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Review ARTICLE
Human rhabdomyosarcoma cell lines for rhabdomyosarcoma research: utility and pitfalls
- 1Department of Pediatrics, Duke University Medical Center, Durham, NC, USA
- 2Duke University School of Medicine, Durham, NC, USA
- 3Department of Pediatrics, University of Virginia School of Medicine, Charlottesville, VA, USA
- 4Laboratory of Pathology, National Cancer Institute, Bethesda, MD, USA
- 5Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC, USA
Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood and adolescence. Despite intergroup clinical trials conducted in Europe and North America, outcomes for high risk patients with this disease have not significantly improved in the last several decades, and survival of metastatic or relapsed disease remains extremely poor. Accrual into new clinical trials is slow and difficult, so in vitro cell-line research and in vivo xenograft models present an attractive alternative for preclinical research for this cancer type. Currently, 30 commonly used human RMS cell lines exist, with differing origins, karyotypes, histologies, and methods of validation. Selecting an appropriate cell line for RMS research has important implications for outcomes. There are also potential pitfalls in using certain cell lines including contamination with murine stromal cells, cross-contamination between cell lines, discordance between the cell line and its associated original tumor, imposter cell lines, and nomenclature errors that result in the circulation of two or more presumed unique cell lines that are actually from the same origin. These pitfalls can be avoided by testing for species-specific isoenzymes, microarray analysis, assays for subtype-specific fusion products, and short tandem repeat analysis.
Rhabdomyosarcoma (RMS) is a malignancy that arises from skeletal muscle precursors (1). It is the most common type of soft tissue sarcoma in children and adolescents less than 20 years old, with an incidence of 4.5 cases per million children/adolescents per year (2). There are two major subtypes of RMS, embryonal and alveolar, which differ markedly in their outcomes. Embryonal RMS usually presents in children less than 10 years old and has a 5-year survival of close to 75%. Alveolar RMS, on the other hand, occurs at about the same incidence throughout childhood and adolescence and is associated with a poorer prognosis with a 5-year survival rate of less than 50%. Patients with refractory or relapsed RMS have an even worse prognosis, with survival ranging from 10 to 30% (3).
Despite numerous clinical trials, outcomes for high risk RMS have not improved significantly in the last 30 years. Given the relatively small population of children diagnosed with this disease annually, and the number and timing of new clinical trials, testing new potential treatment agents is limited. In vitro cell-line research and in vivo xenograft models aid in the basic and preclinical effort to identify potential new treatments. If shown promising, these agents can then be taken into human clinical trials, and compared to standard of care agents. The Pediatric Preclinical Testing Panel (PPTP) is an initiative formed by the National Cancer Institute, working to further characterize and validate available cell lines in multiple kinds of pediatric cancer, including RMS so that preclinical evaluations of new chemotherapeutic agents can be tested (4). Currently, there are 18 embryonal and 12 distinct alveolar human RMS cell lines described in the literature that have been used in more than one study by more than one research group. They differ in their origins, histologies, karyotypes, and methods of validation. They are described below and summarized in Table 1. There are also 16 human RMS cell lines that have been described and used by single research groups (5–17); these are listed in Table 2. [Of note, during revisions of this article an independent list of human and murine RMS cell lines was published (18).] The current article aims to summarize the published RMS cell lines, aid scientists in deciding which lines may be applicable to their research projects, and highlight important historical information and limitations for specific cell lines.
Embryonal RMS Cell Lines
CCA was derived from the biopsy of a "vesical" recurrence of embryonal RMS in an 8-year-old Caucasian male (19). Multiple chromosomal rearrangements were identified upon karyotype analysis, with additional defects on chromosomes 1, 4, 6, 8, 9, 10, 11, 12, and 13 (20). CCA cells express vimentin and desmin. These cells can be used to generate xenografts in nude mice subcutaneously or intramuscularly, and form lung metastases when injected intravenously after pretreatment of the mice with cyclophosphamide. CCA cells harbor a Q61L mutation in KRAS (21). CCA has been grown in modified Dulbecco's medium (DMEM) with 10% fetal bovine serum (FBS) (22). As with cell culture in general, it is up to the investigator whether prophylactic antibiotics penicillin and streptomycin are to be included during routine culture.
This cell line was derived from a primary tumor with an embryonal histology and expresses MyoD, myogenin, and desmin (at very low levels). It was originally developed by Dr. Hajime Hosoi and can be grown in DMEM with 10% FBS (23).
HX170c was established from a paratesticular tumor of a 5-year-old Caucasian male. The patient had been previously treated with vincristine, adriamycin, cyclophosphamide, and radiotherapy. The tumor specimen was designated RMS based on the presence of desmin intermediate filaments, and assigned embryonal histology. HX170c was established simultaneously as a cell line in culture and xenograft directly from the biopsy of a local recurrence 2 months prior to the patient's death. At early passages, HX170c was cultured in vitro on a lethally irradiated layer of mouse fibroblast 3T3 cells; the cell line was later tested and found to contain only human cells. While the tumor biopsy was positive for desmin staining, the HX170c cell line was almost completely negative for this marker when cultured in vitro, except when grown to almost complete confluence. Interestingly, HX170c subcutaneous mouse xenografts regained desmin positivity and also appeared histologically similar to the initial tumor biopsy. HX170c cells are near diploid (50 ± 6) (24).
JR-1 was derived from a lung metastasis of a 7-year-old female with a primary uterine mass, previously treated with vincristine, cyclophosphamide, dactinomycin, and radiation therapy (25). It has a poorly differentiated embryonal histology, based on histological appearance of the biopsy specimen. This cell line was grown in RPMI-1640 medium supplemented with FBS. Chromosome analysis revealed a near tetraploid number of chromosomes, ranging from 44 to100 per cell, with 80% showing chromosomal alterations, the most common of which was 13q−. JR-1 cells have been shown to have mutation of the TP53 gene (26). Antibody staining showed similarities between the cell line and the original tumor, as both stained positive for desmin, vimentin, glycolipid, ganglioside Gq, thy-1 and Gp44; and negative for GFAP, cytokeratin, neurofilament RT97, and myoglobin.
KF-RMS-1 has an embryonal histology (based on the histologic appearance of its tumor source) and is derived from a bone marrow metastasis. It is grown in DMEM with 10% FBS (22).
RD was derived directly from biopsy specimens of a 7-year-old female with a pelvic RMS previously treated with cyclophosphamide and radiation and found to have refractory disease (27). It has an embryonal histology based on histologic appearance of the tumor biopsies and the cultured cells, and 51-hyperdiploid chromosomes. RD has been shown to have amplification of the MYC oncogene (28), Q61H mutation of NRAS (29), and homozygous mutation of TP53 (30, 31). Houghton et al. studied tumor size following injections of various chemotherapy agents and found that RD demonstrated growth inhibition to vincristine and cyclophosphamide, but no other agents tested (32). RD was also used to test tolfenamic acid, and showed decreased tumor size, decreased cell migration, and decreased expression of Sp specificity transcription factors after treatment (33). As mentioned in the Section "Discussion," the cell line TE671, which was originally thought to be a medulloblastoma line, was later shown through cytogenetic analysis and DNA fingerprinting to likely be a subclone of RD cells (34). RD cells are one of the most commonly used cell lines in RMS research, can be obtained from ATCC, and are grown in Eagle's medium with 10% FBS (35).
The RH6 cell line was established in the laboratory of Dr. Edwin Douglass at St. Jude's Children's Research Hospital (SJCRH) in Memphis, TN directly from a 16-year-old male with an infra-renal lymph node (personal communication, Susan Ragsdale, SJCRH). Of note, it has no relation to the Rh6 xenograft listed in (36). When evaluated using cDNA microarray analysis of the cell line versus original tumor, the RH6 cell line did not cluster within the RMS family of tumors and therefore, is not optimal for testing of this cancer type (37). More recently, other studies do mention that this cell line expresses myogenic markers (38).
Of historical and nomenclature import, all of the "RH" cell lines (sometimes denoted "SJRH") were generated at SJCRH and can be obtained from a representative of that institution, or from Dr. Peter Houghton at Nationwide Children's Hospital, Columbus, OH, USA who has maintained these lines. However, during the years in which human RMS cell lines were first being established, two separate laboratories were deriving and naming the cell lines independently. Cell lines established in the laboratory of Dr. Peter Houghton were generated by directly transplanting tumor into mice to establish xenografts, and originally labeled "Rh." Cell lines established in the laboratory of Dr. Edwin Douglass (and subsequently Dr. David Shapiro) were generated directly from patient samples and labeled "RH." The upper or lower case designations for these cell line names have since been interchanged in the literature and are no longer preserved.
RH12 was derived from a xenograft created from a buttock tumor in a previously untreated patient. The original tumor and corresponding xenograft have an embryonal histology based on their spindled appearances (36). Chromosome analysis showed the addition of multiple chromosomes including 2, 3, 7, 8, 9, 10, 11, 12, 13, 18, 21, and dup (1)(q11q31), +del(2)(q23) (39). In a study whereby tumor growth was measured against various chemotherapy agents, RH12 showed volume regression with vincristine, DTIC, cyclophosphamide, doxorubicin, cis-DDP, and busulfan. No growth inhibition was seen with the use of dactinomycin, mitomycin-C, or bleomycin (32). RH12 xenografts were also used to test the effect of vincristine plus topotecan on tumor growth; the combination showed improvement over topotecan alone but was no better than vincristine alone, which had progression of disease in only 1/7 mice tested (40).
The RH14 xenograft was derived from an inguinal tumor in a patient previously treated with vincristine, dactinomycin, and cyclophosphamide, with complete response and no evidence of disease for 2.5 years, then recurrence. It had an embryonal histology based on histologic appearance of the original tumor and the xenograft, both of which showed differentiation and cross striations (36). In evaluations of tumor size injections of various chemotherapy agents, RH14 had complete regression with the use of vincristine and significant regression after cis-DDP, DTIC, and mitomycin-C. No growth inhibition was seen with dactinomycin or doxorubicin (32). It is important to note that RH14 was only established as a xenograft, and to our knowledge no cell line is available.
RH36 (Also Referred to as Birch)
RH36 has an embryonal histology (based on the histologic appearance of the original tumor) and was derived from a paratesticular relapse in a 15-year-old male (41). The PPTP evaluated RH36 and found tumor xenograft inhibition in response to cyclophosphamide (41). RH36 has also been referred to as Birch in the literature (42) and is grown in RPMI-1640 with 10–20% FBS (43).
RMS559 cells were established in the laboratory of Dr. Jonathan A. Fletcher, Department of Pathology, Brigham and Women's Hospital, Boston, MA, USA (44, 45). They can be grown in DMEM with 10% FBS. This cell line was identified to have a V550L FGFR4 mutation by direct sequencing (46).
RMS-YM was derived from a relapsed abdominal mass of 2-year-old Japanese boy, at second relapse (47). The tumor was believed to originate from the urachus and was given an embryonal classification based on histology. The patient was treated with chemotherapy and radiotherapy, but died 3 years later due to multi-organ metastasis. RMS-YM is capable of forming subcutaneous xenografts in nude mice that appear histologically similar to the primary tumor. These cells stain positive for desmin and myoglobin. The karyotype of RMS-YM is 55, XY, −1, −2, −7, +8, −9, −10, −11, −16, −20, −22, +17 mars. Analysis of the MYCN and RAS genes showed no amplification or rearrangement, nor mutations of the 12th, 13th, or 61st codons of KRAS, HRAS, or NRAS. TP53 pathway gene analysis showed no mutation of the TP53 gene, but did show upregulation of MDM2 (31). RMS-YM also shows overexpression of FGFR1, located in 8p11.2 and is grown in RPMI-1640 with 10% FBS (28).
RUCH2 was derived from a 15-month-old female with a botryoid RMS of the vagina (48). It was originally established as a xenograft, then grown in cell culture. Karyotype at passage three revealed 55, X, –X, +7, +inv(7)(q11q32), +8, +8, +12, +13, +13, +19, +20, +21/55, idem, del(20)(q13) [cp40], almost identical to the primary tumor. It can be grown in DMEM supplemented with 10% FBS.
RUCH3 was derived from a patient with relapsed embryonal RMS. This cell line is first mentioned in the original reference for RUCH-2 cell line, and was found to express PAX3 but not MYF3 or MYF5 (48). It can be grown in DMEM supplemented with 10% FBS.
SCMC-RM2 (And Subclone SCMC-RM2-1)
SCMC-RM2 was derived from the bone marrow of an 11-year-old female with relapsed, metastatic embryonal RMS. This patient had a primary tumor in her right abdominal wall with extensive marrow involvement. The tumor was classified as embryonal based on the presence of cytoplasmic longitudinal fibers and cross striations, as well as cell appearance using electron microscopy. She achieved remission with standard chemotherapy but relapsed and died 11 months later after diagnosis. Tumor cells from her bone marrow at relapse were obtained and cultured as SCMC-RM2. Subsequently, at passage 20, a parent cell line was cloned in soft agar and SCMC-RM2-1 was isolated. Both cell lines stain positive for muscle markers including desmin, vimentin, myoglobin, and actin. Interestingly, molecular and cytogenetic evaluation revealed amplification of the MYCN gene with overexpression of MYCN mRNA, which was also found in the primary tumor (49). SCMC-RM2 and its subclone can be grown in RPMI-1640 with 10% FBS (50).
SMS-CTR was derived from an untreated 1-year-old male with a pelvic mass near the prostate. The original biopsy specimen is histologically consistent with a poorly differentiated embryonal RMS. This cell line was established by Dr. Patrick Reynolds in 1979. Cytogenetics were performed on this line in the 10th passage and showed hypertriploid chromosomes, with translocations involving t(1;13)(q21;q14) and t(2;13)(p25;q14) (51). It is important to point out that these are not the characteristic translocations associated with alveolar RMS. Though FOXO1 does localize to the 13q14 region, Northern blot studies of this line with a FOXO1 probe failed to show any evidence of a novel-sized transcript, thus suggesting that FOXO1 is not affected by these translocations (52).
TTC442, also occasionally called A91 in the literature, has an embryonal histology and is grown in RPMI-1640 with 10–20% FBS. This cell line was originally obtained from the laboratory of Dr. Tim Triche of Children's Hospital Los Angeles (CHLA) (43, 53).
TTC516, also known as A995 in the literature, has an embryonal histology and is grown in RPMI-1640 with 10–20% FBS. This cell line was originally obtained from the laboratory of Dr. Tim Triche of CHLA (43, 53).
Alveolar RMS Cell Lines
CW9019 has an alveolar histology and was developed by Dr. Jaclyn Biegel at the University of Pennsylvania, Philadelphia, PA, USA. It can now be obtained from Dr. Frederic Barr at the National Cancer Institute, Bethesda, MD, USA (personal communication, Dr. Frederic Barr). CW9019 has a stable reciprocal translocation of t(1;13), expresses the resulting PAX7-FOXO1 fusion protein and is grown in DMEM with 10% FBS (28). Unlike the typical PAX7-FOXO1-containing tumor, this line does not show amplification of the PAX7-FOXO1 fusion gene (54).
D-RHA1 has an alveolar histology and was originally described in (43), having been obtained from Dr. W. Benedict of M.D. Anderson Cancer Center. It is grown in RPMI-1640 with 10–20% FBS.
KFR was derived from a bone marrow metastasis of a 13-year-old female and designated RMS based on positive staining for vimentin and desmin. Alveolar histology was assigned based on morphology and upon identification of the t(2;13)(q37;q14) translocation, expressing the resulting PAX3-FOXO1 (formerly known as PAX3-FKHR) fusion protein (55). Although there was a time that the 2q breakpoint was assigned to q35 or q37, this has now been resolved as a q35 breakpoint, so that the translocation would now more correctly be stated as t(2;13)(q35;q14). KFR is grown in modified DMEM with 10% FBS (22).
RH5 has an alveolar histology and has been cited in a study of the role of PDGFR-A (56).
RH10 was derived from a 15-year-old female with a perineal tumor at relapse; she had previously received extensive chemotherapy with vincristine, cyclophosphamide, dactinomycin, and doxorubicin. RH10 was first established as a xenograft. The resulting cell line is hyperdiploid and cytogenetic analysis revealed t(2;13)(q35;q14) (57). Although the tumor from which RH10 was originally derived was described as having poorly differentiated embryonal histology, in another publication from the SJCRH group at the same time, this tumor was diagnosed as mixed alveolar/embryonal or monomorphous round cell. With the presence of the t(2;13) translocation, it should not be called embryonal RMS, but either mixed or alveolar RMS (39). After injections of various chemotherapy agents, complete tumor regression was seen after administration of L-PAM, but only transient growth inhibition, if any, to the other agents tested (58). RH10 has also been evaluated by the PPTP and found to have intermediate response activity to vincristine and high response to cyclophosphamide (41).
RH18 (Fusion-Negative and Fusion-Positive Variants)
The RH18 cell line was generated from a xenograft derived from a tumor from a previously untreated 2-year-old patient with a perineal RMS assigned as embryonal histology. Interestingly, there was a small amount of alveolar histology in the tumor (although the xenograft from which the cell line is derived was entirely embryonal) (36). At the time, such a mixed presentation often received a final diagnosis of embryonal RMS, though today the mixed tumors are considered an intermediate subset in which a fraction have the PAX-FOXO1 fusion.
RH18 cell-line chromosome number ranges from 70 to 80 per cell, and cytogenetic abnormalities include del(1)(p22), der(3)t(3;?)(p14;?), del(6)(q24), and der(22)t(1;22)(q31;q11) (57). In the original cytogenetic characterization of this cell line, a t(2;13) translocation was identified in a later passage of a melphalan-resistant population (39). This sub-line also had cytogenetic markers in common with an earlier passage, suggesting that a translocation-bearing clone was selected (or arose) during culture. This finding is consistent with the presence of two available cell lines called RH18 – one fusion-negative (59) and one fusion-positive (60). Indeed an experiment comparing the parent xenograft to tumor selected for resistance after a large single dose of vincristine did show near tetraploidy (with numerous structural abnormalities) in the parent tumor, with all normal diploid cells noted in the resistant xenograft (61). This supports the idea that the original tumor, from which the xenograft was directly derived, had multiple stem lines.
The RH18 cell line also harbors amplification of the 12q13–15 region (including MDM2) (62), although unpublished data shows that the amplicon is present in the fusion-negative, but not fusion-positive, version of RH18 (personal communication, Dr. Frederic Barr). Clearly, Short Tandem Repeat (STR) analysis (see Section "Potential Pitfalls") of the RH18 variants would be helpful to prove their relationship to the original xenograft, and users of these cell lines are well-served to determine which variant they are employing in their experiments.
When tumor volumes were measured following injections of various chemotherapy agents, RH18 showed growth inhibition with vincristine, cyclophosphamide, dactinomycin, doxorubicin, L-PAM, DTIC, cis-DDP, and mitomycin-C. No tumor growth inhibition was seen with bleomycin or busulfan (32). When evaluated by the PPTP, RH18 showed intermediate response activity to vincristine and high response to cyclophosphamide (41).
RH28 (Derived from the Same Patient as RH3)
The RH28 cell line was derived from a xenograft of tumor tissue obtained from an axillary node metastasis in a previously untreated 17-year-old male. The tumor and subsequent cell line have a poorly differentiated alveolar RMS histology. RH28 cells are near tetraploid and harbor a t(2;13)(q35;q14) translocation (57). Evaluation of RH28 proliferation in vitro shows that between passages 59–63, doubling time progressively increases until growth stops completely, and the cells appear to senesce. Further, these cells are multinucleated and show morphologies comparable to normal myoblasts (63). RH28 had complete volume regression with vincristine and L-PAM and significant growth inhibition with cyclophosphamide, doxorubicin, and dactinomycin (58). When tested by the PPTP, RH28 showed high response to both vincristine and cyclophosphamide (41).
There exists a sub-line of RH28, denoted RH28/LPAM, which was generated from RH28 cells in xenografts exposed to increasing doses of melphalan. RH28/LPAM cells are available for use and do not differentiate or stop proliferating with time (61). There are also available cells designated RH3, which have the same DNA breakpoint and pattern of DNA polymorphisms as RH28 cells (64). Indeed the RH3 cell line was developed by the Douglass laboratory from the same patient from which the RH28 xenograft was developed. The RH3 cell line has no relation to the Rh3 xenograft described in (36).
RH30 (Derived from the Same Patient as RMS13) and RH30R
The original Rh30 xenograft was established from an overnight culture of bone marrow obtained from a 16-year-old male with untreated metastatic alveolar RMS. The xenograft tumor was wild-type for TP53. The RH30 cell line was derived from the same patient bone marrow sample maintained in culture in the Douglass laboratory. The RH30 cell line expresses the t(2;13) translocation (39) and chromosomal analysis showed near-triploid chromosomes, between 51 and 87 (65). This line also has amplification of the 12q13–q15 region involving CDK4 and surrounding loci (66–68), and a heterozygous mutation of the TP53 tumor suppressor (30, 31, 65). An independent cell line, RH30R, was derived from a xenograft, made from this same patient, at the time of relapse (41). The relapse xenograft and the cell line from the xenograft both have the same TP53 mutation as the original RH30 cell line. RH30 and RH30R are cultured in RPMI-1640 with 10% FBS.
When the RH30 xenograft was tested with various chemotherapy agents, it showed complete regression using vincristine and L-PAM, significant growth inhibition after cyclophosphamide, but no growth inhibition with dactinomycin or doxorubicin (58). The PPTP evaluated the RH30 cell line and found it to have high response activity to vincristine and cyclophosphamide (41). A third experiment used the RH30 cell line to test the combination of topotecan plus vincristine against tumor growth. The combination showed better tumor control than topotecan alone but was no better than vincristine alone, where 0/5 mice tested had tumor progression (40). The RH30 cell line was also used to investigate the anti-tumor effects of tolfenamic acid. After treatment, RH30 cells showed decreased growth, decreased cell migration, and decreased expression of the PAX3-FOXO1 fusion protein (33).
An alveolar RMS cell-line labeled RMS13 (39, 69) is thought by some investigators to be related to RH30, perhaps derived from the same patient tumor as RH30, but this is not clear. Other studies have presumed RMS13 and RH30 to be independent entities (70). Finally, another cell line has been distributed with the name of RH30 (personal communication, Dr. Frederic Barr). Of interest, this other line still has the 12q amplicon but does not have the PAX3-FOXO1 fusion (unpublished data, Dr. Frederic Barr). STR analysis would help clarify the identity of both the RMS13 and fusion-negative RH30 lines.
RH41 (Derived from the Same Patient as RH4)
RH41 was established by the Houghton laboratory from a xenograft of a lung metastasis from a previously treated 7-year-old female patient with alveolar RMS. RH41 was originally designated as alveolar by histology, and more recently has been shown to harbor the t(2;13) translocation (71). RH41 also harbors mutation of the TP53 gene (26). RH41 was tested by the PPTP and showed intermediate response activity to both vincristine and cyclophosphamide (41).
The cell line designated RH4 was developed from the same patient by another laboratory at SJCRH (personal communication, Dr. Peter Houghton), in which the tumor specimen was placed directly into tissue culture. These cells designated RH4 possess the t(2;13)(q35;q14) translocation (72) and have amplified, overexpression of EYA2 (28). RH4 cells are cultured in RPMI supplemented with 10% FBS.
RH65 was derived from 18-year-old female with a relapsed RMS; it has an alveolar histology based on the original tumor (41).
RMZ-RC2 was established as a subclone of the RMZ cell line, which was derived from a bone marrow metastasis of a 2-year-old male with alveolar RMS; the child had been treated prior with chemotherapy and radiation (73). RMZ-RC2 can be grown in DMEM with 10% FBS. Chromosomal analysis showed near tetraploid range chromosomes with numerous monosomies and double minutes (65). Both the PAX7-FOXO1 fusion gene and MYCN were found amplified (65, 72). RMZ-RC2 cells treated in culture with 2–5 Gy of radiation show dose-sensitive increases in the expression of myosin and the percentage of multinucleated cells, both markers of differentiation (74).
TC212 has an alveolar histology (based on histology of the tumor specimens) and was derived from the bone marrow metastasis of a 16-year-old male who relapsed 5 months after multi-agent chemotherapy and radiation (51). Chromosomal analysis revealed multiple translocations, inversions, and deletions including what was thought to be t(2;13)(q37;q14). Although the 2q breakpoint was assigned to q35 or q37, this has now been resolved as a q35 breakpoint, so the translocation in TC212 should correctly be described as t(2;13)(q35;q14). TC212 cells are grown in RPMI-1640 supplemented with 10% FBS.
RMS Cell Line Imposters
A204 was previously (but no longer) listed in the ATCC catalog as an RMS cell line. However in 1998, A204 cells were found to lack expression of myogenin, MyoD, or desmin, suggesting that either they were not of RMS origin or that with passaging in culture there was down-regulation of myogenic genes (23). These observations were especially compelling, since a prior immunohistochemical study found that most RMS tumor samples (but not normal adult muscle including skeletal muscle) expressed MyoD (75). In 2002, A204 cells were found to have a SMARCB1 (SNF5) mutation, consistent with its classification as a rhabdoid line (76).
A673 was also previously (but no longer) listed in the ATCC catalog as an RMS cell line. However, like A204 cells, in 1998 these cells were found to not express myogenic markers (23). In 2003, A673 cells were found to express the EWS-FLI1 fusion gene, indicating the cell line is derived from a Ewing sarcoma (77).
The RH1 cell line was derived from a patient tumor specimen grown directly into tissue culture. The tumor was of the hand, described in a pathology report to be consistent with alveolar RMS by histologic criteria (personal communication, Dr. Peter Houghton). RH1 has no relation to the Rh1 xenograft described in (36). In 1998, RH1 was noted to lack expression of the myogenic markers myogenin, MyoD, and desmin (23). In 2008, using gene expression profiling the PPTP determined RH1 to cluster with tumors in the Ewing sarcoma family (4). RH1 was later verified to express the EWS-FLI1 fusion characteristic of the Ewing family of tumors (78). In retrospect, it is not clear whether RH1 was contaminated by a Ewing cell line or mislabeled, however the PPTP has excluded RH1 from future RMS studies (4).
Culture of RMS Cell Lines as Rhabdospheres versus Adherent Cells
While all above mentioned studies propagated RMS cell lines as adherent cells or xenografts, Walter et al. recently described a method of culturing rhabdospheres in suspension (79). In this method, spheres are grown by placing human embryonal RMS cell lines into SC serum free medium with neurobasal medium and 10 ng/ml EGF, 20 ng/ml b-FGF, and 2× B27. In this study, RD cells were injected as adherent cells or spheres into mice, whereby tumor growth was measured over weeks. Sphere-derived tumors grew sooner, 40 days after injection, as opposed to 80 days after injection with the adherent cells. Sphere-derived tumors also required fewer cells for injection than adherent cells, indicating that rhabdospheres are more tumorigenic than adherent cell cultures. When stem cell genes associated with the stem cell phenotype were assessed using real-time PCR, sphere cultures had significantly higher expression of these genes than adherent cells, consistent with the premise of stem cell enrichment. Further, when treated with retinoic acid or DMSO, sphere cultures showed greater differentiation toward myocytes, neuronal cells or adipocytes, again indicating higher cancer stem cell potential in lines grown as rhabdospheres versus adherent cells. These data are compelling and may lead to changes in how RMS cell lines are grown for use in preclinical experimentation.
Potential Pitfalls
Several potential problems arise when using cell lines derived from human tumors in vitro or transplanted into mice as xenografts. One possibility is that tumors can be contaminated by murine stromal cells, thereby potentially affecting the outcome of the study. To combat this problem, analysis for species-specific LDH isoenzymes, as well as evaluating for acrocentric mouse chromosomes, can be performed. For example, Hazelton et al. cultured rhabdomyoblasts from the cell lines RH10, RH18, and RH28 and found human specific isoenzymes of LDH present in the lines, without evidence of murine LDH. In this same study, metaphase spreads were analyzed for acrocentric mouse chromosomes, and none of the three cell lines evaluated showed murine cells (57).
A second problem is that there may be discordance between the original tumor and the resulting cell lines or xenografts. To evaluate for this, one can perform gene expression microarray analysis. In 2008, the PPTP published a report using microarray analysis to analyze gene expression of the RMS cell lines RH10, RH18, RH28, RH30, RH41, RH65, and RD and compared expression profiles to a similar number of RMS specimens representing both alveolar and embryonal RMS. After exclusion of immunosurveillance genes (such as macrophage or B-cell markers), the cell lines resembled the other human RMS specimens (4). A similar analysis was performed using cDNA microarray analysis of the RMS cell lines RH1, RH6, RH10, RH12, RH14, RH18, RH28, RH30, RH36, and RH41. These analyses were compared to cDNA of primary tumors and all clustered appropriately by tumor type except RH1 and RH6 (37). Further cDNA microarray analysis done by Khan et al. showed comparisons between alveolar cell lines RMS13, RH3, RH4, RH5, RH18, and RH28 (69). Missiaglia et al. performed cDNA microarray analysis and gene expression profiles, and compared these profiles to primary tumors on the RMS cell lines RD, RMS-YM, RUCH2, RUCH3, RH4, RH18, RH30, RH41, and CW9019 and showed high concordance as well (28). A recent study describes an approach to develop xenografts directly from patient tumors, then test them for genomic similarities (80). In this study, after surgical resection from human subjects, tumors were either implanted into immune-compromised mice as xenografts or analyzed for histology, mutational analysis and gene expression profiles. Xenografts were later surgically resected and implanted into a second set of mice. Xenograft specimens from first and second generation implantations underwent similar analysis as the original patient tumor sample. This approach serves as a good model for evaluating and validating human tumor xenografts, which may help produce preclinical data more closely mirroring later clinical outcomes
A third problem is the situation in which two cell lines with different names are apparently from the same tumor, such as RH36 and Birch, RH30 and RMS13. This situation can represent two different nomenclatures or possibly contamination of one cell line with the other. In addition, although RH3 and RH28 appear genetically identical by fusion breakpoint and STR analysis (64, 81), it is not clear whether the two cell lines were derived from the same tumor from the same patient on the same date. In either case, the two lines may share much of their genetics but may also have diverged biologically due to continued growth as separate cultures. There are also instances where two distinct lines have the same name, but have different genetics. For example, the "RH18" cell line can refer to the version not bearing the fusion gene (59), or the version bearing the fusion gene (60), which may have emerged from the original cell culture.
A fourth problem is unintentional cross-contamination between cell lines. It is estimated that 15–35% of all cell lines are of a different parental origin than assumed, and this results in the misidentification of cell lines and ultimately the misinterpretation of scientific data (82–84). For example, the cell line TE671, which was widely used to study medulloblastoma, was thought to be a misidentified RMS cell line (RD). This statement stemmed from similar phenotypic observations and an activating point mutation in NRAS gene which was found both in TE671 and RD (34). This hypothesis was confirmed with a cytogenetic study of these cell lines (85). In addition, three esophageal adenocarcinoma cell lines were recently found to derive from other tumors (86).
Because of the problems described above, and no historical standard method to validate cell lines, the method of STR profiling was suggested as an international reference standard for cell line authentication (87). STRs (or microsatellite repeats in DNA) consist of 2–6 base pair repeats that differ from individual to individual (84). In this method, commercially available primers are used to amplify a number of polymorphic STR loci, resulting in a cell-line specific "fingerprint" that is recorded as a numerical code in a reference database. The simplicity of the code, reproducibility, and low cost of this method are the strengths supporting its use as a standard of cell line authentication (87). Indeed, several scientific journals including those managed by the American Association for Cancer Research are requiring that articles submitted for peer review contain documentation of cell line authentication (see http://www.aacrjournals.org/site/AuthServCtr/cell_line_auth.xhtml). On the other hand, it is important to acknowledge the limitations of STR in cell line authentication. For example, in a study of four human leukemia cell lines grown under variable culture conditions, STR profiles changed in two of the four cell lines due to subclone outgrowth (84). Thus, STR profiling is an important tool for cell-line validation, but has limitations, especially in tumor cell lines subject to experimental conditions that could alter cellular genetics. Other features including myogenic markers, enzyme isotypes, and cytogenetic analysis can further assist in validation of RMS cells when differences are found in STR loci (57, 84). Table 3 summarizes the validation methods that have been employed for human RMS cell lines. The cell lines with known and published STRs are listed in Table 4.
Numerous RMS cell lines are available for use in basic research and preclinical testing. These cell lines differ markedly in their histologies, origins, karyotypes, degree of treatment prior to development, and the levels to which these lines have each been validated and compared to their tumors of origin. This information is of critical importance when choosing a cell line for study. For example, evaluation of results of studies performed to date on the available cell lines shows a trend that lines derived from tumors previously treated with extensive chemotherapy and/or radiation are then less susceptible (or only transiently so) to standard agents. These previously treated cell lines would therefore be optimal for preclinical testing of new agents proposed for use in relapsed or refractory disease. These further progressed tumors may also be easier to establish in culture than primary tumors. It is necessary to ensure that cell lines used in preclinical trials closely resemble the tumors from which they were derived. While some data is currently available that validates several lines, this work is ongoing and will surely continue to develop as the use of cell lines and RMS xenografts increase in pediatric oncology research.
When examining RMS cell lines as a whole, there is a surprising lack of cell lines derived from primary, untreated RMS tumors. Indeed, the majority of both embryonal and alveolar RMS cell lines are established from tumor relapses or distant metastases. Cells that arise from relapses or metastases (in addition to any adaptations as the result of radiation and/or chemotherapy given to the patient) have genetic changes distinct from the primary tumor, and may be easier to establish in culture than cells from primary tumors. Therefore, applicability of these lines in preclinical studies examining RMS primary tumor biology is questionable. This gap in our understanding of RMS reveals a significant challenge to researchers that routinely use RMS cell lines for preclinical research. How can we make significant strides to identify therapeutic targets when the reagents available do not accurately represent the biology of the primary tumor? Has RMS research been hindered by an excess of false negative hits in pharmacologic and genetic screens due to these cell-line adaptations?
In summary, it will be important for RMS scientists, as a community, to critically evaluate the cell lines we use, and identify approaches to generate improved reagents for future research. Ideally, human cell lines would be established from primary, untreated RMS tumor biopsies. Then, any cell lines generated from subsequent tumor relapse or metastasis could be paired with the primary cell line as a "matched set." To our knowledge there are no matched set cell lines publicly available to RMS researchers. When available, such a resource would support the study of the genetic and biologic changes in the primary tumor, as well as the changes leading to recurrence or metastasis.
Conflict of Interest Statement
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
We thank Charles Keller (Oregon Health and Science University, Portland, OR, USA) for sharing a list of RMS cell lines, an updated version of which is recently published (18) and Peter Houghton (Nationwide Children's, Columbus, OH, USA) for valuable insight and comments. We anticipate that readers of this article will have further insight into the origins of available human RMS cell lines, and look forward to incorporating this information in a future update. This work was supported by a Pfizer Oncology Clinical Fellowship Award (Brian C. Belyea), The Hartwell Foundation (Lisa E. S. Crose), the Intramural Research Program of the NCI (Frederic G. Barr), and the Liddy Shriver Sarcoma Initiative and NCI Award R01-CA122706 (Corinne M. Linardic).
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Keywords: alveolar, embryonal, human cell line, rhabdomyosarcoma, xenograft
Citation: Hinson ARP, Jones R, Crose LES, Belyea BC, Barr FG and Linardic CM (2013) Human rhabdomyosarcoma cell lines for rhabdomyosarcoma research: utility and pitfalls. Front. Oncol. 3:183. doi: 10.3389/fonc.2013.00183
Received: 31 March 2013; Paper pending published: 21 April 2013;
Accepted: 27 June 2013; Published online: 17 July 2013.
Edited by:Stephen Lessnick, University of Utah, USA
Reviewed by:Rimas J. Orentas, National Institutes of Health, USA
Peter J. Houghton, Nationwide Children's Hospital, USA
Copyright: © 2013 Hinson, Jones, Crose, Belyea, Barr and Linardic. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in other forums, provided the original authors and source are credited and subject to any copyright notices concerning any third-party graphics etc.
*Correspondence: Corinne M. Linardic, Departments of Pediatrics, Pharmacology and Cancer Biology, Duke University Medical Center, Box 102382, Durham, NC 27710, USA e-mail: firstname.lastname@example.com


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Dear customer, If you come from United Kingdom and you want to buy wedding dresses bridesmaid dresses or prom dresses from our website, please click here: uk.millybridal.org
This dress was great! Definitely stood out from the crowd. Delivery was very quick as well!
The picture does it no justice!! This dress is the perfect choice and shipping time was perfect, maybe like 20 days from date of purchase. Thank you Millybridal for helping me pick a dress without even trying it on and I have no regrets!!
The dress I ordered came exactly as advertised, no shipping charge and arrived 2 weeks later! I've definitely found a new place for all my dress needs. Thank you!
Dress is really beautiful and it arrives on time. I will definitely order more.
Very nice dress, fit as expected, nice lining, true to size. And it was perfect.
My daughter is so pleased when received your item. Hope you can always provide us the same quality dress in the future. thanks you again !!
Really love the dress. It could not have been better then this, it was the same as described in the picture. Very good !!
I could not love it more! It's perfect. High quality peace as expected.
Extremely pleased with this product.
The quality of the fabric was excellent it fitted perfectly. I was pleasantly surprise by it fitting and how comfortable the gown was. I would recommend customers to buy from this shop. The gown on time for me to wear to a wedding.

subset: commoncrawl


text:
Al Maryah Island will be full of Christmas cheer this December as a winter wonderland comes to town.
Guests will be treated to a world of festive entertainment and attractions for all the family between December 13 and 26.
Highlights include a spectacular Santa's Grotto, live music and dance performances, carnival games and a wide selection of seasonal food and beverage offerings.
And it's the perfect excuse to fit in some last-minute Christmas shopping at The Galleria's dozens of great retail outlets.
It promises to be a treat for all the senses, with gorgeous winter cottages covered in frosty snowflakes hosting kids' activities as the smell of festive favourites fills the air.
More: Abu Dhabi's biggest and best tree-lighting ceremonies for Christmas 2018
Attractions at the Al Maryah Island waterfront promenade include a sliding slope park, a snow zone, train rides, a cookie decorating and face painting.
And there will be some special appearances from popular children's fairytale princesses, with the likes of Snow White, Cinderella and Belle set to mingle among the crowd.
Santa's Grotto is open inside The Galleria from December 6, while the outdoor festivities begin on December 13.
All shows and activities will take place between 4pm and 10pm daily.
December 13-26 4pm-10pm. Waterfront promenade, Al Maryah Island.


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Surprise your spouse with these cheap gifts under $30
Who said that married couples should give each other gifts only on holidays?
Actually, to find a gift for yourself and your spouse is quite easy.
I spent some time on Amazon to explore what they offer. Let's say I made you favour to pick what's the best gifts for married couples. And the best part is that they're all under $30.
Keeping the fire burning after the wedding is twice harder than before the "I Do" ceremony.
The reason is in us. Sadly but many times we decide that the wedding is a stamp on the other's heart that says they belong to us. And many times we forget to cherish this other person.
But because you're reading this, I assume you're not one of those cases. And that's amazing. A small budget is just another reason to be creative with your gifts.
Small gestures will add that spice to your relationship as a married couple. Don't ever miss a chance to show appreciation, to care more about your partner and to make them smile.
There are many things you can get for the two of you, but I had the time and energy to explore Amazon and to select what's best for you. If you liked these suggestions, don't forget to subscribe to our blog so you can be notified when new awesome content is out. We never spam (I know everyone says that, but we mean it)
Related post: LIFE AFTER WEDDING! 13 PIECES OF ADVICE FOR NEWLYWEDS
Gift ideas for married couples if you love to travel and have a sense of humour
If you two love to travel, I've found the sweetest way to show the world you're proud to be married by getting this 2 Passport Wallets (click on the photos to see the products).
I honestly laughed out loud when I saw them. And they're only $21.99, but you could use them for years. On top of that, every time you show them on the airports, the hotels, or anywhere else, you will make other people smile. A great conversation starter and somehow it's charming.
Gift ideas for married couples who love cooking together
Not everyone loves to cook. Well, usually one of you is cooking and the other tries not to ruin everything… But if you both like to spend these cooking moments together, I found the best inspiration to do it today! (click on the photos to see the products)
It's $21.99 and looks extremely sweet. It makes you dream of lazy afternoons when you and your spouse make homemade pizza, grab two beers and spend the rest of the day in the bed. Oh, it can be part of your 14 Day/Mornings to a perfect relationship challenge. Mmm, it smells of well-cooked love. Keeping the promise the gift to be under $30, you could even add this amazing cookbook with recipes for married people – just like you. (It's under $30 if it's Kindle edition).
Gift ideas for married couples who would like this joke
Well, when you see the next gift, you will know exactly what I mean. Let's say it – most of the time we know from the very beginning of the day how it's going to end. You might want to prepare your spouse from the morning to expect naughty evening…or not. (click on the photos to see the products)
For $17.99 that's the best bed joke for months ahead. It will make you smile, it will inspire you, and it will bring some more intimate moments into your life.
Related post: IS MARRIAGE LESS ROMANTIC THAN A RELATIONSHIP?
Gift ideas for married couples to hang things on the wall
Who doesn't love nice message hanging on the wall? When you're tired at the end of the day or feel too sleepy early in the morning, just a small look at one of these messages could bring a smile on your face. (click on the photos to see the products)
It says it all, isn't it. They're all different prices but under $20, so take a pick and inspire your spouse.
Gift ideas for married couples who love to talk and explore each other
Well, of course, you love to talk to your spouse, you're married! But if you're out of ideas or love to find out more about the other's personality, have a look at these 88 Conversation starters for husbands and wives.
For $7.99 you will get 44 cards with questions on each side of the cards. Some of the questions are simple, but others will take you years back in the days before you met. Who knows, you might actually find out something you've never imagined the other had done.
Gift ideas for married couples who want something new in the bed
This Monogamy will be the game you'll probably never finish. But that's the best part of it. With 400 different cards and action to implement while playing, you'll be busy with each other for good hours.
Related post: 7 WAYS TO INITIATE SEX WITH YOUR PARTNER
Gifts under $30 for married couples who love to write
Now is your chance to be poetic and express what you love about your spouse. Actually, even if you're not so gifted as a writer but still don't mind writing your partner how much you love them, this is for you.
It's so easy to get two and inspire each other every single day. Maybe you often forget to tell your spouse why you decided to spend your life together. Perhaps it's too much stress, other tasks, kids… it doesn't matter. Our love for the person next to us came as a blessing, and it should be cherished. If you don't water a plant, it will die. It's the same with love. Feed your love to the other every day, and it will grow and bloom… because of you… for you.
Related post: 50 ROMANTIC MESSAGES FOR YOUR HUSBAND TO TELL HIM YOU LOVE HIM
Gifts for married couples who … have keys
Ok, I was joking with this one. But when you see what I found, you will want it too. These key accessories are the best way to think bright every time you leave the house, or you're coming home. They are gentle, sweet…. and true.
Gifts for married couples who love coffee
Everybody drinks coffee. And the truth is that most of us have a personal coffee mug that no one else touches (please, tell me it's not just me!). So, how about to make the experience even better by sharing a set of two mugs with your loved one. Just have a look at them and tell me you don't smell fresh morning coffee.
Gifts for married couples who are wine and beer lovers
Ok, no way to avoid an evening drink. He loves cold beer; she enjoys sparkling wine? What an amazing combination. So, here's the next suggestion for beer and wine lovers. It is way under the budget. Oh, did you see that each glass has a title? How cool is that?!
Related post: HOW TO STAY MARRIED AND BE HAPPY
Gifts for a married couple who like to be on time
Yes, that's a good way to present the next gift – matching watches for Him and Her. It's innovative accessories for both of you, especially if you already have 13 other watches in the jewellery box. You can get both watches for $16.99 and that leaves you with enough money for something extra.
Gifts for married couples who love Mickey and Mini
I love t-shirts for couples. But that one is one of my favourites as it's with Mickey and Mini. It's an absolute necessity to get a pair of these t-shirts if you love Disney's characters. You can wear them when hiking, or walk in the park. You can wear it every time you're together or separate, although I'm sure the effect won't be the same if you're not together, am I right?
Related post: FAMILY BUDGETING – PROFIT AT THE END OF THE MONTH!


text:
Other conditions originating in the perinatal period P96
- P96 should not be used for reimbursement purposes. Please select a more specific diagnosis below.
- The 2019 edition of ICD-10-CM P96 became effective on October 1, 2018.
- This is the American ICD-10-CM version of P96 - other international versions of ICD-10 P96 may differ.
Codes: = Billable
P96 Other conditions originating in the perinatal period
- P96.0 Congenital renal failure
- P96.1 Neonatal withdrawal symptoms from maternal use of drugs of addiction
- P96.2 Withdrawal symptoms from therapeutic use of drugs in newborn
- P96.3 Wide cranial sutures of newborn
- P96.5 Complication to newborn due to (fetal) intrauterine procedure
- P96.8 Other specified conditions originating in the perinatal period
- P96.9 Condition originating in the perinatal period, unspecified
Reimbursement claims with a date of service on or after October 1, 2015 require the use of ICD-10-CM codes.


text: Editing Master P 0 You do not have permission to edit this page, for the following reasons: The action you have requested is limited to users in one of the groups: Users, FANDOM Staff, FANDOM Helpers, Wiki Managers, Content Team Members. Please log in to edit this wiki.Joining South Park Archives is free, and it only takes a minute.We hope that you sign in, and become a member of the community!Click here to log in or create an account more + You can view and copy the source of this page: {{South Park character |name = Percy Robert Miller |image = Master-p.png |gender = Male |race = African American |hair = Black |age = {{age|1967|4|29}} |dob = April 29, 1967 |job = Singer, Song Artist |appearance = "[[Christian Rock Hard]]" }}'''Master P''' performed the song "[[Kenny's Dead]]" on ''[[Chef Aid: The South Park Album]]''. He also appeared in the accompanying mockumentary, ''[[Chef Aid: Behind the Menu]]'', in which he talked about [[Chef]]'s influence on his career. Master P is one of the musicians who join [[The Boys|the boys]]' band, [[Moop]], in their strike against illegal music downloading in the ''[[South Park]]'' episode "[[Christian Rock Hard]]". He has yet to have any speaking lines. ==Appearance== In "[[Christian Rock Hard]]", he can be seen at his home wearing a black and gold jersey, a white sweat bands, black shoes, and silver chain. He has black hair braided in the style of cornrows, a popular hair style amongst African Americans. In the mockumentary, ''[[Chef Aid: Behind the Menu]]'', he can be seen wearing a black baseball cap backwards, a black leather coat, sunglasses, earrings, and a gold chain. In the music video and cover for "[[Kenny's Dead]]", he can be seen wearing a black leather coat, black fedora, gold chain, sunglasses, and a brass knuckle with his name on it. He appears throughout the video in a car, with [[Chef]], and performs the lyrics in the song. <gallery spacing="small" widths="120" caption="Images of Master P"> Kennysdead.JPG|Master P and Chef in "[[Kenny's Dead]]." R-3881916-1347934739-5404.jpeg.jpg|Master P featured on the cover of the "[[Kenny's Dead]]" single. Masterp2.JPG|Master P in [[Chef Aid: Behind the Menu]]. </gallery> Templates used on the current version of this page: Template:Age (view source) (semi-protected)Template:South Park character (view source) (protected)Return to Master P. Retrieved from "https://southpark.fandom.com/wiki/Master_P" Edit summary Preview Mobile Desktop Show changes


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Palak Paneer Pulavu
Palak paneer pulavu is a traditional Punjabi dish. It is an easy and quick one pot meal; for a healthy lunch box for kids and adults. You can use any choice of your vegetables along with these two ingredients. Check here for the homemade paneer recipe.
- Palak-1cup
- Paneer cubes- ¾ cup
- Basmati rice- 1 ½ cup
- Onion-1
- Grren chilly-1
- Garlic pods-2
- Oil-2tbsp
- Salt-to taste
- water-3 cups
- Cumin seeds- 1/4 tsp
- Bay leaf-1
- Coriander leaves chopped- 2tbsp
- Wash and soak the basmati rice in water for 30 minutes.
- Heat a non-stick pan with 1 teaspoon of oil and add paneer cubes.
- Fry it for a minute until both sides are just golden in color(Don't fry too much).
- Take it out from the pan and keep aside
- In the same pan add garlic, palak, green chilly and coriander leaves.
- Sauté for, just a minute and switch off the flame.
- Grind this mixture in to a smooth paste without adding water (if necessary add, little).
- Add remaining oil in a pressure cooker, splutter cumin seeds and add bay leaf.
- Add onions and sauté until transparent.
- Add blended paste and mix well.
- Add drained basmati rice and sauté for 2 minutes (without cracking the rice).
- Add water and salt; cover the lid and pressure cooks it for 1 whistle on medium-low flame.
- Open the lid only after the whistle goes completely by itself.
- Add paneer cubes on top it and give a gentle mix.
- Serve hot with some pickle, raitha and pappad.
Palak Paneer Pulavu Reviewed by fejnaem on 02:57 Rating:


text:
Studio is zSpace's rich model exploration and presentation tool allowing students to compare, dissect, analyze, measure, annotate and explore 1500+ 3D models from zSpace Model Gallery or 3D models created by students.
Teachers will find Studio a natural fit for creating complex scenes of content. It's easy to add labels, make annotations, take measurements, integrate multiple models to build a rich scene and create video tours of their models to share.
For more information please visit our zSpace EDU website.
Complete list of included activities
For a complete description of all activities, including support materials and standards, refer to edu.zspace.com/activities.
zSpace Studio, activity pack, and model packs are now available via the zSpace App Manager. Please contact your zSpace Authorized Partner or sales representative to purchase a license.
Release Notes
Current release notes for Studio can be found here.
Complete archive release notes can be found here.


text:
+966 12 676 0000 Ext.2403, 2405, 5557
email: firstname.lastname@example.com
Al-Madinah newspaper
IT is unfair that some people describe the process of Saudization and putting Saudi citizens first as racism. It is as if they are only looking at the issue from one side. However, the population in Saudi Arabia is increasing and young males and females, who make up 70 percent of the population, are in a serious need for jobs.
Saudi citizens do not want jobs to have fun or pass time, rather they need jobs to live and survive. They want jobs because they do not want to be poor but in order to support their families, get married, have children and support their own homes. As a result of the large number of expatriates who occupy a large number of jobs and the loopholes in employment that have not yet been fixed, many of these young males and females are unemployed.
If this were to happen in any other country, then no one would call it racism. This is the system followed in other countries, so why are we called racists when they are not?
I understand that expatriates have contributed to the advancement of our country. Honestly, I am forever grateful for the contributions of these expatriates, however, the citizens of countries should have priority in jobs over everybody else. Every expatriate is welcome, but citizens should be employed in the main.


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Chocolate Dipped Candy Cane Fudge: Minty candy cane flavored fudge made with white chocolate and dipped in semisweet chocolate and sprinkled with candy cane pieces.
My dad loves mint.
Favorite ice cream is Mint Chip.
His favorite part of Christmas was candy canes.
So whenever he comes out for Christmas I make sure that I have peppermint desserts.
I made the Peppermint Bark Red Velvet Cupcakes for him.
As well as this fudge.
Which is a combo of white chocolate and white peppermint bark.
But to take it up a notch, just like my Boston Cream Pie Fudge I dipped these in chocolate.
It's my new favorite thing to do to fudge.
Because how can you go wrong with more chocolate?
Chocolate Dipped Candy Cane Fudge
- For the Fudge:
- 2 cups granulated sugar
- 4 oz. unsalted butter
- ⅓ cup heavy cream
- 1 cup Ghirardelli Peppermint Chunks or Wilton Candy Cane Melts
- 1 cup white chocolate chips
- 7-ounce jar marshmallow crème
- For the topping:
- 6 oz. semisweet chocolate, finely chopped
- 1 1/2 TBSP vegetable oil
- 3 TBSP candy cane pieces
- Line an 8×8 inch pan with parchment with an overhang so you can lift the fudge out later and coat with baking spray.
- Combine sugar, butter, heavy cream, and liquor into a large heavy bottomed sauce pan and bring to a boil over medium heat.
- Once boiling, attach a candy thermometer and reduce the heat to a hair above medium low and cook for 7-10 minutes until your thermometer reads 234°F (soft ball stage), stirring constantly.
- Once you reach 234F remove from heat and add the white chocolate chips and Peppermint Chunks, stirring vigorously with a wooden spoon until the chocolate has melted and the mixture is smooth.
- Add the marshmallow crème and mix until well blended.
- Pour into prepared pan.
- Let cool to room temperature. Then lift out of the pan and onto a cutting board.
- Cut into whatever amount you want. I think I got about 24 pieces.
- For the Topping:
- Combine chocolate and oil in a medium heat-proof bowl set over a medium saucepan of barely simmering water; stir until melted and smooth.
- Transfer to a small bowl, and let cool about 15 minutes.
- Dip the top of each piece of cut fudge into the chocolate.
- Sprinkle with candy cane pieces.
- Let the chocolate set up, about an hour and serve.


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I completely understand your points about how much time and energy it would take to implement first person mode, I do realize it's asking for almost an entirely new game. And I also realize that for the good of the project this would be, and should be, one of the first things cut so that the rest of the game systems can come out polished, workable, and fun. But, maybe way later down the road, when the game is completed, keep it in mind for a potential update maybe
thanks for listening!


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« Make that 3000 release a printer grip | Main | Two very tough days for 3000 customers »
July 16, 2019
Wayback: Security boosts as enhancements
They weren't called enhancements at the time, but 13 years ago this month some security patches to MPE represented internal improvements that no company except HP could deliver to 3000s. Not at that time, anyway. This was the era when the 3000 community knew it needed lab-level work, but its independent support providers had no access to source code.
Just bringing FTP capability up to speed was a little evidence the vendor would continue to work on MPE/iX. For the next few years, at least; HP had halted OpenMPE's dreams to staff up a source code lab by delaying end of support until 2008. The vendor announced a couple more years of its support to 3000 customers.
In doing that, though, HP made an assignment for itself with the support extension, the first of two given to the 3000 before the MPE lab went dark in 2010. That assignment was just like the one facing today's remaining HP 3000 customers: figure out how to extend the lifespan of MPE expertise in a company.
FTP subsequently worked better in 2006 than it had in the years leading up to it. It's not an arbitrary subject. FTP was the focus of a wide-ranging online chat in May. Did you know, for example, that FTP has a timeout command on MPE/iX?
The connection time-out value indicates how long to wait for a message from the remote FTP server before giving up. The allowable range is 0 to 3000. A value from 1 to 3000 indicates a time-out value in seconds. A value of 0 means no time-out (i.e., wait forever). If num-secs is not specified, the current time-out value will be displayed. Otherwise, this command sets the connection time-out to num-secs seconds.
When an FTP job gets stuck, using timeout can help.
MPE/iX engineers and systems managers were working more often in 2006 than they do today. When anybody who uses MPE/iX finds a 3000 expert still available, they need to get in line for available work time. It remains one good reason to have a support resource on contract. A company relying on a 3000 shouldn't be thinking a mailing list or a Slack channel represents a genuine support asset. Even if that FTP tip did arrive via the 3000-L.
The resource of good answers for crucial questions gets ever more rare. The 3000-L mailing list has rarely been so quiet. There are information points out there, but gathering them and starting a discussion is more challenging than ever. File Transfer Protocol is pretty antique technology for data exchange. It turns out to be one of the most current standards the 3000 supports.Before the boosts, FTP services had lagged behind the rest of the world's FTP for some time. The final patches for MPE/iX 7.5, 7.0 and 6.5 improved FTP in several areas. HP said in 2006 it made SOX compliance easier. It was an era when people cared about SOX and HP still wanted 3000 customers to be able to meet the Sarbanes-Oxley protocols.
The final security enhancements to FTP/iX are in patches FTPHDG9 7.5, FTPHDH0 7.0 and FTPHDH1 6.5. The patches include:
- deny del, overwrite, rename
- chroot limiting cd, dir, put, get, mput, mget
- new enhanced semantics for 'noretrieve'
Patch IDs are:
FTPHDH1(A) for 6.5
FTPHDF5(A) for 7.0 ** note, this patch is for some general FTP fixes
FTPHDH0(A) for 7.0
FTPHDG9(A) for 7.5
08:26 PM in Hidden Value, History, News Outta HP | Permalink
Use our search engine to find 20 years
of HP 3000 news and articles
The comments to this entry are closed.


text:
from mangolds to whisky – Nant EstatePosted: May 8, 2013 Filed under: agriculture, beverages, humour | Tags: Bothwell, distillery, Hobart, mangold, mangold hurling, Nant Estate, whisky Leave a comment
Via The Mercury (Hobart) – 6 August 1889, a story of a large mangold (root vegetable) which had apparently taken the form of a human hand. The mangold photo here is from an English website dedicated to the fine sport of mangold hurling. Keep your tongue firmly in your cheek as you read!
The Nant Estate (where the disfigured mangold was discovered) is now home to the Nant Distillery, home of the only commercially operating water-powered flour mill in Australia. This now heritage site, an hour north of Hobart, produces single malt whisky.
Probably not a mangold in sight these days!


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When a global speciality chemicals and sustainable technologies organisation needed a safe fume cupboard solution to manage high heat loads it turned to Clean Air. The specialist in containment, fume cupboards and extract systems designed customised high heat load fume cupboards for Johnson Matthey's Technology Centre in Reading, UK. These bespoke fume cupboards exceeded the requirements for BS EN 14175 Parts 3, 6 and 7.
The brief
The analytical team at the Johnson Matthey Technology Centre regularly prepares samples using strong acids to achieve dissolution and enable precise and accurate determination of the sample composition. Large industrial hot plates are located in their fume cupboards and the heat from these disturbs the airflow in a standard fume cupboard, compromising containment.
"The safety of our scientists is of premium importance to us," says Martin Allwright, Technology Centre Site Services Manager. "We're creating the sustainable technologies of the future. The research, development and quality control we conduct in our laboratories often involves boiling solutions and evaporating concentrated acids.
"We needed a solution that would protect our scientists, offer them safe, generous working space and contribute to a healthy laboratory environment."
The Clean Air solution
Applying its considerable industry experience and technology expertise, the Clean Air design team created a solution that offers excellent fume containment. Modifying the premium APEX-2 fume cupboards to contain any potential leakage caused by buoyant hot air, the combination sash option was incorporated for added operator protection and flexible access.
Representatives from the Johnson Matthey Technology Centre were invited to Clean Air's in-house type-testing facility to witness the testing of their bespoke fume cupboards. Their own industrial hot plates were used during many of the tests to ensure the conditions in their laboratories were accurately replicated.
The result
The results of the fume cupboard type-testing exceeded BS EN 14175 Parts 3 and 6 when tested at ambient temperature and Part 7 for High Heat and Acidic Load. The key Robustness of Containment Protection Factor (PFR) reading was 926,000 at 5kw, and 674,000 using the Technology Centre's equipment.
"Working with us, Clean Air designed fume cupboards to address our needs and deliver evidence-based safety assurance," adds Martin. "Our scientists have confidence that they are operating in safe conditions in a potentially hazardous environment. The choice of combination sashes has provided an improved operator experience with more flexible and safer access into the working space and contributes to lowering our energy consumption. The system as installed has also reduced noise levels over the previous installation."
For more information on high heat load fume cupboards click here.
"Working with us, Clean Air designed fume cupboards to address our needs and deliver evidence-based safety assurance. Our scientists have confidence that they are operating in safe conditions in a potentially hazardous environment."
Martin Allwright, Johnson Matthey Technology Centre Site Services Manager
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