpers and so on. Double D swims free! Heh heh. Even though Piranha 3DD is more boring than entertaining for a long time, it still entertains (sometimes).
Garbage horror in 3D has always been an attracting concept to me, but 99% of such films I've seen have no balls at all. Piranha 3DD is no different. I can only wonder who had the idea of having a cheap 3D film with cheap 3D effects spending half of the time in dim and dark surroundings? How…
Full to the tits with dicks.
Piranha 3DD - This is pure trash cinema and an exploitation extravaganza of hammy acting, bad jokes, blood and guts, and female flesh. (C+)
O primeiro já era meio bizarro, mas esse conseguiu ser mais bizarro ainda!
Mas por algum motivo, talvez pela bizarrice, tive vontade de ver, e se tiver uma sequência (como deixaram a entender que teria) verei.
More fun than I remembered , seen part of the Cleveland 12 hours of terror!
The story could have been whipped up in an afternoon. The acting by the main characters is amateurish, but there are some great cameos by Christopher Lloyd, Ving Rhames and one or two others that I won't spoil. The titular piranhas (pun intended) have no sense of menace about them, although there are a few gross out scenes that may titillate those looking for cheap thrills. And yes, as per the 2nd part of the title. there are some big tits, but they are not as central to the plot as the name may lead one to think.
"How the hell did you buy that shotgun leg?"
"With the money I saved on socks."
No, it's not quite as good as the beautifully crass B-movie that spawned it, Piranha 3D, but is just as ridiculously absurd and funny. In the crassest way possible, of course. I liked it better on this viewing due to the looks and laughter from Julie as we watched it.
a list that is trying to contain every horror film made that is not lost and is found on the…


The International Tennisorganisatie (ATP) has confirmed to open an investigation into racist allegations at the address of the American Donald Young (ATP 65). He should be in the match of the first round of the ATP tournament in New York castigated by his fellow countryman Ryan Harrison (ATP 44).
During the first set were both players of words and had to be taken apart by the referee.
“The ATP takes any racist allegations very seriously,” said the international tennis federation in a press release. “We will review the video thoroughly and not comment on the incident before we reach a conclusion.”
On Twitter were the two fighting cocks after the game. “I am shocked and disappointed in you, Ryan Harrison, that you are in the middle of our duel told me how you about me thought as a black tennis player. I thought that this was a ‘gentleman’s sport’ was”, told the 28-year-old Young again.
Harrison denied any racist statement. “The accusations by Donald Young to my address totally wrong. I am very disappointed that a player reacts after a match to have lost. The audio and video will be me one hundred percent exonerated.”
The 25-year-old Harrison won the match finally in two sets (6-3, 7-6 (7/4)).


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Vince Gilligan, the creator of Breaking Bad, and Miranda Hart have signed up to speak at the 2013 Guardian Edinburgh International Television Festival.
Gilligan will deliver a masterclass on his critically acclaimed US drama. The final eight episodes of Breaking Bad's fifth and final series will begin broadcasting shortly before the festival in August.
Hart, the creator and star of BBC1 sitcom Miranda, will be taking part in the Richard Dunn Memorial interview.
The festival, which this year has a line-up of more than 50 sessions, takes place from Thursday 22 August to Saturday 24 August.
• To contact the MediaGuardian news desk email email@example.com or phone 020 3353 3857. For all other inquiries please call the main Guardian switchboard on 020 3353 2000. If you are writing a comment for publication, please mark clearly "for publication".


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Located 2.5 km from Miracle Mile Miami, 3.2 km from Marlins Park and 5 km from Vizcaya Museum, Large Comfortable beautiful home features accommodation situated in Miami. This holiday home offers air-conditioned accommodation with a terrace and free WiFi.
The holiday home is equipped with 2 bedrooms, a flat-screen TV and a fully equipped kitchen that provides guests with a dishwasher, a microwave, and a washing machine.
An American breakfast is available every morning at the holiday home.
Cocowalk Shopping Center is 5 km from Large Comfortable Beautiful Home. Miami International Airport is 4 km away.

domain: travel


Kawasaki’s New Z1000
Posted at 06:15:00 PM
File under Kawasaki
Author: Mike Werner
Location: Normandy, France
Kawasaki have released the specs of their 2010 Z1000 motorcycle, and it's a monster. The 2010 model has got 136 bhp and 81 lbs/ft of torque (up from 72.8 of last year's model). This has been achieved thanks to an increase in engine size, from 953 cc to 1043 cc.
The styling of the motorcycle is very modern and eye-catching.Aluminium frame, large air intake ducts and massive 4-into-2 exhaust pipes.
Some key features:
- High visual impact
- High riding impact
- Completely designed from a clean sheet
- All-new 1043cc engine delivers more power, more torque
- All-new chassis for superb handling
- Pearl Stardust White / Candy Burnt Orange
- Metallic Chestnut Brown
- Metallic Spark Black / Atomic Silver
Engine Type: Four-stroke, liquid-cooled, DOHC, four valves per cylinder, inline-four
Displacement: 1043 cc
Bore & Stroke: 77.20 x 50.9 mm
Compression Ratio: 11.8:1
Fuel Injection: DFI with four 38mm Keihin throttle bodies, oval sub-throttles
Ignition: TCBI Digital Advance
Final Drive: X-Ring Chain
Frame Type: Aluminum Backbone
Rake/Trail: 24.5 deg. / 4.1 in.
Front Suspension/Wheel Travel :41 mm inverted cartridge fork with stepless compression and rebound damping, adjustable spring preload / 4.7 in.
Rear Suspension/Wheel Travel: Horizontal monoshock with stepless rebound damping, adjustable spring preload / 5.4 in.
Front Tire Size: 120/70 ZR17
Rear Tire Size: 190/50 ZR17
Wheelbase: 56.7 in.
Front Brake Type: Dual 300mm petal-type rotors with radial-mount four-piston calipers
Rear Brake Type: Single 250mm petal-type rotor with single-piston caliper
Fuel Tank Capacity : 4.9 gal.
Seat Height: 32.1 in.
Overall Length: 82.5 in.
Overall Width: 31.7 in.
Overall Height: 42.1 in.
Color: Pearl Stardust White, Metallic Spark Black
Is it me, or does this look like the new V-max? This bike will have to compete with the likes of the Honda CB1000R. As naked bikes go, this one has reached what it should have been in the beginning. It is one motorcycle that I'll be looking at very closely at EICMA next month.


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What exactly is a Sphongledroid, you ask? In October, the psytrance music of Shpongle (real name Simon Posford) will join forces with the visionary art of Andrew “Android” Jones. The Fillmore Auditorium will host the shows, featuring a live set by Posford paired onstage with the iconic visuals of Jones. Expect an extravaganza of sights and sounds when Shpongledroid comes together!
Shpongle Returns to Colorado
Posford (also known by the stage name Hallucinogen), who handles synths and production for the group Shpongle, will be headlining the show. Shpongle last visited Colorado in 2019 at the legendary Red Rocks Amphitheatre. The immersive, full-band live performance featured dancers, guest singers, and trippy art galore.
Since that epic back-to-back bonanza at Red Rocks, Shpongle has toured sparingly and released one EP to keep fans wanting more. Carnival of Peculiarities dropped in 2021, offering listeners 25 minutes of psychedelic bliss. Posford remained busy during that time, releasing a studio album of his own. Flux & Contemplation – Portrait of an Artist in Isolation offers his take on the struggles of dealing with the COVID lockdown. This toned-down masterpiece takes listeners on a triumphant if somber voyage.
Android Jones Takes Art Worldwide
Born and raised in Boulder and residing in Lyons, Colorado, Jones got his artistic start in game development. Having worked on several titles in the Metroid series, he eventually became disillusioned with developer culture. Beginning a new journey into digital visual arts, Jones founded the production company Obscura Digital. The group achieved notoriety by projecting digital images onto worldwide landmarks like the Sydney Opera House and Empire State Building.
After attending a 2016 SynerGenesis party, Jones found a creative home within the visionary arts movement. He flourished leaning into a more psychedelic-friendly environment. His artwork adorns the album covers of numerous influential musicians, including STS9, Tipper, and Beats Antique to name a few. His works include digital art like NFTs and projection demonstrations. He also creates physical pieces like posters, pins, and murals. Expect amazing items to be available at the shows to complement his artistic performance.
A Spectacular Sensory Showcase
For two nights in October — Friday, the 10th, and Saturday, 1the 28th — the Fillmore Auditorium in Denver will be a haven of art and music. Beyond the feature pairing of Shpongledroid, you’ll see additional performances that partner musicians and artists each night. Jam to sets by Ultrasloth, Freddy Todd, and Cosmic Trigger on night one. Night two, The Trifinity, Ahee, and Miraja will bring the noise. Attendees can check out live painting, an art gallery, crystals and more! Don’t miss what will be an unforgettable event, and secure your tickets today!

domain: artistic


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Wedding ceremonies are all about the entrance. Sure, there's the actual marriage part, but really it's that triumphant bride "reveal" and the reception that we all secretly attend weddings for.
This tone-deaf, rhythm-challenged trumpeter has single-handedly ruined that special moment. This baffling performance of 'Mendelssohn's Wedding March', played on the brass instrument, has gone viral since being uploaded on YouTube a few days ago.
It really is astounding just how bad this rendition is, but we have to hand it to the trumpeter; while he lacks any actual talent he still fully commits to the song.


Required permissions in the Meapp – (iPhone)February 10, 2021
Me Shortcuts – (Android)February 13, 2021
We on ME app work every day to bring you new and innovative features to improve your ME experience on your daily use.
Following new features and operating system requirements, new permissions are needed.
In this article, we will list all the necessary permissions we need from you for the new features. which ones are mandatory and why, and which ones are not mandatory, and yet, why are they needed.
For ME to be able to present to you how your friends have called you in their contacts list, the operating system requires access to your contacts list, also this permission is required to backup your contacts.
To enable real-time caller ID, phone permission is required.
For a caller ID to receive the incoming call phone number, the operating system requires your permission. Without it, it won’t detect the incoming call and it will not work!
For your call log to always show identified numbers even if they’re not in your contacts we need for the App to detect the incoming call
And now for the Non-Mandatory permissions at your discretion:
Google requires caller ID to add a default dialer option
it does not have to be used for caller ID to work!
To select a profile picture, we must have access to your pictures.
If you want to use the search numbrs from camera and detection caller id from camera with the help of photocopying phone numbers, the operation system needs your permission to access the camera so we can use it.
In the new version, there is a new feature in profile and call recognition – that presents you the location distance between you and your friends. To use this feature, it is necessary to permit an access to your location settings.
Only contacts you authorized will be able to see the location distance between you and them!! This datum will appear in the caller ID and profile and will stay private only between you and the contacts you approved.
We will soon upload more content, please feel free to write us questions and comments. We are always happy to hear from you 🙂
FYI, We are fully GDPR compliant and you have the right to be forgotten, if you wish to remove all your data from our servers, you can simply delete your profile, Tap on Me logo (enter app settings) scroll down to Profile section -> Delete Profile -> Approve and all your data will be no longer exists in our services.


THE Merry Christmas Charity Ball being hosted by Haven Hub and Limerick Land Search Team, is set to take place on Thursday December 14 at the waterfront Clayton Hotel in Limerick City.
Guests will savour a four-course feast with live music from 17Ten and DJ Katie, with complimentary drinks reception to boot.
Every ticket purchase directly supports the Haven Hub and Limerick Land Search Team in delivering their new shared base, which aims to be Ireland’s first mental health community centre.
For ticket information and event details, find out more on HavenHub.ie.
Interested sponsors can contact the Hub’s fundraising co-ordinator at email@example.com.


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Body Cleanse – Detox Program - 21 Day Body Makeover
Fake Health Care & weight loss myths
Dr. Tim Church, M.D., M.P.H, Ph.D. is one of the country’s leading physicians in exercise and obesity research.
Dr Church shares 3 myths of health you're not likely to won't hear in your doctors office
1) Does having breakfast every morning help or hurt fat loss?
Where did this notion begin?
Aren't we suppose to break-the-fast?
Should we eat breakfast before exercise?
Is genetics a factor?
2) Do activity monitors (Fitbit, Jawbone) really prevent weight loss?
How accurate is the calorie count on these devices
3) How do you separate fake health care news from real health news?
Do you want the truth?
Or news with fake studies that suit your belief system?
- 150lb Loss - Fat to Fit150lb Loss - Fat to Fit
- Eating Health is a Disorder?
- Sinusitis cause stroke?
- Weight gain May not be your Fault
- Did You Hear Your Body Complain?
- Tricks to Slow Aging
- Food, Mood, Outcome
- Diet Exercise and Heart Attack
- Shirtless Cold = Health
- Fitbit Fails
- Training Ecosystems
- Exercise Can Make You Smarter
- Selective Mating
- MCT Oil Profound Effects
- Balance Strength and Support
- Increase Metabolism
- Health? HIIT or Aerobic
- Pokemon GO Trouble or Beneficial
- Sugar, IQ and Hormone Disrupter
- Sports nutrition with Jill Lane
- Responsibilities of Being Healthy
- Addiction Relapsing Brain Disease
- Benefits of Fasting
- Are you a Push-over
- Men & Women Exercise Differences
- NFL's Dave Vobora Training
- Kimyla 140lb Loss Don't Diet Decide
- Fundamentals of Success

domain: medical


Last September, a local soccer team made up of homeless and previously homeless men played their way to Rio de Janeiro, Brazil. It was the prototypical underdog story: their participation in the 2010 Homeless World Cup was the culmination of many months of work creating a soccer program where there wasn’t one before, and recruiting and training players to get to the level required for international competition.
It was more than worth it though, as the team returned home proudly sporting the tournament’s Fair Play Award. “Rio was an amazing experience for everyone involved,” says Sarah Blyth, one of the organizers and volunteers behind Portland FC, a street soccer team in Vancouver whose players partly made up the national squad.
Since then, the street soccer movement has grown in the city. The excitement behind the team’s participation has led to more players, more teams and more games. Practices that were once held weekly have doubled, as coaches and players now meet every Thursday (at Andy Livingstone Park in Chinatown) and Sunday (at Creekside Community Centre). There is a palpable excitement in the practices, and the players who participated in Rio are happy to mentor newcomers.
However, for Blyth and Kailin See—both support staff at New Fountain Shelter where the Portland FC team originated—there was a huge gap in who they were reaching and supporting with the street soccer program.
“There were no women from Vancouver playing in Brazil,” says See. “When we saw what teams from other countries were doing with their women’s street soccer program, we were inspired to bring that home.”
And so they started recruiting, canvassing the various women’s centres and shelters in the Downtown Eastside to try to get players out to practices. A key part of the recruitment process was understanding that in order to get women interested in participating, they needed to offer something different from the men.
“We’d had a few women come out to practice [before], but they rarely seemed to become regulars like the men,” says Blyth. “Life is hard on the streets for everyone, but in many ways women have their own set of challenges and needs that are unique from men. Part of what we’re doing is trying to meet those needs.”
That has meant focusing on the social aspect of the game, providing a supportive community, a safe haven and food and drinks for every practice. They also separate the men’s and women’s teams during practices and have a female-only coaching staff so that new players are more comfortable and less overwhelmed.
And it’s been working. On the day I attend their practice, there are a dozen players and volunteers on the women’s side of the gym. They practice and laugh with each other genially.
One player named Erin had been participating for a little over a month. She sits on the bench beside me, and proudly laces up a pair of new Puma indoor trainers, shoes she says were donated to the team. She then rushes off to join her teammates in drills.
While the men engage in loud and robust drills (their thunderous kicks sound throughout the entire space), the women go about their practice more quietly. There was a clear focus on instruction and mechanics, like simple drills on passing and receiving a pass. Many of the players here have never really played soccer before, or even participated in organized sports. As a result, the atmosphere is intentionally positive and supportive, and the volunteers spend a lot of time on one-on-one instruction.
“We’re trying to break the game down to its simplest of forms so that everyone can succeed,” explains Simone Aardema, who has volunteered as a coach since the beginning of the New Year. “It’s important to get the basics down. We focus in on the positive, because sometimes this game can be very challenging.
“In a very short period of time, I’ve seen incredible improvement, especially in the ones that are absolute beginners.”
It’s an approach that Paula Armstrong certainly appreciates. At 54 years old, she is older than most of the other players. But she is bubbly and exuberant, clearly excited to be playing regularly. Armstrong, a resident at the Rainier Hotel, spoke about how she’d quit crack and has now been sober for over six months, and the role soccer has played in her recovery.
“It’s the activity and the camaraderie,” she says. “I wouldn’t be able to motivate myself on my own, but with these other women, there’s a support group.
“When you’re trying to change your life, something like this really helps. It helps you focus your mind and your body, and keep positive. And it’s led to these great relationships.”
Armstrong races off to the far end of the gym to practice on her own after the other players have left. She says she’s tired from the scrimmage and drills, yet continues to kick the ball into an open net long after most others have left.
The women’s team now has a name separate from the men’s—the Portland Phoenix—and new red and black jerseys to go with it. For See, the name has special meaning to the women, and captures the inspiration she herself felt from watching the other women’s squads in Brazil. “It’s about redemption, about rising up again,” she says.
And with their new name and new uniforms, they also have a specific goal in mind—to play in the Homeless World Cup in Paris later this year. For these women, soccer is a kick start to dreaming big.


Updated: Apr 14, 2021
I remember so clearly watching the news in January 2020 and learning of Wuhan, the capital of Hubei Province in the People's Republic of China, experiencing the most aggressive virus with devastating consequences. I raise my hand and confess while being struck by how awful the situation was, I was absolutely in the mind-set of ‘they are experiencing a terrible time’. At no point, naively, did it enter my head that this would become a global pandemic.
But here we are, over a year later and I think it’s fair to say that life has changed for most of us. Personally I am experiencing nervousness as a parent, a business owner and a family member - I truly believe we don’t know what the long term impact will be on each and every one of us.
What can we learn from history? If we go back to the great plague in 1665 we know that the business and economy suffered to an extent. We learned of grass growing in London due to the fact it was so quiet and everyday ‘felt like a Sunday’. The loss of life was significant in proportion and crime increased with the shortage of food and resources. The situation also resulted in broken relationships where Samuel Pepys wrote in his diary “The plague is making us cruel as dogs to one another”
I think we can all think of someone who has bubbled with excitement over the ‘report someone on the Covid-19 line’ where you can do your duty and make sure any breaking of the rules is notified to the higher authorities. To be clear I'm not saying this is the wrong action to take, but the interesting observation is the low status high power mentality where there is almost a thrill in the process.
Then we consider Social Media where you almost feel the rubbing of hands with glee when someone might post something controversial which then becomes the catalyst for a keyboard war. Cruel and disparaging responses that would never be spoken face to face are suddenly acceptable under the banner of 'free speech'. A beautiful quote to consider, 'life is short - be sure to spend lots of time arguing with total strangers on social media' - Bizarre when we are all too aware how quickly time can be taken from us.
The actions and behaviours of some individuals unquestionably leave us scratching our heads in puzzlement.. The ‘this is my human right’ scenario for those pushing the boundaries at least, and quite frankly rebelling at most, against the parameters our governments have set out to try and deal with this situation in the best way they can considering the unfamiliar territory.
Wear a mask, don’t wear a mask, Open the borders, close the borders, Vaccinate or don’t, Stay at home or party to your hearts content - so many situations we all interpret differently. Whatever the guidance is from the authorities there will be followers who agree and detractors who don't, each with very strong opinions exasperated by social media.
Turning now to the workplace. Teams who thrived together not only throughout the working week, but socially afterwards. Consider companies like Zappos or Southwest airlines who have relied on the culture developed by teamwork to deliver outstanding service and set them apart from their competition. Our colleagues who work with gusto knowing that a vacation is ‘just around the corner’ and thinking of the sand between their toes and the sun at their back. (Suddenly going to the supermarket can be the biggest adventure you might experience all year!)
Many colleagues will have worked through the change curve as companies have moved into a ‘working from home’ culture. The devastation at the start, how will my IT work? how will I manage remotely? who is there to help and support me? Who will I talk to?
Then in the middle, this is great, I can work to my own schedule, I can make a brew when I like, I have no commute and look at the money I am saving for my lunch. To suddenly, I haven’t left the house all week. There is no differentiation between work and home, it’s all just home. My personal space is now intruded by my work and my interactions are all on video.
There is so much time we could spend considering the emotional impacts on individuals, to name a few:
Fathers missing the birth of their children due to hospital restrictions
Family members sadly passing away and no opportunity to ‘say goodbye’
·Children missing the most critical social time where they should be learning how to engage and interact
Burnout – Covid Fatigue
Major surgeries being deferred where the long term impacts of this are still being realised
Increase in depression and anxiety, especially for those who live on their own
Redundancy or Furlough
The reality is we just do not know how this will play out over the next few years, Will we 'just ‘get back to normal'?
I honestly don’t think so, some of this culture is transformational.
Will we ever work together as teams again or will the new agile culture put an end to that? Will we trust getting back on that plane and flying for our vacations? Will masks become a permanent fixture as it does already in some parts of the world? How will our communities come back together as we can see ‘be kind’ has certainly not been at the forefront of everyone’s heart and mind. For those who have learned to be ‘solo beings’ will they ever truly be comfortable back in company again?
So many questions and we don’t have the answers, but we do have a responsibility for sure.
Open your eyes to how people around you are feeling. Listen, love and support - help others on their journey. As a line manager take the time to care. Colleagues tell us they will give you more loyalty and commitment if they truly believe you value them. As part of the community whether virtually or physically remember we are all in this together. While we read it repeatedly and probably now are a little immune to the impact of it, we genuinely do not know what someone else is going through, so be kind, always.
And finally – consider educating yourself. Absolutely we don’t have the answers but there is help and guidance we can turn to which will ensure we are in the best position possible to support and help others, whatever their experience. Simply doing nothing with what we are facing is no longer enough.


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Since 1981, Columbia, Missouri area congregations have supported low-income families through the Faith in Service to Humanity (FISH) Fund of Interfaith Council. Voluntary Action Center is the sole administrator of this fund, providing this service at no charge to the Interfaith Council. Every year the FISH Fund provides over $30,000 in goods and services to needy people in the Columbia and Boone County area.
Giving to the FISH Fund avoids duplication of services to those in need by offering assistance at one location. It increases overall accountability by restricting coordination of available service to one organization. Donating to the FISH Fund saves time by offering resources through one agency, allows you to work with social service professionals and helps to ensure that the social service system is not being abused.
The needs of low-income families continue to grow each year and your church can help! The FISH Fund allows your church's charitable giving to be used effectively and efficiently, and ensures that needed funds are readily accessible to those in need.
All Columbia and Boone County faith communities are invited to help in this much needed, much appreciated effort. Churches, area congregations, faith communities, and individuals are invited to contribute to the FISH Fund.
Send your donations to VAC, 403A Vandiver Drive, Columbia, MO 65202 or donate now by credit card.

domain: sociology


It's difficult to recall the last time a band received a standing ovation at the Jazz Showcase, a tough room that sets exalted standards for those who take its stage.
But the crowd shot to its feet on Wednesday night, after the Dave Holland Quintet finished the first set of a weeklong engagement. There simply was no arguing that one of the most dynamic small units in jazz had performed near the top of its considerable form.
Certainly listeners would have been even more delighted if Holland had brought his octet, which plays robustly on Holland's latest CD, "Pathways" (Dare2 Records). But the quintet at the core of that recording has been Holland's primary expressive outlet for years, and he has honed it into an organization without peer.
Just listen to these five musicians working together, Steve Nelson's buoyant phrases on vibraphone powered by Nate Smith's incendiary drum work and Holland's tonally resplendent bass lines. Somehow, these three musicians know how to share a rhythmic pulse without getting locked into predictable patterns. The elasticity of their approach to beats and offbeats cannot be taught or mimicked – it's uniquely theirs, developed through years of performance and several enlightening recordings.
Then there's the front line, Robin Eubanks' full-and-rounded tone on trombone facing off – or partnering – with Chris Potter's slightly acidic, slightly abrasive sound on tenor saxophone. When blasting against each other, the musicians produce palpable sonic tension. When working in tandem, they suggest four horns rather than two.
Remarkably, though, the combined force of these five musicians owes not to volume but to musical incident. Rather than bray for all they're worth, in other words, the players craft multiple melodic lines, these strands interwoven in often delicate and intricate ways. En masse, this band never plays at maximum decibel levels, focusing instead on the luster of its sound and the flow of its ideas.
The Holland Quintet opened its engagement with a tour de force, an extended version of the title track to "Pathways." Immediately, each of the players announced his intentions, Potter's heroic tenor solo enriched by Smith's sharp-edged percussive attacks, Holland's insistent bass lines, Eubanks' declamatory trombone shouts and Nelson's sonorous chords on vibes. It's not easy to sustain this much energy without playing too fast or loud, but the Holland Quintet somehow did it.
Vibist Nelson attained some of his best work in "Claressence," his hard-hitting lines and sharply defined accents reminding listeners that he can do much more than fill out ensemble textures. Elsewhere in the set, Nelson shifted from vibes to marimba, bringing out the yin and the yang of his distinctive tintinnabulation.
For those who wondered, the quintet also can produce music of real austerity, as it proved in the most haunting work of the night, "Veil of Tears." The meditative tone and stripped-down textures of this music left each musician tremendously exposed, to sometimes wondrous effect. Potter's tenor saxophone never has sounded more mystical or incantatory, while the whispered utterances of the rest of the band showed what carefully controlled ensemble playing is all about.
The quintet closed the set on an exuberant note, but even here, in "What Goes Around" (the title track of a Holland Big Band recording), the entire ensemble – and Eubanks in particular – emphasized musical substance over mere bravura.
No wonder everyone stood up.
Dave Holland Quintet
When: 8 p.m. and 10 p.m. Saturday; 4 p.m. and 8 p.m. Sunday
Where: Jazz Showcase, 806 S. Plymouth Ct.


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Keep track of your favorite shows and movies, across all your devices.
A look at some extreme floating homes, including a former French coal barge.
Season 1, Episode 7
Featured: a salvaged shipwreck in Bermuda; a floating sauna in Sweden; and houseboat with an oceanview worth millions.
Season 1, Episode 6
Aquatic homes that are swoonworthy are toured during visits to Key West, Fla., and Paris. Also: a floating cottage on the Atlantic Coast.
Season 1, Episode 5
Featured: boat homes in Paris; a tailgate party on water; and a psychedelic submarine trip.
Season 1, Episode 4
Out-of-the-ordinary houseboats are featured, including a fishing vessel that has been converted into a spa. Also: a floating residence with a $100,000 bathroom; and a redesigned warship.
Season 1, Episode 3
Houseboats featured include: a million-dollar manor decked out in marble and crystals; a 5500-square-foot yacht complete with a heliport; and a floating residence that belongs to a TV personality.
Season 1, Episode 1
Luxurious floating homes are spotlighted.
Because it's never too early to plan Thursday night... two months from now.
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