text:
MicroRNA expression profile in chronic nonbacterial prostatitis revealed by next-generation small RNA sequencing
Li Zhang1,2,3, Yi Liu1,2,3, Xian-Guo Chen1,2,3, Yong Zhang1, Jing Chen1, Zong-Yao Hao1,2,3, Song Fan1,2,3, Li-Gang Zhang1, He-Xi Du1, Chao-Zhao Liang1,2,3
1 Department of Urology, The First Affiliated Hospital of Anhui Medical University, Hefei 230022, China
2 Institute of Urology, Anhui Medical University, Hefei 230022, China
3 Anhui Province Key Laboratory of Genitourinary Diseases, Anhui Medical University, Hefei 230022, China
Date of Submission 25-Apr-2018
Chao-Zhao Liang
Department of Urology, The First Affiliated Hospital of Anhui Medical University; Institute of Urology; Anhui Province Key Laboratory of Genitourinary Diseases, Anhui Medical University, Hefei 230022
Keywords: chronic nonbacterial prostatitis; immune; inflammation; miRNA; next-generation sequencing
Zhang L, Liu Y, Chen XG, Zhang Y, Chen J, Hao ZY, Fan S, Zhang LG, Du HX, Liang CZ. MicroRNA expression profile in chronic nonbacterial prostatitis revealed by next-generation small RNA sequencing. Asian J Androl 2019;21:351-9
Zhang L, Liu Y, Chen XG, Zhang Y, Chen J, Hao ZY, Fan S, Zhang LG, Du HX, Liang CZ. MicroRNA expression profile in chronic nonbacterial prostatitis revealed by next-generation small RNA sequencing. Asian J Androl [serial online] 2019 [cited 2019 Jul 19];21:351-9. Available from: http://www.ajandrology.com/text.asp?2019/21/4/351/248810 - DOI: 10.4103/aja.aja_97_18
Inflammation is regarded as the driving factor in many diseases, including atherosclerosis, cancer, autoimmunity, and infections.[1] It is broadly defined as a protective response by the organism in the presence of invaded pathogens or endogenous stress signals that subsequently results in the elimination of initial injury causes, necrotic cell clearance, and eventually facilitates tissue repair. Immunological processes are often involved in inflammatory responses.[2] The pro-inflammatory cytokines and chemokines released by host cells such as tumor necrosis factor (TNF) and interleukin-1β (IL-1β) have autocrine and paracrine effects, leading to the local activation of macrophages and neutrophils. Meanwhile, cytokine-mediated activation of endothelial cells can increase vascular permeability and elicit the entrance of immune cells into tissues.[3] Next, infiltrated type 1 helper T (TH1) cells secrete interferon-γ (IFN-γ) to activate neutrophils. Meanwhile, IL-17-producing helper T cells (TH17 cells) and innate lymphoid cells could secrete IL-22 to further stimulate the production and release of antimicrobial peptides.[4] For instance, autoimmune diseases such as rheumatoid arthritis[5] and inflammatory skin diseases[6] caused by immunological imbalances have been frequently linked with chronic lesion inflammation. Likewise, pro-inflammatory cytokines can promote the progression of immune diseases by generating pathogenic B- and T-cells.[7] Therefore, immune responses are important parts of inflammation.
Chronic nonbacterial prostatitis (CNP) is a commonly observed distress in male patients that is characterized by genital or pelvic discomfort, voiding and pain symptoms, and even by sexual dysfunction;[8] it is much more common than chronic bacterial prostatitis (CBP), comprising more than 90% of chronic prostatitis.[9] To date, the etiology of CNP remains largely unknown. During the past decades, several hypotheses have been proposed to explain CNP pathogenesis including defective urothelial integrity and function, cryptic infections, autoimmunity, endocrine imbalances, neuroplasticity, and psychosocial conditions.[10] Recently, accumulating evidences from studies in patients and animal models have raised the possibility that CNP is a consequence of dysregulated inflammation associated with autoimmune disorders.[11] Immunization with prostate gland homogenates or purified prostate proteins such as prostate–steroid binding protein and prostate acid phosphatase or peptides could induce prostatic inflammation and chronic pelvic pain.[11] Consistently, T-cells with self-reactivity to prostate-specific antigen (PSA), prostatic acid phosphatase, and other prostatic and seminal plasma proteins have been successfully isolated from CNP patients.[12]
MicroRNAs (miRNAs) are posttranscriptional repressors of gene expression that play critical roles in a variety of physiological and pathological processes including development, differentiation, aging, and cancer. Notably, miRNAs are also widely accepted to participate in inflammation and lymphocyte homeostasis and allergic responses through coordinated modulation of multiple target genes. For example, miRNAs can execute the functional regulation of immune cells such as Th cells[13] or inflammatory signal pathways such as the nuclear factor kappa-light-chain enhancer of activated B-cell (NF-κB) network[14] in inflammatory or autoimmune diseases, indicating the crucial role of miRNAs in both inflammation and immune responses.
Recently, few studies have focused on the miRNA in CNP. One study revealed a decreased risk of erectile dysfunction (ED) in patients with CNP who were carriers of the miR-146a rs2910164 C allele,[15] while another study found that N-acetylcysteine could ameliorate carrageenan-induced prostatitis and prostatic pain through miR-141-regulated Kelch-like ECH-associated protein-1/nuclear factor erythroid-2-related factor 2 (Keap1/Nrf2) signaling pathways.[16] In 2018, a study evaluated the miRNA expression profile in expressed prostatic secretion (EPS).[17] However, EPS is limited when illustrating the integral immune response and inflammation status of prostate tissue. Therefore, we comprehensively evaluated the dysregulated expression profile of miRNAs in CNP prostate samples using next-generation sequencing (NGS) technology, aiming to fully understand the role of miRNAs in CNP initiation and progression. Furthermore, bioinformatics analyses were applied to enrich immune-related biological processes, molecular functions, cellular components, and signaling pathways, the efforts of which would help explore the immune regulatory mechanisms in CNP.
Experimental animals
Eighteen healthy adult male Wistar rats (aged 8 weeks, 250 ± 20 g) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., Beijing, China. The animals were housed in groups of three in plastic cages with soft bedding and free access to food and water under a 12/12 h reversed light-dark cycle. All animals were acclimated for 1 week before experimental procedures. All experimental protocols were approved by the Animal Research Ethical Committee of Anhui Medical University, Hefei, China. All surgeries were performed under chloral hydrate anesthesia, and all efforts were made to minimize suffering.
Carrageenan-induced CNP model establishment
Nine Wistar rats were randomly divided into the following three groups: (1) blank control (without any interference, BC) group, (2) normal saline injection (NS) group, and (3) carrageenan injection (CAR) group. For injection of carrageenan, rats were anesthetized with an intraperitoneal injection of 4% chloral hydrate (A600288; Sangon Biotech, Shanghai, China) and fixed in a supine position. Then, the lower abdomen above the rats' penis was shaved and the skin in this area was disinfected using three applications of 10% povidone-iodine solution (A606166; Sangon Biotech). A small midline incision was made in the disinfected area, and the bladder and prostate were carefully exposed. Using a microsyringe, 50 μl of sterile suspension of 5% carrageenan suspension (22049; Sigma-Aldrich, St. Louis, MO, USA) was injected into each ventral lobe in the prostate gland.[18] A commensurate amount of sterile normal saline was injected in the NS group and nothing was injected in the BC group. After checking for bleeding at the dissection site, the wound was closed in layers.
One week later, all the rats were deprived of food and water for 12 h and then sacrificed under anesthesia with chloral hydrate. Prostate samples were collected and rinsed with phosphate-buffered saline (PBS) to remove adherent blood and fat tissues. One ventral lobe was formalin fixed for hematoxylin and eosin (H&E) and immunohistochemistry (IHC) staining and the other ventral lobe was cut into pieces, stored in RNAlater solution (AM7020; Ambion, Carlsbad, CA, USA), and frozen at −80°C according to a standard operation procedure.
Histological analyses
The rat prostate tissues were fixed in 10% formalin, dehydrated, and then embedded in paraffin. Sections about 4-μm thickness were sliced from each embedded tissue and stained with HE. For HE staining, the standard protocol was followed. Briefly, 4 μm-sized paraffin-embedded tissue slides were dried at 65°C for 2 h and de-paraffinized with xylene (Sinopharm Chemical Reagent, Shanghai, China) for four consecutive times (10 min each time). The sections were then dipped into 100%, 95%, 80%, and 70% ethanol for 5 min each time. The sections were rinsed with tap water for 2 min to remove ethanol and then stained with hematoxylin for 5 min, followed by tap water washing for 3 min. Subsequently, the slides were dipped into 0.1% HCl (Sinopharm Chemical Reagent) for three times and washed with tap water, followed by 50°C water incubation for 5 min. Later on, the slides were stained with eosin for 2 min and then washed with tap water for 2 min. Finally, after dehydration and dipping the slides into xylene, 2–3 drops of mountant were added onto the slide for observation under a microscope (IX 71; Olympus, Tokyo, Japan).
For cyclooxygenase-2 (COX-2) expression evaluation, IHC analysis was performed with COX-2 antibody (E303; Spring Bioscience, Pleasanton, CA, USA) on whole-tissue sections. Briefly, after routine deparaffinization and rehydration, the slides were treated with 10 mmol l−1 sodium citrate buffer (pH = 6.0; ZLI-9065; Zhongshan Golden Bridge Biotechnology Co., Ltd., Beijing, China) and boiled for 10 min to retrieve antigen. The endogenous peroxidase activity was quenched by 3% hydrogen peroxide (Sinopharm Chemical Reagent) for 15 min. After PBS (E607008; Sangon Biotech) washing for 15 min, the slides were blocked by bovine serum albumin (BSA, 37520; Thermo Fisher Scientific, Waltham, MA, USA) in a wet box and incubated for 10 min. BSA was then removed and the slides were incubated with primary COX-2 antibody with 1:100 dilutions overnight at 4°C. The next day, the incubated slides were washed with PBS for three times (5 min each time) and further incubated with poly-horseradish peroxidase (HRP) goat IgG (SP-9000; Zhongshan Golden Bridge Biotechnology Co., Ltd) for 15 min. After 15-min PBS washing (5 min each time), streptavidin/HRP was added to incubate for 15 min, and then washed with PBS for 15 min (5 min each time). Subsequently, detection was made using 3, 3′-diaminobenzidene as instructed (DAB kit, ZLI-9018; Zhongshan Golden Bridge Biotechnology Co., Ltd). Finally, the slides were counterstained with hematoxylin before microscopic observation. Cytoplasmic staining was scored as negative (-), weak (+), moderate (++), or strong (+++). A positive result required both cytoplasmic staining in >10% of cells and moderate-to-strong intensity, as previously described.[19] Quantitative analysis of the brown positive staining was performed using Image-Pro Plus 6.0 (Media Cybernetics, Rockville, MD, USA).
Enzyme-linked immunosorbent assay (ELISA)
Serum IFN-γ concentrations were determined by using rat ELISA kit assay (E-EL-R0009C; Elabscience, Wuhan, China) according to the manufacturer's protocol. Briefly, 48-well plates were incubated with 100 μl standard solution or serum samples obtained from rats (three rats for each group) at 37°C for 90 min and the liquid was removed. After 100-μl biotinylated detection antibody incubation for 1 h at 37°C, each well was washed three times. Subsequently, 100-μl HRP conjugate was added and incubated at 37°C for 30 min, and then washed five times. Then, 90-μl substrate reagent was added and reacted at 37°C for 15 min. Finally, 50-μl stop solution was added. Absorbances were measured at 450 nm immediately. The sensitivity limit of the method was 18.75 pg ml−1.
RNA quality check, amplification, and NGS
Total RNA was collected from the three groups of tissues using the TRIzol reagent (15596-026; Life Technologies, Carlsbad, CA, USA). RNA quality check was assessed using an A260/A280 ratio (A: absorbance) between 1.8 and 2.2, an OD260/230 radio (OD: optical density) more than 2.0, a sample concentration >200 ng μl−1, and an RIN over 7 using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Then, ligation of 3'- and 5'-adapters was completed for each qualified RNA with T4 RNA Ligases (Thermo Fisher Scientific) with the presence of a phosphate group on the 5'-end and a hydroxyl group on the 3'-end of mature miRNAs. Next, reverse transcription (RT) was performed to make cDNA with the SuperScript IV revertase (Thermo Fisher Scientific), followed by PCR amplification. The acquired miRNA library was separated and purified by high-resolution polypropylene acyl ammonia gel electrophoresis (PAGE), and finally the miRNA library quality check was completed with a Qubit and an Agilent 2100 Bioanalyzer before NGS (Hiseq 2500; Illumina, San Diego, CA, USA).
NGS data analyses and novel miRNA identification
The miRNA sequencing data were further analyzed according to the methods described in a previous publication.[20] Briefly, clean reads were acquired via the following steps: removing adaptor sequences, deleting sequences without 3'-adapters and inserting sequences, and omitting sequences with a Q20 ratio <60% and a length >18–36 bp using the FASTX-Toolkit software (http://hannonlab.cshl.edu/fastx_toolkit/). The qualified sequences were analyzed based on their length peak and underwent homogeneous BLAST alignment (http://blast.ncbi.nlm.nih.gov/) to mature miRNAs in miRBase (http://www.mirbase.org/),[21] as well as reference genome alignment using Rfam (http://rfam.xfam.org/).[22] Identification of known miRNAs was conducted by using the miRDeep2 tool (https://www.mdc-berlin.de/8551903/en/),[23] which was also routinely used for novel miRNA exploration.
Bioinformatics analyses for differentially expressed miRNAs
The approaches described for the enrichment of functions and signaling pathways were frequently used in the present study. Briefly, target genes of differentially expressed miRNAs from three groups were predicted using RNAhybrid (http://bibiserv.techfak.uni-bielefeld.de/rnahybrid/)[24] and miRanda (http://www.microrna.org/microrna/home.do)[25] and analyzed with R package clusterProfiler (http://bioconductor.org/packages/release/bioc/html/clusterProfiler.html) before undergoing Gene Ontology (GO; http://www.geneontology.org) and Kyoto Encyclopedia of Genes and Genomes (KEGG; http://www.genome.jp/kegg/)[26] enrichment analyses. After mapping the potential target genes to the dataset of GO annotations and KEGG pathway datasets, the enriched biological processes, molecular functions, cellular components, and signaling pathways were eventually sorted.
Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) verification for altered miRNA and gene expression
cDNA synthesis was performed using a FastQuant RT kit (with gDNAase) following the manufacturer's instructions (KR10; Tiangen Biotech, Beijing, China). The abundance of individual miRNA was detected via an Applied Biosystems 7500 PCR System (Applied Biosystems, Foster City, CA, USA) using SuperReal PreMix Plus (SYBR Green; FP215, Tiangen Biotech). The specific RT and qPCR primers for all miRNAs and genes were purchased from Sangon Biotech and listed in [Supplementary Table 1] [Additional file 1]. The qPCR procedures were performed according to the protocols provided by the manufacturers. Briefly, triplicate reactions were performed at 95°C for 15 min, and the subsequent 40 amplification cycles were conducted at 95°C for 10 s and 60°C for 32 s. Meanwhile, U6 snRNA or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as an internal normalized control. Relative miRNA abundances were calculated using 2−ΔΔCt (threshold cycle) formula, where Δ Ct = CtmiRNA/gene− CtU6 snRNA/GAPDH and ΔΔ Ct =Δ CtCAR−Δ CtBC/NS. The miRNA/gene abundance differences between CAR and BC or NS group were analyzed by using unpaired t-tests. P < 0.05 indicated a statistically significant difference.
The differences among groups were analyzed using a randomized block design analysis of variance with SPSS version 10.0 software (SPSS Inc., Chicago, IL, USA). P < 0.05 was considered statistically significant.
Inflammatory cell accumulation and COX-2 expression in CNP tissues
Intraprostatic carrageenan injection induced prostatic edema and increased inflammatory cell accumulation. The majority of inflammatory cells were neutrophils and presented to the interstitial space in the CAR group, while no inflammatory cell infiltration was observed in the BC or NS group [Supplementary Figure 1] [Additional file 9]. Consistently, COX-2 protein levels were significantly higher (P < 0.01) in the prostates from the CAR group compared with those of the BC and NS groups [Supplementary Figure 2] [Additional file 10]. The HE and IHC staining results indicated that the CNP models were successfully established.
Serum IFN-γ levels were elevated in CNP rats
IFN-γ has been widely recognized as a signature pro-inflammatory cytokine that plays a central role in inflammation, and several studies have suggested a crucial role for IFN-γ in the pathogenesis of experimental prostatitis in animal models.[27],[28] Thus, we have evaluated the serum concentration of the pro-inflammatory cytokine in our carrageenan-induced prostatitis model. As a result, concentrations of IFN-γ in BC and NS groups were 24.41 ± 1.50 pg ml−1 and 19.55 ± 1.13 pg ml−1, respectively, while CAR group showed a significantly increased level of IFN-γ at 44.11 ± 1.22 pg ml−1 (mean ± standard error of the mean [s.e.m.]; CAR vs BC, P = 0.0005; CAR vs NS, P = 0.0001). The elevated serum levels verified that CAR injection indeed elicited CNP in rats [Supplementary Figure 3] [Additional file 11].
Overview of miRNA-NGS data from CNP prostate specimens
The majority of the clean sequencing reads were 22 nucleotides (nt) in size, followed by 21-nt or 23-nt RNA fragments, which were within the typical size range of registered miRNAs [Figure 1]a, [Figure 1]b, [Figure 1]c. The 18–36-nt qualified sequences were further analyzed based on their length peak. The counts of mature miRNAs aligned to miRBase for each sample were as follows: 626, 602, and 564 for the BC group; 243, 632, and 608 for the NS group; and 425, 453, and 525 for the CAR group. After comparing the sequences from each sample to the Rfam database, the miRNA sequence number and proportion of the genome for miRNAs are presented in [Figure 1]d, [Figure 1]e, [Figure 1]f, revealing that the ratio of miRNAs in each sample was >85%.
Figure 1: Length distribution of clean reads from small RNA NGS and Rfam alignment analyses in different sequencing samples. Length distribution (nucleotides) of clean reads from small RNA NGS of prostatic tissues (a) without any treatment (BC), (b) treated with NS, and (c) treated with CAR. The ratio of different smRNAs (miRNAs, rRNAs, scRNAs, snoRNAs, snRNAs, and tRNAs) compared with Rfam database from small RNA NGS of prostatic tissues (d) without any treatment (BC), (e) treated with NS, and (f) treated with CAR. NGS: next-generation sequencing; BC: blank control group; NS: normal saline group; CAR: carrageenan group.
Characteristics of top abundant known and novel miRNAs
To analyze the most abundant miRNAs in the prostate tissues, we listed the top twenty highly expressed miRNAs with relatively more read count in decreasing order from the known miRNAs in the CAR, NS, and BC groups, respectively, whose expression profiles were indicated by the average read counts. In the inflammatory prostate tissues, rno-miR-10a-5p, rno-miR-148a-3p, rno-miR-21-5p, rno-miR-10b-5p, rno-miR-143-3p, rno-miR-99a-5p, rno-miR-99b-5p, rno-miR-200b-3p, rno-miR-100-5p, and rno-miR-183-5p were the top ten abundant miRNAs. The highly expressed miRNAs in the NS group were rno-miR-148a-3p, rno-miR-10a-5p, rno-miR-200b-3p, rno-miR-143-3p, rno-miR-99a-5p, rno-miR-10b-5p, rno-miR-21-5p, rno-miR-100-5p, rno-miR-183-5p, and rno-miR-99b-5p. Correspondingly, the miRNAs possessing the highest expression levels in the BC group were rno-miR-148a-3p, rno-miR-200b-3p, rno-miR-10a-5p, rno-miR-143-3p, rno-miR-99a-5p, rno-miR-21-5p, rno-miR-10b-5p, rno-miR-152-3p, rno-miR-100-5p, and rno-miR-200c-3p. From these data, we found that the most abundantly expressed miRNAs in the prostate tissues from the three groups were approximately the same [Supplementary Table 2][Additional file 2], indicating the identical histological origin of the paired samples undergoing NGS. Furthermore, 115 novel miRNAs were identified and presented in [Supplementary Table 3] [Additional file 3], whose roles in the development and progression of prostatitis require further investigation.
Differentially expressed profile of miRNAs in CNP tissues
When comparing the distinct levels of miRNA abundance among the three groups, two quantities were of great importance: one is the P value and another is the log2(fold change) value. The P value responses to whether the difference in expression levels was statistically significant, while the log2(fold change) value could illustrate the magnitude of the miRNA abundance differences. A log2(fold change) value above 0 indicates upregulation and a log2(fold change) value below 0 indicates decreased expression. Notably, even very large log2(fold change) values may have insignificant P values caused by considerable variabilities. Similarly, relatively small magnitude in the differences may also possess tiny P values due to the highly consistent technical replicates within each sample. Thus, a useful visualization method called a "volcano plot," which can simultaneously exhibit log2(fold change) and P values on the X- and Y-axes, respectively, has been introduced to conduct our analysis. Generally, points located in the upper-left or upper-right regions of the plot and indicated with green or red color possess both small P values (<0.05) and high log2(fold change). In this comparative study, the volcano plot generated by the miRNA profiles from the three groups is shown in [Figure 2]a and [Figure 2]b. As a result, 84 (CAR vs BC) and 70 (CAR vs NS) significantly dysregulated known miRNAs were found after adjusting for multiple testing. Among them, the top twenty upregulated and downregulated miRNAs are shown in [Table 1] (CAR vs BC) and [Table 2] (CAR vs NS), respectively. Meanwhile, hierarchical clustering analysis was performed to cluster the same or similar differentially expressed miRNAs [Figure 2]c and [Figure 2]d.
Figure 2: The overview of the volcano plot generated by miRNA profile and clustering heat maps of differentially expressed miRNA. The volcano plot generated by miRNA expression profile from small RNA NGS between prostatic tissues (a) treated with CAR or without any treatment (BC) and (b) treated with CAR or NS. The red points in the plot represent significantly upregulated miRNAs and the green points represent significantly downregulated miRNAs, while the blue points show no statistically significant differences due to either small fold change (<2) or insignificant P values. The heat map of miRNA expression profile between prostatic tissues (c) treated with CAR or without any treatment (BC) and (d) treated with CAR or NS. For c and d, each column represents a certain sample and lines represent different genes; the corresponding color represents the amount of gene expression in the sample: red color indicates higher expression and green represents lower expression. DEG: differentially expressed genes; BC: blank control group; NS: normal saline group; CAR: carrageenan group.
Table 1: A collection of top twenty upregulated and downregulated miRNAs detected by next-generation sequencing in prostate tissues (carrageenan group vs blank control group)
Table 2: A collection of top twenty upregulated and downregulated miRNAs detected by next.generation sequencing in prostate tissues (carrageenan group vs normal saline group)
As expected, the twenty upregulated and downregulated miRNAs between the CAR versus BC groups and the CAR versus NS groups mostly overlapped [Table 1] and [Table 2], verifying the significant regulatory roles of miRNAs in the progression of inflammation in CNP. Meanwhile, the normalized expression levels of aforementioned top differentially expressed miRNAs in BC, NS, and CAR groups are shown in [Supplementary Table 4] [Additional file 4]. The most abundantly upregulated miRNAs in the prostate tissues were rno-miR-155-5p, rno-miR-501-3p, rno-miR-1956-3p, rno-miR-212-5p, and rno-miR-146a-3p for CAR versus BC and rno-miR-155-5p, rno-miR-184, rno-miR-132-3p, rno-miR-501-3p, and rno-miR-1956-3p for CAR versus NS. Meanwhile, rno-miR-33-5p, rno-miR-136-5p, rno-miR-19a-3p, rno-miR-150-5p, and rno-miR-130a-3p were the top five most abundantly downregulated miRNAs in the prostate tissues for CAR versus BC, while rno-miR-33-5p, rno-miR-136-5p, rno-miR-130a-3p, rno-miR-19a-3p, and rno-let-7g-3p were the five most decreased miRNAs for CAR compared with NS. Besides, we have compared the differential expression between BC and NS groups, the results showed almost no significant difference of the miRNA expression levels between the two groups except for rno-miR-124-3p and rno-miR-150-5p, indicating that the altered expression between CAR versus BC and CAR versus NS was reliable [Supplementary Table 5] [Additional file 5] and [Supplementary Table 6] [Additional file 6].
Validation of differentially expressed miRNAs revealed by NGS
To better evaluate the accuracy of NGS data, we have established a new batch of CNP model rats and corresponding control groups. Validation of altered miRNA expression in CNP tissues profiled by NGS was performed by qRT-PCR in BC, NS, and CAR groups. We randomly picked five upregulated and five downregulated miRNAs among the differentially expressed miRNAs to conduct the analysis, and each expression abundance was presented using the normalized fold change of CAR group versus BC and NS groups. As a result, the altered expression tendency of all the ten deregulated miRNAs was consistent with the data obtained from NGS technology. Besides, overwhelming majority of the tested miRNAs showed significant differences between CAR versus BC and CAR versus NS. Among all the compared miRNAs, significant differences were only not found in miR-130a-3p and miR-26a-3p between CAR and NS, which together indicated that the expression levels of miRNAs detected by NGS were reliable [Figure 3].
Figure 3: Relative expression of (a) five upregulated and (b) five downregulated miRNAs in BC, NS, and CAR groups at day 7 after model establishment (n =3 for each group). Data are presented as mean ± s.e.m.*P < 0.05,**P < 0.01 BC: blank control group; NS: normal saline group; CAR: carrageenan group; s.e.m: standard error of the mean.
GO enrichment analysis of differentially expressed genes in the CNP prostate tissues
The essential biological functions of the putative target genes were classified via the GO analysis. To better understand the biological behaviors of the known deregulated miRNAs, potential target genes and biological functions targeted by these miRNAs were predicted. The top ten most enriched GO terms for biological processes (BPs), cellular components (CCs), and molecular functions (MFs) are presented in [Supplementary Table 7] [Additional file 7], [Supplementary Table 8] [Additional file 8] and [Supplementary Figure 4] [Additional file 12]. Of these genes, the putative target genes of deregulated miRNAs appeared to be mostly enriched for CC such as cell, cell part, intracellular, intracellular part, and organelle, when comparing the CAR group with either the BC or NS group. Meanwhile, the most enriched MFs were binding, protein binding, ion binding, organic cyclic compound binding, and heterocyclic compound binding, and the most enriched BPs were cellular process, single-organism process, metabolic process, organic substance metabolic process, and cellular metabolic process for the CAR versus BC or CAR versus NS comparison. Correspondingly, the putative target genes of deregulated miRNAs between CAR and NS appeared to mostly overlap with the CAR versus BC comparison.
KEGG pathway analysis of differentially expressed genes in the CNP prostate tissues
After GO analysis, the putative target genes of the differentially expressed miRNAs in the prostate specimens were examined by KEGG for pathway enrichment analysis. The top ten enriched KEGG pathways are presented in [Figure 4]. Based on the proportion of differentially expressed genes in all the annotated genes, Q-values (adjusted P values), and number of genes enriched in specific pathways, we have annotated the most enriched pathways. When comparing CAR with BC, these pathways were tightly linked to cancer, endocytosis, proteoglycans in cancer, axon guidance, and forkhead box O (FoxO). When comparing CAR with NS, these pathways were tightly linked to cancer, mitogen-activated protein kinase (MAPK), proteoglycans in cancer, cyclic adenosine monophosphate (cAMP), and calcium signaling pathways. Furthermore, to confirm that the aforementioned deregulated miRNAs could contribute to affecting the enriched KEGG pathways, we have selected two essential genes cAMP and mammalian target of rapamycin (mTOR) for mRNA expression validation from "cAMP signaling pathway" and "mTOR signaling pathway." As a result, cAMP showed significant increased expression in CAR group, while mTOR presented significant decreased expression in CAR group, compared with BC and NS groups (P < 0.01; [Supplementary Figure 5] [Additional file 13]). These results indicated that miRNA expression changes in fact caused altered gene expression in enriched pathway, which may further initiate the activation or depression of the following signaling transduction involved in inflammation and immune response.
Figure 4: KEGG enrichment dot plot for target genes of differentially expressed miRNAs (CAR vs BC and CAR vs NS). The top ten enriched KEGG pathways are presented. The X-axis represents enrichment factor, indicating the proportion of differentially expressed genes in all annotated genes on certain signaling pathway. The Y-axis represents log10(Q-values). Enriched pathways with higher X and Y values possessed better statistical significances. (a) CAR versus BC; (b) CAR versus NS. BC: blank control group; NS: normal saline group; CAR: carrageenan group; KEGG: Kyoto Encyclopedia of Genes and Genomes; FoxO: forkhead box O; AGE-RAGE: advanced glycation end products-receptor for advanced glycation end products; MAPK: mitogen-activated protein kinase; cAMP: cyclic adenosine monophosphate; mTOR: mammalian target of rapamycin; Wnt: wingless/int 1; Q-values: adjusted P values.
CNP is a complex and frustrating syndrome caused by unknown pathological mechanisms with a lack of effective therapeutics. Massive efforts have been made to uncover the pivotal factors that promote the occurrence and development of CNP. Although only one-third have inflammation on biopsy,[29] and how chronic pelvic pain develops and whether it could be mechanistically linked with inflammation were still controversial, the crucial role of inflammation in CNP and symptoms such as chronic pelvic pain should not be unnoticed, as unresolved chronic inflammation may potentiate tissue injury resulting in central sensitization and eventually cause chronic pelvic pain.[10],[30],[31] Recently, researches in biomedical science studies have indicated that inflammation and immune responses coexist in various kinds of diseases.[32] Correspondingly, immune mechanisms have been reported to be involved in CNP etiology, pathogenesis, and symptom development.[10] However, full understanding of the exact molecular mechanisms, especially the immune regulatory networks involved in CNP, remains largely absent. Notably, an increasing number of studies have shown that microRNAs, a class of evolutionally conserved and endogenously expressed RNAs, could be considered as critical regulators of gene expression at posttranscriptional levels.[33] MiRNAs are involved in the pathogenesis of a broad spectrum of human disorders, including metabolic and inflammatory diseases.[34] Accordingly, as CNP has shown complex symptom features such as prostate inflammation and immune disorders, it is of great importance to generate the differentially expressed miRNA profiles in prostatitis tissues to further understand the miRNA regulatory networks in CNP initiation and progression, especially to clearly understand the possible role of the immune system in CNP.
Recently, a newly developed NGS technology has been widely used for miRNA quantitative studies, due to the higher test specificity and sensitivity to detect extremely low-abundance miRNAs that may not be detected by the traditional quantitative assays. Based on NGS, bioinformatics analyses of deregulated miRNA expression signatures have further enriched several significant GO terms and signaling pathways, which are considered to be related to inflammatory diseases such as osteoarthritis[35] and rheumatoid arthritis (RA).[36] However, no systematic studies have been conducted on miRNAs profiling in CNP tissues by NGS.
In the present study, we used NGS technology to identify miRNAs prepared from prostate tissues from a CNP animal model. We quantified a total of 20 048 744, 15 070 023, and 9 183 863 sequencing read counts with typical 18–36 nts length for the BC, NS, and CAR groups, respectively, following alignment to the rat genome sequence dataset, which represented 15 526 924, 12 752 646, and 7 918 591 read counts aligned to miRBase for each group (n = 3), respectively. Among these alignments, 1224, 1039, and 1029 genome-wide expressed known miRNAs accompanied by 257, 204, and 190 novel miRNAs were in BC, NS, and CAR groups, respectively. As the histological origins of paired samples undergoing NGS were identical, most of the top twenty highly expressed miRNAs in the three groups showed similar expression pattern, while rno-miR-146a-5p was found to be differentially expressed between BC and CAR groups (P < 0.001) and also between NS and CAR groups (P < 0.001). Interestingly, [Figure 3]showed that CAR treatment group had a far higher abundance of tRNAs identified (7.54%) than BC (2.60%) or NS (1.48%) groups. tRNAs can be processed to distinct small RNA species commonly referred to as tRNA-derived fragments (tRFs or tDRs). Based on the origin from 5' or 3' end, tRFs were divided into tRF-5 and tRF-3.[37] It has been revealed that a particular tRF-3 (named CU1276 or tRF-3027) was downregulated in human malignant B-cells and lymphoma biopsies compared with naive, germinal, and memory B-cells. It interacted with argonaute (AGO) proteins, and its overexpression could repress the endogenous abundance of the tumor suppressor replication protein A1 (RPA1) in a miRNA-like manner.[38] These results indicated that CAR treatment might cause the deregulated expression of several posttranscriptional modulators including not only miRNAs but also some tRNAs.
Furthermore, based on a "volcano plot" that simultaneously defined small P values and high fold changes, we focused on exploring miRNAs that showed significantly different expression patterns. The comparative analysis identified 84 and 70 dysregulated miRNAs between the CAR group and BC group, and between CAR group and NS group, respectively. Notably, of the top twenty differentially upregulated or downregulated miRNAs in CAR versus BC and CAR versus NS comparisons, 14 miRNAs were overlapped either in upregulated or downregulated group, indicating the consistent tendencies after the injection of carrageenan.
To explore the concrete regulatory roles of the overlapped and dysregulated miRNAs in CNP, we firstly compared the expression patterns between our results and those of previous publications. As mentioned before, it has been reported that miR-146a rs2910164 C allele could compromise miR-146a expression and thereby increase the expression of nitric oxide synthase 1 (NOS1) for decreased risk of ED.[15] In consideration of that ED was substantially increased in patients with CP, these findings were consistent with our data that miR-146a-5p/3p was upregulated in carrageenan-induced prostatitis. Besides, miR-141 was addressed to be downregulated in CP patients,[16] again verifying our similar findings in carrageenan-induced prostatitis model [Supplementary Table 4]. Later on, we have also attempted to link our results to those reports regarding other inflammatory diseases. For example, miR-146b-5p has been reported to be highly expressed in inflamed temporal artery biopsies from patients with giant cell arteritis;[39] it was also significantly elevated in CNP tissues in our NGS data. miR-511-3p, which is associated with macrophage-mediated pro-inflammatory cytokine production and contributed to inflammation,[40] was significantly upregulated in our investigations. A similar result was obtained for upregulated miR-155-5p in endomyocardial patients with inflammatory cardiomyopathy.[41] Notably, miR-155-5p was regarded as a critical component in an inflammation-induced regulatory loop that controlled T-cell function and peripheral immune tolerance.[42] Thus, upregulated miR-155-5p may promote the progression of CNP inflammation through the enhancement of peripheral immune tolerance.
Among the representative downregulated miRNAs in the CAR group, miR-139-5p was also significantly decreased in clinically advanced primary biliary cholangitis.[43] It was also found that miR-200b was significantly downregulated in early pulmonary inflammation and fibrosis development, which was the same as its expression pattern reported in CNP.[44] Specifically, miR-150-5p, which was decreased in enterovirus-induced inflammatory injury in central nervous system,[45] was significantly downregulated in the CAR group. Given that extracellular vesicles released by CD4+ T helper (Th) lymphocytes such as T regulatory (Treg) cells contained enriched miR-150-5p levels to inhibit CD4+ T-cell proliferation,[46] the decreased exocrine miR-150-5p levels in Treg cells could be one of the possible mechanisms leading to inflammation. More importantly, we have identified several new miRNAs with altered expression, including miR-1956-3p, miR-6215, miR-883-5p, and miR-1247-5p, whose roles in inflammation were largely unknown before.
Intriguingly, there were certain dysregulated miRNAs identified in the current work that showed inconsistent expression patterns with published data. For example, miR-19b-3p was upregulated in plasma from knee osteoarthritis patients and was positively correlated with disease severity.[35] However, the expression of miR-19b-3p in CNP was decreased. The probable explanation was that miRNAs might exert distinct expression levels in plasma or local tissue; additionally, the complex pathological mechanisms and regulatory networks of miRNAs may not be uniform in different kinds of inflammatory diseases.
Following the differential expression analysis, we performed GO and KEGG bioinformatics analyses. The significantly enriched GO terms revealed the participation of target genes in several biological processes such as regulation of immune system process, immune response, immune effector process, inflammatory response, innate immune response, and activation of immune response, indicating the regulatory effects of miRNAs in inflammation progression and immune responses. KEGG pathway analysis enriched several immune-related pathways, such as "MAPK signaling pathway," "cAMP signaling pathway," "endocytosis pathway," "mTOR signaling pathway," and "FoxO signaling pathway." Generally, MAPK signaling pathway is thought to be involved in innate immune Toll-like receptor (TLR) expression[47] and is important in the T-cell antigen receptor (TCR) signaling negative feedback loop.[48] The cAMP signaling network is necessary for inducing the catalytic activity of protein kinase A (PKA) in splenocytes, lymphocytes, and thymocytes, which is important for regulating innate as well as adaptive immune sensitivities.[49] cAMP was the first identified intracellular second messenger of extracellular ligand action. Within the immune system, elevated intracellular cAMP levels generally reduce pro-inflammatory mediators and increase anti-inflammatory factor production,[50] while among T-cells, Treg produce and respond to prostaglandin E2(PGE2), which is modulated by COX-2 and acts as an autocrine factor to increase intracellular cAMP. It has been reported that nonsteroidal anti-inflammatory drugs (NSAIDs) and COX-2 inhibitors could block PGE2 synthesis, eventually downregulating intracellular cAMP levels in T-cells, suggesting the crucial role of cAMP in maintaining the immune balance.[51] We found in our study that cAMP mRNA expression and pro-inflammatory miR-155-5p were both upregulated in CNP after CAR treatment, this was consistent with a previous report that cAMP and miR-155-5p may modulate inflammatory process.[52] Similarly, the enriched endocytosis pathway also has been reported to participate in enhanced major histocompatibility complex II (MHC II) presentation mediated by CD4+ T-cells.[53] mTOR complex 1 (mTORC1)-induced glycolytic reprograming can activate human monocytes and macrophages and subsequently promote pro-inflammatory cytokine production.[54] mTOR is a serine/threonine kinase that regulates cellular growth, proliferation, inflammation, and immune in response to various environmental stimuli. Although the concrete roles of mTOR in inflammation and immune were largely controversial, a central role for mTOR in innate immunity has been recently defined by its ability to restrain pro-inflammatory factors. It has been reported that inhibition of mTOR by rapamycin could augment inflammation and pulmonary injury through NF-κB activity enhancement.[55] Similarly, in myeloid immune cells, mTOR inhibition was demonstrated to effectively block the anti-inflammatory effects of glucocorticoids.[56] In our current investigation, as expected, mTOR downregulation was observed with upregulated miR-155-5p in CAR-induced CNP, which was supported by the fact that miR-155-5p could inhibit mTOR expression,[57] together indicating that mTOR and miR-155-5p may coordinately modulate CNP through complex network involved in immune cells. Meanwhile, the FoxO pathway is considered to promote immune activity by negatively regulating the expression of immunosuppressive proteins including programmed death receptor 1 ligand (PD-L1).[58] Thus, our results suggest that immune factors are probably involved in CNP regulation networks facilitated by miRNAs.
We have obtained the first data for the comprehensive characterization of miRNAs in CNP prostate tissues with NGS technology and identified several differentially expressed miRNAs that are potentially associated with prostatic inflammation progression. We have also enriched several immune-associated pathways, indicating the possible immunological regulatory functions in CNP progression. Our data provide an important platform for future investigations aiming to characterize the concrete roles of specific miRNAs and immune factors in CNP initiation, progression, and the development of effective theragnosis.
CZL and LZ designed this experiment. LZ, YL, XGC, YZ, JC, LGZ, and HXD performed the research and analyzed the data. YL and ZYH wrote the manuscript, and then LZ, SF, and CZL revised the manuscript. All authors read and approved the final manuscript.
We are grateful to Dr. Yu Yin at theFirst Affiliated Hospital of Anhui Medical University (AHMU) for her technical assistance in histological research. This work was supported by the National Natural Science Foundation of China (Grant No. 81630019, 31430028, 81401518, and 81470986), Anhui Provincial Institutes for Translational Medicine (Grant No. 2017ZHYX02), Cultivation Project of Young Top-Notch Talent Support from Anhui Medical University (AHMU), and Funding for Distinguished Young Scientists of theFirst Affiliated Hospital of AHMU.
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Zhang L
Liu Y
Chen XG
Zhang Y
Chen J
Hao ZY
Fan S
Zhang LG
Du HX
Liang CZ
chronic nonbacterial prostatitis; immune; inflammation; miRNA; next-generation sequencing


Recommended Best Uses: Fenugreek supports healthy lactation, digestive wellness, blood sugar balance, heart health, and optimal liver and kidney function.
Warnings: Keep your licensed health care practitioner informed when using this product. Do not use if you are pregnant.


Westbury Amateur Swimming Club - Why Join?
If you enjoy swimming or want to learn to swim then joining Westbury Swimming Club is a great idea. Not only will you make loads of new friends but you'll receive the best coaching so you can improve your technique. Also the club has Swim21 accreditation which is the ASA's 'quality mark'.
Swimming is great exercise and the best sport for improving heart and lung power and muscle tone in general. It is also low impact and so puts little stress on growing joints.
You will learn all four strokes and also other skills like diving, turns, skulling etc.
Swimming is the best way to improve your general fitness for other sports like football, netball, athletics etc.
Knowing how to swim could save your life!
So contact us today to find out more about joining Westbury Swimming Club or drop by the pool to take a look.


Now is the best time to refresh your home and wardrobe. Hudson's Bay makes it possible for you to do just that without going broke especially when you log onto https://www.groupon.com/coupons/stores/thebay.com first. Shop today and enjoy more when you refresh with Hudson's Bay.


text:
Q: Opposite of aspnet_regiis -ga  I am looking to deny access using aspnet_regiis and was hoping if anyone knew how to do this. So the opposite of this:
aspnet_regiis -ga <UserName>

A: According to
http://forums.asp.net/t/1307213.aspx
this is not possible.
If this is a common requirement, use the -ga switch with a group that You then can add/remove users to/from.
/Andreas

redpajama_set_name: RedPajamaStackExchange


A local retailer has landed on the Texas Alcoholic Beverage Commission's (TABC) most recent Credit Law Delinquent List.
According to the TABC website liquor wholesalers are required to report any retailers with delinquent accounts; that information is used to compile the Credit Law Delinquent List. After the effective date of the list, wholesalers are not allowed to make sales or deliveries to these retailers until their accounts are current and TABC's records are cleared.
The full TABC Credit Law Delinquent List can be viewed HERE.


All data on the HR Portal is confidential and must only be used for the purpose for which it was generated.
The responsibility for inappropriate disclosure rests with the individual member.
Do not exclude or offend people or discriminate in any way and do not use abusive or offensive terminology.
A Member breaching these Conditions of Use may be subject to disciplinary action.


A short circuit at just 1.2 miles, the Indy circuit is challenging nonetheless.
Turn 1 is Paddock Hill bend and requires careful attention in order to achieve the best line. It's deceptive as the track goes up, then drops away from you sharply. The trick is to aim for the Marshal's hut to maximise your time upright for braking.
Turn 2, Druids, is the slowest corner. Braking is up-hill, so gravity aids you slowing the bike down. It can take a while to get your braking marker here but perseverance is worthwhile. The key here is a late turn in to allow you to set the bike up right for the acceleration down the hill to Graham Hill Bend.
Turn 3. Graham Hill bend is the first left-hander and it's always important to remember that the left hand side of your tyre will be a lot cooler than the right. In the wet, it's not the grippiest corner on the exit, so watch out for the rear end losing traction. The key here is to use ALL the track.
Cooper straight is fairly short but you can achieve a good acceleration by getting the bike upright as soon as possible after exiting Graham Hill.
Turns 5, 6 and 7 McLaren, Clearways and Clarke Curve. The initial right-hander of the series of turns is McLaren and the complex is often simply reffered to people as "Clearways". Depending on your bike, you'll be looking to enter this corner slower than might seem possible. This will allow you to take a tighter line through the first part of the right hander and then maximise the straight at the end. As you reach the point of the track where it goes downhill, allowing the bike to use all of the track, by gently gassing it will help you get the best run up onto the straight.
Remember, minimise the corners to maximise the straights!
Brabham straight. This is hardly worth calling a straight! However, on the entry you'll want to be as far left as you can as you pass the start of the Pit Wall (certainly on a bigger bike) as you will be able to use full throttle for longer.


text:
Lack of Clarity on Preventive Services Recommendations May Create Access Barriers
Vaccines / Preventive Services
Richard Hughes IV
Neil Rosacker
Reed Maxim
Ambiguous recommendations for preventive services are causing confusion among plans and providers on the clinically recommended preventive services that should be covered without cost sharing. A recent USPSTF recommendation on HIV prevention offers an opportunity to urge a recommending body to provide further clarity in its recommendation. Comments are due December 26.
Under Section 2713 of the Public Health Service Act as amended by the Affordable Care Act (ACA), commercial plans must cover and are prohibited from imposing cost sharing on preventive services and products receiving recommendations from federal bodies, including:
Health Resources and Services Administration (HRSA): provides coverage guidelines for access to preventive services such as contraceptives, as directed by the ACA
Advisory Committee of Immunization Practices (ACIP): provides advice and guidance on effective control of vaccine-preventable diseases
U.S. Preventive Services Task Force (USPSTF): reviews the evidence of effectiveness and develops recommendations for clinical preventive services
Although Congress intended for Section 2713 to ensure coverage without cost sharing for recommended services and products, payers may inconsistently apply this directive to coverage policies in practice. CMS has promulgated basic implementing rules for Section 2713; however, it has provided no overarching guidance or further specificity to the recommending bodies or agencies on how their respective and varied recommendations should be put into effect by payers. Consequently, payers may not implement recommendations as intended by Congress, potentially resulting in inconsistent coverage of preventive services.
Under Section 2713's implementing rules, plans must cover all contraceptive services without cost sharing. In 2015, CMS clarified that plans may choose to cover only 1 form of contraception without cost sharing in each of the FDA-approved methods. This additional guidance provided payers and providers with clear coverage directives for HRSA-approved contraceptives.
ACIP
By contrast, plans must cover without cost sharing all ACIP-recommended vaccines listed on the CDC's immunization schedule, which, unlike HRSA's comprehensive guidelines, includes multiple products across disease categories.
Additionally, ACIP's recommendations are often nuanced, referring to specific subpopulations or patient risk factors, or permissive, deferring to provider recommendations. As such, commercial plans have reported significant confusion regarding whether private insurance would cover category "B" vaccines, which are those recommended for individual clinical decision-making (e.g., meningococcal B vaccine).
Because of this lack of clarity, vaccine coverage under a category "B" ACIP recommendation can result in variability in patient access across health plans. For example, some large commercial payers limit coverage for vaccines given a category "B" recommendation. In some cases, payers provide vaccine coverage with no cost sharing only when an ACIP recommendation includes definitive language (e.g., "should", "shall", "is"), instead of a category "B" ("may") recommendation, despite the Section 2713 requirement that plans cover all ACIP-recommended vaccines.
Without further regulatory guidance or clarity from the ACIP in future recommendations, payers will remain uncertain about how to appropriately cover vaccines.
USPSTF
Plans are prohibited from imposing cost sharing on recommended services receiving an "A" or "B" rating from the USPSTF.
A recent draft recommendation on HIV prevention and screening from USPSTF illustrates the ambiguity that payers may face in making coverage policy based on USPSTF's recommendations. On November 20, the USPSTF issued draft "A" grade recommendations on both the prevention of human immunodeficiency virus (HIV) with PrEP and screening for HIV. It remains unclear whether the ancillary diagnostic tests that accompany PrEP and screening (e.g., renal function testing, serologic testing for hepatitis B and C virus, testing for other sexually transmitted infections, pregnancy testing when appropriate, and regular blood testing) would also be covered without cost sharing. Although the USPSTF acknowledges that these services are generally suggested, the draft recommendation does not explicitly include them under the PrEP "A" grade recommendation.
Moreover, if a service is billed together with an office visit and is the "primary purpose" of the patient's stay, then the plan may not impose cost-sharing on the office visit itself. However, if a service is billed together with an office visit but is not the "primary purpose," then the plan may impose cost-sharing requirements on the visit. This distinction demonstrates the challenge of managing ancillary services without clearer guidance, potentially creating access barriers that may undermine the intent of the grade "A" recommendation.
The ACA's guiding regulatory language does not require the USPSTF to be more explicit in its language nor does it clarify which components of a recommendation are subject to Section 2713, leading to further payer confusion regarding coverage requirements.
USPSTF is accepting comments on its proposed HIV recommendations until December 26. Stakeholders should urge USPSTF to provide further clarity on whether ancillary services are included in its final recommendations.
To receive more expert insights on preventive services, connect with us.
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redpajama_set_name: RedPajamaCommonCrawl


"All the things people should expect from an attorney."
Thank you Giovanna. I appreciate your advice. You were good at listening, maintaining a professional manner, and giving me advice. All the things people should expect from an attorney. If I decide to pursue legal remedy and need a lawyer, you will be hearing from me.


In an exclusive interview Monica Bedi's entire family based in Norway spoke freely to Star News' Jitendra Dixit. Monica is alleged to have married underworld don Abu Salem and is currently in jail in Hyderabad facing forgery charges.
Star News traveled across the world and tracked down Monica's family. In our endeavor to understand her personality we spoke to the people closest to her. This is the first and only ever media interview given by Monica Bedi's family. Just two weeks ago Star News was the only channel to speak with Samira Jumani, the first wife of underworld don Abu Salem.
This report provides an insight into the childhood and upbringing of Monica Bedi, the woman who for many years trotted to globe with Salem, and who is alleged to have married him. We visited her home in Norway, went to her school and spoke to people in Norway who knew her the best.
Monica's family has been living in Norway since 1975. Star News tracked them down and spoke with the entire family for several hours. During this time Star News tried to understand Monica's motivations for leaving her secure and comfortable home in Norway to try her luck in Mumbai's uncertain film world. He attempted to unravel the reasons for her getting involved with the dangerous underworld gangster.
Monica's family lives in Drammen, which is 45km from Norway's capital Oslo. Her father and brother together run a clothing store. Monica's father Dr. Prem Bedi was a doctor in Punjab's Hoshiarpur, before migrating to Norway thirty years ago.
Monica studied in Norway till college, before moving to England in 1992 for studies.
Star News tracked down the family gave them the opportunity to present their side of the story before the media.
Monica's father Prem Bedi at first insisted that he had "never met Abu Salem". He later conceded that he met him only while he was in prison, and the first time that he met him was after Monica and Salem's arrest in Portugal in 2002.
According to Prem Bedi, his daughter had referred to Salem in the past; however she never revealed his real name or identity to them. Instead she introduced them to him as 'Sanjay'. According to him, Salem insisted that his name was Sanjay when he met him in prison.
Monica according to her parents was an "innocent child", and was mislead by Salem. Her father insisted that "Monica never knew Abu Salem's true identity until she moved to Dubai".
Strongly denying the possibility of his daughter having married the infamous don, Prem Bedi insisted that his daughter had never married. She however mentioned that 'Sanjay' was a friend who had "helped her get a few films".
Prem Bedi said that he was coming to India next week to help his daughter, and in the meantime his brother based in Hoshiarpur was chipping in with the help that he could provide.
Monica's father said that while his daughter may have used a fake passport, she never did so willingly. Prem Bedi insisted that she only used a fake passport under pressure from Abu Salem.
The last time Prem Bedi met his daughter Monica, she was crying and saying "Papa I have got stuck".
Monica's father said that he met Salem for the first time when he was in Jail in Portugal. Salem respectfully said "Pranaam" to him, and introduced himself as Sanjay. Prem Bedi said that Salem never gave him any impression of being a notorious underworld gangster, but instead insisted that he was innocent and was being framed. Prem added that Salem was extremely courteous during his meeting.
During his conversation with Bedi, Salem respectlfully called Prem Bedi "Papa". He maintained that he was "framed" perhaps because of his being "connected with bad people". Salem however maintained that "he was innocent". In conversation Salem told Bedi that he had no involvement in the Sanjay Dutt case, as alleged by the police.
A clearly disturbed Bedi said that he would "call his daughter home, because she had no future left in India".
He also reminisced of her childhood days, and said that "Monica was a sharp child" and that "she never did anything wrong." He said that she was loved and regarded by people in Norway.
Jitendra also spoke to Monica's mother, Shakuntala, who was suffering from partial paralysis. Shakuntala spoke about how she often accompanied Monica during her shoots and also about how she put her through dance and driving classes. Obviously disturbed by the unfortunate turn of events, Shakuntala broke down on more than one occasion while speaking with Jitendra. She also insisted that Monica had not married Salem, saying that her daughter just got trapped, saying that children growing up in Norway were shielded from the harsh realities of the world and were thus innocent and easily misled.
Monica's sister in law also spoke of how Monica was left all alone in India ever since 1995 when her mother in law fell sick.
Star News spent a large part of two days in Drammen interacting with the Bedis. We visited their shop in the city, in which they sell Indian clothes and also film CDs to the 500 strong expatriate Indian community.
STAR NEWS, the first Indian news channel, is a joint venture between ABP TV - a 100 % subsidiary of ABP Pvt. Ltd. - and STAR India Pvt. Ltd., two of the biggest brands in Indian media. The Channel is best defined by a fair and balanced approach that combines prompt reporting with insightful analysis of news and current affairs. Living upto its motto of "Aapko Rakhe Aagey', STAR NEWS is a people's channel that focuses on relevant news in a cutting-edge format. Since its conception, the Channel has grown to command a viewership of over 20 million Indian homes. Today, STAR NEWS is available in the Indian subcontinent, South-East Asia, Central Asia, UK, USA and Australia, making it the first and only Global Indian News Channel.


text:
This administration is unfair to Christians, says CAN Youth President
From Musa Solomon, Yola.
Barely one week after the Director of Muslim Rights Concern (MURIC) Professor Ishad Akintola accused Christian Association of Nigeria (CAN) of sheltering corrupt Christians, the national president youth wing CAN, Daniel David Kadzai,has said that the dangerous line where Muslims are favoured over Christians in the body polity may not augur well for the system.
"It is sad and unfortunate that some Nigerians either due to sentiments, bigotry or cronyism chose to be blinded to the obvious reality of evident Islamic agenda and interest exhibited by the APC administration," he stated.
Daniel in a press statement made available on Saturday in Yola, Adamawa State, said that CAN as a body is not against fighting corruption, but that the war against corruption should be wholistic and not selective as the present situation.
"We are watching the outcome of the SGF and NIA Director General's drama and perhaps who takes over from them. We simply demand equity, social justice and the rule of law."
"To the pundits of Islamization or superiority of Isla, let us put the records straight. Christianity has never been known for mediocrity from the ancestry of the Jewish religion. From Abraham, Moses, Jacob, King David and the divine legate Jesus Christ the source of Christianity, there is no trace of mediocrity," he said.
He maintained that CAN will not be distracted by people he described as lacking in ability, objectivity to critically and deductively analyse the dangers inherent in government's approach to national issues.
"We are aware of the age long denigration of Christian leaders in Nigeria while promoting Muslim leaders at all cost," he stated.
The CAN youth president maintained further that the modesty and decency of Christian leaders over the years is evident in the leadership of General Yakubu Gowon, exhibited by the likes of Ernest Shonekan, balanced by Obasanjo's statesmanship and confirmed by Goodluck Jonathan who rather than resisting the conspiracy and manipulation to unseat him handed over to avoid a planned blood bath.
"The APC administration shamelessly truncated the trust of Christians to appoint over 90% Muslims in key positions. Is Nigeria for the Muslims? Are the Muslims the most qualified to rule Nigeria? Had Gowon in the 60s' or Obasanjo and Goodluck contemplated what Buhari did, would the Muslims have tolerated it…," he asked.
According to him the present administration has furthered the palpable divide and distrust between the Christians and Muslims.
"This administration and its Islamic protégés have not only marginalized Christians especially of the North but have also used its anti-curruption war to systematically shield Muslims, past and present corrupt leaders.," he said.
"We are aware some few Muslims are used as sacrificial lambs to deceive Christians. We refuse to be deceived.Unless the appointments are balanced, unless the corrupt Muslims are no longer tactically shielded, we can no longer trust this administration no matter the rhetorics of paid praise singers," he maintained.
Daniel pointed out that true nationalists are blind to religious, tribal and regional interests. "A reminder is that, despite the favor against Christians, the economy remains in shambles and Muslims are not immune to the hunger and poverty in Nigeria," he said.
UPDATE: President Buhari speaks on 82 freed Chibok school girls
40 years after, govt abandons communities that gave way for Kainji Dam


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Home — Essay Samples — Philosophy — Philosophers — Friedrich Nietzsche
Essays on Friedrich Nietzsche
Concepts: Morality, Beyond Good and Evil, Thus Spoke Zarathustra, On the Genealogy of Morality, Existentialism, Plato, God is dead
Freud's and Nietzsche's Views on Human Morality
Friedrich Nietzsche and Sigmund Freud offer bold critiques of human morality that greatly differ from the commonly accepted views of virtue and ethics. Both reject the idea of morality as an instinctive or natural element of human life. Rather, they contend that morality has been...
Friedrich Nietzsche 3 Pages
The Reactive Nietzsche: Contradictions in the Genealogy of Morals
In his Genealogy of Morals Nietzsche censures the members of the Judeo-Christian tradition for their "impotence." As a result of their impotence the descendents of this tradition (slaves, as I will call them to maintain some modicum of political correctness), have developed a hatred "to...
Negative Impacts of Science Proved by Friedrich Nietzshe
In The Gay Science, Friedrich Nietzsche critiques the shortcomings and possibilities of modern science. In this critique, Nietzsche analyzes the limits of science, the ways in which science falsifies life, and the motivation for a scientific pursuit of knowledge. Although Nietzsche does not categorically reject...
Friedrich Nietzsche 1 Page
Nietzsche's Assault on Modern Morality: the Kamehameha II Connection
German philosopher Friedrich Nietzsche is most remembered for the jarring statement, "God is dead," but to reduce him to such a slogan would be to truncate an intricate and complex critique of morality into just three short words. Nietzsche saw the morality of his own...
The All-Encompassing Will of Power
This world is the will to power and nothing besides. And you yourself are also this will to power and nothing besides. — Friedrich Nietzsche Human beings have struggled for centuries to explain their existence: some have thought it meaningless, others God-given. But since the...
Comparison Of The Views On Social stratification By Friedrich Nietzsche And W.E.B. DuBois
Social stratification does more than distinguish people by wealth and occupation; it also impacts the way people view themselves and analyze others around them. The mental workings of people are of particular interest to philosophers who propose theories on the holistic psychology of different demographics,...
Friedrich Nietzsche Philosophy Social Stratification 6 Pages
The fury of history: The Sound and The Fury from the perspective on Henri Bergson and Friedrich Nietzsche
I remember the first time I really heard classical music. As long as I can remember I have loved music, but growing up, no matter how many times my parents dragged me, kicking and screaming, to the symphony, or my piano teacher tried to teach...
Friedrich Nietzsche The Sound and The Fury 8 Pages
Actions done out of love are always beyond good and evil
Critical Analysis of Nietzsche's Claim: Actions Done out Of Love Are Always beyond Good and Evil Friedrich Nietzsche is one of the renowned German philosophers, cultural critics, and an essayist. Nietzsche's works on aesthetics, realization, truth, cultural theory, the meaning of existence, language, decency, history,...
Lightening Is Not a Wolf
The pattern of Nietzsche's On the Genealogy of Morals revolves around the deconstruction and consequent rebuilding of common thought relationships; within these differentiations we find both the thematic basis for and the huge diversity of Nietzsche's philosophic scope. Deleuze and Guattari assert that Nietzsche constructed...
On the Proper Uses and Improper Abuses of History for the Existing Individual
Friedrich Nietzsche's On the Advantage and Disadvantage of History for Life reads as a polemic against German historicism, the prevailing attitude of his time with respect to the value of history. Originally published as the second of four Untimely Meditations, this work offers a cultural...
Language and Nietzsche's Critique of Suffering
In his book On the Genealogy of Morality, Friedrich Nietzsche explores the relationship between suffering and guilt. Nietzsche argues that humans react to suffering by thinking that "someone or other must be guilty" (Nietzsche 94) for their suffering. Nietzsche critiques this phenomenon — that a...
Motives vs Principles of Morality in 'Gay Science'
In The Gay Science, Friedrich Nietzsche advocates skepticism and rejection of many traditional beliefs and values. This dismissal of commonly accepted societal norms is evident in his attack on morality and virtue in section 21 of the book. In this section, Nietzsche argues that the...
Friedrich Nietzsche: Were His Views Straightforward?
In Genealogy of Morals, Nietzsche details the shortcomings of the Judeo-Christian tradition. He focuses on the fact that the followers of this tradition have developed in such a way that impedes happiness and the normal will power of a strong individual. While Nietzsche mentions some...
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Q: Need help with GetShortPathName I have used GetShortPathName frequently with no problem.  However, now I'm having a problem.
In the past I have done, for example, @"C:\LongFoldername\LonfolderName\"
Now I'm using UNC like this @"\\MyServerName\TheLongFolderName"
But it does not get shortened.  It stays the same.
I have tried @"\\?\MyServerName\TheLongFolderName"
But that returns "".
I have read GetShortPathName Function But it did not help.
What am I missing?
Thanks!

A: I doubt very much that GetShortPathName will work on network names because they wouldn't be unique anymore and who would manage the mappings.
In a filesystem the short path name in guaranteed unique on the whole file system and it is created when the file with the long name is created or renamed. You cannot make ensure this in a network.
But even on a file system it is not guaranteed that a given file has a short file name, this may depend on system settings.

A: THe share name is too long - it must be under 11 chars for compability with GetShortPathName.  I think you may be warned of this when creating long share names in some instances.
http://groups.google.co.uk/group/microsoft.public.win32.programmer.kernel/msg/88454e39076262ab


I remember whenever I would try out a new recipe on my own or with my friends or kaki, we would follow the recipe word to word. But that was when we were learning. Now its getting the main ingredients and the method correct and then the recipe turns into my way, my method etc. Now, I have the confidence to explore and find easier ways. Don't get me wrong, certain recipes do have to be followed precisely. Can you imagine not adding baking powder or soda to the cake, over fermenting the bread dough, adding more liquid to the roti or paratha dough, adding more salt to the curry, etc etc. There are so many possibilities for a recipe to turn disastrous. I have had my share of failures and some have been beyond repair.
During my Polytechnic days, my friends and I would dine at each others place quite often. The fun part was that we would also prepare the dinner for us and the rest of the family. At that time, Tarla Dalal's books were our Bible. We would decide on the recipe, shop and cook.
One of the recipes we would make was her baked spaghetti with paneer balls in spinach sauce. So when Lakshmi Vimala suggested spinach and cream as my 2 secret ingredients for the shhh cooking secretly challenge, I wanted to make baked spaghetti with paneer balls in spinach sauce. However, as I planned out the recipe it took a whole new conversion as I didn't want to fry the balls. Also didn't want to make the spinach sauce as I didn't have enough spinach, but spinach I had to use. So from the basic recipe, I turned mine into different dish. Of course hubby dear would have loved it if I had fried the balls.
Place the washed spinach in the tray of the steamer and let it steam for 2-3 minutes or until it becomes a little soft.
Remove the spinach from the steamer and let it cool a bit.
In the meantime add the rest of the paneer ball ingredients to the paneer.
Squeeze out the water from the steamed spinach and add it to the paneer.
Mix and make it into a dough.
Grease your hands with little oil and make balls. I took about a tablespoonful of the mixture and got about 16 -18 balls.
Boil water in the steamer. Grease the steamer tray and place all the balls in the tray. Steam the paneer balls for 10 minutes.
Remove the balls from the steamer and keep it on the side till required.
Add the chopped onion and stir fry till it is soft and cooked.
Add garlic and stir fry for a few seconds.
Add the tomato puree and salt.
Cover the pan and cook till the sauce becomes thick.
Add pepper and let the sauce cool down a bit.
Add the fresh cream and mix well.
If you need to heat up the sauce again, then use low heat and stir the sauce frequently.
Boil water in a deep saucepan.
Add the pasta and boil for 8-10 minutes or as instructed on the package.
Stir occasionally while its cooking.
Drain out the pasta into a colander.
Sprinkle a bit of olive oil over the pasta and mix.
Spoon the pasta into the serving plates.
Top it with the sauce.
Sprinkle cheese over it and serve.
Fry the paneer balls if you like.
Can add the paneer balls to the sauce if you like, once the sauce is ready.
Use white or spinach sauce if you like.
Use spaghetti instead of tagliatelline.
Always boil pasta with plenty of water to avoid it from sticking to each other.
spinach balls look to good…hey dear i can't comment on some of your recent post… why?


Please find below details of the websites of suppliers such as nurseries, horticultural services and local links which I find very useful in my day to day work.
Specialised plant nursery based in Wenhaston, Suffolk.
Small local nursery offering an excellent range of plants, based near Saxmundham, Suffolk.


JRDN, an upscale surf 'n' turf spot located inside Tower23 Hotel in Pacific Beach, is the perfect spot for dining and exploring the numerous options for libations. Watch as the lead bartender Armando Garcia makes this creamy, gin-based cocktail, Slo Mo Fizz.
I did not grow up in San Diego. I grew up in very small town called, Coalinga, which is located in central California.
I've been in San Diego for 10 years now, working at JRDN most of that time and I don't plan on leaving anytime soon. I got into bartending because I was chasing after a girl. We are now married, with an awesome pup, and a home in Clairemont (insert boy girl holding hands with dog).
When I'm not bartending, I'm usually at the local gym tossing the weights around.
What's the origin of the Slo Mo Fizz?
The Slo Mo Fizz is actually a fruit forward cocktail, but also a balanced one. Making a balanced cocktail is essentially what separates a good cocktail from a great one. That is something I learned from great bartenders that I've worked with in the past such as, Juan Sanchez, Ryan Walsh, and Steven Tuttle. Not only are they incredibly talented bartenders, they are also very easy on the eyes. If there were to be some type of bartending beauty pageant we would have a very close 1st, 2nd, and 3rd- and that is one hell of a trifecta!
When I tell people the ingredients of the Slo Mo Fizz, I sometimes get a not so pleasant look, gin and egg whites? Which only adds to my enjoyment of seeing someone have a cocktail with egg mixed in for the first time. They think the cocktail is going to taste like egg, but it really doesn't. The egg just adds body to the drink, and it also gives a beautiful layer of froth. The key component in this cocktail is definitely the Slo Gin. Even though there's only half an ounce, it goes a long way. Slo Gins by nature, don't have much alcohol, usually 15 to 30 percent by volume. Therefore, I also used Plymouth gin, to give the cocktail some backbone, and also to raise the alcohol levels (insert smile emoji).
I would pair this cocktail with the pork belly. The acidity in the cocktail will cut through some of the richness in the pork belly. Also, the dark berry and plum flavors in the dish will also compliment the juniper, citrus and the herbaceous finish.
Tower23 Hotel, 723 Felspar St., Pacific Beach.


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Akwa Ibom gets new commissioner of police
TV Continental
The Inspector General of Police, Ag. IGP Mohammed Abubakar Adamu, has approved the posting of a new commissioner of police to Akwa Ibom State.
The newly posted police commissioner, CP. Zaki Ahmed, who is the 27th commissioner of police in Akwa Ibom State, took over from CP Bashir Makama who has been redeployed to Ogun State.
The command's public relations officer DSP MacDon Ogbeche who disclosed this in a press statement issued over the weekend said CP. Zaki until his redeployment to Akwa Ibom state was the commissioner of police, Rivers State.
Ogbeche in reeling out the CP's profile said, "The Police Boss for the State holds a B.Sc (Hons) degree in Sociology from the Usman-Danfodio University, Sokoto and a Masters Degree in Law Enforcement and Criminal Justice (MLC) from the prestigious Ahmadu Bello University, Zaria.
"He was appointed into the Nigeria Police Force as a cadet Assistant Superintendent of Police in 1988 and has served the Force in various capacities. Before his present appointment, he was the Assistant Commissioner of Police, Operations, Rivers State Police Command.
"Deputy Commissioner of Police. Operations, Delta State Police Command. Commandant, Police Detective College, Enugu. Commissioner of Police, Bauchi State Police Command.
"Before his present appointment, he was the Commissioner of Police, Rivers State Police Command. He has assumed duty as the 27th Commissioner of Police, Akwa Ibom State."
The command's spokesperson, who described CP Ahmed as a team player, said the commissioner has expressed his readiness to fight crime and criminality in the state and urged the people to cooperate with him by remaining law abiding.
FRSC warns drivers against reckless driving
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The Big VR Thread
By Lu , March 4, 2015 in Video Gaming
I only got to tune in for a little bit (looking forward to the VoD) but the frame rate on some of the stuff Dan was looking at was pretty lousy looking. I wonder if it was only on the stream, the second video output, or if the headset was showing the same unevenness. That seems like an issue if it's on the headset.
Dr Wookie
Zombie Thumb
They said that it felt fine when actually playing; this might be thanks to asynchronous time warp (which apparently smooths out juddering at less than optimal frame rates).
Hmm, it looks like Adr1ft is going to be compatible with Vive after all... how many petals are left? Apparently the gameplay isn't great, but I plan to get it anyway for the awesome space views .
Fingus
Thumb Citizen
So yesterday we announced the project (yeah, on April Fools Day, and on the Giant Bomb UPF stream at that) that Will Smith left Tested to work on. FooVR.
https://medium.com/will-smith/what-i-ve-been-working-on-84871ac38cea#.5jhbwk15j
What we've been tinkering on the last several months has been a framework for recording and publishing "shows"/"podcasts" with multiple people in the same VR space. Of special interest to us Thumbs readers; Episode 0 is an interview with our very own Jake and Sean about Firewatch, complete with a trip to VR version of Henry's lookout tower.
It's currently live on Oculus Home, and we're working on kicking out a final Vive SteamVR build. I think that version is going to be really impressive due to room tracking and the hand controllers, and internally we're developing and recording with the Vive so the project is tuned for it.
a wooden leg named "Smith"
I'm excited about this. I hope the episode is still up by the time I get some hardware that can run it.
Ben X
Formerly bbX1138
Can I just confirm that as a VR viewer of these recordings, you can walk around in them and stuff? Can you tea-bag the guests?
osmosisch
Quit gettin' mad
Location: Onnen
This sounds really cool, but I'm also quite sad about all the 'person X hasn't slept in days' toxic crunch BS.
@clyde
We have no plans of taking Ep0 down, and in case we do I'm sure I could kick you a build.
@Ben
Oculus doesn't do room tracking the way Vive does, so we had to settle with teleporting between preset points in the environment. But you can pick up a copy of Dangerous Hunks and slap Sean in the face with it if you so desire, or any number of other props around in Henry's Tower. (There may even be a Christmas Duck hidden somewhere in there)
@Osmosisch
Yeaaah, we're all kinda short on sleep. It ain't great. But that's the reality of 4 people with no money trying to make something ambitious that's really reliant on getting eyes on it early. In a perfect world we would have enough time and money to take it at a comfortable pace and make sure everyone's well rested and everything is well polished, but them's the breaks. I'd argue that us busting our ass on something we care about and want to make great is different from a AAA producer telling everyone to stay the weekend, or an art student mistakenly thinking the only way to be successful is to paint for 18 hours straight every day.
Some kind soul recorded Ep0 for the youtubes. Enjoy our hilariously janky demo vicariously though someone else's eyes.
Joewintergreen
Location: The Australias
https://twitter.com/joewintergreen/status/716455121190674432
Can you dig it?
Very cool, fun pilot episode and the VR interactions made it more enjoyable than I would have expected. You've probably considered it but you could have your own version on Youtube with like 3D and 360 video support. It's nice that someone uploaded episode zero but in the future you and other content creators would want to control that distribution.
Also reminds me of this thing that Microsoft released recently:
I understand. For short projects and such like this it can make sense to go above & beyond for a short period. I don't think that makes it OK to glorify that kind of thing, or frame it as normal. And yeah, of course it's different for a small outfit - but not in all ways. If through cultural osmosis this is the way all small studios operate, it makes it harder and harder for anyone to get into gamedev as a career while living a 'normal' life.
Anyway I'm sorry to add a sour note to what looks like a lovely moment for you. Congratulations again.
Awesome, I was worried this was just going to end up being some sort of boring conferencing solution.
Cordeos
Probable Regicide
In anticipation of my Vive shipping soon I broke out my old Virtual Boy.
manny_c44
I really enjoyed Mario tennis on virtual boy.
I borrowed the office DK2 last weekend and checked out the stuff in Oculus Home, including the Foo Show. It was pretty damn cool. Standing next to Sean Vanaman as he reads the blurb on the back of an in-game book and talks about the writing of said blurb was pretty out there. It feels like being there in the room with the guys, but then you realize that the room doesn't actually exist outside of the simulation. The mix of reality, Video game and podcast is unlike anything I've experienced before. A new medium. I look forward to future episodes.
Also played a whole bunch of Lucky's Tale. Very solid, fun platformer, tho for some reason the performance goes to hell on the first level of Chapter 4(Magma Mambo, I think it's called). The game was buttery smooth up till that point but then it immediately became unplayably laggy. :/
Pinball FX2 on the Rift. The footage is very shaky, but this video shows me some stuff I want to know about.
A demo of an Oculus Social prototype
Here is the other side of the conversation:
https://www.facebook.com/mike.booth/videos/vb.1369360149/10209079379126175/
Dragonfliet
Okay, so this is a silly implementation, but that they have a very functional, interesting and pretty good, actually, prototype of walking in VR spaces like this is really exciting: http://arstechnica.com/gaming/2016/04/for-vr-walking-forget-treadmills-just-stick-a-vive-wand-down-your-pants/
This article suggests that there may be significant VR news at the upcoming Google conference. A smart-phone that is built with the capability to capture 3D meshes of spaces would certainly perk my ears up.
http://arstechnica.com/gadgets/2016/04/the-google-io-2016-schedule-is-packed-with-virtual-reality-talks/
BTW: I just found out that I can make stereoscopic 360 captures of any scene in a Unity project using a free prefab from the asset store. That is awesome. I have only done a few tests, nothing that I'm especially proud of. But it's cool to be able to see 3d, panoramic captures of things I drop into Unity using the GearVR. 4096x4096 seems to be the best resolution for it. I've also been experimenting with using the ios Street View app to make photospheres for use in the GearVR. It's pretty fun.
Got my Vive shipping notice finally. Tuesday!
Dinosaursssssss
Got my Vive set up last night! Wasn't too bad to get going, had to update the controller firmware but after that things were pretty smooth. The area I have juuust barely fits the minimum for room scale (about 5' by 6' I think?)
So here's me playing Space Pirate Trainer in my pajamas and grinning like an idiot.
I've got an Oculus order in for the beginning of August. I'm paying attention to news about things like Project Tango, Samsung's dedicated device, and details about the Touch controllers in the meantime. If any dramatically benificial alternatives arrive before my creditcard gets charged, I'll jump ship. Either way, I hope the platform-companies prioritize cross-platform support between the devices because I'd love to see what hanging out with Thumbs in VR would be like.
kickinthehead
I just got my Vive this week and I'm loving it. I can't wait for the full version of Budget Cuts to come out, I had several moments where by the skin of my teeth I managed to take out a robot and it was that much more exciting because it involved me crouching on the ground, turning around and quickly throwing a knife at it.
FOO Show was a lot of fun to interact with, can't wait for more of that. The novelty of being next to the people overrides the jankiness of it that you see when watching a video replay of it.
But for the time being I'm actually most excited just to show the Vive to people. I showed one friend yesterday and it just blew his mind. Watching people play VR games is just a delight.
Got my Vive set up on saturday and am loving it. But just wanted to let people know that there is a really solid user mod for Minecraft that makes it playable with the vive. Highly recommended https://www.reddit.com/r/Vive/comments/4f5upo/minecraft_for_vive_in_vr_is_our_first_aaa_title/
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