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PubMedDate not supplied

Gene expression profiles of human BPH (II): Optimization of laser-capture microdissection and utilization of cDNA microarray.

Laser capture microdissection (LCM) enables the dissection of heterogeneous components of tissues, helping to investigate the molecular properties of these tissues. We have reported gene expression profiles in human benign prostatic hyperplasia (BPH) using LCM and quantitative real-time PCR. In the current work, we extended the previous observations. Firstly, we studied the relationship between the number of dissected acini and amplification by PCR, and found that androgen receptor (AR) and 18S rRNA transcripts were successfully amplified from total RNA obtained from one prostate acinus. Furthermore, LCM-dissected samples were applicable to methylation-specific PCR of E-cadherin promoter gene after bisulfite modification of genomic DNA. Next, we performed cDNA microarray analysis to screen gene expression profiles in the epithelium and stroma. RNA was amplified by T7-RNA polymerase and labeled with Cy3 and Cy5. Epithelium-related or stroma-related genes were identified through cDNA microarray. We confirmed true gene expression levels by quantitative real-time PCR. In epithelium, E-cadherin and serine protease 2, Kunitz-type gene expression levels were significantly elevated, while the connective tissue growth factor gene expression level was significantly elevated in stroma. Thus, LCM-dissected samples were applicable to various molecular examinations including methylation-specific PCR and cDNA microarray, and this will contribute to a precise understanding of the molecular profiles of prostate glands.

DNA↗
PubMed1991

CO2 laser used through the operating channel of laser laparoscopes: in vitro study of power and power density losses.

The purpose of this study was to measure the effects of several variables on energy transmission and power density through the CO2 laser laparoscope. The factors studied included the laser beam diameter, coupler optics, laparoscope lumen size, and absorption of the laser wavelength by the insufflation gas. The addition of CO2 insufflating gas to the operating channel at higher power settings not only reduced the energy transmitted to tissue by 35-58% with a 7.2-mm laparoscopic operating channel and by as much as 61% with a 5-mm operating channel, but also increased the spot size. This "blooming" of the laser beam definitely reduced power density at tissue and eliminated the pinpoint spot size needed for microdissection. Even under optimal conditions regarding lumen size and beam diameter, our data indicate a point of diminishing returns for power density above 40 W for the systems tested. Power densities obtainable at laparotomy were not possible. Clinically, this effect resulted in optimal cutting (vaporization) at low power settings and coagulation accompanying cutting at higher settings.

Carbon Dioxide↗
PubMed1990

Production of collagenase and tissue inhibitor of metalloproteinases (TIMP) by rat growth plates in culture.

Growth plate cartilage from normal and vitamin D-phosphate deficient (-VDP) rats was cultured to study the production of collagenase and tissue inhibitor of metalloproteinases (TIMP) in vitro. All tissues secreted latent collagenase into the medium at a constant rate during the 5 days in culture. Microdissected-VDP growth plates, containing predominatly hypertrophic cells, released up to 8-fold more collagenase into the medium than either intact-VDP or normal growth plates. TIMP was also secreted during the culture, but its rate of production was not as dependent on tissue type as collagenase. The tissue level of collagenase and TIMP before culture was compared with that found in conditioned medium and remnant tissue after culture. During the 5 day culture period microdissected-VDP growth plates, containing predominatly hypertrophic cells, produced 3-times more collagenase/microgram DNA over the starting level than either intact-VDP or normal growth plates. TIMP was never found in tissues after they had been cultured, but was present in all tissues before culture except those containing predominatly hypertrophic cells. The amount of TIMP required to block collagenase was calculated. Growth plates in culture produced enough TIMP to block all collagenase found in the medium and remnant tissue, while extracts of uncultured intact -VDP growth plates, and those divided to contain hypertrophic cells, had an excess of collagenase over TIMP. The results suggest that hypertrophic cells produce far more collagenase than other cells in the growth plate, but all cell types have about the same capacity to synthesize TIMP. As a result, increased collagenase synthesis by hypertrophic cells may surpass increases in TIMP synthesis and lead to collagen removal. This would allow for thinning of the longitudinal septa and expansion of the hypertrophic cells.

Animals↗
PubMed2004

Laser microdissection and DNA typing of cells from single hair follicles.

Isolation and identification of single cells from tissue samples or smears assume a great relevance in pathological and forensic applications; in this latter field, the possibility to identify a specific genetic profile can be obtained by short tandem repeat (STR) typing, allowing to achieve a scientific proof important in law courts. It is well known that DNA extraction may be performed from several tissue fragments, blood traces, spermatozoa as well as telogen hair. However, in the last case, few follicle cells are coupled to a great amount of keratin reducing the efficiency of DNA amplification. Recently, the introduction of laser microdissection technique has greatly improved the capability to select single cells without any cross-contamination. In the present report, we have performed a laser microdissection using a Leica AS LMD (Leica Microsystems, Germany), utilized on cutting the telogen hair in order to exclusively collect the lower part of the follicle and reduce keratin contamination. In this way we can accurately extract an adequate amount of DNA, successfully typed by STR profile.

DNA↗
PubMed2000

Detection of SYT-SSX fusion transcripts in both epithelial and spindle cell areas of biphasic synovial sarcoma using laser capture microdissection.

To investigate the distribution of tumour cells expressing the SYT-SSX fusion gene in biphasic synovial sarcoma, modified reverse transcription polymerase chain reaction (RT-PCR) analysis was performed using microdissected specimens from haematoxylin and eosin stained sections of archival paraffin wax embedded tissues. This modified RT-PCR included a stage with degenerate oligonucleotide primed (DOP) PCR, which randomly amplified cDNA after reverse transcription. SYT-SSX fusion transcripts were detected in both epithelial and spindle cell areas of all three biphasic synovial sarcomas examined. Subsequent sequence analysis confirmed that the detected messages were derived from the SYT-SSX1 fusion gene in two cases and from SYT-SSX2 in one. These results indicate that SYT-SSX fusion transcripts are found in both epithelial and spindle cell areas of biphasic synovial sarcoma, and RT-DOP-PCR-PCR analysis is a useful method for detection of extremely small amounts of mRNA in microdissected samples from archival formalin fixed, paraffin wax embedded tumour tissues.

Adenocarcinoma↗
PubMed1997

A new method for the isolation of myenteric plexus from the newborn rat gastrointestinal tract.

The myenteric plexus is not only essential for gastrointestinal functions, but it is also a very interesting model for the study of neuronal circuits and neuron-glial interrelationships and may be a valuable source of donor tissue, for grafting into different regions of the central nervous system. For both grafting and culture procedures it is a great advantage to obtain the maximum amount of tissue. To date, most studies have isolated the myenteric plexus by manual microdissection after collagenase digestion. Using this method, it has only been possible to obtain relatively small amounts of the myenteric plexus, mostly from the cecum and proximal colon of the guinea-pig or rat. We present here a new method, which enables much greater quantities of the plexus from the small intestine and colon to be obtained. The myenteric plexus of the entire small intestine can be isolated by a combination of enzymatic digestion and mechanical agitation. The method works from birth up to 3 week old pups, and with some modifications tissue from older or even adult animals can also be processed. Another advantage over the microdissection method is that the myenteric plexuses of the different parts of the intestine can be cultured and studied separately.

Animals↗
PubMed1986

Alpha 2-adrenoceptor stimulation and cellular cAMP levels in microdissected rat glomeruli.

A functional role for the numerically predominant glomerular alpha 2-adrenoceptors is unknown. In other tissues, activation of alpha 2-adrenoceptors inhibits adenylate cyclase activity. We therefore examined the effect of alpha 2-adrenoceptor stimulation with (-)-epinephrine (E) on the cellular cAMP concentration in glomeruli isolated by microdissection. Parathyroid hormone (1-34 PTH), prostaglandin E2 (PGE2), histamine, serotonin, or adenosine, in the presence of 3-isobutyl-1-methylxanthine (phosphodiesterase inhibitor) and propranolol, was used to activate adenylate cyclase in single intact rat glomeruli. alpha 2-Adrenoceptors were activated with varying concentrations of E (37 degrees C, 2 min). In the presence of PTH-stimulated cAMP production, alpha 2-adrenoceptor activation with E (5 X 10(-7) to 5 X 10(-6) M) suppressed cellular cAMP levels in a dose-dependent fashion with the maximum at 30%. This suppression by E was inhibited by 5 X 10(-6) M yohimbine but not by 5 X 10(-6) M prazosin, confirming alpha 2-adrenoceptor mediation of this effect of E. Consistent with the above findings, the specific alpha 2-adrenoceptor agonist BHT933 inhibited PTH-stimulated cAMP accumulation. E also inhibited cAMP accumulation stimulated by serotonin. However, E did not suppress the PGE2-, histamine-, or adenosine-stimulated increase in cellular cAMP in the glomerulus. Activation of alpha 2-adrenoceptors inhibits cAMP formation stimulated by PTH or serotonin but not by PGE2, histamine, or adenosine in the rat glomerulus. Thus, the ability of alpha 2-adrenoceptors to inhibit adenylate cyclase appears to be hormone and probably function specific.

Adenosine↗
PubMed2006

Epigenetic heterogeneity of high-grade prostatic intraepithelial neoplasia: clues for clonal progression in prostate carcinogenesis.

High-grade prostatic intraepithelial neoplasia (PIN) is the most likely precursor of prostate adenocarcinoma, but the frequency and timing of epigenetic changes found in prostate carcinogenesis has not been extensively documented. Thus, the promoters of three genes (APC, GSTP1, and RARbeta2) involved in prostate carcinogenesis were tested by quantitative methylation-specific PCR in tissue DNA from 30 prostate carcinomas, 128 high-grade PIN lesions, and 30 normal prostate tissue samples dissected from 30 radical prostatectomy specimens using laser capture microdissection. The percentage of methylated alleles (PMA) was calculated for each gene, and hierarchical cluster analysis was used to define the degree of similarity of epigenetic alterations among the various samples. We found that PMA values of APC and RARbeta2 were higher than those of GSTP1 in all three types of tissue samples and median PMA values for all three genes were higher in prostate cancer. By cluster analysis, 26 of 30 prostate carcinomas and 82 of 128 high-grade PIN lesions were grouped in the "high methylation" branch, whereas 24 of 30 normal prostate tissue samples were allocated in the "low methylation" branch. Although high-grade PIN lesions are epigenetically more similar to prostate carcinoma than to normal prostate tissue, paired prostate carcinoma and high-grade PIN lesions did not always segregate together. We concluded that APC and RARbeta2 hypermethylation is frequent in normal prostate tissue and the progressive enrichment in cells carrying methylated alleles observed in high-grade PIN and prostate carcinoma is consistent with clonal progression. Because GSTP1 promoter methylation is mainly observed in prostate carcinoma and some high-grade PIN lesions, it represents an important marker for the transition of in situ to invasive neoplasia.

Aged↗
PubMed2003

High frequency of human herpesvirus 6 DNA in multiple sclerosis plaques isolated by laser microdissection.

The frequency of human herpesvirus 6 (HHV-6) DNA was assessed in autopsy material from multiple sclerosis (MS) plaques and normal-appearing white matter (NAWM) from brains of persons with MS, healthy brains, and brains of persons with other neurologic diseases. Specific areas from formalin-fixed, paraffin-embedded brain tissue samples were isolated by laser microscope. DNA was extracted from laser microdissected brain material, and HHV-6 genomic sequences were amplified by nested polymerase chain reaction. We analyzed 44 NAWM samples and 64 MS plaques from 13 patients with MS, 46 samples from 13 patients with non-MS neurologic disorders, and 41 samples from 12 healthy control brains. Of the 44 NAWM samples, 7 (15.9%) were positive for HHV-6 DNA sequences, versus 37 (57.8%) of 64 MS plaques (P<.0005). HHV-6 DNA was detected in 10 (21.7%) of 46 samples from patients with non-MS neurologic disorders and in 11 (26.8%) of 41 samples from patients without known neurologic disease. Although the frequency of HHV-6 DNA did not differ significantly by sample type, HHV-6 DNA was significantly more common in MS plaques, suggesting that HHV-6 may play a role in MS pathogenesis.

Adolescent↗
PubMed1999

Characterization of CD44v6 isoforms in head-and-neck squamous-cell carcinoma.

CD44 splice variants, especially those containing the v6 domain, are assumed to play a critical role in the malignant progression of many human tumors. This concept was based on (i) the aberrant expression of CD44v6 in malignant cells, often encoded by alternatively spliced transcripts, and (ii) the absence of CD44v6 expression in corresponding normal tissues. Remarkably, data on CD44v6 expression in squamous cells do not support this hypothesis: the v6 domain is highly expressed in normal squamous tissues and down-regulation has been described in the majority of squamous-cell carcinomas of the head and neck (HNSCC). In this study, we have compared the expression of v6 in normal oral mucosa and HNSCC in a qualitative and quantitative way. Immuno-histochemistry was performed with 3 different anti-v6 antibodies (U36, U39 and VFF18) on a large panel of HNSCC cell lines and tumors. The v6-encoding splice variants were characterized by screening a cDNA library of a human HNSCC cell line and by RT-PCR on HNSCC cell lines, microdissected normal mucosa and primary as well as metastatic HNSCC tissue. The results revealed that there is no, or only marginal, down-regulation of CD44v6 in HNSCC. About 97% of the primary HNSCC tumors exhibited a high and homogeneous staining pattern (U36, 270/277; U39, 268/277 tumors with more than 50% positive cells). Furthermore, the v6-containing CD44 splice variants present in HNSCC primary tumors and metastases were identical to those expressed in normal mucosa. Our data indicate that v6-containing CD44 splice variants do not play a role in the malignant progression of HNSCC.

Alternative Splicing↗
PubMed2000

PSGR, a novel prostate-specific gene with homology to a G protein-coupled receptor, is overexpressed in prostate cancer.

PSGR, a new prostate tissue-specific gene with homology to the G protein-coupled odorant receptor gene family, has been identified. Here we report the characteristics of the predicted protein sequence of PSGR and its prostate tissue specificity and expression profile in human prostate cancer and matched normal tissues. Using multiple tissue Northern blots from over 50 different tissues, PSGR expression was restricted to human prostate tissues. Paired normal and tumor specimens from 52 primary prostate cancers, obtained by laser capture microdissection or manual microdissection, were analyzed for PSGR expression by semiquantitative and real-time PCR assays. The differential expression of PSGR between normal and tumor tissues was highly significant (P < 0.001), and 32 of 52 (62%) matched prostate specimens exhibited tumor-associated overexpression of PSGR. Of note, there was very little or no expression of PSGR in many normal specimens in comparison with the generally high expression of PSGR seen in matched tumor specimens. In situ hybridization assays showed restricted PSGR expression in the epithelial cells of the normal and tumor tissue sections. Restricted expression of PSGR in prostatic epithelial cells, overexpression of the PSGR in a significant percentage of prostate cancers, and the predicted protein sequence of PSGR with seven transmembrane domains provide a foundation for future studies evaluating the potential of PSGR as a prostate cancer gene expression marker and the utility of PSGR protein as a novel target for developing immunotherapeutic strategies for prostate cancer.

Amino Acid Sequence↗
PubMed2005

Mutations of mitochondrial 12S rRNA in gastric carcinoma and their significance.

AIM: To detect the variations of mitochondrial 12S rRNA in patients with gastric carcinoma, and to study their significance and the relationship between these variations and the genesis of gastric carcinoma. METHODS: PCR amplified mitochondrial 12S rRNA of 44 samples including 22 from gastric carcinoma tissues and 22 from adjacent normal tissues, was detected by direct DNA sequencing. Then laser capture microdissection technique (LCM) was used to separate the cancerous cells and dysplasia cells with specific mutations. Denaturing high performance liquid chromatography (DHPLC) plus allele-specific PCR (AS-PCR), nest-PCR and polyacrylamide gel electrophoresis (PAGE) were used to further evaluate this mutant property and quantitative difference of mutant type between cancerous and dysplasia cells. Finally, RNAdraw biosoft was used to analyze the RNA secondary structure of mutant-type 12S rRNA. RESULTS: Compared with Mitomap database, some new variations were found, among which np652 G insertion and np716 T-G transversion were found only in cancerous tissues. There was a statistic difference in the frequency of 12S rRNA variation between intestinal type (12/17, 70.59%) and diffusive type (5/17, 29.41%) of gastric carcinoma (P<0.05). DHPLC analysis showed that 12S rRNA np652 G insertion and np716 T-G transversion were heteroplasmic mutations. The frequency of 12S rRNA variation in cancerous cells was higher than that in dysplasia cells (P<0.01). 12S rRNA np652 G insertion showed obviously negative effects on the stability of 12S rRNA secondary structure, while others such as T-G transversion did not. CONCLUSION: The mutations of mitochondrial 12S rRNA may be associated with the occurrence of intestinal-type gastric carcinoma. Most variations exist both in gastric carcinomas and in normal tissues, and they might not be the characteristics of tumors. However, np652 G insertion and np716 T-G transversion may possess some molecular significance in gastric carcinogenesis. During the process from normality to dysplasia, then to carcinoma, 12S rRNA tends to convert from homoplasmy (wild type) to heteroplasmy, then to homoplasmy (mutant type, np717 T-G).

Base Sequence↗
PubMed2005

Laser-assisted microdissection, techniques and applications in pathology (review).

Innovations in molecular medicine provided sophisticated tools for analysis of the pathogenesis of diseases. Much emphasis is put on examination of alterations in affected organ systems. However, since in vivo tissues are inherently complex mixtures of different cell types, specific molecular data on individual cell populations are difficult to obtain. The advent of laser assisted microdissection (LAM) now allows efficient isolation of pure cell populations and even of single cells from mixed tissues. The most promising aspect of LAM is its combination with different molecular downstream analyses of microdissected cells at the levels of the genome, the transcriptome and the proteome. This review focuses on the two basic techniques of laser-assisted microdissection and on its applications in molecular pathology.

Genomics↗
PubMed1999

Differential expression and activity of tissue-nonspecific alkaline phosphatase (TNAP) in rat odontogenic cells in vivo.

Among the four existing isoforms of alkaline phosphatase (AP), the present study is devoted to tissue-nonspecific alkaline phosphatase (TNAP) in mineralized dental tissues. Northern blot analysis and measurements of phosphohydrolase activity on microdissected epithelium and ectomesenchyme, in situ hybridization, and immunolabeling on incisors confirmed that the AP active in rodent teeth is TNAP. Whereas the developmental pattern of TNAP mRNA and protein and the previously described activity were similar in supra-ameloblastic and mesenchymal cells, they differed in enamel-secreting cells, the ameloblasts. As previously shown for other proteins involved in calcium and phosphate handling in ameloblasts, a biphasic pattern of steady-state TNAP mRNA levels was associated with additional variations in ameloblast TNAP protein levels during the cyclic modulation process. Although the association of TNAP upregulation and the initial phase of biomineralization appeared to be a basic feature of all mineralized tissues, ameloblasts (and to a lesser extent, odontoblasts) showed a second selectively prominent upregulation of TNAP mRNA/protein/activity during terminal growth of large enamel crystals only, i.e., the maturation stage. This differential expression/activity for TNAP in teeth vs bone may explain the striking dental phenotype vs bone reported in hypophosphatasia, a hereditary disorder related to TNAP mutation. (J Histochem Cytochem 47:1541-1552, 1999)

Aging↗
PubMed2002

Optimizing gene expression analysis in archival brain tissue.

Analysis of gene expression in the brain is a valuable tool to study the function of the brain under normal and pathological conditions. Although there are many techniques used to measure gene expression the validity of any such experiment is directly related to the quality of the RNA in the samples. The most readily available source of human brain tissue is post-mortem and while frozen tissue is sometimes available, most archived tissue is fixed and paraffin-embedded. The use of fixed tissue for expression analysis introduces variables, which must be considered in the experimental design. In addition, factors associated with clinical variability of the patient and with tissue procurement can affect RNA transcript levels. In order to illustrate the effects of two common tissue fixatives, formalin and ethanol, on the quality of RNA for expression analysis we compare RNA extracted from these fixed tissues to the gold standard, flash-frozen tissue. We describe RNA extraction from fixed tissue and ways to assess the quality or intactness of the RNA using reverse transcription combined with polymerase chain reaction amplification. An advantage of using archived tissue is the ease with which single cells or subpopulations of cells can be obtained by laser microdissection. The successful isolation of RNA from microdissected cells is also presented. From our results and a review of the literature we conclude that RNA from fixed tissues is a viable source of RNA for expression analysis which should enable new experimental approaches and discoveries as long as attention is given to variables that can affect RNA at all levels of analysis.

Archives↗
PubMed2000

Detection of gene amplification in archival breast cancer specimens by laser-assisted microdissection and quantitative real-time polymerase chain reaction.

Gene amplification is one of the most important mechanisms leading to deregulated gene expression in cancer. The exact quantitative detection of this frequent genomic alteration in solid tumors is often hampered by an admixture of nonneoplastic bystander and stroma cells. To overcome this obstacle and to develop an objective quantitative method we have combined laser-assisted microdissection of tumor cells with the novel 5'-exonuclease-based real-time polymerase chain reaction (PCR) assay. The latter method enables the highly reproducible exact quantification of minute amounts of nucleic acids. As a model system amplification of c-erbB2/Her-2/neu gene and the adjacent topoisomerase IIalpha gene was determined in paraffin-embedded breast cancer specimens (n = 23) after immunohistochemical labeling and laser-based microdissection of tumor cells. The high sensitivity of real-time PCR enabled the reliable and objective detection of low-level amplifications in as few as 50 cells from archival tissue sections. Low-level amplifications were shown to escape from detection unless tumor cells were isolated by microdissection. In selected cases intratumor heterogeneity was demonstrated using areas of approximately 50 to 100 cells. This novel approach combining immunohistochemistry, laser microdissection, and quantitative kinetic PCR allows morphology-guided studies in archival tissue specimens and will enable the exact quantification of gene copy numbers in even small and precancerous lesions.

Antigens, Neoplasm↗
PubMed2006

Transplantation of human embryonic stem cells to the chick embryo.

The traditional methods of studying the differentiation of human embryonic stem cells (hESCs) are to differentiate them in vitro or in immune-deficient mice as teratomas. The chick embryo is a well-studied and accessible experimental system that has been shown to permit the development of mammalian cells, including murine embryonic stem cells. We therefore performed experiments transplanting colonies of hESCs into organogenesis-stage chick embryos, hoping this might provide a novel system for studying the developmental programs and decisions of these important cells. hESCs, constitutively expressing green fluorescent protein or labeled with the dye CFDA, were used to allow the following the hESC in living embryos. As a first step, we chose to transplant hESCs into the trunk of chick embryos, both into and instead of developing somites. Our first results showed that hESCs survive, migrate, and integrate into the tissues of the chick embryo. Some of the hESCs differentiate and the type of embryonic microenvironment that the implanted cells were exposed to modified their differentiation. Therefore, this hESC-chick embryo system has potential for complementing studies in rodents and in vitro, and uniquely, to shed light on early processes in the development of human cells in the embryonic context.

Animals↗
PubMed2004

In situ-RT and immunolaser microdissection for mRNA analysis of individual cells isolated from epilepsy-associated glioneuronal tumors.

Analysis of gene transcription patterns in complex tissues with multiple cell types is a major challenge. Examination of cellular subpopulations for molecular expression patterns requires their isolation from other surrounding cells. We performed single-cell mRNA analysis to study gangliogliomas obtained from patients with pharmacoresistant epilepsy (n = 6), in order to characterize CD34 expressing cells found in these tumors. Fresh-frozen biopsy tissue was analyzed by initial in situ-reverse transcription (in situ-RT) with oligonucleotides, subsequent immunohistochemistry (IHC) to identify specific cell types, and laser-capture microdissection (LCM, herein termed immuno-LCM) to obtain antigen-expressing cell subpopulations. Isolated complementary DNAs (cDNAs) were then quantified by real time-polymerase chain reaction (RT-PCR). We found that short- vs long-term incubation time for the IHC step did not adversely affect cDNA abundance obtained by subsequent RT-PCR, either for high-abundance (glyceraldehyde dehydrogenase; GAPDH), medium-abundance (glial fibrillary acidic protein; GFAP), or low abundance (neurofilament; NFM) gene transcripts. We also determined that the cellular specificity of capture was excellent, as determined by lack of contamination between different immuno-LCM cell isolates. We were therefore able to examine the lineage expression markers of isolated CD34-expressing cells. We observed coexpression of CD34 and NFM, suggesting neuronal differentiation of the CD34 expressing cellular elements in gangliogliomas. Expression markers for other cellular types (myelin basic protein for oligodendroglia; GFAP for astrocytes) were negative. Our findings support the hypothesis that gangiogliomas contain neuronal elements with compromised or atypical differentiation. We consider that this in situ-RT/immuno-LCM protocol is of general applicability, whereby virtually any primary antibody can be used to facilitate capture of individual cells in tissue sections for molecular analysis.

Adolescent↗